[Mg2+]o/[Ca2+]o determines Ca2+ response at fertilization: tuning of adult phenotype?

in Reproduction

Correspondence should be addressed to J-P Ozil; Email: Jean-Pierre.Ozil@inra.fr

Alteration of the postnatal phenotype has sparked great concern about the developmental impact of culture media used at fertilization. However, the mechanisms and compounds involved are yet to be determined. Here, we used the Ca2+ responses from mouse eggs fertilized by ICSI as a dynamic and quantitative marker to understand the role of compounds in egg functioning and establish possible correlations with adult phenotypes. We computed 134 Ca2+ responses from the first to the last oscillation in media with specific formulations. Analyses demonstrate that eggs generated two times as many Ca2+ oscillations in KSOM as in M16 media (18.8 ± 7.0 vs 9.2 ± 2.5). Moreover, the time increment of the delay between two consecutive oscillations, named TIbO, is the most sensitive coefficient characterizing the mechanism that paces Ca2+ oscillations once the egg has been fertilized. Neither doubling external free Ca2+ nor dispermic fertilization increased significantly the total number of Ca2+ oscillations. In contrast, removing Mg2+ from the M16 boosted Ca2+ oscillations to 54.0 ± 35.2. Hence, [Mg2+]o/[Ca2+]o appears to determine the number, duration and frequency of the Ca2+ oscillations. These changes were correlated with long-term effects. The rate of female’s growth was impacted with the ‘KSOM’ females having only half the fat deposit of ‘M16’ females. Moreover, adult animals issued from M16 had significantly smaller brain weight vs ‘KSOM’ and ‘control’ animals. TIbO is a new Ca2+ coefficient that gauges the very early functional impact of culture media. It offers the possibility of establishing correlations with postnatal consequences according to IVF medium formulation.

Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/154/5/675/suppl/DC1.

Abstract

Alteration of the postnatal phenotype has sparked great concern about the developmental impact of culture media used at fertilization. However, the mechanisms and compounds involved are yet to be determined. Here, we used the Ca2+ responses from mouse eggs fertilized by ICSI as a dynamic and quantitative marker to understand the role of compounds in egg functioning and establish possible correlations with adult phenotypes. We computed 134 Ca2+ responses from the first to the last oscillation in media with specific formulations. Analyses demonstrate that eggs generated two times as many Ca2+ oscillations in KSOM as in M16 media (18.8 ± 7.0 vs 9.2 ± 2.5). Moreover, the time increment of the delay between two consecutive oscillations, named TIbO, is the most sensitive coefficient characterizing the mechanism that paces Ca2+ oscillations once the egg has been fertilized. Neither doubling external free Ca2+ nor dispermic fertilization increased significantly the total number of Ca2+ oscillations. In contrast, removing Mg2+ from the M16 boosted Ca2+ oscillations to 54.0 ± 35.2. Hence, [Mg2+]o/[Ca2+]o appears to determine the number, duration and frequency of the Ca2+ oscillations. These changes were correlated with long-term effects. The rate of female’s growth was impacted with the ‘KSOM’ females having only half the fat deposit of ‘M16’ females. Moreover, adult animals issued from M16 had significantly smaller brain weight vs ‘KSOM’ and ‘control’ animals. TIbO is a new Ca2+ coefficient that gauges the very early functional impact of culture media. It offers the possibility of establishing correlations with postnatal consequences according to IVF medium formulation.

Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/154/5/675/suppl/DC1.

Introduction

In humans and animals, several reports demonstrate that the culture media used at fertilization affects the developmental potential and may have postnatal consequences (Fernandez-Gonzalez et al. 2004, Watkins et al. 2007, Donjacour et al. 2014, Kleijkers et al. 2014, Morbeck et al. 2014). Given the considerable use of in vitro fertilization (IVF) technologies in human, the elucidation of the developmental influence of the culture media remains a critical challenge.

In mammals, fertilization triggers a series of Ca2+ oscillations, the number, frequency and duration of which are highly variable (Saunders et al. 2002, Kurokawa & Fissore 2003, Vanden Meerschaut et al. 2013) and which have an impact on the developmental processes (Ozil et al. 2006, Yu et al. 2008).

In the present study, we used the ultra-sensitive nature of Ca2+ oscillations triggered by fertilization as a quantitative marker to understand how media formulation alters egg functioning and could correlate with postnatal outcome.

The frequency of the Ca2+ oscillations has been extensively used to characterize the Ca2+ response at fertilization in mammals. This variable is however not accurate enough to describe the egg's response because the occurrence of Ca2+ oscillations is neither strictly periodic nor chaotic. To assess the evolution of the time interval better, we used a linear regression method to define a new coefficient called TIbO (for time increments between oscillations) that most closely quantifies the time interval increase between two consecutive oscillations.

Considerable progress has been made in understanding how these Ca2+ oscillations are generated, maintained and terminated following fertilization (Miyazaki 1995, Kono et al. 1996, Politi et al. 2006, Swann & Lai 2013, Kashir et al. 2014, Dupont & Combettes 2016). It is well established that the Ca2+ response triggered by fertilization is determined by two decisive factors: firstly the dose of PlCζ brought into play by the incoming sperm that confers calcium-induced Ca2+ release (CICR) excitability to the egg (Rice et al. 2000, Saunders et al. 2007, Sanusi et al. 2015) and secondly the transmembrane Ca2+ influx that supports the persistence of Ca2+ oscillations (Mohri et al. 2001, Miao et al. 2012, Takahashi et al. 2013, Wakai et al. 2013).

In this context, to explore the sensitivity of the Ca2+ responses to various media formulations, we managed to fertilize a large number of mouse eggs by intra-cytoplasmic sperm injection (ICSI). We systematically recorded the whole Ca2+ response in the presence of M16 or KSOM medium, which have the same chemical compounds but in different proportions (Whittingham 1971, Biggers 1998). We then varied the concentration of a series of compounds such as [CaCl2]o, [MgSO4]o, [KH2PO4]o and [BSA]o to identify their impacts on the time interval between oscillations. We conducted analysis to detect if the evolution of the time interval measured by TIbO is correlated with the total number of Ca2+ oscillations, which provides a quantitative estimate of the work carried out by the egg (Ozil & Huneau 2001, Ducibella et al. 2002, Ozil et al. 2005, 2006, Ducibella & Fissore 2008) according to medium formulation.

Finally, we explored whether the Ca2+ responses set by M16 and KSOM media correlate with changes in postnatal growth and animal phenotypes.

Materials and methods

Ethics statement

Animal experiments were carried out in strict accordance with the recommendations of the Code for Methods and Welfare Considerations in Behavioral Research (Directive 86/609EC) and were approved by the Committee on the Ethics of Animal Experiments of the author’s institution, permit number 12165 INRA.

Gametes production and collection

Female mice (C57BL/6 × CBA; 7–11 weeks old, Janvier, Le Genest, France) were superovulated by i.p. injection of 8 IU of PMSG (Chronogest Intervet) followed 48 h later by injection of 7.5 IU of hCG (Chorulon Intervet). Metaphase II oocytes were collected from the oviducts 15–16 h after hCG injection, and then placed in 0.1% hyaluronidase (Sigma H-3506) in Hepes buffered M2 medium (Sigma M-7167) in a Petri dish, to remove the cumulus cells. After washing and selection, oocytes were put into M16 medium under mineral oil (Sigma-M8410) and incubated under an atmosphere of 5% CO2 in air at 37°C. When fertilized eggs were needed, females were caged overnight with F1 males. In vivo fertilized eggs were recovered 17 h after hCG injection and eggs that displayed two pronuclei (PNs) were subjected to experimental protocol. Spermatozoa from C57BL/6 × CBA males (8–12 weeks old, Janvier) were collected from the caudae epididymides and suspended in 2 mL of M16 (Kuretake et al. 1996).

Culture media

M16 and KSOM media were made without EDTA, antibiotics or amino acid (Table 1). ICSI media was made up of 134.0 mM KCl, 2.6 mM NaCl, BSA (4 g/L) and 0.6 µM cytochalasin D (Sigma C-8273). All inorganic and organic components were purchased from Sigma Chemical Company. When needed, NaCl was substituted for MgSO4 or KH2PO4 in the M16 formulation (Table 1).

Table 1

M16 and KSOM culture media formulation in mM.

Compounds
NaClCaCl2NaHCO3KClMgSO4KH2PO4NaLacNaPyrGlucoseBSA (g/L)
M16951.7254.81.21.2230.335.64
KSOM951.7252.50.20.35100.20.21
Difference0002.310.85130.135.43

M16 and KSOM have close formulations, but in M16, some components have higher concentration than that in KSOM. These might be responsible for the differences in Ca2+ responses between the two media. In bold, the differences in the concentration of media tested.

Intra-cytoplasmic sperm injection

Sperm heads were separated from their tails by applying ultrasonic pulse with the digital sonicator (Model DS 250 Ultrasonic Corporation, USA) (Kuretake et al. 1996). Before ICSI, MII oocytes were maintained for 15 min in M16 with cytochalasin D to make the plasma membrane more malleable during ICSI intervention. Oocytes and sperm heads were placed together into ICSI media deprived of Ca2+ ions under the stage of a NIKON TMD at room temperature. The micropipette containing one or two sperm head(s) were advanced through the oocyte plasma membrane and a puncture was made by using a piezo-pulse (Piezodrill Burleigh, USA) (Kimura & Yanagimachi 1995). After release of the sperm head(s) into the cytosol, just as the pipette was being pulled back out of the zona, the part of the plasma membrane, made fluent by cytochalasin, was pulled into the pipette and squeezed against the zona with the tip of the pipette to mechanically seal the plasma membrane in the absence of external Ca2+ (Fig. 1). Eggs were replaced in culture medium at 37°C in less than 3 min following ICSI. When Ca2+ recording was carried out, sperm injections were given in ICSI medium supplemented with 500 µM of Fura 2 Dextran (F3029 Molecular Probes Fisher Technology Lot No. 983882 with 1.6 dye/mol of Dextran).

Figure 1
Figure 1

Schematic representation of the experimental design. (Part 1) Oocytes were fertilized by ICSI. Ca2+ responses were recorded inside a microfluidic device until the cessation of Ca2+ oscillations. Eggs were checked for the presence of 2 or 3 PNs and then discarded. Ca2+ records were automatically analyzed. The photo shows details of the ICSI procedure. After sperm deposition, the plasma membrane, made fluent by cytochalasin, was squeezed against the zona in order to close the plasma membrane onto itself by mechanical pressure and thus avoid any ions influx into the cytosol during the membrane healing period. After ICSI, eggs are immediately placed in the culture medium at 37°C. (Part 2) Oocytes were fertilized by ICSI and cultured in the incubator in the presence of M16 or KSOM for 4 h. Eggs with 2 PNs were cultured before being transferred into female recipients at the 1-cell at D0 or 2-cell stage at D1 according to the availability of recipients. Control eggs were fertilized in vivo and collected at the time of PN formation i.e. 3–4 h after fertilization. They were cultured in KSOM and transferred into female recipients as described for experimental eggs. The rate of animal growth was recorded up to the 8th week of age.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Level of [free Ca2+] in culture media

The free Ca2+ concentrations were measured with a perfect ION Ca2+ ion-selective electrode connected to the Ph/Ionomètre S500 device from Mettler Toledo SAS France. Results are expressed as mean ± s.d. of at least 9 measurements of different aliquots of the same medium formulation.

Ca2+ signal recording

Two eggs in turn, held opposite one another in the microfluidic chamber by suction pipettes (Ozil & Swann 1995), were subjected to ICSI and then immediately washed by a super flux of M16 medium (10 µL/s) to remove the ICSI medium. The chamber was positioned in the viewing field of a Nikon TE2000 inverted microscope fitted with a Fluor 40× oil immersion objective (NIKON). The optical field was illuminated by a 75-W Xenon arc lamp and wavelengths were selected at 340 ± 5 nm and 380 ± 5 nm by using a Cairn OptoScan Monochromator. Light emitted from the Fura 2 Dextran loaded eggs was transmitted to an EMCCD Photometric camera (EVOLVE Roper Scientific, USA). Intracellular [Ca2+] levels were recorded in terms of a ratio of fluorescence (F340/F380) at 0.5 Hz from the minute following their deposition in the microfluidic chamber until the cessation of Ca2+ oscillations. The intensity of UV light was highly attenuated by neutral dense (ND) filters (×32) to avoid cell heating and perturbing the permeation of Ca2+ ions through the plasma membrane that manifests itself by a progressive acceleration of the Ca2+ oscillation during the time course of recording (personal observation). The permanent flow of culture media in the microfluidic chamber, 1.4 µL/s at 37°C ± 0.1, ensured constant refreshment of O2, CO2, pH, salt composition and carbohydrates under the zona pellucida. This makes it possible to keep eggs spiking throughout the period of egg activation with the [Ca2+]i resting line remaining horizontal and flat between two successive oscillations. This is a mandatory condition for automatic detection of the first rising data point at the root of every [Ca2+]i rise before Ca2+ release takes place. Eggs were sometimes observed for PN formation before cessation of Ca2+ oscillations and always after cessation. The number of independent replicates is half the total number of Ca2+ records, i.e. 67.

Automated Ca2+ signal analysis: coefficients and parameters definition

An exploratory data analysis of the Ca2+ response (EDA, NIST/SEMATECH e-Handbook of Statistical Methods http://www.itl.nist.gov/div898/handbook/eda/eda.htm) was conducted to find out what Ca2+ parameter was the most sensitive to changes in media formulation.

Every Ca2+ record was automatically analyzed using SigmaPlot 13.0 homemade macro scripts. Two parameters and one coefficient were used to characterize the whole Ca2+ response:

  • The total number of Ca2+ oscillations was counted by automatic peaks detection (Fig. 2A).
  • The duration of the Ca2+ response was computed by number of the data points between the initial rise of the first oscillation and the peak of the last oscillation divided by the acquisition frequency (0.5 Hz; Fig. 2A).
  • The linear regression coefficient ‘a’ between the row order of Ca2+ oscillations and the time interval between n and n + 1 oscillation as detailed in Fig. 2B (y = ax + b, with y = time interval between oscillations; x = order). The regression coefficient, named TIbO, most closely estimates the step of time that is added after every Ca2+ oscillation before the next oscillation pops up (Fig. 2B). The details of the algorithm will be published elsewhere.
Figure 2
Figure 2

Automated analysis of the Ca2+ response. (A) Example of the decomposition of a Ca2+ response following ICSI in a series of critical points. Every Ca2+ peak represents a single oscillation and is automatically labeled by a red star and counted (here 9 oscillations). The time interval between two consecutive oscillations corresponds to the number of data points counted between two consecutive minima identified by the macro script (red downward triangle) divided by the frequency of acquisition (0.5 Hz) (e.g. see the 6th time period). The total duration of the Ca2+ response is defined as the time elapsed between the lowest data point at the root of the first Ca2+ oscillation (first red downward triangle) and the last maximum (last red star). (B) Linear regression line (y = ax + b) between the rank order of the Ca2+ oscillations (x) and their respective time interval (y) shown by a red circle symbol. The regression coefficient ‘a’ is the coefficient that best reflects the increments of the time interval between two consecutive oscillations. It is called TIbO. The y-intercept b corresponds to the initial time interval. Given its linearity, TIbO and y-intercept can be estimated on a short time window encompassing a few number of oscillations. It can be used to predict the total number of oscillations (NofOp), the total duration (TDp) and the predicted frequency (Fp). For example, with TIbO = 187.533 s; y-intercept = 262.778; NofOp = f(TIbO) = 9.029 (computed with the parameters of the empirical function Fig. 7). Then, TDp =  = 10,803 s = 3 h 0 minwith Fp = (NofOp/TDp) * 3600 = 3.0 oscillations/h. The accuracy of the prediction relies on the R value of the linear regression and the quality of the Ca2+ recording (frequency of data point acquisition).

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Culture media and embryo treatment

Monospermic eggs fertilized by ICSI were immediately put into 40-µL drops of either M16 or KSOM and cultured for 4 h (Fig. 1). After 4 h, eggs with 2 PNs were cultured further in M16 until their transfer into pseudo-pregnant recipients either the day of the experiment at (D0) or the next day at the 2-cell stage (D1) depending on the number of recipients available at D0 and D1. In vivo–fertilized eggs used as control were cultured in KSOM until their transfer to a recipient at D0 or D1.

Embryo transfer

Female mice (SWISS, CD1, 8–12 weeks old, Janvier) were used as recipients. They were mated to vasectomized males and the morning of the experimental day or the next day, the recipients were checked for the presence of a vaginal plug. Eggs showing 2 PNs were transferred in groups of 8 to the left oviduct of female recipients either at the one-cell stage (D0) or at the two-cell stage (D1).

Postnatal phenotype

Offspring were weighed weekly beginning the first week after the day of birth. The pups were weaned at the 4th week and males and females were separated and placed in groups at a maximum of 5 in independent cages. At the 8th week of age, animals were killed by cervical dislocation. The livers, kidneys, hearts, lungs, brains and fat were dissected out and weighed.

Statistical analysis

The data were analyzed using SigmaPlot automation analysis procedures (SigmaPlot 13.0 software). The sizes of the groups in Tables 2 and 3 are fairly small (less than 30). Approximate normality of observations was assumed for the purposes of this exploratory analysis. For data Table 2, we applied unpaired t-test to test the differences between means without any correction for multiple comparisons in order to let the data speak for themselves and reveal their structure. For Table 3, we applied chi-square (χ2) test for analysis of contingency tables and Z-test analysis to compare the proportion of males vs females as indicated in the footnote. To detect whether two factors such as the culture media (three levels: M16, KSOM and control) and the day of transfer (two levels: D0 and D1) affect the animal growth until the 8th week of age, we used two-way ANOVA with all pairwise comparisons with Bonferroni t-test correction for every week and female and male animals separately (Table 4). The difference in mean of organ weights and organ:bodyweight ratios according to the medium formulations were analyzed by one-way ANOVA (three levels: M16, KSOM and control) with pairwise multiple comparison procedures and Bonferroni t-test correction for female and male animals separately (Table 5). Since the determinism of the long-term impact is not known, the results and the signification of the statistical tests in Tables 4 and 5 should be regarded as a trend that merits further researches peering into the programming role of the fertilization process.

Table 2

Calcium response parameters following fertilization by ICSI (monospermic or dispermic) in presence of M16, KSOM and various declinations of these two culture media formulations.

Culture media[Ca2+]free (mM)n of eggsMeann of oscillationsMean durationMean frequency (spike/h)Mean TIbO
M16 (1)1.16 ± 0.10a,c179.2 ± 2.5d,i,w3 h 19 ± 36 minj2.7 ± 0.4e,h,k3 min 18 ± 2 min 38f, l
KSOM (1)1.39 ± 0.11a,b1818.8 ± 7.0d,p,m,u3 h 35 ± 36 min5.1 ± 1.5e,g,n,q46 s ± 32 sf,o,r,v
M16 2 × [Ca2+]o (1)2.44 ± 0.23b,c1011.3 ± 4.2u3 h 25 ± 54 min3.6 ± 2g2 min 47 ± 2 min 11v
M16 (2)1.16 ± 0.10910.3 ± 3.02 h 49 ± 33 min3.6 ± 0.5h2 min 18 ± 1 min 24
KSOM (2)1.39 ± 0.111420.3 ± 7.43 h 32 ± 46 min5.6 ± 1.656 s ± 47 s
M16-MgSO4(1)1.12 ± 0.1171054.0 ± 35.2i4 h 14 ± 31 minj12.3 ± 7k12 s ± 9 sl
M16-KH2PO4(1)1.13 ± 0.11399.1 ± 2.13 h 0 ± 30 min3.0 ± 0.83 min 43 ± 2 min 39
KSOMX-M16(1)1.27 ± 0.1591410.6 ± 3.8m3 h 8 ± 46 min3.4 ± 1.0n3 min 20 ± 2 min 14o
M16X-KSOM(1)1.15 ± 0.1421412.3 ± 3.5p,w3 h 50 ± 35 min3.2 ± 0.7q2 min 11 ± 1 min 10r
M16-0-0-4(1)1.14 ± 0.1271057.8 ± 22.2s3 h 59 ± 45 min15.2 ± 7.78.2 s ± 3.8 st
M16-0-0-1(1)1.25 ± 0.132983.2 ± 28.2s4 h 16 ± 42 min19.3 ± 5.74 s ± 1.9 st

(1): monospermic, (2): dispermic; M16X-KSOM, corresponds to a M16 formulation with the [MgSO4] and [KH2PO4] from the KSOM. KSOMX-M16, corresponds to a KSOM formulation made with the [MgSO4] and [KH2PO4] from the M16. M16-0-0-4, corresponds to a M16 formulation without ([MgSO4] and [KH2PO4]) and 4 g/L BSA. M16-0-0-1, corresponds to a M16 formulation without ([MgSO4] and [KH2PO4]) and 1 g/L BSA. The same letter is used to denote statistical difference. a(***P < 0.001); b(***P < 0.001); c(***P < 0.001); d(***P < 0.001); e(***P < 0.001); f(***P < 0.001); g(*P = 0.031); h(***P < 0.001); i(***P < 0.001); j(*** P < 0.001); k(***P < 0.001); l(***P < 0.001); m(***P < 0.001); n(***P < 0.001); o(***P < 0.001); p(**P = 0.004); q(***P < 0.001); r(***P < 0.001); s(*P = 0.042); t(**P = 0.009); u(**P = 0.005); v(***P < 0.001); w(**P < 0.008). Diagrams Fig 4 and 6 reveal the enslavement of the parameters upon themselves.

Table 3

Survival rate to term for ‘M16’, ‘KSOM’ and ‘control’ groups.

Eggs transferredCleavage (%)No. recipientsNo. pregnant (%)Live pups from transferred (%)Live pups from pregnant (%)Gestational length ± s.d.Litter size ± s.d.♂/♀ ratioNo dead (%)
‘M16’ D0 (2PN)5674 (57.1)14 (25.0)14 (43.7)19.5 ± 0.53.5 ± 1.60.51
‘M16’ D1 (2C)160209 (45.0)28 (17.5)28 (38.9)20.2 ± 0.43.1 ± 0.71.171
‘M16’total216(98.2)2713 (48.2)42 (19.4)a42 (40.4)c20 ± 0.53.2 ± 1.10.902 (4.7)
‘KSOM’ D0 (2PN)104137 (53.8)24 (23.1)24 (42.9)19.9 ± 0.43.4 ± 2.311
‘KSOM’ D1 (2C)96128 (66.7)17 (17.7)17 (26.6)20.5 ± 0.52.1 ± 1.31.51
‘KSOM’total200(97.0)2515 (60.0)41 (20.5)b41 (34.2)d20 ± 0.52.7 ± 2.01.172 (4.8)
Control D0 (2PN)2433 (100)18 (75)18 (75)20 ± 06.0 ± 1.60.630
Control D1 (2C)2432 (66.6)8 (33.3)8 (50)20 ± 04 ± 11.670
CONTROL48(100)65 (83.3)26 (54.2)a, b26 (65.0)c, d20 ± 05.2 ± 1.90.860
Total4645833 (56.9)109 (23.5)109 (43.9)20 ± 0.53.3 ± 1.81.024 (3.7)

The same letter is used to denote statistical difference. a(***P < 0.001); b(***P < 0.001); c(*P = 0.014); d(***P < 0.001) by χ2 test. The proportion of litter size were not significantly different between groups, is presented in bold, with Z-test and Yates correction applied for calculations.

Table 4

Animal weight from the 1st to the 8th week of age according to 3 media, 2 days of transfer and 2 genders.

Stage/week1st2nd3rd4th5th6th7th8th
M16 femalesD0 (2PN); n = 96.30 ± 0.4210.17 ± 1.9713.19 ± 1.8717.15 ± 1.8419.67 ± 1.8620.94 ± 1.4522.33 ± 1.1422.56 ± 1.49
D1 (2C); n = 126.16 ± 0.9710.55 ± 1.3813.00 ± 1.7517.68 ± 1.6920.43 ± 1.5621.35 ± 1.3322.25 ± 1.3023.49 ± 1.81
Total; n = 216.22 ± 0.810.38 ± 1.613.09 ± 1.717.45 ± 1.720.10 ± 1.721.18 ± 1.422.28 ± 1.223.09 ± 1.7
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
KSOM femalesD0 (2N); n = 125.29 ± 0.498.79 ± 1.0011.36 ± 1.4415.28 ± 1.8618.29 ± 1.3819.49 ± 1.4620.19 ± 1.7821.08 ± 1.76
D1 (2C); n = 65.32 ± 0.478.61 ± 0.7210.96 ± 1.2115.28 ± 1.2118.2 ± 0.8018.77 ± 0.6819.67 ± 0.8720.36 ± 0.96
Total; n = 185.30 ± 0.58.73 ± 0.9a11.23 ± 1.315.28 ± 1.618.27 ± 1.219.25 ± 1.3b20.03 ± 1.520.84 ± 1.5
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
Control femalesD0 (2PN); n = 115.82 ± 0.569.48 ± 0.6812.35 ± 0.9116.56 ± 1.1819.55 ± 2.0020.93 ± 2.0621.69 ± 2.4822.57 ± 2.52
D1 (2C); n = 35.45 ± 0.469.69 ± 0.3912.31 ± 0.9916.88 ± 0.8719.74 ± 0.3520.80 ± 0.5921.74 ± 0.9222.08 ± 1.94
Total; n = 145.74 ± 0.59.52 ± 0.6a12.34 ± 0.916.63 ± 1.119.59 ± 1.820.90 ± 1.8b21.71 ± 2.222.46 ± 2.3
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
Statistical ≠ between media into female groupsM16 > KSOM (***P < 0.001)M16 > KSOM (***P < 0.001)M16 > KSOM (***P < 0.001)M16 > KSOM (***P < 0.001)M16 > KSOM (**P = 0.004)M16 > KSOM (***P < 0.001)M16 > KSOM (***P = 0.001)M16 > KSOM (**P = 0.002)
M16 malesD0 (2PN); n = 56.30 ± 0.3310.22 ± 1.0012.84 ± 1.1817.90 ± 2.1620.83 ± 2.2423.25 ± 2.5824.65 ± 2.9325.46 ± 3.16
D1 (2C); n = 145.69 ± 1.179.95 ± 1.4713.21 ± 1.8919.06 ± 2.0622.90 ± 2.5025.20 ± 2.6726.92 ± 2.9627.79 ± 3.19
Total; n = 195.86 ± 1.010.02 ± 1.313.11 ± 1.718.76 ± 2.122.36 ± 2.524.69 ± 2.726.32 ± 3.027.18 ± 3.3
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
KSOM malesD0 (2P); n = 125.69 ± 1.059.47 ± 1.9612.76 ± 2.8318.17 ± 3.2423.26 ± 2.0625.02 ± 2.4526.65 ± 2.3428.49 ± 2.18
D1 (2C) n = 94.99 ± 0.818.85 ± 1.2411.52 ± 1.9016.85 ± 1.8921.88 ± 1.2323.69 ± 1.5325.45 ± 1.8226.38 ± 1.87
Total; n = 215.39 ± 19.22 ± 1.612.23 ± 2.517.60 ± 2.822.67 ± 1.824.45 ± 2.226.13 ± 2.127.59 ± 2.3
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
Control malesD0 (2PN); n = 75.94 ± 0.629.84 ± 0.4713.43 ± 0.8319.79 ± 1.8824.30 ± 2.2726.60 ± 1.7527.49 ± 1.7829.11 ± 1.90
D1 (2C); n = 55.87 ± 0.5110.22 ± 0.3413.62 ± 0.5219.88 ± 1.0123.64 ± 1.7226.35 ± 1.6528.14 ± 1.0528.92 ± 1.15
Total; n = 125.92 ± 0.610.0 ± 0.513.50 ± 0.719.83 ± 1.524.02 ± 2.026.49 ± 1.627.76 ± 1.529.03 ± 1.6
Statistical ≠ between stage D0 and D1nsnsnsnsnsnsnsns
Statistical ≠ between media into male groupsnsnsnsCtrl > M16 (*P = 0.025)Ctrl > M16 (*P = 0.042)Ctrl > M16 (*P = 0.043)

Data were analyzed with two way ANOVA with all pairwise comparison with Bonferroni t-test correction for every week and female and male animals separately.

Table 5

Mean organ weight (g) and organ/body weight ratios of female (n = 12 for control; n = 7 for M16 and 13 for KSOM) and male offspring (n = 12 for control n = 7 for M16 and 10 for KSOM) at 8th week of age.

HeartLiverLungKidneyBrainFat
A. Female
 Organ weight (mg) noticed
  ‘Control’109.0 ± 10.81263.5 ± 306a140.9 ± 15.3285.0 ± 43.0483.4 ± 17.7193.3 ± 85.3
  ‘M16’115.2 ± 13.51188.9 ± 139148.9 ± 10.2305.2 ± 44.8449.5 ± 59.0285.2 ± 140.9b
  ‘KSOM’105.0 ± 6.11024.5 ± 107a136.1 ± 13.7281.0 ± 20.3473.7 ± 18.1135.3 ± 49.5b
 Organ:body weight ratio (×100)
  ‘Control’0.490 ± 0.0525.600 ± 0.9590.633 ± 0.0561.272 ± 0.1032.184 ± 0.2340.844 ± 0.300
  ‘M16’0.503 ± 0.0495.183 ± 0.4360.650 ± 0.0281.333 ± 0.1821.972 ± 0.300c1.233 ± 0.551d
  ‘KSOM’0.507 ± 0.0344.94 ± 0.410.658 ± 0.0741.357 ± 0.1052.292 ± 0.169c0.643 ± 0.200d
B. Male
 Organ weight (g)
  ‘Control’149.5 ± 10.71612.7 ± 234.5152.5 ± 13.8 g470.1 ± 68.9499.5 ± 25.8f, g549.7 ± 199.7
  ‘M16’136.0 ± 18.71510.4 ± 261.2166.4 ± 13.9e435.6 ± 82.7435.5 ± 40.8f607.6 ± 363.4
  ‘KSOM’137.3 ± 7.21564.7 ± 140.3150.1 ± 8.7e430.9 ± 43.05462.83 ± 17.76g481.7 ± 176.5
 Organ:body weight ratio (×100)
  ‘Control’0.515 ± 0.0245.552 ± 0.7220.525 ± 0.036h1.618 ± 0.2061.724 ± 0.104i1.903 ± 0.731
  ‘M16’0.480 ± 0.0485.300 ± 0.2650.590 ± 0.050h1.529 ± 0.1511.550 ± 0.195i2.049 ± 0.992
  ‘KSOM’0.504 ± 0.0395.726 ± 0.3930.546 ± 0.0191.581 ± 0.1851.698 ± 0.1061.756 ± 0.628

Statistical analysis for organ weight was performed by one-way ANOVA with pairwise multiple comparison procedures and Bonferroni t-test correction for female and male animals separately. Letters in bold indicate significant difference between groups; a(*P = 0.025); b(*P = 0.03); c(*P = 0.016); d(**P = 0.002); e(*P = 0.038); f(***P < 0.001); g(*P = 0.015); h(**P = 0.002); i(*P = 0.029). See the Radar Plot Fig. 9.

Results

Impact of the culture media formulation on the Ca2+ response

The Ca2+ responses in M16 and KSOM culture media were different

Two typical Ca2+ responses following monospermic fertilization by ICSI in M16 and KSOM are shown on Fig. 3A and B. The first oscillation always showed a series of very rapid dampened sinusoidal oscillations after the peak (Fig. 3).

Figure 3
Figure 3

Typical mono and dispermic Ca2+ responses in M16 and KSOM. Time zero corresponds to the start of Ca2+ recording in the microfluidic chamber occurring in less than 3 min following ICSI. The oscillations usually started in less than 1 min after the egg was held in the microfluidic chamber by pipette. The first oscillation is shown at higher temporal magnification on the left side of every record. A series of dampened sinusoidal super-oscillations can always be seen on the top of the first oscillation after an initial shoulder. We used the pattern of this first oscillation as a quality check of ICSI (see text). (A) Monospermic egg response in M16; (B) in KSOM; (C) dispermic egg response in M16; (D) dispermic egg response in KSOM. After a few hours, oscillations stop abruptly, sometimes with a slight increase of [Ca2+]i that appears insufficient to trigger the n + 1 oscillation. This is called abortion of the last oscillation on the record. The number of oscillations, TIbO parameter with R and the total duration are shown for every single record.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

The mean number of Ca2+ oscillations was twice as high in KSOM as in M16, i.e. 18.8 ± 7.0 vs 9.2 ± 2.5 respectively (P < 0.001; Table 2). The mean durations of the Ca2+ response were not significantly different (3 h 35 ± 36 min vs 3 h 19 ± 36 min respectively, Fig. 4A) but the mean frequencies were significantly different (5.1 ± 1.5 vs 2.7 ± 0.4; P < 0.001, Table 2).

Figure 4
Figure 4

Functional linkage between the mono and dispermic Ca2+ response parameters in M16 and KSOM (exploratory data analysis of the Ca2+ responses): (A) KSOM vs M16 responses. (B) M16 2 × [Ca2+] vs M16 and KSOM. (C) Dispermic and monospermic responses in M16. (D) Dispermic and monospermic responses in KSOM. The axes of the Radar Plot are in arbitrary units for each of the four parameters. The color lines represent the average values while the color areas represent the standard deviations. Stars represent the significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; nsP > 0.05. The key figures are detailed in Table 2. Note that the duration is the less variable parameter.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Regarding the evolution of the time interval between oscillations, Figs. 3A and B show that the time intervals between two consecutive oscillations were regularly incremented by a rather constant value. The mean value of TIbO coefficient (described above) most closely estimates this constant value, which corresponds to the mean step of time that regularly increments the time interval between two successive oscillations (TIbO value is shown for every record Fig. 3). The mean values of the TIbO coefficient were significantly different between the Ca2+ responses in KSOM and in M16 (46 s ± 32 s vs 3 min 18 ± 2 min 38; P < 0.001; Table 2). So, in average, the mean time interval between two consecutive oscillations was increased by a time step that is 4 times less in KSOM than in M16. The relative functional dependence of the parameters upon themselves according to the culture media is shown on the diagram (Fig. 4A).

Doubling external [Ca2+]o did not significantly change the Ca2+ response in M16

Doubling extracellular [Ca2+]o to 3.4 mM in M16 (Table 1) essentially doubled the free Ca2+ concentration (2.44 ± 0.23 mM vs 1.16 ± 0.10 mM; P < 0.001, Table 2). However, the mean number of Ca2+ oscillations was not significantly increased (11.3 ± 4.2 vs 9.2 ± 2.5, Fig. 4B), nor was the mean total duration of the responses (3 h 25 ± 54 min vs 3 h 19 ± 36 min, Fig. 4B) or the mean frequency that increased to 3.6 ± 2 from 2.7 ± 0.4 but still remaining significantly below the 5.1 ± 1.5 mean frequency in KSOM (P = 0.031; Fig. 4A and B).

Regarding the evolution of the time interval between oscillations, the means of TIbO were not significantly different (2 min 47 ± 2 min 11 vs 3 min 18 ± 2 min 38, Fig. 4B). The higher free Ca2+ concentration in KSOM was therefore not responsible for the difference in mean number of Ca2+ oscillations between KSOM and M16.

Dispermic fertilization had limited effect on the Ca2+ response in M16 or KSOM

With dispermic eggs, the mean number of Ca2+ oscillations and the mean duration of the Ca2+ responses (Fig. 3C and D) were not statistically different from their respective monospermic counterparts in M16 or KSOM (Fig. 4C, D and Table 2). However, in M16, dispermic fertilization increased significantly the mean frequency (3.6 ± 0.5 vs 2.7 ± 0.4; Fig. 4C), which is associated with a slight reduction of the mean duration (2 h 49 ± 33 min vs 3 h 19 ± 36 min; Fig. 4C). In both cases, the means of TIbO were not significantly different from their respective monospermic counterparts. Therefore, M16 and KSOM media both appear capable of cushioning the effect of dispermic ICSI but with higher intensity in M16 than in KSOM (Fig. 4C and D).

Effects of deprivation of Mg2+ or H2PO4 on the Ca2+ response are different

In M16 deprived of MgSO4 (M16-MgSO4), the mean number of Ca2+ oscillations increased dramatically from 9.2 ± 2.5 to 54.0 ± 35.2 (P < 0.001; Figs 5A and 6A) along with an increase in mean duration from 3 h 19 ± 36 min to 4 h 14 min ± 31 min (P < 0.001; Fig. 6A) and a significant increase in mean frequency from 2.7 ± 0.4 to 12.3 ± 7.1 (P < 0.001; Fig. 6A). The TIbO was decreased from 3 min 18 ± 2 min 38 to 12 s ± 9 s (P < 0.001, Fig. 6A). Therefore, the deprivation of Mg2+ in M16 increased by almost 5.7 times the number of Ca2+ oscillations, 1.27 times the total duration and decreased by 16 times the mean TIbO coefficient (Fig. 6A and Table 2).

Figure 5
Figure 5

Six typical Ca2+ signal responses following monospermic ICSI in various media formulations. (A) Upon replacement of M16 by M16-MgSO4, oscillations frequency starts rising with a concomitant decrease in Ca2+ peak amplitude, before slowing progressively with a coincident increase in Ca2+ peak amplitude and finally ending with a drop in Ca2+ peak amplitude. (B) Ca2+ response in M16-KH2PO4. The response was not different from the response in standard M16 (Table 2). (C) Ca2+ response in M16X-KSOM. The number of Ca2+ oscillations was slightly increased (Table 2). (D) Ca2+ response in KSOMX-M16, the number of Ca2+ oscillations was significantly reduced (Table 2). (E) Ca2+ response in M16-0-0-4. (F) Ca2+ response in M16-0-0-1. Note that in the absence of Mg2+ responses, (A, E and F) show higher number of oscillations and similar typical M-shape formats.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Figure 6
Figure 6

Functional linkage between the Ca2+ response parameters in various media formulations (exploratory data analysis of the Ca2+ responses): (A) M16 vs M16 without Mg2+. (B) M16 vs M16 without KH2PO4. (C) M16X-KSOM vs M16 and KSOM. (D) KSOMX-M16 vs KSOM and M16. Stars represent the significant differences; * P < 0.05; **P < 0.01; ***P < 0.001; nsP > 0.05. The key figures are detailed in Table 2.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Moreover, we can see that the pattern of the time courses of the peak amplitude of Ca2+ oscillations (Fig. 5A) took an M-shape with three phases: (i) the peak amplitude decreased while the frequency was rising; (ii) the peak amplitude re-increased while the frequency slowed down; (iii) the peak amplitude decreased again while the frequency continued to slow down before stopping. This pattern appeared typical of media deprived of Mg2+ (Fig. 5A, E and F), and in this case, PNs were observed before oscillations stopped (data not shown).

In contrast, deprivation of [KH2PO4]o in M16 (M16-KH2PO4) made no difference (Fig. 6B).

Reciprocal changes of [MgSO4]o and [KH2PO4]o between M16 and KSOM had different effects

To reveal any complex interactions between external Mg2+ and H2PO4 with some others components, we switched their concentrations between M16 and KSOM (Table 1). A new M16 was made with the [MgSO4] and [KH2PO4] from the KSOM that is called M16X-KSOM. Conjointly a new KSOM was made with the [MgSO4] and [KH2PO4] from the M16, that is called KSOMX-M16.

In KSOMX-M16, the means of the number of Ca2+ oscillations, frequency, duration and TIbO were not significantly different from those obtained in M16 (Fig. 6C and Table 2) but were significantly different from those obtained in KSOM (the mean number of Ca2+ oscillations decreased from 18.8 ± 7.0 to 10.6 ± 3.8; (P < 0.001); the mean frequency decreased from 5.1 ± 1.5 to 3.4 ± 1.0 (P < 0.001) and the mean of TIbO increased from 46 s ± 32 s to 3 min 20 ± 2 min 14 (P < 0.001); Fig. 6C). Hence, the combined increase of MgSO4 and KH2PO4 in KSOM to their M16 levels was sufficient to restore almost the typical M16 Ca2+ response (Fig. 6C).

In contrast with M16X-KSOM, the mean number of Ca2+ oscillations remained significantly lower than that in KSOM (12.3 ± 3.5 vs 18.8 ± 7.0) (P = 0.004, Fig. 6D) and significantly higher than that in M16 (12.3 ± 3.5 vs 9.2 ± 2.5 (P = 0.008), Fig. 6D). The mean duration was not significantly different (Fig. 6D), which made the frequency significantly lower than that in KSOM (3.2 ± 0.7 vs 5.1 ± 1.5; P < 0.001) but not significantly different from the frequency obtained in M16 (3.2 ± 0.7 vs 2.7 ± 0.4; P = 0.054). The mean TIbO remained significantly higher than the mean TIbO in KSOM (2 min 11 ± 1 min 10 vs 46 s ± 32 s (P < 0.001) but not significantly different from the TIbO obtained in M16 (2 min 11 ± 1 min 10 vs 3 min 18 s ± 2 min 38 s, Fig. 6D). Hence, the combined decrease of MgSO4 and KH2PO4 in M16 to their KSOM levels was insufficient to restore the typical KSOM Ca2+ response (Fig. 6D).

Thus, the reciprocal changes in [MgSO4] and [KH2PO4] between M16 and KSOM had different effects and suggest that other components might impact the Ca2+ response.

Change of [BSA]o impacted the Ca2+ response

To test the possible impact of BSA, the concentration of which is 4 times higher in M16 than that in KSOM (Table 1), we firstly removed both MgSO4 and KH2PO4 from the M16 in order to exacerbate the Ca2+ response and then we decreased the [BSA]o from 4 g/L to 1 g/L. We shall henceforth refer to such formulations as M16-0-0-4 and M16-0-0-1 respectively throughout the text.

The mean number of Ca2+ oscillations obtained with M16-0-0-4 (see record Fig. 5E) was not statistically different from those obtained with M16-MgSO4 (57.8 ± 22.2 vs 54.0 ± 35; Table 2). However, with M16-0-0-1 (see record Fig. 5F), the number of Ca2+ oscillations increased significantly from 57.8 ± 22.2 to 83.2 ± 28.2 (P = 0.042, Table 2); the duration and the frequency were not significantly different but the TIbO decreased significantly from 8.2 s ± 3.8 s to 4 s ± 1.9 s (P = 0.009; Table 2). These results suggest that [BSA]o has a slight but significant effect on the Ca2+ response when the Ca2+ response is exacerbated.

The TIbOs were always highly correlated with the number of Ca2+ oscillations whatever the interindividual variabilities of the Ca2+ responses

To better envision whether the TIbO coefficient correlates with the number of Ca2+ oscillations and can describe egg functioning whatever the culture media, we plotted a fitting line through all data (n = 134) with TIbO on the X-axis and the numbers of Ca2+ oscillations on the Y-axis (Fig. 7). The plot shows that the numbers of Ca2+ oscillations was a decreasing function of the TIbO with a high correlation coefficient (R = 95.3%). It showed a constant relationship between TIbO and the number of Ca2+ oscillations for every egg whatever their interindividual Ca2+ response variabilities (Fig. 7).

Figure 7
Figure 7

The TIbO coefficient is always correlated with the number of Ca2+ oscillations whatever the interindividual variability among the Ca2+ responses. All data were plotted on a Cartesian graph with TIbO coefficient on the X-axis and the total number of Ca2+ oscillations on the Y-axis. Every color-coded dot point is the cross point between the TIbO and the number of oscillations for every single egg (n = 134 eggs). For every experimental group (n = 11), the range of TIbO coefficients is represented in the bottom part of the graph by a color-coded line with the mean value as a dot point and the standard error of the mean by upward triangles. On the right side of these lines at the same level, are the name of the group and the average TIbO values. We can see that the projection of the TIbO coefficient along the X-axis for any egg inside any group, on the regression line (y = (a + b × x)/(1 + c × x + d × x ^ 2) with a = 191.455, b = 5.834, c = 0.494, d = 0.001) predicts with accuracy the number of Ca2+ oscillations that are displayed on the first column on the left to the Y-axis. The second column represents the experimental values ordered by increasing value from the bottom. We can see that the predictive values are very close to the experimental data. This graph makes it possible to see that the TIbO coefficient has predictive power whatever the interindividual variabilities of the egg functioning. In every culture medium formulation, the Ca2+ response appears determined by a common functional logic.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Developmental responses following ICSI in the presence of M16 and KSOM

Rate of egg activation and cleavage following ICSI

Among 777 oocytes subjected to monospermic ICSI and cultured in vitro in M16 or KSOM media, 653 survived (84%). No eggs swelled or fragmented when replaced immediately after ICSI in M16 or KSOM at 37°C. All surviving eggs formed PNs by 4 h following ICSI, but 53 of them (8.1%) displayed a single PN. The cleavage rate from the ‘M16’ and ‘KSOM’ groups to the 2-cell stage was respectively equal to 98.2% (160/163) and 97.0% (96/99) (Table 3). The cleavage rate of in vivo fertilized eggs cultured in KSOM was 100% (24/24; Table 3).

Rate of survival to term

Among ICSI eggs cultured in M16 (‘M16’ eggs), 216 eggs (2-PN or 2-cell) were transferred into 27 recipients. Thirteen of them (48.2%) gave birth to 42 live pups (40.4%) but 2 died within the first week.

Among ICSI eggs cultured in KSOM (‘KSOM’ eggs), 200 eggs (2-PN or 2-cell) were transferred into 25 recipients. Fifteen of them (60%) gave birth to 41 live pups (34.2%) but 2 died within the first week (Table 3).

Regarding in vivo–fertilized egg (‘control’ eggs), 48 eggs (2-PN or 2-cell) were transferred into 6 recipients. Five of them (83.3%) gave birth to 26 live pups (65.0%) (Table 3).

The rates of live pups from the number of eggs transferred were not significantly different between ‘M16’ and ‘KSOM’ but both of them were significantly lower than the ‘control’ (19.4% and 20.5% vs 54.2%; P < 0.001; χ2 test, Table 3), as well as the rate against the number of eggs transferred into the pregnant recipients (40.4% and 34.2% vs 65%; P < 0.001; χ2 test, Table 3). The mean litter sizes at birth between experimental ‘M16’, ‘KSOM’ and ‘control’ were not statistically different (3.2 ± 1.1; 2.7 ± 1.9 and 5.2 ± 1.7 respectively by Z-test; Table 3). Regarding the sex ratio, there is no significant difference between the proportions of males and females in every group (Z test). No statistical impact of the day of egg transfer i.e. D0 at 2PN stage or D1 at 2C stage on the rate of live pups or the pregnancy rates was detected (Table 3).

Postnatal growth until the 8th week of age

The growth of the ‘M16’ females from the 1st to the 8th week of age was always significantly higher than that of the ‘KSOM’ females whatever the day of transfer (D0 or D1; Table 4). However, the females issued from ‘control eggs’ that were fertilized in vivo but cultured in KSOM before their transfer had a higher growth rate than the females issued from the ‘KSOM eggs’ (see diagram Figs 1, 8A and Table 4). Regarding the males, the ‘control eggs’ tended to have a higher growth than ‘M16 eggs’ after week 3, but no statistical difference were detected between ‘KSOM’ and ‘M16’ groups (Fig. 8B and Table 4).

Figure 8
Figure 8

The postnatal growths. (A) The weights of female offspring are plotted with vertical bars representing the standard deviation of the data. The medium is represented by a color code. Solid lines represent the average growth of animals issued from transfer at both D0 and D1. Dashed lines represent the growth of animals issued from D0 or D1. Dashed lines and solid lines of the same color are confounded, showing that the prolonged culture period from D0 to D1 has no impact on the growth whatever the culture media. (B) Male growth with the same color code as in (A). Statistical tests were done for females and males separately with 2-way ANOVA (3 media × 2 stages) for every week with all pairwise multiple comparison procedures (Bonferroni t-test). Stars represent the level of statistical significance (α = 5%). The plots are from Table 4 data.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Organs allometry of mice at the 8th week of age

Post-mortem examination of selected organs (hearts, livers, lungs, kidneys, brains) and white fat around the kidneys and gonads at the 8th week showed that ‘M16’ females had more than twice the fat deposit than that measured in ‘KSOM’ females at the 8th week, i.e. 285.2 ± 140.9 vs 135.3 ± 49.5 mg respectively (Table 5). In contrast, we found that the ‘M16’ males had an average of 26% more fat deposit than the ‘KSOM’ males (but not statistically significant), and a significant increase in the lung:bodyweight ratio (Table 5). Interestingly, ‘M16’ females displayed significantly smaller brains and smaller brains relative to their body mass than ‘KSOM’ females while ‘M16’ males also displayed significantly smaller brains and smaller brains relative to their body mass than the ‘control’ (Fig. 9A, B and Table 5).

Figure 9
Figure 9

Normalized and centered polar plots of the relative organ:bodyweight ratio. (A) Female organs: ‘M16’ females display a significant increase in fat weight associated with a decrease in brain weight in comparison with the ‘KSOM’. (B) Male organs: ‘M16’ males display a significant increase in lung and a significant decrease in brain in comparison to the ‘Control’. The medium is represented by a color code. Data from females and males were centered-reduced together.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

Discussion

The near linear increment of the time delay between oscillations appears to be a common characteristics of the Ca2+ responses at fertilization in mice

Experimental requirements for analyzing the sensitivity of the Ca2+ response to external media

Exploiting the parameters of Ca2+ signaling as a quantitative measurement of egg functioning confronts, the high plasticity of the Ca2+ signaling machinery (Yu 2008, Wakai et al. 2011, Swann 2013) and its extreme sensitivity to perturbations due to methodologies and technical settings that can lead to misinterpretations (Swann 2013). We used a microfluidic chamber (Ozil & Swann 1995, Ozil et al. 2006) to keep eggs spiking in a constantly refreshed environment throughout the period of egg activation. Moreover, fertilization by ICSI is known to perturb Ca2+ homeostasis by causing extra Ca2+ influx due to the puncture of the plasma membrane (Tesarik & Sousa 1995, Sato et al. 1999, Yanagida et al. 2001). As a quality check of ICSI procedure, we systematically caught the initial rise of the Ca2+ oscillation. Then, if the initial Ca2+ oscillation appeared normal (Deguchi et al. 2000, Saunders et al. 2002, Ozil et al. 2005), i.e. always occurring within a similar time span observed after fertilization, i.e. 3–4 min following ICSI (Figs 3 and 5) (Lawrence et al. 1997), Ca2+ recording was validated and continued. With our ICSI protocol, this first large Ca2+ oscillation was always followed by a series of shorter transient Ca2+ oscillations (Figs 3 and 5) that also displayed a couple of super-oscillations on the top (data not shown). This experimental setting makes it possible to record the whole series of Ca2+ oscillations and discard artifactual responses due to ICSI or malfunctioning of the technical set-up.

Analysis of the Ca2+ responses in the presence of different media makes it possible to identify a new metrics: the TIbO parameter

The near linear increment of the time delay between oscillations appears to be a common characteristic of the Ca2+ responses at fertilization in mice, whatever the culture media diversity. This finding (see examples Fig. 2) suggests that the Ca2+ responses, once fertilization has occurred, have a predictable nature, which is dependent on the culture media. Furthermore, the fact that all eggs were activated clearly shows that medium formulation is capable of supporting higher numbers of Ca2+ oscillations than the minimum needed for the activation of the developmental processes (i.e. 4 oscillations in M16, data not shown). Moreover, the fact that an empirical regression line most closely describes the association between TIbO and the total number of Ca2+ oscillations (Fig. 7) strongly suggests that Ca2+ influx, which is known to be the trigger of Ca2+ oscillations (Miyazaki 2007, Takahashi et al. 2013) is faithfully coupled with an unknown mechanism that adds a delay in ascending order before triggering the next oscillation and causes cessation of oscillations when the time interval reaches a maximum. In addition, we can see that the characteristics of the TIbO coefficient with its y-intercept makes it possible to predict not only the total number of oscillations but also the total duration of the Ca2+ response and therefore the average frequency for every single record without the need of recording the whole Ca2+ response (see legend Fig. 2 for an example of calculation).

We exploited this new metrics to firstly gauge the functional changes induced by different medium and secondly to better understand what sort of Ca2+ mechanism determines the linkage between the TIbO, the number of Ca2+ oscillations, the total duration, the frequency and some compounds of the medium.

External [Mg2+] has a major effect on the time increment between Ca2+ oscillations (TIbO)

Surprisingly, doubling the free [Ca2+] in M16 from 1.16 to 2.44 mM (Table 2) did not significantly increase the mean frequency to the level displayed in KSOM because the means remained significantly different (3.6 ± 2 vs 5.6 ± 1.6 (P = 0.031); Fig. 4B). Moreover, dispermic fertilization, which is thought to double the amount of the PlCζ and increase the frequency as previously shown by Faure and coworkers did not significantly increase the mean number of oscillations and the mean duration in both the M16 and the KSOM (Fig. 4C and D); it increased the mean frequency in M16 (2.7 ± 0.4–3.6 ± 2; P < 0.001; Fig. 4C) but not in KSOM (Fig. 4D) (Faure et al. 1999). This shows that the effect of polyspermy on the Ca2+ response is media formulation dependent (see further below). However, regarding the pace, some reports have shown that whatever the degree of polyspermy, the frequency of Ca2+ oscillations tends to increase during Ca2+ recording (Faure et al. 1999). At this stage, it is difficult to understand why the Ca2+ response would accelerate before stopping after some time. In our hand, acceleration of Ca2+ oscillations only occurred when eggs were exposed to UV light that was not sufficiently attenuated by ND filters. In this case, Ca2+ oscillations started to accelerate slowly, they never stopped and very often the resting [Ca2+]i level increased and eggs ended up by dying suggesting that acceleration could originate from a detrimental effect of the UV light when the frequency of data acquisition is high; here, 0.5 Hz for several hours. Therefore, comparison of frequency with previous reports can be done when records did not display acceleration of Ca2+ oscillations. For example, our results fit well with a recent report showing that when ICSI eggs were subsequently injected with a single equivalent dose of PlCζ, about doubling the dose of PlCζ, the increase in number of Ca2+ oscillations in KSOM was only marginal (Sanusi et al. 2015).

Therefore, we can see that despite the extreme sensitivity of PlCζ to tiny [Ca2+]i elevation (Rice et al. 2000, Kouchi et al. 2005, Nomikos et al. 2005, Yu 2008, Swann 2013), neither the doubling of the free [Ca2+]o nor dispermic fertilization are capable of significantly getting over some unknown functional constraints imposed by the formulation of M16 or KSOM medium that make the Ca2+ responses different.

In contrast, when the Mg2+ ion was removed from the M16, the Ca2+ responses were dramatically changed (Table 2). These results draw attention to the role of external [Ca2+]o and [Mg2+]o and their ratio in controlling the Ca2+ response. Mg2+ is known to be a natural weak Ca2+ flux blocker (Iseri & French 1984, Lansman et al. 1986, Hartzell & White 1989) and well-known inhibitor of some members of the TRP cationic non-selective plasma membrane channels, which are expressed in mouse oocytes (Carvacho et al. 2013, 2016).

It has been proposed that Ca2+ influx mostly supports Ca2+ oscillations by ensuring the replenishment of the Ca2+ stores in advance of the next oscillation suggesting that refilling the Ca2+ stores during the time course of the Ca2+ response is the pacemaker of the oscillations (Takahashi et al. 2013, Wakai et al. 2013). The M-shape pattern of the time course of [Ca2+]i oscillations seen in Fig. 5A, E and F clearly shows that high frequency runs down the Ca2+ oscillation peak amplitude while subsequently, when the frequency is slowed down, the peak amplitude is restored. It thus appears that the mechanism that sets the pace might not require the full replenishment of the Ca2+ stores. In addition, the M-shape pattern suggests that when the intensity of the Ca2+ influx is not inhibited by external Mg2+, it can trigger oscillations with partially empty stores that release little Ca2+. It might also override the downregulation of the InsP3R channels by persistent Ca2+ oscillations that have been shown to be responsible of the cessation of Ca2+ oscillations (Brind et al. 2000, Malcuit et al. 2005, Lee et al. 2006) because here the total duration increased from 3 h 19 ± 36 min in M16 to 4 h 14 min ± 31 min in M16 without Mg2+ (Fig. 6A).

From these observations, we can conclude that the external [Mg2+]o/[Ca2+]o ratio has a critical function in controlling the pace of oscillations probably through the intensity of transmembrane Ca2+ influx.

In contrast, removing external H2PO4, which is a powerful Ca2+ complexing agent (Zoeteweij et al. 1993, Dutka et al. 2005, Allen et al. 2011), has no apparent impact on either the free Ca2+ in M16 or the Ca2+ response (Fig. 6B and Table 2), even when the Ca2+ response is exacerbated by the lack of Mg2+ in media (Fig. 5A, E and F and Table 2).

However, switching both [Mg2+]o and [KH2PO4]o between M16 and KSOM shows that M16X-KSOM cannot mimic KSOM response (Fig. 6D) while KSOMX-M16 does mimic M16 response (Fig. 6C). Therefore, it is possible that high external (BSA), which is 4 times higher in M16 than that in KSOM, can contribute to slow down the pace of the Ca2+ response by a still unknown mechanism (Fig. 5E, F and Table 2).

Overall, these observations strongly suggest that the free external [Mg2+]o/[Ca2+]o ratio is a strong determinant of the evolution in the time increment between oscillations and their number. It remains to be understood the mechanism by which external [Mg2+]o/[Ca2+]o ratio determines the parameters of the Ca2+ response.

External [Mg2+]o/[Ca2+]o ratio appears responsible for causing a cumulative delay in triggering Ca2+ oscillations

Regarding the role of Mg2+, some reports have demonstrated that Mg2+ ions that penetrate into the pore when the plasma membrane Ca2+ channel is activated can experience stabilization from ligand groups inside the channel (Lansman et al. 1986). Such intra-channel stabilization of Mg2+ ions might be responsible for the progressive blockage of Ca2+ permeation each time Ca2+ entry is stimulated following every Ca2+ oscillation (Mohri et al. 2001, Takahashi et al. 2013). Therefore, we hypothesize that accumulation of Mg2+ inside the channel lumen after every Ca2+ influx might cause a cumulative blockage of Ca2+ permeation from outside. More time would then be required to elevate the [Ca2+]i toward the threshold level above which Ca2+ oscillation is triggered (see diagram Fig. 10). The cessation of the Ca2+ oscillations might also correspond to the time when the intra channel pores have accumulated a sufficient amount of Mg2+ to fully block any Ca2+ permeation into the cytosol (diagram Fig. 10). In the absence of external Mg2+, the Ca2+ influx is not inhibited (Fig. 5A, E and F) and thus might override several mechanisms that are known to stop Ca2+ oscillations such as: (i) the depletion of Ca2+ stores (Takahashi et al. 2013, Wakai et al. 2013); (ii) the progressive downregulation of the desensitization of IP3R (Jellerette et al. 2000) and (iii) sequestration of PLCζ in PN (Marangos et al. 2003). Evidence of this precedence is the fact that oscillations still occurred when PNs were visible (data not shown).

Figure 10
Figure 10

A schematic diagram illustrating how external Mg2+ could progressively run down Ca2+ influx during the time course of the Ca2+ oscillations. In gray the diagram proposed by Swann (2013), Yu (2008) shows a positive feedback loop taking over the mechanism of oscillation when PLCζ is introduced into the egg at fertilization. In black two additional feedbacks upstream of PLCζ activation by the rise in [Ca2+]i; (1) a Ca2+ influx following the Ca2+ release from intracellular stores increases the [Ca2+]i. (2) Accumulation of external Mg2+ inside the lumen of the open Ca2+ channels might cause a progressive blockage of Ca2+ permeation, which postpones the occurrence of the next oscillation. We hypothesize that the downregulated intensity of the Ca2+ influx after every Ca2+ oscillation slows down the rise of the [Ca2+]i before the next oscillation. More time is required for the [Ca2+]i to reach the threshold beyond which Ca2+ oscillation is triggered. As a consequence, the pacemaker phase (in black) increases over time as shown by decomposing every phase of the Ca2+ oscillations from the Ca2+ record taken from Fig. 2 (see text). When Ca2+ permeation blockage through the channels reaches a maximum, Ca2+ oscillations stop. The hypothetical mechanism of ABCI might constitute a counting mechanism that is [Mg2+]o/[Ca2+]o ratio and Ca2+ channel property dependent.

Citation: Reproduction 154, 5; 10.1530/REP-16-0057

In mice, studies have indicated that divalent-selective ion channels such as TRPM7 and TRPM6 are involved in the control of Mg2+ homeostasis (Komiya et al. 2014) and are required for early development in the mouse (Liu et al. 2011, Carvacho et al. 2016). The overall results suggest that an accumulative blockage of Ca2+ influx (here named ABCI) induced by external Mg2+ might explain the time step that builds on the TIbO coefficient (see diagram Fig. 10). Moreover, it is possible that this singular phenomenon helps interpreting the fact that the slight increase in frequency caused by dispermic fertilization (Fig. 4C and D) correlates with a slight decrease in total duration. It is possible that any increase in frequency due to higher dose of PLCζ, other things being equal, will accelerate the termination of the Ca2+ response because the full blockage of the Ca2+ influx by Mg2+ will occur earlier due to the hypothetic ABCI mechanism as can be seen on Fig. 4C and D. It would be interesting to see whether the termination of the Ca2+ oscillations is associated with the saturation of divalent-selective ion channels by Mg2+. Another possible demonstration of the ABCI model will require measuring the evolution of the duration of the pacemaker phase that precedes every Ca2+ oscillation during the time course of the Ca2+ response (Fig. 10). However, regarding the large interindividual variabilities of the Ca2+ responses in all groups (Table 2), further studies should determine what the respective roles played by the sperm in delivering various dose of PLCζ are (Kashir et al. 2014) and that of the mature oocytes with their varying number of functional divalent-selective ion channels (Carvacho et al. 2016) that might contribute to building up the diversity of the Ca2+ responses at fertilization.

Do the development stages and adult phenotype correlate with the Ca2+ response at fertilization?

Developmental stages

In contrast to the change of the Ca2+ responses between M16 and KSOM, no difference was found in the rate of egg activation and cleavage to 2-cell or in the rate of live pups by pregnant recipients between the media (Table 3). Despite the fact that these rates of live pups were comparable with those obtained when ICSI eggs were transferred at the 2-cell stage (Kuretake et al. 1996, Lacham-Kaplan et al. 2003), the survival rate by eggs transferred remains significantly low compared with eggs fertilized in vivo but cultured in KSOM (19.4% and 20.5% vs 54.2% respectively, P < 0.001,Table 3) as well as the survival rate among the pregnant recipient (40.4% and 34.2% vs 65% respectively, Table 3). However, given the range of the number of Ca2+ oscillations in ‘M16’ (4–14) and ‘KSOM’ (9–31), it is impossible to determine if ‘M16’ or ‘KSOM’ offspring are issued from similar or different Ca2+ responses (the TIbOs overlap, Fig. 7). The lower survival rates are not in either case due to aberrant Ca2+ signaling induced by ICSI (Table 2). We assume that the use of cytochalasin for a couple of minutes at room temperature before ICSI that made the plasma membrane malleable or the short time spent in ICSI media (see ‘Materials and methods’ section) were not detrimental because the effect of cytochalasin is highly reversible (Balakier & Tarkowski 1976) and ICSI medium was instantly removed when eggs were placed in the microfluidic chamber at 37°C. It remains possible that other phenomena related to ICSI, not mirrored in the Ca2+ response, such as the delay between the full disintegration of the plasma and nuclear membranes of the sperm once injected into the cytosol (Kuretake et al. 1996, Knott et al. 2003, Yanagimachi 2005, Morozumi et al. 2006) have an impact. Further studies focusing on synchronization of Ca2+ oscillations at different frequencies with the remodeling of the sperm nucleus by using sequential media with different [Mg2+]o/[Ca2+]o ratios during the fertilization period might help understand the role of Ca2+ oscillations in sperm cell remodeling and perhaps improve the survival rate to term after ICSI.

Adult phenotype

The growth rates (Fig. 8 and Table 4) clearly show sex-specific effects of culture media, which have already been widely described and studied in the mouse (Fernandez-Gonzalez et al. 2004, Watkins et al. 2007, Banrezes et al. 2011, Donjacour et al. 2014, Feuer et al. 2014a) and in some reports in human following IVF (Ceelen et al. 2008a,b, Kleijkers et al. 2014). Though the present results contribute to narrowing the time window down to the first 4 h during which culture media affects the postnatal response in a sex-specific manner.

The experimental design (Fig. 1) demonstrates that the differences in the female growth rates were mostly induced by the nature of the external media during the short period between fertilization and PNs formation for the following reasons:

  • The differences in growth between ‘M16’ and ‘KSOM’ were due neither to the litter size, which were similar (Table 3) nor to the duration of the culture period in M16 after the PN stage because the growth rate issued from eggs transferred at D0 or D1 are similar in all cases (Table 4).
  • The differences between ‘M16’ and the ‘control’ were small (Fig. 8), showing that after PNs formation, the use of the M16 for experimental eggs or KSOM for ‘control’ for a limited time in culture had no apparent impact on the growth.
  • The difference between ‘M16’ and ‘KSOM’ groups is highly significant. Therefore, given points 1 and 2, it appears that the most sensitive time window to the formulation of the culture medium is the short time period between fertilization and the PNs stage.

Hence, these results confirm an earlier study that the period of egg activation is a very sensitive time window where any functional perturbation might have developmental consequences (Ozil et al. 2006, Ducibella & Fissore 2008) including the survival rate to term.

Regarding organ allometry, the present results show that the weight of several organs and the organ:bodyweight ratios issued from animals of the ‘M16’ group, in particular the brain, were significantly lower than the ‘control’ and the ‘KSOM’ group (Fig. 9 and Table 5). Regarding the female-specific fat deposit in the presence of M16 (Fig. 9 and Table 5), our observations are consistent with other reports on IVF of mice issued from eggs cultured in Whitten media (Feuer et al. 2014b). This group has shown that IVF and prolonged in vitro culture to blastocyst stage in Whitten media increases postnatal fat accumulation and glucose metabolism in female mice. They revealed broad changes in metabolic homeostasis characterized by systemic oxidative stress and mitochondrial dysfunction (Feuer et al. 2014b), but they did not record Ca2+ signaling at fertilization. At this stage, we can only suggest that these long-term impacts are mostly linked with the initial 4-h period following fertilization. A preliminary study on differential miRNA expression at PNs stage following fertilization by ICSI suggests that early transcription of maternal mRNA is indeed associated with the intensity of Ca2+ activity (Barrey et al. 2014) and reinforces previous suspicions regarding the role of Ca2+ oscillations at fertilization in gene expression (Ozil et al. 2006).

For this reason, any empirical intervention at fertilization in human IVF or prolonged in vitro culture should be avoided as previously mentioned (van Blerkom et al. 2015) until extensive scrutiny of long-term phenotypic and epigenetic consequences are carry out in mice or other animal models.

Conclusion and perspectives

The major findings of this study are: (i) the time intervals between oscillations increase almost linearly during the time course of egg activation; (ii) the TIbO coefficient that measures the time increments between two successive oscillations varies according to the external [Mg2+]o/[Ca2+]o ratio; (iii) the external Mg2+ might cause an accumulative blockage of Ca2+ influx (ABCI) during the time course of Ca2+ oscillations; (iv) the ABCI might constitute a reliable counting mechanism that stops oscillations when the incremental phenomena has totally blocked Ca2+ ions permeation by stabilization of Mg2+ inside the lumen of divalent-selective ion channels; (v) the adult phenotypes after fertilization by ICSI appear highly sensitive to culture media formulation during the short time window between the time of ICSI and the time of PNs formation.

In addition to providing new insight into the mechanism of Ca2+ oscillations, the apparent faithful mechanism of ABCI that sets the total numbers of Ca2+ oscillations, offers a physiological mean to tune at will the number of Ca2+ oscillations by varying external [Mg2+]o/[Ca2+]o ratio in media during egg activation. For this aim, we looked at the Ca2+ response induced by human IVF commercial media such as COOK and VITROLIFE. These modern media have differences in pyruvate, lactate and amino acids but higher [Mg2+] and lower [Ca2+] than M16 or KSOM (i.e. 1.5 mM Mg2+ and 1.1 mM Ca2+ for Cook and 1.7 mM Mg2+ and 1.1 mM Ca2+ for VITROLIFE (Morbeck et al. 2014). In our hands, COOK and VITROLIFE media generated 5 ± 0.8 oscillations (n = 12) and 5 ± 1.4 oscillations (n = 15) respectively (unpublished data). Therefore, it clearly appears that high concentration of Mg2+ locks up the number of Ca2+ oscillations to few Ca2+ oscillations with low variability, whereas removing Mg2+ will unleash the regime of Ca2+ oscillations with greater interindividual variability. Since the redox parameters have been shown to have postnatal impacts (Banrezes et al. 2011), the present results highlight the critical role of medium formulation during the period of fertilization. Yet, we do not know what the range of Ca2+ responses is when eggs are fertilized in vivo and why they have higher survival rate (Table 3). A very recent report shows that some in vivo–fertilized eggs with sperm derived from Plcz1/− males that fail to trigger Ca2+ oscillations can develop to term albeit at greatly reduced efficiency (Hachem et al. 2017). However, since it has been shown that low ionic strength media has the capability to trigger monotonic large Ca2+ release from internal stores in non-fertilized eggs (Ozil & Swann 1995), it might be possible that the oviductal fluid has some unknown but specific properties that make the Ca2+ response different from in vitro fertilization. As we have no access to the Ca2+ response in vivo, testing the survival rate and health of mice fertilized in vitro in media with various combinations of [Mg2+]o/[Ca2+]o and pyruvate/lactate ratio could make it possible to determine the best combination of compounds that optimizes the production of ATP (Campbell & Swann 2006) and sets the best NADH and FADH2 redox potential (Dumollard et al. 2009, Banrezes et al. 2011). Media formulations gauged by TIbO could become an active means to better understand the role of early Ca2+ oscillations and the mitochondrial response in the developmental processes, and probably improve the survival rate and health of ART children across the life course (Feuer & Rinaudo 2017).

It remains to be learned whether the ABCI mechanism is present in other animal and human species at fertilization.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research was supported in part by INRA department PHASE, Agence de Biomedicine (AMP, diagnostic prénatal et diagnostic génétique 2014 ‘Measurement of the metabolic impact of the culture media used for in vitro fertilization’) and FRM (Physiopathologie mitochondriale N° DPM 20121125544 to C Wrutniak).

Acknowledgments

The authors would like to thank Ryuzo Yanagimachi for his interest and stimulating discussions, Corinne Cotinot for critical reading of the manuscript and Donald White for English revision. They thank Jerome Pottier from the unit of Infectiologie Experimentale des Rongeurs et Poissons (UE IERP INRA Jouy) for excellent animal care. The authors are members of the COST Epiconcept Action FA1201 http://cost-epiconcept.eu.

References

  • AllenDGClugstonEPetersenYRoderIVChapmanBRudolfR 2011 Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue. Journal of Applied Physiology 111 358366. (doi:10.1152/japplphysiol.01404.2010)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • BalakierHTarkowskiAK 1976 Diploid parthenogenetic mouse embryos produced by heat-shock and Cytochalasin B. Journal of Embryology and Experimental Morphology 35 2539.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • BanrezesBSainte-BeuveTCanonESchultzRMCancelaJOzilJP 2011 Adult body weight is programmed by a redox-regulated and energy-dependent process during the pronuclear stage in mouse. PLoS ONE 6 e29388. (doi:10.1371/journal.pone.0029388)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • BarreyEBanrezesBSainte-BeuveTVaimanAOzilJ-P 2014 Early differential miRNA expression following in vitro fertilization of mouse eggs incubated in two standard culture media. Abstract book in FEBS Journal 281 (Supplement 1) 264; EMBO Meeting 30th August–4th September 2014 Paris.

    • Search Google Scholar
    • Export Citation
  • BiggersJD 1998 Reflections on the culture of the preimplantation embryo. International Journal of Developmental Biology 42 879884.

  • BrindSSwannKCarrollJ 2000 Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca(2+) or egg activation. Developmental Biology 223 251265. (doi:10.1006/dbio.2000.9728)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • CampbellKSwannK 2006 Ca2+ oscillations stimulate an ATP increase during fertilization of mouse eggs. Developmental Biology 298 225233. (doi:10.1016/j.ydbio.2006.06.032)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • CarvachoILeeHCFissoreRAClaphamDE 2013 TRPV3 channels mediate strontium-induced mouse-egg activation. Cell Reports 5 13751386. (doi:10.1016/j.celrep.2013.11.007)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • CarvachoIArdestaniGLeeHCMcGarveyKFissoreRALykke-HartmannK 2016 TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse. Scientific Reports 6 34236. (doi:10.1038/srep34236)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • CeelenMvan WeissenbruchMMVermeidenJPvan LeeuwenFEDelemarre-van de WaalHA 2008a Cardiometabolic differences in children born after in vitro fertilization: follow-up study. Journal of Clinical Endocrinology and Metabolism 93 16821688. (doi:10.1210/jc.2007-2432)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • CeelenMvan WeissenbruchMMVermeidenJPvan LeeuwenFEDelemarre-van de WaalHA 2008b Growth and development of children born after in vitro fertilization. Fertility and Sterility 90 16621673.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • DeguchiRShirakawaHOdaSMohriTMiyazakiS 2000 Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs. Developmental Biology 218 299313. (doi:10.1006/dbio.1999.9573)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • DonjacourALiuXLinWSimbulanRRinaudoPF 2014 In vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model. Biology of Reproduction 90 80.

    • Search Google Scholar
    • Export Citation
  • DucibellaTHuneauDAngelichioEXuZSchultzRMKopfGSFissoreRMadouxSOzilJP 2002 Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. Developmental Biology 250 280291. (doi:10.1006/dbio.2002.0788)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • DucibellaTFissoreR 2008 The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Developmental Biology 315 257279. (doi:10.1016/j.ydbio.2007.12.012)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • DumollardRCarrollJDuchenMRCampbellKSwannK 2009 Mitochondrial function and redox state in mammalian embryos. Seminars in Cell and Developmental Biology 20 346353. (doi:10.1016/j.semcdb.2008.12.013)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • DupontGCombettesL 2016 Fine tuning of cytosolic Ca (2+) oscillations. F1000Research 5 F1000.

  • DutkaTLColeLLambGD 2005 Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle. American Journal of Physiology: Cell Physiology 289 C1502C1512. (doi:10.1152/ajpcell.00273.2005)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • FaureJEMylesDGPrimakoffP 1999 The frequency of calcium oscillations in mouse eggs at fertilization is modulated by the number of fused sperm. Developmental Biology 213 370377. (doi:10.1006/dbio.1999.9388)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fernandez-GonzalezRMoreiraPBilbaoAJimenezAPerez-CrespoMRamirezMARodriguez De FonsecaFPintadoBGutierrez-AdanA 2004 Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. PNAS 101 58805885.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • FeuerSKDonjacourASimbulanRKLinWLiuXMaltepeERinaudoPF 2014a Sexually dimorphic effect of in vitro fertilization (IVF) on adult mouse fat and liver metabolomes. Endocrinology 155 45544567. (doi:10.1210/en.2014-1465)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • FeuerSKLiuXDonjacourALinWSimbulanRKGiritharanGPianeLDKolahiKAmeriKMaltepeE 2014b Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis. Endocrinology 155 19561969.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • FeuerSKRinaudoPF 2017 Physiological, metabolic and transcriptional postnatal phenotypes of in vitro fertilization (IVF) in the mouse. Journal of Developmental Origins of Health and Disease 8 403410.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • HachemAGodwinJRuasMLeeHCBuitragoMFArdestaniGBassettAFoxSNavarreteFde SutterP 2017 PLCzeta is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but offspring can be conceived in its absence. Development 144 29142924.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • HartzellHCWhiteRE 1989 Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes. Journal of General Physiology 94 745767. (doi:10.1085/jgp.94.4.745)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • IseriLTFrenchJH 1984 Magnesium: nature’s physiologic calcium blocker. American Heart Journal 108 188193. (doi:10.1016/0002-8703(84)90572-6)

  • JelleretteTHeCLWuHParysJBFissoreRA 2000 Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation. Developmental Biology 223 238250. (doi:10.1006/dbio.2000.9675)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KashirJNomikosMLaiFASwannK 2014 Sperm-induced Ca2+ release during egg activation in mammals. Biochemical and Biophysical Research Communications 450 12041211. (doi:10.1016/j.bbrc.2014.04.078)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KimuraYYanagimachiR 1995 Intracytoplasmic sperm injection in the mouse. Biology of Reproduction 52 709720. (doi:10.1095/biolreprod52.4.709)

  • KleijkersSHvan MontfoortAPSmitsLJViechtbauerWRoseboomTJNelissenECCoonenEDerhaagJGBastingsLSchreursIE 2014 IVF culture medium affects post-natal weight in humans during the first 2 years of life. Human Reproduction 29 661669. (doi:10.1093/humrep/deu025)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KnottJGKurokawaMFissoreRA 2003 Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization. Developmental Biology 260 536547. (doi:10.1016/S0012-1606(03)00251-3)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KomiyaYSuLTChenHCHabasRRunnelsLW 2014 Magnesium and embryonic development. Magnesium Research 27 18.

  • KonoTJonesKTBos-MikichAWhittinghamDGCarrollJ 1996 A cell cycle-associated change in Ca2+ releasing activity leads to the generation of Ca2+ transients in mouse embryos during the first mitotic division. Journal of Cell Biology 132 915923. (doi:10.1083/jcb.132.5.915)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KouchiZShikanoTNakamuraYShirakawaHFukamiKMiyazakiS 2005 The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Czeta. Journal of Biological Chemistry 280 21015–21021. (doi:10.1074/jbc.M412123200)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • KuretakeSKimuraYHoshiKYanagimachiR 1996 Fertilization and development of mouse oocytes injected with isolated sperm heads. Biology of Reproduction 55 789795. (doi:10.1095/biolreprod55.4.789)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • KurokawaMFissoreRA 2003 ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development. Molecular Human Reproduction 9 523533. (doi:10.1093/molehr/gag072)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lacham-KaplanOShawJSanchez-PartidaLGTrounsonA 2003 Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains. Biology of Reproduction 69 16831689. (doi:10.1095/biolreprod.103.018184)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • LansmanJBHessPTsienRW 1986 Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore. Journal of General Physiology 88 321347. (doi:10.1085/jgp.88.3.321)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • LawrenceYWhitakerMSwannK 1997 Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse. Development 124 233241.

  • LeeBVermassenEYoonSYVanderheydenVItoJAlfandariDDe SmedtHParysJBFissoreRA 2006 Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway. Development 133 43554365. (doi:10.1242/dev.02624)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • LiuWSuLTKhadkaDKMezzacappaCKomiyaYSatoAHabasRRunnelsLW 2011 TRPM7 regulates gastrulation during vertebrate embryogenesis. Developmental Biology 350 348357. (doi:10.1016/j.ydbio.2010.11.034)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MalcuitCKnottJGHeCWainwrightTParysJBRoblJMFissoreRA 2005 Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium release in type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine eggs. Biology of Reproduction 73 213. (doi:10.1095/biolreprod.104.037333)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MarangosPFitzHarrisGCarrollJ 2003 Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei. Development 130 14611472. (doi:10.1242/dev.00340)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MiaoYLSteinPJeffersonWNPadilla-BanksEWilliamsCJ 2012 Calcium influx-mediated signaling is required for complete mouse egg activation. PNAS 109 41694174. (doi:10.1073/pnas.1112333109)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • MiyazakiS 1995 Calcium signalling during mammalian fertilization. Ciba Foundation Symposium 188 235247.

  • MiyazakiS 2007 Intracellular calcium oscillations in mammalian eggs at fertilization. Journal of Physiology 584 713714. (doi:10.1113/jphysiol.2007.144238)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • MohriTShirakawaHOdaSSatoMSMikoshibaKMiyazakiS 2001 Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract. Cell Calcium 29 311325. (doi:10.1054/ceca.2000.0196)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • MorbeckDEKrisherRLHerrickJRBaumannNAMaternDMoyerT 2014 Composition of commercial media used for human embryo culture. Fertility and Sterility 102 759.e759766.e759. (doi:10.1016/j.fertnstert.2014.05.043)

    • Search Google Scholar
    • Export Citation
  • MorozumiKShikanoTMiyazakiSYanagimachiR 2006 Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development. PNAS 103 1766117666. (doi:10.1073/pnas.0608183103)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • NomikosMBlayneyLMLarmanMGCampbellKRossbachASaundersCMSwannKLaiFA 2005 Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations. Journal of Biological Chemistry 280 3101131018. (doi:10.1074/jbc.M500629200)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • OzilJPSwannK 1995 Stimulation of repetitive calcium transients in mouse eggs. Journal of Physiology 483 331346. (doi:10.1113/jphysiol.1995.sp020589)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • OzilJPHuneauD 2001 Activation of rabbit oocytes: the impact of the Ca2+ signal regime on development. Development 128 917928.

  • OzilJPMarkoulakiSTothSMatsonSBanrezesBKnottJGSchultzRMHuneauDDucibellaT 2005 Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse. Developmental Biology 282 3954. (doi:10.1016/j.ydbio.2005.02.035)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • OzilJPBanrezesBTothSPanHSchultzRM 2006 Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term. Developmental Biology 300 534544. (doi:10.1016/j.ydbio.2006.08.041)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • PolitiAGaspersLDThomasAPHoferT 2006 Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks. Biophysical Journal 90 31203133. (doi:10.1529/biophysj.105.072249)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • RiceAParringtonJJonesKTSwannK 2000 Mammalian sperm contain a Ca(2+)-sensitive phospholipase C activity that can generate InsP(3) from PIP(2) associated with intracellular organelles. Developmental Biology 228 125135. (doi:10.1006/dbio.2000.9929)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • SanusiRYuYNomikosMLaiFASwannK 2015 Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Czeta. Molecular Human Reproduction 21 783791. (doi:10.1093/molehr/gav042)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • SatoMSYoshitomoMMohriTMiyazakiS 1999 Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI). Cell Calcium 26 4958. (doi:10.1054/ceca.1999.0053)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • SaundersCMLarmanMGParringtonJCoxLJRoyseJBlayneyLMSwannKLaiFA 2002 PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 129 35333544.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • SaundersCMSwannKLaiFA 2007 PLCzeta, a sperm-specific PLC and its potential role in fertilization. Biochemical Society Symposia 74 2336.

  • SwannKYuY 2008 The dynamics of calcium oscillations that activate mammalian eggs. International Journal of Developmental Biology 52 585594. (doi:10.1387/ijdb.072530ks)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • SwannK 2013 Measuring Ca2+ oscillations in mammalian eggs. Methods in Molecular Biology 957 231248.

  • SwannKLaiFA 2013 PLCzeta and the initiation of Ca(2+) oscillations in fertilizing mammalian eggs. Cell Calcium 53 5562. (doi:10.1016/j.ceca.2012.11.001)

  • TakahashiTKikuchiTKidokoroYShirakawaH 2013 Ca(2)(+) influx-dependent refilling of intracellular Ca(2)(+) stores determines the frequency of Ca(2)(+) oscillations in fertilized mouse eggs. Biochemical and Biophysical Research Communications 430 6065. (doi:10.1016/j.bbrc.2012.11.024)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • TesarikJSousaM 1995 Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca2+ fluxes and oocyte cytoplasmic dislocation. Fertility and Sterility 64 770776. (doi:10.1016/S0015-0282(16)57853-4)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • van BlerkomJCohenJJohnsonM 2015 A plea for caution and more research in the ‘experimental’ use of ionophores in ICSI. Reproductive BioMedicine Online 30 323324. (doi:10.1016/j.rbmo.2015.02.002)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vanden MeerschautFLeybaertLNikiforakiDQianCHeindryckxBDe SutterP 2013 Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure. Human Reproduction 28 8798. (doi:10.1093/humrep/des368)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • WakaiTVanderheydenVFissoreRA 2011 Ca2+ signaling during mammalian fertilization: requirements, players, and adaptations. Cold Spring Harbor Perspectives in Biology 3 a006767.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • WakaiTZhangNVangheluwePFissoreRA 2013 Regulation of endoplasmic reticulum Ca(2+) oscillations in mammalian eggs. Journal of Cell Science 126 57145724. (doi:10.1242/jcs.136549)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • WatkinsAJPlattDPapenbrockTWilkinsAEckertJJKwongWYOsmondCHansonMFlemingTP 2007 Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure. PNAS 104 54495454.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • WhittinghamDG 1971 Culture of mouse ova. Journal of Reproduction and Fertility 14 7–21.

  • YanagidaKKatayoseHHirataSYazawaHHayashiSSatoA 2001 Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI. Human Reproduction 16 148152. (doi:10.1093/humrep/16.1.148)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • YanagimachiR 2005 Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals. Reproductive BioMedicine Online 10 247288. (doi:10.1016/S1472-6483(10)60947-9)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • YuYSaundersCMLaiFASwannK 2008 Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta. Human Reproduction 23 365373. (doi:10.1093/humrep/dem350)

    • Search Google Scholar
    • Export Citation
  • ZoeteweijJPvan de WaterBde BontHJMulderGJNagelkerkeJF 1993 Calcium-induced cytotoxicity in hepatocytes after exposure to extracellular ATP is dependent on inorganic phosphate. Effects on mitochondrial calcium. Journal of Biological Chemistry 268 33843388.

    • Search Google Scholar
    • Export Citation

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    Schematic representation of the experimental design. (Part 1) Oocytes were fertilized by ICSI. Ca2+ responses were recorded inside a microfluidic device until the cessation of Ca2+ oscillations. Eggs were checked for the presence of 2 or 3 PNs and then discarded. Ca2+ records were automatically analyzed. The photo shows details of the ICSI procedure. After sperm deposition, the plasma membrane, made fluent by cytochalasin, was squeezed against the zona in order to close the plasma membrane onto itself by mechanical pressure and thus avoid any ions influx into the cytosol during the membrane healing period. After ICSI, eggs are immediately placed in the culture medium at 37°C. (Part 2) Oocytes were fertilized by ICSI and cultured in the incubator in the presence of M16 or KSOM for 4 h. Eggs with 2 PNs were cultured before being transferred into female recipients at the 1-cell at D0 or 2-cell stage at D1 according to the availability of recipients. Control eggs were fertilized in vivo and collected at the time of PN formation i.e. 3–4 h after fertilization. They were cultured in KSOM and transferred into female recipients as described for experimental eggs. The rate of animal growth was recorded up to the 8th week of age.

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    Automated analysis of the Ca2+ response. (A) Example of the decomposition of a Ca2+ response following ICSI in a series of critical points. Every Ca2+ peak represents a single oscillation and is automatically labeled by a red star and counted (here 9 oscillations). The time interval between two consecutive oscillations corresponds to the number of data points counted between two consecutive minima identified by the macro script (red downward triangle) divided by the frequency of acquisition (0.5 Hz) (e.g. see the 6th time period). The total duration of the Ca2+ response is defined as the time elapsed between the lowest data point at the root of the first Ca2+ oscillation (first red downward triangle) and the last maximum (last red star). (B) Linear regression line (y = ax + b) between the rank order of the Ca2+ oscillations (x) and their respective time interval (y) shown by a red circle symbol. The regression coefficient ‘a’ is the coefficient that best reflects the increments of the time interval between two consecutive oscillations. It is called TIbO. The y-intercept b corresponds to the initial time interval. Given its linearity, TIbO and y-intercept can be estimated on a short time window encompassing a few number of oscillations. It can be used to predict the total number of oscillations (NofOp), the total duration (TDp) and the predicted frequency (Fp). For example, with TIbO = 187.533 s; y-intercept = 262.778; NofOp = f(TIbO) = 9.029 (computed with the parameters of the empirical function Fig. 7). Then, TDp =  = 10,803 s = 3 h 0 minwith Fp = (NofOp/TDp) * 3600 = 3.0 oscillations/h. The accuracy of the prediction relies on the R value of the linear regression and the quality of the Ca2+ recording (frequency of data point acquisition).

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    Typical mono and dispermic Ca2+ responses in M16 and KSOM. Time zero corresponds to the start of Ca2+ recording in the microfluidic chamber occurring in less than 3 min following ICSI. The oscillations usually started in less than 1 min after the egg was held in the microfluidic chamber by pipette. The first oscillation is shown at higher temporal magnification on the left side of every record. A series of dampened sinusoidal super-oscillations can always be seen on the top of the first oscillation after an initial shoulder. We used the pattern of this first oscillation as a quality check of ICSI (see text). (A) Monospermic egg response in M16; (B) in KSOM; (C) dispermic egg response in M16; (D) dispermic egg response in KSOM. After a few hours, oscillations stop abruptly, sometimes with a slight increase of [Ca2+]i that appears insufficient to trigger the n + 1 oscillation. This is called abortion of the last oscillation on the record. The number of oscillations, TIbO parameter with R and the total duration are shown for every single record.

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    Functional linkage between the mono and dispermic Ca2+ response parameters in M16 and KSOM (exploratory data analysis of the Ca2+ responses): (A) KSOM vs M16 responses. (B) M16 2 × [Ca2+] vs M16 and KSOM. (C) Dispermic and monospermic responses in M16. (D) Dispermic and monospermic responses in KSOM. The axes of the Radar Plot are in arbitrary units for each of the four parameters. The color lines represent the average values while the color areas represent the standard deviations. Stars represent the significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; nsP > 0.05. The key figures are detailed in Table 2. Note that the duration is the less variable parameter.

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    Six typical Ca2+ signal responses following monospermic ICSI in various media formulations. (A) Upon replacement of M16 by M16-MgSO4, oscillations frequency starts rising with a concomitant decrease in Ca2+ peak amplitude, before slowing progressively with a coincident increase in Ca2+ peak amplitude and finally ending with a drop in Ca2+ peak amplitude. (B) Ca2+ response in M16-KH2PO4. The response was not different from the response in standard M16 (Table 2). (C) Ca2+ response in M16X-KSOM. The number of Ca2+ oscillations was slightly increased (Table 2). (D) Ca2+ response in KSOMX-M16, the number of Ca2+ oscillations was significantly reduced (Table 2). (E) Ca2+ response in M16-0-0-4. (F) Ca2+ response in M16-0-0-1. Note that in the absence of Mg2+ responses, (A, E and F) show higher number of oscillations and similar typical M-shape formats.

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    Functional linkage between the Ca2+ response parameters in various media formulations (exploratory data analysis of the Ca2+ responses): (A) M16 vs M16 without Mg2+. (B) M16 vs M16 without KH2PO4. (C) M16X-KSOM vs M16 and KSOM. (D) KSOMX-M16 vs KSOM and M16. Stars represent the significant differences; * P < 0.05; **P < 0.01; ***P < 0.001; nsP > 0.05. The key figures are detailed in Table 2.

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    The TIbO coefficient is always correlated with the number of Ca2+ oscillations whatever the interindividual variability among the Ca2+ responses. All data were plotted on a Cartesian graph with TIbO coefficient on the X-axis and the total number of Ca2+ oscillations on the Y-axis. Every color-coded dot point is the cross point between the TIbO and the number of oscillations for every single egg (n = 134 eggs). For every experimental group (n = 11), the range of TIbO coefficients is represented in the bottom part of the graph by a color-coded line with the mean value as a dot point and the standard error of the mean by upward triangles. On the right side of these lines at the same level, are the name of the group and the average TIbO values. We can see that the projection of the TIbO coefficient along the X-axis for any egg inside any group, on the regression line (y = (a + b × x)/(1 + c × x + d × x ^ 2) with a = 191.455, b = 5.834, c = 0.494, d = 0.001) predicts with accuracy the number of Ca2+ oscillations that are displayed on the first column on the left to the Y-axis. The second column represents the experimental values ordered by increasing value from the bottom. We can see that the predictive values are very close to the experimental data. This graph makes it possible to see that the TIbO coefficient has predictive power whatever the interindividual variabilities of the egg functioning. In every culture medium formulation, the Ca2+ response appears determined by a common functional logic.

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    The postnatal growths. (A) The weights of female offspring are plotted with vertical bars representing the standard deviation of the data. The medium is represented by a color code. Solid lines represent the average growth of animals issued from transfer at both D0 and D1. Dashed lines represent the growth of animals issued from D0 or D1. Dashed lines and solid lines of the same color are confounded, showing that the prolonged culture period from D0 to D1 has no impact on the growth whatever the culture media. (B) Male growth with the same color code as in (A). Statistical tests were done for females and males separately with 2-way ANOVA (3 media × 2 stages) for every week with all pairwise multiple comparison procedures (Bonferroni t-test). Stars represent the level of statistical significance (α = 5%). The plots are from Table 4 data.

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    Normalized and centered polar plots of the relative organ:bodyweight ratio. (A) Female organs: ‘M16’ females display a significant increase in fat weight associated with a decrease in brain weight in comparison with the ‘KSOM’. (B) Male organs: ‘M16’ males display a significant increase in lung and a significant decrease in brain in comparison to the ‘Control’. The medium is represented by a color code. Data from females and males were centered-reduced together.

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    A schematic diagram illustrating how external Mg2+ could progressively run down Ca2+ influx during the time course of the Ca2+ oscillations. In gray the diagram proposed by Swann (2013), Yu (2008) shows a positive feedback loop taking over the mechanism of oscillation when PLCζ is introduced into the egg at fertilization. In black two additional feedbacks upstream of PLCζ activation by the rise in [Ca2+]i; (1) a Ca2+ influx following the Ca2+ release from intracellular stores increases the [Ca2+]i. (2) Accumulation of external Mg2+ inside the lumen of the open Ca2+ channels might cause a progressive blockage of Ca2+ permeation, which postpones the occurrence of the next oscillation. We hypothesize that the downregulated intensity of the Ca2+ influx after every Ca2+ oscillation slows down the rise of the [Ca2+]i before the next oscillation. More time is required for the [Ca2+]i to reach the threshold beyond which Ca2+ oscillation is triggered. As a consequence, the pacemaker phase (in black) increases over time as shown by decomposing every phase of the Ca2+ oscillations from the Ca2+ record taken from Fig. 2 (see text). When Ca2+ permeation blockage through the channels reaches a maximum, Ca2+ oscillations stop. The hypothetical mechanism of ABCI might constitute a counting mechanism that is [Mg2+]o/[Ca2+]o ratio and Ca2+ channel property dependent.