The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P4 affects bovine embryo development in vitro and in vivo. mRNA for P4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P4 on the embryo. Exposure to P4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.
The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle (Lamming & Royal 1999, Mann & Lamming 2001, Inskeep 2004, Stronge et al. 2005, McNeill et al. 2006) and sheep (Ashworth et al. 1989, Satterfield et al. 2006).
The effects of elevated P4 shortly after conception on the advancement of conceptus elongation have been convincingly demonstrated in cattle and sheep. Garrett et al. (1988) administered 100 mg P4 on Days 1, 2, 3, and 4 of pregnancy which resulted in an increased peripheral plasma P4 concentration on Days 2–5 and significantly larger conceptuses on Day 14. Using a P4 implant on Day 3 of pregnancy, Carter et al. (2008) significantly elevated P4 concentrations until Day 8 and this was associated with a larger conceptus recovered at slaughter on Day 16. Similarly, when ewes received daily injections of 25 mg P4 from 36 h postmating, blastocyst diameter increased by 220% on Day 9 and the time of elongation of blastocysts to a filamentous conceptus on Day 12 was advanced (Satterfield et al. 2006); these effects of P4 treatment on blastocyst development were blocked by administration of RU486, a P4 receptor (PGR) antagonist.
From the above, it is clear that the concentration of circulating P4 has an effect on the developing embryo. This effect is likely as a result of downstream effects of P4-induced changes in gene expression in the tissues of the uterus (Bauersachs et al. 2006, Satterfield et al. 2006, Forde et al. 2009) resulting in changes in the composition of histroph to which the developing embryo is exposed. Whether any of the effects of P4 is directly on the embryo or whether the observed advancement of development is mediated solely through changes induced in the endometrium is, as yet, unknown. In an attempt to answer this question, some authors have added P4 to embryo culture medium in vitro and examined development to the blastocyst stage. Results, however, have been varied and contradictory with some authors reporting positive effects of P4 (Ferguson et al. 2005, Merlo et al. 2007) while others have reported no effect (Reggio et al. 1997, Goff & Smith 1998). These contradictory observations may be partially explained by the culture system used, as steroids are known to be lipophilic and culture under oil, the system used in many laboratories, may result in depletion from the medium (Shimada et al. 2002).
Many of the actions of P4 are mediated through binding to its nuclear receptor (PGR). This classical PGR was originally characterized in the rodent uterus (Milgrom et al. 1970), and then shortly after in humans (Rao et al. 1974). It was subsequently shown to exist in two isoforms, PGRA and PGRB, both of which similarly bind P4 with high affinity (Lessey et al. 1983). Selective knockout experiments in mice have indicated that PGRA is both necessary and sufficient to elicit the P4-dependent reproductive responses necessary for female fertility while PGRB is required to elicit normal proliferative responses of the mammary gland to P4 (Conneely et al. 2002). Two related proteins with progestin binding activity were subsequently described, namely PGR membrane component (PGRMC) 1 and 2 (Falkenstein et al. 1996, Meyer et al. 1996, Gerdes et al. 1998). While only minimal information is available for PGRMC2, PGRMC1 has both high- and low-affinity P4 binding sites (Meyer et al. 1996), has a single transmembrane spanning domain, and attenuates apoptosis in cultured rat (Peluso et al. 2006) and human (Engmann et al. 2006) granulosa cells in response to P4 treatment. Whether these receptors function in bovine embryos is unknown.
Therefore, using ∼8500 bovine oocytes across six experiments, this study was designed to answer the following questions: 1) is there mRNA for the PGR on bovine embryos during early preimplantation development, a time when elevated P4 in vivo is associated with an advancement in post-hatching development? 2) does addition of P4 to the culture medium of bovine embryos under controlled conditions affect blastocyst development in vitro and subsequent embryo survival and elongation in vivo following transfer to synchronized recipient heifers? and 3) does elevated P4 in the early days of the estrous cycle prepare the endometrium and uterine environment to advance the elongation of the conceptus following transfer of blastocysts produced in vitro in the absence of P4?
PGR mRNA relative abundance in bovine embryos
PGR mRNA was detected at all stages of early embryo development except the morula stage (Fig. 1A). The relative abundance of PGR mRNA was highest in immature oocytes and lowest at the 2- to 4-cell and 16 cell stages. Both PGRMC1 and 2 were detected at all stages of development, decreasing from oocyte to embryonic genome activation and increasing at the blastocyst stage (Fig. 1B and C). A small number of mRNA copies of PGR were detected per blastocyst (10±2.7). For PGRMC1, the number of mRNA copies per blastocyst was higher (10 111±2548) while for PGRMC2 the number was intermediate (1366±160).
P4 concentration in the mid-luteal bovine oviduct
Mean P4 concentration in the oviduct flushings was 4.0±0.44 ng/ml (n=23). P4 was undetectable in the PBS used for flushing. In the stock solution used as a control for the assay, 17.29±1.02 ng/ml P4 was detected from the expected 20 ng/ml concentration.
Disappearance of P4 during culture
P4 concentration in synthetic oviduct fluid (SOF) on Day 1 (first day of in vitro embryo culture) was as expected (∼35 ng/ml). Culture in 25 μl droplets under mineral oil resulted in the rapid depletion of P4 such that by Day 3 it was barely detectable (0.1–1.6 ng/ml). When culture took place in a larger volume of medium (500 μl) in the absence of oil, P4 concentrations remained unchanged (39–40 ng/ml) throughout the 7 days of culture (Fig. 2A); however, in the presence of embryos the concentration decreased gradually over the subsequent 7 days such that by Day 7 approximately half of the original concentration remained (∼15 ng/ml; Fig. 2A). Similarly, the presence of a bovine oviduct epithelial cell (BOEC) monolayer resulted in the rapid depletion of P4 from the medium (Fig. 2B); when the medium was replenished every 2 days by removing 250 μl and replacing it with 250 μl medium containing the original concentration of P4, concentrations temporarily increased but rapidly returned to low levels (Fig. 2B). Based on these results, all embryo culture in subsequent experiments took place in 500 μl medium without oil overlay.
Effect of P4 supplementation on embryo development in vitro
Ethanol at the concentrations used to prepare the working solutions of P4 had no effect on any of the parameters studied. Addition of exogenous P4 at any of the concentrations tested did not affect cleavage rate or blastocyst development, irrespective of the absence (Table 1) or presence (Table 2) of a BOEC monolayer. Similarly, there was no evidence of any P4-induced difference in the relative transcript abundance of any of the genes studied in either blastocysts or the cultured oviduct cells (Fig. 3). The cell number and allocation (inner cell mass (ICM), trophectoderm (TE)) within Day 7 blastocysts was not affected by culture with P4 (Table 3).
Effect of supplementation with progesterone (P4) during in vitro culture in synthetic oviduct fluid on the cleavage rate and blastocyst yield of bovine embryos in vitro (Experiment 4, 9 replicates).
|N||Cleaved, n (mean±s.e.m.%)||Day 6, n (mean±s.e.m.%)||Day 7, n (mean±s.e.m.%)||Day 8, n (mean±s.e.m.%)|
|Control||410||315 (77.1±3.1)||58 (16.2±4.2)||121 (31.6±4.7)||138 (36.2±5.6)|
|0.1×10−7 M P4||402||323 (80.9±4.3)||66 (17.5±2.4)||118 (31.0±3.2)||147 (38.2±3.4)|
|1×10−7 M P4||408||329 (81.8±3.7)||61 (16.8±2.9)||127 (33.8±4.3)||152 (40.0±4.0)|
|10×10−7 M P4||407||329 (82.1±2.7)||71 (18.8±1.8)||138 (35.3±3.1)||154 (39.36±3.5)|
|Control (ethanol)||194||161 (82.9±2.7)||19 (9.5±4.5)||57 (29.1±5.7)||68 (34.9±4.5)|
Effect of supplementation with progesterone (P4) during in vitro culture in synthetic oviduct fluid in the presence of a bovine oviduct epithelial cell monolayer on the cleavage rate and blastocyst yield of bovine embryos in vitro (Experiment 4, 7 replicates).
|N||Cleaved, n (mean±s.e.m.%)||Day 7, n (mean±s.e.m.%)||Day 8, n (mean±s.e.m.%)||Day 9, n (mean±s.e.m.%)|
|Control||328||245 (74.3±2.6)||74 (25.6±4.9)||99 (30.2±5.2)||102 (31.2±4.9)|
|0.1×10−7 M P4||328||248 (75.1±3.0)||68 (20.6±1.1)||92 (28.1±1.7)||97 (29.6±1.6)|
|1×10−7 M P4||328||259 (78.5±1.9)||89 (27.3±2.0)||108 (33.1±2.0)||109 (33.4±1.8)|
|10×10−7 M P4||328||253 (76.8±1.9)||75 (22.8±1.9)||98 (29.9±2.3)||101 (30.9±2.2)|
Total, inner cell mass (ICM) and trophectoderm (TE) cell numbers (mean±s.e.m.) in Day 7 bovine blastocysts produced in vitro in the absence or presence of progesterone (1×10−7 M).
|No. of blastocysts||42||32|
|No ICM nuclei||34.33±1.15||36.19±1.83|
|No TE nuclei||110.95±4.30||101.72±5.30|
Effect of exposure to P4 in vitro on blastocyst elongation in vivo following embryo transfer
Data on the production of embryos in vitro in the absence or presence of progesterone for embryo transfer and on the recovery of embryos after transfer are shown in Table 4. As expected, there was no difference in the P4 profile of the recipient animals which received control or P4-treated embryos (Fig. 4A). The recovery rate of embryos on Day 14 was similar for both groups (control: 50.4±5.35% versus P4-treated: 65.0±6.75%; Table 4). There was large variation in conceptus size on Day 14, even within the same recipient (Fig. 5). Exogenous P4 in culture up to Day 7 had no effect on embryo dimensions on Day 14 (length: control: 3.80±0.46 mm, n=121 versus P4-treated: 4.46±0.53 mm, n=130; Fig. 4B).
Development of bovine blastocysts produced in vitro in the absence or presence of progesterone (P4, 10×10−7 M) following transfer (in groups of 20 blastocysts) to synchronized recipients on Day 7 and recovery on Day 14 (Experiment 5).
|N||Cleaved, n (mean±s.e.m.%)||Blastocyst yield, n (mean±s.e.m.%)||No. of recipients||No. of embryos recovered (recovery rate %)|
|Control||1659||1208 (72.7±1.91)||412 (24.6±1.60)||12||121 (50.4±5.35)|
|+P4||1876||1372 (72.9±2.08)||486 (25.7±1.46)||10||130 (65.0±6.75)|
Effect of elevated P4 in vivo prior to embryo transfer on blastocyst elongation
Data on the production of embryos in vitro for transfer to progesterone-primed recipients and on the recovery of embryos after transfer are shown in Table 5. Insertion of a P4 releasing intravaginal device (PRID) on Day 3 increased P4 concentration four- to fivefold within 24 h (Fig. 6A). The concentration remained significantly higher than control animals until Day 7 after which time concentrations were no longer different between control and treated heifers. Elevation of P4 prior to embryo transfer on Day 7 resulted in a significant increase in mean conceptus length and area on Day 14 compared to those recovered from control animals (length: control: 4.27±0.42 mm, n=112 versus P4-treated: 15.4±1.61 mm, n=79, P<0.001; Fig. 6B).
Development of bovine blastocysts produced in vitro in the absence of progesterone (P4) following transfer on Day 7 (in groups of 20 blastocysts) to synchronized recipients with normal or elevated P4 and recovery on Day 14 (Experiment 6).
|N||Cleaved, n (mean±s.e.m.%)||Blastocyst yield, n (mean±s.e.m.%)||No. of recipients||No. of embryos recovered, n (recovery rate %)|
|1817||1527 (84.2±0.92)||527 (29.7±1.53)||Normal P4||9||112 (61.7±5.89)|
|High P4||9||79 (43.9±7.49)|
Elevated progesterone was achieved by insertion of a PRID on Day 3 of the estrous cycle. See Materials and Methods for a detailed description.
The main findings of this study are i) mRNA for PGRs exists on the bovine embryo, raising the possibility of a direct effect of P4 on the embryo ii) supplementation with P4 during in vitro embryo culture does not affect the proportion of zygotes reaching the blastocyst stage in vitro, cell number and allocation within the blastocyst, mRNA abundance of selected transcripts or post-hatching elongation in vivo, iii) elevation of P4 in vivo prior to embryo transfer leads to a dramatic increase in conceptus length on Day 14. Taken together, these results suggest that the well described association between early elevation of P4 post conception and advanced conceptus length (Garrett et al. 1988, Satterfield et al. 2006, Carter et al. 2008) is not due to a direct effect of P4 on the embryo but rather due to P4-induced changes in the environment in which the embryo is developing. Furthermore, the early embryo does not need to be present in the uterus when P4 concentrations are elevated in order to benefit from their consequences.
Several studies have reported lower P4 concentrations in cows that failed to become pregnant although the degree and timing of the association between P4 concentrations and embryo survival are still unclear. For example, some authors have reported a positive association between milk or plasma P4 concentration and embryo survival on Days 4–6 of the estrous cycle (Green et al. 2005, McNeill et al. 2006) while others found no association until Days 14–17 (Chagas e Silva & Lopes da Costa 2005). Despite these inconsistent results there is an indication that low P4 concentrations during the early post-ovulatory luteal phase, or a delay in the normal rise in P4 at this time are associated with reduced embryo survival (Diskin & Morris 2008).
Expression of PGRs in embryos has been demonstrated in several species including mouse (Hou & Gorski 1993), pig (Ying et al. 2000) and horse (Rambags et al. 2008). To our knowledge, the present study shows for the first time PGR mRNA expression in bovine preimplantation embryos. However, the number of transcripts per embryo was low and the functionally is, as yet, unclear. Expression of PGRMC1 has previously been shown in bovine preimplantation embryos (Dode et al. 2006) but, to date, little is known about its function.
Culture of embryos in vitro in droplets of medium necessitates the use of oil to prevent evaporation. The problem of absorption of steroids by the oil overlaying the culture media drops is well known (Shimada et al. 2002, Zheng et al. 2003). One approach to overcome this problem is the pre-equilibration of oil with the appropriate steroid (Zheng et al. 2003). Alternatively, by using a high concentration of P4 at the beginning of incubation (almost 3000 ng/ml), the same authors reported that the capacity of the oil overlay to absorb the steroid was close to being saturated before the oocytes were added, thus sustaining an adequate concentration of between 211 and 48 ng/ml for the duration of oocyte culture (Zheng et al. 2003). In the present study, having established that P4 was depleted rapidly from the medium in the presence of oil, we chose to eliminate the oil completely to avoid any ambiguity in the results.
Irrespective of the concentration used, P4 supplementation during in vitro culture had no effect on the proportion of zygotes developing to the blastocyst stage or on the quality of the blastocysts as assessed in terms of cell number and allocation within the blastocyst or relative transcript abundance for a selected number of genes. Similarly, Pereira et al. (2009) failed to demonstrate a direct association between high P4 concentrations and embryo survival. This apparent lack of an effect on blastocyst development is consistent with previous observations from our group. For example, Carter et al. (2008), reported no effect on the recovery of blastocysts on Day 7 from single-ovulating animals with normal or elevated P4 concentrations despite a clear advancement of post hatching elongation. In addition, Rings et al. (2008) reported no effect on blastocyst yield following the endoscopic transfer of in vitro produced zygotes to the ipsilateral oviduct of heifers with normal or elevated P4 concentrations. Taken together, these results would strongly suggest that P4 is not associated with the ability of the oocyte to form a blastocyst.
Extending the above data, we addressed the question of whether an effect of P4 could be manifested post hatching by transferring blastocysts produced in the absence or presence of P4 to synchronized recipients. We chose Day 14 to slaughter the animals as it represents the initiation of elongation in cattle, a critical checkpoint in development. In addition, previous personal experience has shown that in the case of multiple transfers of embryos as carried out here, separating rapidly elongating conceptuses after Day 14 can be extremely difficult. Lastly, but perhaps most importantly, it is known that very little embryonic loss occurs after Day 14 in cattle (Dunne et al. 2000) and thus survival to Day 14 represents a useful measure of developmental competence. Consistent with the data on blastocyst formation described above, culture in the presence of P4 to the blastocyst stage and subsequent transfer to recipients did not lead to an advancement in conceptus length by Day 14. Blood samples from all recipients from Day 0 to Day 14 indicated no difference between those receiving control or P4-treated embryos, thus ruling out any potential effect of recipient P4 concentration on embryo development as previously described (Lonergan et al. 2007). These observations are perhaps not that surprising when one considers that it is possible to transfer embryos produced in vitro in the absence of P4, and achieve acceptable pregnancy rates.
Given that we have previously shown that elevating P4 in heifers in a manner identical to that described in the present study leads to a significant alteration in the transcriptome of the endometrium (Forde et al. 2009), the final experiment addressed the question of whether elevating P4 in vivo for a short window (Day 3–7) prior to embryo transfer would create an environment capable of advancing the development of blastocysts transferred to the uterus on Day 7. In other words, does the embryo need to be present during the period of elevated P4 in vivo in order to benefit from it? In contrast to the results of Experiment 5, transfer of blastocysts to a high P4 environment on Day 7 lead to a fourfold increase in conceptus length on Day 14. This provides clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle (Garrett et al. 1988, Carter et al. 2008) and sheep (Satterfield et al. 2006) and, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.
In conclusion, taking these results together with those from previous studies, it would appear that P4 either in vivo (Carter et al. 2008, Rings et al. 2008) or in vitro (present study) does not affect the ability of embryos to develop to the blastocyst stage in cattle. Furthermore, exposure to P4 in vitro does not affect the post-hatching elongation of the embryo following transfer to a recipient. In contrast, however, transfer of a blastocyst to a uterine environment previously primed by elevated P4 results in a significant advancement in conceptus elongation consistent with an increase in interferon-τ production and a stronger signal around the critical time of maternal recognition of pregnancy. Given our knowledge of P4-induced changes in endometrial gene expression, future studies will focus on the composition of uterine histotroph in an effort to identify secreted factors associated with the stimulation of conceptus growth.
Materials and Methods
All experimental procedures involving animals were licensed by the Department of Health and Children, Ireland, in accordance with the Cruelty to Animals Act (Ireland 1897) and European Community Directive 86/609/EC.
In vitro embryo production
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Company. The techniques for producing embryos in vitro have been previously described in detail (Rizos et al. 2002). Immature cumulus oocyte complexes (COCs) were obtained by aspirating follicles from the ovaries of heifers and cows collected at slaughter. COCs were matured for 24 h in TCM-199 supplemented with 10% (v/v) FCS and 10 ng/ml epidermal growth factor at 39 °C under an atmosphere of 5% CO2 in air with maximum humidity. For IVF, matured COCs were inseminated with frozen–thawed percoll-separated bull sperm at a concentration of 1×106 spermatozoa/ml. Gametes were co-incubated at 39 °C under an atmosphere of 5% CO2 in air with maximum humidity. Semen from the same bull was used for all experiments.
At ∼20 h post insemination (pi), presumptive zygotes were denuded, divided into groups of 40–50, and transferred to 500 μl culture wells. The basal medium for all embryo culture was SOF supplemented with 5% FCS. Depending on the experiment (see below), this was supplemented or not with P4 and/or with BOECs. Cleavage rate was recorded at 48 hpi and blastocyst development recorded at Days 6, 7, and 8 pi. All Day 7 blastocysts were snap frozen in groups of ten in liquid nitrogen and stored at −80 °C for mRNA extraction and real-time quantitative RT-PCR.
Preparation of BOEC monolayer
Ipsilateral oviducts were collected from slaughtered heifers at the mid-luteal phase of the estrous cycle and transported to the laboratory on ice. Each oviduct was flushed with 10 ml of SOF+5% FCS. Oviduct cells were centrifuged at 750 g for 9 min and the resulting pellet was washed by centrifugation at 100 g for 5 min in 5 ml of the same medium. The final pellet was re-suspended in culture medium and the cells separated by pipetting. Oviduct cells were counted in a hemocytometer and diluted in the appropriate volume of culture medium to give a final concentration of 1×106 cells/ml. Culture wells (500 μl) were then prepared from this solution and used for embryo culture 4–5 days later. Half of the media was replenished every 48 h before and during embryo culture.
Differential staining of blastocysts
Differential staining of ICM and TE cells was carried out using published procedures (Thouas et al. 2001) with minor modifications. For partial permeabilization and staining of TE, blastocysts were incubated in 300 μl of PBS with 0.2% Triton X-100 and 100 μg/ml propidium iodine in the dark for 50 s at room temperature. For fixation and total cell staining, blastocysts were then immediately transferred into 300 μl 100% ethanol containing 25 μg/ml bisbenzimide (Hoechst 33342) for 3 min at room temperature. Fixed and stained blastocysts were then transferred directly into glycerol, taking care to avoid carryover of excessive amounts of fixative solution. Finally, blastocysts were mounted onto a glass microscope slide in a drop of glycerol, gently flattened with a cover slip and visualized for cell counting under a fluorescence microscope.
Following collection, blood samples were refrigerated (4 °C) for 12–24 h before being centrifuged at 1500 g at 4 °C for 20 min. Serum was separated and stored at −20 °C until assayed to determine P4 concentration by solid phase RIA using a Coat-A-Count Progesterone kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). The sensitivity of the assay was 0.03 ng/ml. The intra- and interassay coefficients of variation for assays of P4 (low, medium, and high samples) were 8.5, 4.8, 4.9, 10, 4.9, and 5% respectively.
In order to determine P4 concentrations in spent culture media and in oviductal flushings an ELISA kit, prepared for human serum or plasma, (DE1561 DIMEDITEC Diagnostics GmbH, Kiel, Germany) was used. Inter and intra-assay coefficients of variation were 6.63 and 6.42% respectively.
RNA extraction, RT, and quantification of mRNA transcript abundance
For the analysis of the expression of mRNA for PGRs across development, RNA was extracted from pools of ten immature oocytes, 2- to 4-cell, 8-cell, 16-cell, morula and blastocyst stage embryos. To assess the effect of culture with P4 on mRNA abundance of candidate transcripts in blastocysts and in co-cultured oviduct cells, three pools of ten Day 7 blastocysts or oviduct cells from each experimental group were snap frozen.
Poly(A) RNA was prepared following the manufacturer's instructions using the Dynabeads mRNA Direct Extraction KIT (Dynal Biotech, Oslo, Norway). To take account of potential differences in RNA content between different developmental stages, after adding the lysis buffer, 0.1 pg rabbit globin mRNA (Sigma) was added to each tube as an internal standard. The RT reaction was carried out following the manufacturer's instructions (Promega) using poly(T) anchored primers and AMV reverse transcriptase enzyme in a volume of 20 μl to prime the RT reaction and to produce cDNA. Tubes were heated to 70 °C for 5 min to denature the secondary RNA structure and then the RT mix was completed with the addition of 5 units of Superscript RT enzyme. They were then incubated at room temperature for 10 min and then at 42 °C for 60 min to allow the RT of RNA, followed by 70 °C for 10 min to denature the RT enzyme.
The quantification of all mRNA transcripts was carried out by real-time quantitative PCR (qPCR) as previously described (Bermejo-Alvarez et al. 2008). Three replicate qPCR experiments were conducted for all genes of interest. To examine mRNA expression in blastocysts and BOEC, experiments were conducted to contrast relative levels of each transcript and histone (H2AFZ) in every sample. qPCR was performed by adding a 2.5 μl aliquot of each sample to the PCR mix containing the specific primers to amplify H2AFZ, BCL2-associated X protein (BAX), BCL2-like 1 (BCL2L1), Src homology 2 domain containing transforming protein C1 (SHC1SHC or P66), tumor protein p53 (TP53), genes related to apoptosis, thioredoxin (TXN), aldo-keto-reductase family 1-member 1 (AKR1B1), prostaglandin-endoperoxidase-2 (COX-2 or PTGS2), genes related to metabolism, placenta-specific 8 (PLAC8), related to implantation, and POU class 5 homeobox 1 (OCT3/4 or POU5F1) related to pluripotency. To compare mRNA expression of PGRs (PGR, PGRMC1, and PGRMC2) in embryos at different developmental stages, the experiments were conducted to contrast relative levels of each transcript and rabbit globin (HBA) in each sample. Primer sequences and the sizes of the amplified fragments of all transcripts are shown in Table 6. The comparative CT method was used to quantify expression levels (Rizos et al. 2007). Quantification was normalized to the endogenous control, H2AFZ (when expression between blastocysts and BOEC monolayer was carried out) or to an exogenous control (HBA; when expression between embryos at different developmental stages was carried out). Fluorescence was acquired in each cycle to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence increased above background for each sample. Within this region of the amplification curve, a difference of one cycle is equivalent to doubling of the amplified PCR product. According to the comparative CT method, the ΔCT value was determined by subtracting the control gene (H2AFZ or HBA) CT value for each sample from each gene CT value of the sample. Calculation of ΔΔCT involved using the highest sample ΔCT value (i.e. the sample with the lowest target expression) as an arbitrary constant to subtract from all other ΔCT sample values. Fold changes in the relative gene expression of the target were determined using the formula 2−ΔΔCT.
Details of primers used for quantitative real-time PCR.
|Gene symbol||MGI official name||Primer sequence (5′–3′)||Fragment size||GenBank accession no.|
|H2AFZ||H2A histone family, member Z||5′-AGGACGACTAGCCATGGACGTGTG||212|
|BAX||BCL2-associated X protein||5′-CTACTTTGCCAGCAAACTGG||158|
|SHC1SHC||Src homology 2 domain containing transforming protein c1 (P66)||5′-GTGAGGTCTGGGGAGAAGC||334|
|TP53||Tumor protein p53||5′-CTCAGTCCTCTGCCATACTA||364|
|AKR1B1||Aldo-keto-reductase family 1-member 1||5′-CGTGATCCCCAAGTCAGTGA||152|
|PTGS2||Prostaglandin-endoperoxide synthase 2 (COX2)||5′-ATCTACCCGCCTCATGTTCCT||187|
|POU5F1||POU class 5 homeobox 1 (OCT3/4)||5′-CGAGTATCGAGAACCGAGTG||440|
|PGRMC1||Progesterone receptor membrane component 1||5′-TGTGTGTCACAAATCCAGAAAG||265|
|PGRMC2||Progesterone receptor membrane component 2||5′-AGGAGAGGTGAGGGTGTCCATC||206|
PGR mRNA copy number quantification in Day 7 blastocysts
In addition to the above, an absolute quantification method was used to measure the presence and copy number of PGR, PGRMC1, and PGRMC2 in Day 7 blastocysts. Three groups of ten blastocysts from different experimental replicates were used. Quantification of PGR mRNA copy number in each sample was done using a standard curve for each receptor type. To generate the copy number standard curve for cDNA standards, PGR cDNA was amplified by PCR from mRNA purified from BOECs. PCR products were purified from an agarose gel according to the manufacturer's protocol (Elu-quick DNA purification kit, Whatman, Stanford, ME, USA). Following the confirmation of the PCR product by ethidium bromide-stained 2% agarose gel electrophoresis, the expected fragments were eluted and the concentration of the PCR fragments was determined spectrophotometrically by measuring absorbance at 260 nm. The standards in triplicate and cDNA samples (equivalent to 0.66 embryos per sample) were then co-amplified in the same reaction prepared from a master mix. Copy numbers of fragments were empirically determined using the following equation: copy number of PCR standard=mole×Avogadro Constant=grams/fragment molecular weight×Avogadro Constant (Avogadro Constant=6.0221367×1023). The quantified PCR fragments were later serially diluted in five 10-fold serial-dilutions increments and were used as known copy-number template standards in qPCR. Fluorescence was acquired at each cycle in order to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence rises above background for each sample. The Rotor-Gene quantification software generates a best-fit line and determines unknown concentrations by interpolating the noise-band intercept of an unknown sample against the standard curve of known concentrations. Reverse-transcriptase negative controls (samples containing RNA to which reverse transcriptase was not added) were included to exclude PCR amplification of contaminating genomic DNA.
Data were analyzed using the SigmaStat (Jandel Scientific, San Rafael, CA, USA) software package. One-way repeated-measures ANOVA (followed by multiple pair-wise comparisons using Student–Newman–Kleus method) was used for the analysis of differences in embryo development (cleavage rate, blastocyst yield), ratio of ICM to TE cells, recovery rate of embryos on Day 14 and also for the analysis of differences in mRNA expression. Blastocyst cell numbers, serum P4, and embryo dimensions on Day 14 were compared with Students t-test.
Experiment 1: PGR mRNA in bovine embryos
The aim of this experiment was to determine the presence of mRNA for three PGRs (PGR, PGRMC1, and PGRMC2) during early bovine embryo development. Three groups of ten immature oocytes and in vitro produced bovine embryos were removed from culture at the 2- to 4-cell, 8-cell, 16-cell, morula and blastocyst stage, snap-frozen in groups of 10 in liquid nitrogen and stored at −80 °C until analysis. In addition, three groups of ten blastocysts from different experimental replicates were used to determine the copy number of PGR, PGRMC1, and PGRMC2 in Day 7 blastocysts.
Experiment 2: P4 concentration in bovine oviduct flushings
In order to get an estimate of the concentration of P4 to which the embryo might normally be exposed in vivo (as opposed to serum concentrations), bovine oviducts (n=23) were collected from animals slaughtered at the mid-luteal phase of the estrous cycle (assessed by the presence of a large functional corpus luteum) and transferred on ice to the laboratory. All oviducts were trimmed and flushed slowly with 2 ml PBS; to maximize P4 recovery, the PBS was left in the oviduct for up to 5 min before recovery. P4 concentration was determined as described above.
Experiment 3: effect of culture system on P4 concentration in culture medium
It is known that steroids are lipophilic in nature and use of oil during culture can result in their depletion from the medium (Shimada et al. 2002). Before progressing to experiments examining the addition of P4 to culture on embryo development, the aim of this experiment was to assess the effect of culture system on the rate of disappearance of P4 from culture medium. Following addition of a known quantity (1×10−7 M) of P4 to the culture medium on Day 1, the concentration remaining on Days 3, 5, and 7 of culture was assessed. Culture conditions tested included: i) 25 μl droplets of SOF under mineral oil, the system of culture routinely used on our laboratory ii) 500 μl SOF in wells (without embryos), iii) 500 μl SOF in wells with embryos, iv) 500 μl SOF in co-culture with BOEC (without embryos) and v) 500 μl SOF in co-culture with BOEC with replacement of half of the media every 2 days (without embryos).
Experiment 4: effect of P4 supplementation of culture medium on embryo development and quality
The aim of this experiment was to determine the effect of exogenous P4 added during in vitro culture on the proportion of embryos reaching the blastocyst stage and on blastocyst quality measured as the relative mRNA abundance of selected genes related to quality. Bovine zygotes produced by IVM/IVF were cultured in SOF alone (n=1627; 9 replicates) or in co-culture with BOEC (n=1312; 7 replicates) in the absence or presence of P4 at one of three concentrations (0.1, 1 or 10×10−7 M, equivalent to 3.15, 31.5, and 315 ng/ml respectively). A vehicle control containing an equivalent amount of ethanol used to prepare the P4 stock solution was included initially to eliminate the effect of ethanol (n=194). Day 7 blastocysts from each group were snap-frozen in liquid nitrogen in groups of 10 for mRNA extraction. Also, BOEC from each group were isolated on Day 7 using 100 μl lysis buffer and snap-frozen for mRNA extraction. Quantification of transcripts for BAX, BCL2L1, SHC1SHC, TP53, TXN, AKR1B1, COX2, PLAC8, and POU5F1 was carried out by real-time qRT-PCR. These genes have been previously associated with likelihood of pregnancy after embryo transfer in cattle (El-Sayed et al. 2006).
Experiment 5: effect of exposure to P4 in vitro on survival and elongation of blastocysts in vivo following embryo transfer
As mentioned above, there is clear evidence for an association between circulating P4 concentration and advanced conceptus elongation (Garrett et al. 1988, Carter et al. 2008). In order to test the hypothesis that exposure to P4 during early development in vitro (zygote to blastocyst stage) would affect post-hatching elongation, zygotes produced by IVM/IVF were cultured in the absence (n=1659) or presence (n=1876) of 10×10−7 M P4 (4 replicates). Although higher than normal physiological concentrations, this dose was chosen to maximize the likelihood of observing an effect if one should exist. Based on the results of Experiment 3, medium was replenished every 2 days to maintain P4 concentrations. Blastocyst development was recorded and Day 7 blastocysts (n=440) were transferred to the uteri of synchronized recipients (20 blastocysts per recipient). Twenty-two crossbred beef heifers were used as recipients (predominantly Charolais or Limousin cross; 18–24 months old). Animals were housed indoors on slats for the duration of the experiment and were fed a diet consisting of grass and maize silage supplemented with a standard beef ration. Estrous cycles were synchronized by administration of two i.m. injections of the prostaglandin F2α analogue, cloprostenol (PG; Estrumate; Chanelle, Loughrea, Galway, Ireland), 11 days apart. Standing estrus was defined as Day 0. A total of 20 in vitro-produced blastocysts produced in the presence or absence of P4 were transferred to each recipient on Day 7 (control, n=12; P4-treated, n=10) and all recipients were slaughtered on Day 14 to assess embryo survival. A daily blood sample was taken from all animals by jugular venipuncture from Day 0 to Day 14 to establish the recipient P4 profile in order to ensure that all animals had similar endogenous P4 concentrations. Following slaughter, the reproductive tract was removed, sealed in a plastic bag and placed in a sealed polystyrene box for transportation to the laboratory (within 60 min). After removal of the ovaries and the oviducts the uterine horns were trimmed free of excess tissue before being flushed with 40 ml PBS. Embryos were located under a stereomicroscope, measured and then snap frozen individually in liquid nitrogen and stored at −80 °C. From each replicate a representative number of Day 7 blastocysts from both groups was processed as described above for assessment of cell number (control: n=42; P4: n=32).
Experiment 6: effect of elevated P4 in vivo on survival and elongation of bovine blastocysts following embryo transfer
In order to test the hypothesis that a modified uterine environment induced by artificially elevated circulating P4 is capable of advancing the post-hatching elongation of Day 7 blastocysts, groups of 20 blastocysts produced in vitro in the absence of P4 were transferred to synchronized recipient heifers with normal (n=9) or elevated (n=9) P4 concentrations. To obtain divergent P4 concentrations in heifers, a validated model was used where approximately half of the animals received a PRID (containing 1.55 g P4; CEVA Animal Health Ltd, Chesham, UK) on Day 3 of the estrous cycle as previously described (Carter et al. 2008). A daily blood sample was taken from all animals by jugular venipuncture from Day 0 to Day 14 to establish the recipient P4 profile. All heifers were slaughtered on Day 14 and elongating conceptuses were recovered and measured as described above.
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.
This work was supported by Science Foundation Ireland, Grant 06/IN1/B62 (the opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Science Foundation Ireland). Dimitrios Rizos is supported by the Spanish Ministry of Science and Technology (AGL2006-05616) and INIA (AT2006-003). Maria Clemente was supported by a scholarship from INIA.
The authors thank M Wade, P Duffy, and Penny Furney for their excellent technical assistance.
FordeNCarterFFairTCroweMAEvansACSpencerTEBazerFWMcBrideRBolandMPO'GaoraP2009Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle. Biology of Reproduction[in press]. DOI: 10.1095/biolreprod.108.074336.
Lamming GE & Royal MD 1999 Ovarian hormone patterns and subfertility in dairy cows. Proceedings of an International Symposium Organized by the British Society of Animal Science Entitled ’Fertility in the High-Yielding Dairy Cow’ Galway Ireland September 1999. BSAS Occasional Publication 2001. 26 105–118.