Abstract
Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.
Introduction
Full developmental competency of the oocyte is defined as the ability to develop into a viable embryo following fertilization. It is acquired gradually during follicular growth and completed following the preovulatory LH surge (Mehlmann 2005, Gilchrist & Thompson 2007). The processes involved in maturation of mammalian oocytes are not fully understood. In mono-ovulatory species such as the sheep, the initiation of follicular growth, which occurs in a sequential manner, gives rise to a hierarchical pattern of growing follicles (Edwards et al. 1970, Sawyer et al. 2002, McNatty et al. 2014) and, on average, only one follicle will reach the preovulatory stage at every reproductive cycle. Even superovulation procedures do not overcome this inherent hierarchy of follicles within the ovary (Blondin et al. 1996, Patrizio & Sakkas 2009, McNatty et al. 2010).
It is now widely accepted that, within each follicle, the oocyte secretes growth factors to regulate both granulosa (GC) and cumulus cell number as well as their function. In turn, the increasingly complex interactions among the oocyte and adjacent cell-types are critical for the attainment of developmental competency of the oocyte. In sheep, two oocyte-derived growth factors, namely bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for follicular growth beyond the primary stage of development (Galloway et al. 2000, Hanrahan et al. 2004). Homozygous BMP15-/- and GDF9-/- sheep are sterile (Galloway et al. 2000, Hanrahan et al. 2004) due to a lack of GC proliferation, and thus insufficient numbers of GC, to support further follicular development (Juengel et al. 2004, Fabre et al. 2006). In contrast, sheep heterozygous for an inactivating mutation in either the GDF9 or BMP15 genes, or heterozygous or homozygous for a mutation in the specific Type 1 receptor for BMP15 (BMPR1B), have increased ovulation rates (OR) and are highly prolific (Galloway et al. 2000, Mulsant et al. 2001, Hanrahan et al. 2004, McNatty et al. 2005, 2016). Moreover, the phenotype of combined heterozygous mutations, such as in the I+B+ ewe, which carries only one functional copy of both BMP15 (single copy Inverdale; I+) and of BMPR1B (Booroola; B+) results in an even higher number of preovulatory follicles that are typically smaller in diameter due to fewer GC (Davis et al. 1999, McNatty et al. 2016, Juengel et al. 2017). Currently, there is no evidence to confirm that I+B+ oocytes that develop in these morphologically divergent preovulatory follicles gain full developmental competency, similar to that observed in preovulatory follicles of ++ ewes.
In this study, the developmental competency of oocytes from preovulatory follicles from I+B+ ewes was confirmed by embryo transfer. Furthermore, we examined the characteristics and biochemical microenvironments of all follicles ≥1 mm in diameter in ovaries of I+B+ and wild-type (++) ewes, as well as both cumulus cell- and oocyte-derived molecular markers of developmental competency. The rationale for comparing these genotypes was to identify the key biochemical and molecular events necessary for producing a developmentally competent oocyte in preovulatory follicles, despite marked differences in follicular morphology and OR (McNatty et al. 2016, Juengel, 2018). Thus, the similarities and/or differences resulting in both follicular maturation and oocyte developmental competence between these two sheep genotypes will provide new insights into how follicles with markedly fewer GC than the norm for their species (e.g. I+B+) can achieve a higher level of prolificacy.
Materials and methods
Experimental design
All experiments were performed with the approval of the AgResearch Invermay Animal Ethics Committee in accordance with the Animal Welfare Act of New Zealand.
The objective of Experiment 1 was to confirm the developmental competency of oocytes of naturally mutated I+B+ animals using embryo transfer. Embryos from 37 I+B+ donor ewes were transferred into 55 recipient ewes using standard embryo transfer procedures. Further details regarding this experiment can be found in Supplementary data 1 (see section on supplementary materials given at the end of this article).
The objective of Experiment 2 was to elucidate the intra-follicular microenvironment and associated molecular events within growing and presumptive preovulatory follicles. The experiment included 20 ewes that were 3–4 years of age and had access to pasture and water ad libitum. The genotype of naturally mutated heterozygous Inverdale (FecXI /+ ; I+) – Booroola (FecXB /+ ; B+) cross (FecXI /B ; I+B+) and wild-type (FecX+/+ ; ++) ewes were confirmed by genotyping (n = 9 I+B+ and n = 11 ++). Estrous cycles were synchronized by insertion of controlled internal drug release devices (CIDR-G; SVS Veterinary Supplies Ltd; Christchurch, New Zealand) for 10 days. Twenty-four hours prior to CIDR-G removal, a prostaglandin F2α analogue (Cloprostenol; Bayer Animal Health; Auckland, New Zealand) was administered intramuscularly and ewes were euthanized ~52 h later for ovary extraction. This timing ensured that tissues were collected sometime between the preovulatory LH surge and ovulation (Boland et al. 1978, Crawford et al. 2011), thereby permitting the collection of expanded cumulus cell-oocyte complexes (COC). A blood sample was also collected at the time of killing by jugular venipuncture. All ovarian follicles ≥1 mm in diameter were individually assessed for genotype differences between follicular microenvironment, gonadotropin-responsiveness of GC, COC maturation and putative markers of oocyte developmental competency. Gonadotropin-responsiveness and concentrations of constituents within follicular fluid together with gene expression were only measured in non-atretic follicles from which an intact COC and sufficient number (at least 1.2 × 105) of GC were retrieved. A summary of the samples collected from this study is included in Supplementary Table 1.
Ovarian tissue collections
Individual follicles of ≥1 mm in diameter (n = 668) were extracted from ovaries and individually transferred to a clean, dry petri dish. For each follicle, the diameter and degree of vascularization were noted and following puncture, follicular fluid was collected, if recoverable, into 150 µL of PBS and stored at −20°C. Each COC was transferred into 1 mL of dissection media (DMEM containing 25 mM HEPES, 0.2 mM 3-isobutyl-1-methylxanthine and 0.1% (w/v) BSA (ICPbio, Auckland, NZ)) and its morphology (i.e. compact or expanded) was recorded. Following two wash steps in PBS, the cumulus cells were removed from the oocyte and stored separately at −80°C for gene expression analyses. The follicular wall was transferred into 1 mL of dissection media, and GC were scraped from the follicle using a nichrome loop.
The status of each follicle was assessed retrospectively using criteria previously published for ++, I+, B+ and BB ewes (McNatty et al. 1986, 2009, 2016). Briefly, atretic follicles were defined as those that exhibited one or more of the following: absence of visible blood capillaries in the theca interna; presence of debris in the follicular fluid; asymmetry of the oocyte, a thin or ruptured zona pellucida, the absence of, or very few, cumulus cells surrounding the oocyte and <25% of the maximum number of GC. The latter criteria is based on the observation that follicles containing <25% of the maximum number of GC for a given follicle size, regardless of genotype, are destined for atresia (McNatty et al. 1986). In this study, the maximum number of GC recorded in 1, 2, 3, 4, 5, 6, 7 and 8 mm diameter follicles was 1.5, 2.3, 2.4, 1.7, 7.7, 5.3, 10.5 and 7.9 × 106 in ++ ewes. In I+B+ ewes, the maximum number of GC recorded in 1, 2, 3, 4 and 5 mm diameter follicles was 1.0, 1.3, 1.8, 1.6 and 1.6 × 106, respectively.
Gonadotropin-responsiveness studies
The total numbers of GC in each follicle were counted using a hemocytometer and centrifuged at 300 g for 10 min before being gently re-suspended in 1 mL of dissection media. For those follicles that contained sufficient GC numbers (i.e. enough for at least one replicate of each treatment and control), gonadotropin-responsiveness assays were performed. In brief, 60,000 GC were incubated in triplicate in 48-well plates in a final volume of 600 μL in the absence (control) or presence of purified ovine FSH (100 ng/mL; oFSH Wal; bioactivity of 1.4× USDA-oFSH-19-SIAFP or 33,000 IU/mg using human FSH International RP as standard; <0.002% (w/w) LH contamination as determined by bioassay; Moore et al. 1997, McNatty et al. 2009) or hCG (1000 ng/mL; CR121; 13,450 IU/mg, NICHD, MD, USA) for 45 min in a 37°C water bath. These doses were known to elicit maximal cAMP responses (Henderson et al. 1985, Shackell et al. 1993, McNatty et al. 2009, 2016). The samples were then incubated at 80°C for 15 min, cooled on ice and stored at −20°C.
A previously described cAMP RIA (Jolly et al. 1997, McNatty et al. 2009) with a detection limit of 0.3 pmol/106 GC and cross-reactivity with cGMP of 0.5% was performed. The intra-assay coefficients of variation for low, medium and high controls were 8.1, 5.9 and 9.3%, respectively. The inter-assay coefficients of variation for low, medium and high controls were 15.4, 6.1 and 10.5%, respectively. Follicles were termed FSH- or LH-responsive if cAMP production was ≥5 pmol/106 GC following FSH or hCG treatment, respectively. With the exception of two individual follicles from one sheep, all LH -responsive follicles contained an expanded COC.
Measurement of follicular fluid and plasma constituents
Those follicles that contained follicular fluid but were unresponsive to LH were termed growing follicles. This group included follicles that were either unresponsive to both FSH and LH (n = 121 ++ and 46 I+B+), or just LH (n = 42 ++ and 41 I+B+): all these follicles contained compact COC. In contrast, LH-responsive follicles (n = 16 ++ and 45 I+B+) containing expanded COC were termed presumptive preovulatory (PPOV) follicles.
Measurements of glucose, cholesterol, amino acids and steroid hormone concentrations in plasma and follicular fluid were undertaken using methods described elsewhere (Bloomfield et al. 2002, Green et al. 2011, Thorstensen et al. 2012, Hudson et al. 2014). The limit of detection for both glucose and cholesterol was 0.11 mM. Due to low plasma concentrations of progesterone, samples were also measured using the autoanalyser with a detection limit of 0.025 ng/mL. Plasma concentrations of cortisone, cortisol, corticosterone, 11-deoxycortisol, testosterone and oestradiol and follicular fluid-derived progesterone concentrations were measured using a HPLC-mass spectrometer system consisting of a Waters Alliance 2690 Separations module (Waters Corporation, Milford, MA, USA) followed by an Ion Max APCI source on a Finnigan TSQ Quantum Ultra AM triple quadrapole mass spectrometer, controlled by Finnigan Xcalibur software (Thermo Electron Corporation, San Jose, CA, USA). Detection limits for cortisone, cortisol, corticosterone, 11-deoxycortisol, testosterone, oestradiol and progesterone were 0.025, 0.050, 0.025, 0.025, 0.010, 0.020 and 0.100 ng/mL, respectively. The L-forms of alanine, arginine, asparagine, aspartic acid, citrulline, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, methionine, ornithine, phenylalanine, proline, serine, taurine, threonine, tyrosine and valine were measured using a modified fluorescence-tagged HPLC method as described previously (Bloomfield et al. 2002, Thorstensen et al. 2012). The detection limits for aspartic acid, hydroxyproline, glycine and the remainder of the amino acids were 1 µM, 1 µM, 10 µM and 2 µM, respectively.
Gene expression measurement in cumulus cell masses and denuded oocytes
The cumulus cell-derived genes investigated in this study were hyaluronan synthase 2 (HAS2), versican (VCAN) heat shock protein 90B1 (HSP90B1), follicle stimulating hormone receptor (FSHR), progesterone receptor (PGR), epiregulin (EREG), bone morphogenetic protein receptor 2 (BMPR2), and connexin 43 (GJA1). The oocyte-derived genes investigated were pituitary tumor-transforming gene (PTTG1), heat shock factor 1 (HSF1), zona pellucida glycoprotein 3 (ZP3), phosphodiesterase 3A (PDE3A), poly (A) polymerase alpha (PAPOLA), ataxia telangiectasia and Rad3 related (ATR), BMP15 and GDF9.
Total RNA was extracted from single cumulus cell masses and their denuded oocytes, and cDNA was synthesized as previously published (Crawford et al. 2011). Primer and TaqMan probes were designed using Beacon Designer software (Premier Biosoft International, CA, USA; Supplementary Table 2). Gene expression levels were quantified by preparing a reaction mix containing the optimized concentrations of primers and TaqMan probes together with reagents supplied in the Brilliant Multiplex QPCR Mastermix kits (Agilent Technologies) as described previously (Crawford et al. 2011). A calibrator sample was included in every run to correct against inter-assay variation. Controls included samples in which template or reverse-transcriptase enzyme had been omitted from the cDNA synthesis step to check for genomic DNA contamination. Amplification efficiencies for all genes were >80%, and efficiencies were similar within each multiplex gene set.
Two genes, RPL19 and PPIA, were selected as reference genes. Multiplex gene sets for the oocyte were: RPL19, PPIA, BMP15 and GDF9; RPL19, PTTG1, HSF1 and ZP3 and RPL19, PDE3A, PAPOLA and ATR and for the cumulus cells were RPL19 and PPIA; RPL19, HAS2, VCAN and HSP90B1; RPL19, FSHR, PGR and GJA1 and RPL19, EREG and BMPR2. The reference genes were validated by comparing cumulus cell number and expression levels (R2 = 0.948 for RPL19 and 0.909 for PPIA). Quantification of cumulus cell-derived mRNA expression levels were calculated using the 2(-ΔΔCT) method (Livak & Schmittgen 2001). Quantification of oocyte-derived mRNA expression levels were corrected only to the calibrator sample (2(-ΔCT)) to determine expression levels per oocyte (Livak & Schmittgen 2001).
Statistical analyses
For Experiment 1, data from 55 recipient ewes that received three embryos from I+B+ ewes were available for analyses. The number of recovered embryos from donor ewes included unfertilized oocytes (Supplementary data 1).
For Experiment 2, genotype differences in OR, numbers of follicles with gonadotropin-responsive GC, GC number and plasma concentrations of amino acids, glucose, cholesterol, creatinine, triglyceride and steroid hormones were analysed using a t-test, and Welch’s correction was applied where unequal sample sizes or variances existed. Where data were not normally distributed and one main effect was to be investigated, Mann–Whitney U tests were used. When the effect of both genotype and follicular stage were investigated, a generalized linear mixed model (GLMM; SPSS) was employed. Sheep were included as a random effect. Due to the small and unbalanced nature of the samples, the post-estimation settings were altered, and the Satterthwaite Approximation option was selected as the method for determining the degrees of freedom for significance tests. Where the assumptions of the model were violated, the robust estimates option was also selected as the method for testing fixed effects and coefficients. Follicular fluid amino acid, glucose and cholesterol concentrations were also normalised such that the amount of a constituent available per million GC were determined. To calculate this, the number of GC (106) was corrected for the volume of follicular fluid in each follicle (GC/vol) and then constituent concentration was corrected for GC/vol. These data were also analysed using GLMM.
Cumulus cell-derived gene expression data were analysed using Mann–Whitney U tests. For these data, genotype differences were investigated first. If no genotype difference was detected, data were combined and differences between follicular stage were assessed. Where significant genotype differences were identified, differences between follicular stages were investigated separately for each genotype. Additionally, genotypic differences within the growing and PPOV follicles were also investigated.
Results
Experiment 1: Developmental competency of I+B+ oocytes measured by embryo survival
The mean ± s.e.m. OR of the I+B+ ewes was 6.6 ± 0.3. An average of 5.1 ± 0.3 embryos/unfertilized ova were recovered per ewe giving a recovery rate of 77%. There was an average of 4.4 ± 0.4 Grade 1 or 2 embryos recovered per ewe, with 0.2 ± 0.2 unfertilized/uncleaved ova and 0.5 ± 0.2 poor quality embryos recovered per ewe. Therefore, of the embryos recovered, 4% failed at fertilization/cleavage, 10% were poor quality and 86% were of transferable (good-excellent) quality. A total of 51 (93%) recipient ewes were pregnant, and the mean number of lambs born per recipient ewe was 2.1 ± 0.1.
Experiment 2: Follicular characteristics
These data are summarized in Table 1. The OR was determined by calculating the number of follicles containing expanded COCs (i.e. PPOV) per ewe. By this definition, two ++ ewes did not have a PPOV follicle present and were excluded from the OR analyses. The I+B+ ewes exhibited a higher OR, and follicles were smaller and contained fewer GC, compared to ++ ewes.
Ovulation rate and diameter (mm) and number of granulosa cells (106) in non-responsive, FSH-responsive and LH-responsive follicles in ++ and I+B+ ewes.
| ++ | I+B+ | Significance | |||
|---|---|---|---|---|---|
| n* | Value | n* | Value | ||
| Ovulation rate, mean ± s.e.m. | 9 | 1.7 ± 0.2 | 9 | 4.6 ± 0.7 | ** |
| Range | 1-2 | 1-7 | |||
| Diameter (mm; mean (95% CI))† | |||||
| Non-responsive | 121 | 2.07 (1.95, 2.18) | 46 | 1.64 (1.49, 1.80) | *** |
| FSH responsive | 42 | 2.12 (1.94, 2.32) | 41 | 1.54 (1.43, 1.65) | *** |
| LH-responsive | 16 | 5.33 (4.24, 6.71) | 45 | 3.47 (3.17, 3.79) | *** |
| Granulosa cells (106; mean (95% CI))† | |||||
| Non-responsive | 121 | 0.73 (0.66, 0.82) | 46 | 0.40 (0.34, 0.46) | *** |
| FSH responsive | 42 | 0.69 (0.57, 0.85) | 41 | 0.33 (0.29, 0.38) | *** |
| LH-responsive | 16 | 2.70 (1.67, 4.34) | 45 | 0.70 (0.58, 0.84) | *** |
†Geometric mean (95% confidence limits) where data were non-normally distributed. *Number of observations for each genotype; **P < 0.01; ***P < 0.001.
When cAMP production was normalized per 1 × 106 GC, non-responsive follicles in I+B+ ewes appeared to produce more cAMP, compared to ++ ewes (Fig. 1A and C). Moreover, FSH-responsive follicles in I+B+ ewes also produced more cAMP when treated with FSH, compared to ++ ewes (Fig. 1B). With the exception of the non-responsive follicles, cAMP production in LH-responsive follicles was similar between genotypes (Fig. 1C).
Effect of genotype on cAMP production of granulosa cells from non-, FSH- and LH-responsive follicles. Data are presented as standardised cAMP production (A, B and C) and as cAMP production per follicle (D, E and F) following incubation with media alone (control; A and D), FSH (B and E) or hCG (C and F). Values represent geometric mean and 95% confidence limit. (Number of observations: 121 non-responsive ++ follicles; 42 FSH-responsive ++ follicles; 16 LH-responsive ++ follicles; 46 non-responsive I+B+ follicles; 41 FSH-responsive I+B+ follicles and 45 LH-responsive I+B+ follicles). Statistically significant differences between genotypes are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Effect of genotype on cAMP production of granulosa cells from non-, FSH- and LH-responsive follicles. Data are presented as standardised cAMP production (A, B and C) and as cAMP production per follicle (D, E and F) following incubation with media alone (control; A and D), FSH (B and E) or hCG (C and F). Values represent geometric mean and 95% confidence limit. (Number of observations: 121 non-responsive ++ follicles; 42 FSH-responsive ++ follicles; 16 LH-responsive ++ follicles; 46 non-responsive I+B+ follicles; 41 FSH-responsive I+B+ follicles and 45 LH-responsive I+B+ follicles). Statistically significant differences between genotypes are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Effect of genotype on cAMP production of granulosa cells from non-, FSH- and LH-responsive follicles. Data are presented as standardised cAMP production (A, B and C) and as cAMP production per follicle (D, E and F) following incubation with media alone (control; A and D), FSH (B and E) or hCG (C and F). Values represent geometric mean and 95% confidence limit. (Number of observations: 121 non-responsive ++ follicles; 42 FSH-responsive ++ follicles; 16 LH-responsive ++ follicles; 46 non-responsive I+B+ follicles; 41 FSH-responsive I+B+ follicles and 45 LH-responsive I+B+ follicles). Statistically significant differences between genotypes are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Due to larger numbers of GC present in ++ follicles, cAMP production was also calculated per follicle. In this case, cAMP production was higher in follicles of ++, compared to I+B+, ewes regardless of stage or treatment (Fig. 1D, E and F). An exception was the hCG-treated GC from non-responsive follicles (Fig. 1F).
Experiment 2: Amino acid, energy and steroid constituents in plasma
These results are presented in Table 2. Plasma concentrations of two amino acids, out of the 22 tested, differed between genotypes. Glycine and methionine concentrations were higher and lower in I+B+ ewes, respectively, compared to that in ++ ewes. There were no genotype differences in any other constituent measured. The concentrations of 11-deoxycortisol, androstenedione, testosterone and progesterone in plasma were below the limits of detection.
Mean ± s.e.m. plasma constituent concentrations in ++ and I+B+ ewes.
| Plasma constituent concentration | ++ (n = 9) | I+B+ (n = 9) | Significance |
|---|---|---|---|
| Amino Acid (mM) | |||
| Alanine | 0.28 ± 0.017 | 0.269 ± 0.022 | NS |
| Arginine | 0.118 ± 0.010 | 0.124 ± 0.007 | NS |
| Asparagine | 0.037 ± 0.002 | 0.036 ± 0.003 | NS |
| Aspartic acid | 0.008 ± 0.001 | 0.010 ± 0.001 | NS |
| Citrulline | 0.126 ± 0.016 | 0.132 ± 0.014 | NS |
| Glutamic acid | 0.166 ± 0.009 | 0.177 ± 0.013 | NS |
| Glutamine | 0.139 ± 0.011 | 0.134 ± 0.008 | NS |
| Glycine | 0.490 ± 0.035 | 0.577 ± 0.021 | * |
| Histidine | 0.023 ± 0.002 | 0.023 ±0.002 | NS |
| Hydroxyproline | 0.010 ± 0.001 | 0.009 ± 0.001 | NS |
| Isoleucine | 0.084 ± 0.005 | 0.079 ± 0.004 | NS |
| Leucine | 0.134 (0.114, 0.158)† | 0.121 (0.109, 0.133)† | NS |
| Lysine | 0.137 ± 0.009 | 0.129 ± 0.013 | NS |
| Methionine | 0.014 ± 0.001 | 0.010 ± 0.001 | * |
| Ornithine | 0.066 ± 0.004 | 0.069 ± 0.008 | NS |
| Phenylalanine | 0.049 ± 0.003 | 0.044 ± 0.002 | NS |
| Proline | 0.152 ± 0.010 | 0.151 ± 0.015 | NS |
| Serine | 0.100 ± 0.006 | 0.099 ± 0.006 | NS |
| Taurine | 0.056 ± 0.006 | 0.055 ± 0.006 | NS |
| Threonine | 0.156 (0.124, 0.197)† | 0.131 (0.116, 0.148)† | NS |
| Tyrosine | 0.052 ± 0.005 | 0.048 ± 0.003 | NS |
| Valine | 0.175 ± 0.011 | 0.200 ± 0.016 | NS |
| Energy substrate (mM) | |||
| Glucose | 3.951 ± 0.165 | 4.092 ± 0.115 | NS |
| Cholesterol | 2.112 ± 0.090 | 2.002 ± 0.089 | NS |
| Creatinine | 0.110 ± 0.005 | 0.107 ± 0.117 | NS |
| Triglycerides | 0.323 ± 0.017 | 0.313 ± 0.011 | NS |
| Steroid hormones (ng/mL) | |||
| Cortisone | 2.146 ± 0.237 | 2.113 ± 0.301 | NS |
| Cortisol | 17.30 ± 3.478 | 9.932 ± 1.612 | NS |
| Corticosterone | 0.161 (0.051, 0.502)† | 0.111 (0.077, 0.160)† | NS |
| Estradiol | 0.049 ± 0.004 | 0.043 ± 0.004 | NS |
| Testosterone | ND | ||
| Androstenedione | ND | ||
| Progesterone | ND | ||
†Geometric mean (and 95 % confidence limits) where data were non-normally distributed; *P < 0.05.
n, number of ewes; ND not detected (below the limit of quantification); NS, not statistically significant.
Experiment 2: Amino acid, energy and steroid constituents in follicular fluid
Regardless of follicular stage, there were clear genotype differences in arginine, citrulline, glutamine, isoleucine, leucine, lysine, methionine, taurine, tyrosine and valine, where concentrations were higher in I+B+, than ++, follicles (Table 3). With the exception of tyrosine and valine, the concentrations of all other amino acids shown were higher in the growing, compared to PPOV, follicles. While there were no genotype differences in alanine and proline concentrations, clear differences were observed between the follicular stages, with concentrations being higher in PPOV and growing follicles, respectively. Genotype by follicular stage interactions were also observed for asparagine, aspartic acid, glutamic acid, glycine, histidine, hydroxyproline, phenylalanine and serine. In all cases, these interactions appeared to be due to genotype differences in PPOV follicles only, where concentrations were higher in I+B+ ewes. Threonine was the only amino acid in which concentrations were not affected by genotype or follicular stage. Similarly, ornithine did not differ between genotypes but was only measurable in some PPOV suggesting a follicular stage difference.
Mean ± s.e.m. concentrations of constituents in follicular fluid extracted from individual growing and PPOV follicles of ++ and I+B+ ewes.
| Growing | PPOV | Significance | |||||
|---|---|---|---|---|---|---|---|
| ++ (n = 13) | I+B+ (n = 7) | ++ (n = 11) | I+B+ (n = 26) | Foll | Gen | Int | |
| Amino acid (mM) | |||||||
| Alanine | 0.56 ± 0.11 | 0.50 ± 0.19 | 0.69 ± 0.06 | 0.95 ± 0.11 | * | ||
| Arginine | 0.43 ± 0.04 | 0.63 ± 0.07 | 0.14 ± 0.02 | 0.45 ± 0.07 | ** | * | |
| Asparagine | 0.13 ± 0.01 | 0.12 ± 0.01 | 0.04 ± 0.04 | 0.09 ± 0.02 | ** I | * | |
| Aspartic acid | 0.21 ± 0.06 | 0.22 ± 0.05 | 0.04 ± 0.01 | 0.25 ± 0.06 | * | ||
| Citrulline | 0.71 ± 0.08 | 0.99 ± 0.10 | 0.25 ± 0.03 | 0.64 ± 0.06 | *** | ** | |
| Glutamic acid† | 0.87 (0.64, 1.18) | 0.92 (0.60, 1.42) | 0.50 (0.38, 0.64) | 1.07 (0.83, 1.38) | * | ||
| Glutamine | 1.87 ± 0.19 | 2.50 ± 0.38 | 0.26 ± 0.04 | 1.20 ± 0.09. | *** | *** | |
| Glycine | 2.56 ± 0.24 | 2.51 ± 0.27 | 0.85 ± 0.05 | 1.80 ± 0.16 | *** | * | |
| Histidine† | 0.68 (0.46, 0.99) | 0.66 (0.42, 1.02) | 0.07 (0.05, 0.09) | 0.30 (0.25, 0.37) | *** | b | |
| Hydroxyproline† | 0.12 (0.08, 0.17) | 0.13 (0.09, 0.19) | 0.02 (0.01, 0.02) | 0.06 (0.05, 0.08) | *** | * | |
| Isoleucine | 0.51 ± 0.05 | 0.76 ± 0.10 | 0.17 ± 0.03 | 0.43 ± 0.03 | *** | *** | |
| Leucine | 0.46 ± 0.04 | 0.66 ± 0.07 | 0.22 ± 0.03 | 0.48 ± 0.04 | *** | ** | |
| Lysine | 0.49 ± 0.05 | 0.75 ± 0.11 | 0.25 ± 0.03 | 0.53 ± 0.03 | *** | *** | |
| Methionine | 0.14 ± 0.01 | 0.18 ± 0.02 | 0.05 ± 0.01 | 0.11 ± 0.01 | *** | *** | |
| Ornithine | ND | ND | 0.02 (0.014 0.03) | 0.03 (0.02, 0.05) | NS | ||
| Phenylalanine† | 0.14 (0.11, 0.17) | 0.17 (0.13, 0.23) | 0.04 (0.03, 0.06) | 0.10 (0.09, 0.12) | *** | * | * |
| Proline† | 1.69 (1.14, 2.52) | 2.00 (1.39, 2.90) | 0.29 (0.22, 0.37) | 0.72 (0.55, 0.93) | *** | ||
| Serine | 0.68 ± 0.14 | 0.72 ± 0.12 | 0.16 ± 0.02 | 0.49 ± 0.07 | *** | * | |
| Taurine | 1.15 ± 0.21 | 1.70 ± 0.44 | 0.23 ± 0.03 | 0.59 ± 0.06 | *** | * | |
| Threonine | 0.18 ± 0.06 | 0.11 ± 0.01 | 0.13 ± 0.01 | 0.10 ± 0.01 | NS | NS | NS |
| Tyrosine | 0.18 ± 0.03 | 0.21 ± 0.03 | 0.12 ± 0.01 | 0.18 ± 0.02 | * | ||
| Valine† | 0.32 (0.24, 0.43) | 0.54 (0.43, 0.68) | 0.27 (0.21, 0.34) | 0.42 (0.37, 0.49) | b | ** | |
| Energy substrate (mM) | |||||||
| Glucose | 5.71 ± 0.73 | 5.70 ± 0.91 | 1.01 ± 0.20 | 3.05 ± 0.35 | *** | * | |
| Cholesterol† | 6.77 (4.66, 9.82) | 7.71 (5.23, 11.36) | 1.11 (0.92, 1.34) | 3.41 (2.79, 4.18) | *** | b | * |
| Creatinine | NM | NM | NM | NM | |||
| Triglycerides | NM | NM | NM | NM | |||
| Steroid hormones (ng/mL) | |||||||
| Cortisone | 2.02 ± 0.41 | 2.06 ± 0.33 | 1.58 ± 0.32 | 2.21 ± 0.27 | NS | NS | NS |
| Estradiol† | 1.09 (0.73, 1.61) | 1.39 (0.92, 8.63) | 2.85 (0.94, 8.63) | 3.20 (2.37, 4.32) | * | ||
| Testosterone† | 13.30 (8.50, 20.81) | 18.20 (3.14 105.70) | 0.54 (0.13, 2.29) | 0.75 (0.42, 1.35) | *** | ||
| Androstenedione | 7.15 ± 2.29 | 54.66 ± 23.18 | 4.44 ± 3.00 | 3.11 ± 1.45 | ** | ** | |
| Progesterone† | 6.89 (5.08, 9.35) | 7.46 (4.73, 11.77) | 75.84 (19.75, 291.2) | 36.23 (26.56, 49.42) | * | ||
†Values presented are geometric mean (and 95% confidence limits) where data were non-normally distributed; *P < 0.05; **P < 0.01; ***P < 0.001; aP = 0.053; bP = 0.070–0.079.
Foll, Significant main effect of follicular stage; Gen, Significant main effect of genotype; Int, Significant interaction between follicle stage x genotype; n, number of follicular fluid samples; ND, not detected (below the limit of quantitation); NM, not measured; NS, no significant effects.
There were no clear genotype effects of glucose or cholesterol concentrations, but they were higher in growing follicles. However, concentrations in PPOV follicles were higher in I+B+, compared to ++, ewes leading to an interaction for both constituents.
Given that the follicles were different sizes, with different numbers of GC, substrate availability was examined following calculations of total amount of available substrate per million GC. Many amino acids (arginine, asparagine, aspartic acid, citrulline, glutamic acid, glycine, isoleucine, leucine, lysine, methionine and serine) had a higher availability to GC in I+B+ follicles, regardless of follicular stage (Supplementary Table 3). Several amino acids, as well as cholesterol and glucose, also had a higher availability to GC in growing compared to PPOV follicles, as well as I+B+ compared to ++ follicles. Alanine was more available to GC of PPOV compared to growing follicles, regardless of genotype. Threonine, tyrosine and valine were not affected by either genotype or follicular stage.
There were no clear genotype differences in steroid concentrations (Table 3). As expected, both oestradiol and progesterone concentrations were higher in PPOV, than growing, follicles. Inversely, testosterone and androstenedione concentrations were lower in PPOV, than growing, follicles. Interestingly, androstenedione concentrations were markedly higher in growing follicles of I+B+, compared to ++, ewes.
Experiment 2: Cumulus cell-derived gene expression
The expression levels of key cumulus cell-derived genes involved in candidate pathways for the attainment of oocyte maturation are depicted in Fig. 2.
Relative mRNA expression levels in cumulus cell masses from ++ and I+B+ ewes (A, B, C and D) and in growing and PPOV follicles (E, F, G and H). Data represent the geometric mean and 95% confidence limit of the linearized CT values (Number of observations in A, B, C and D: 256 growing follicles; and 53 PPOV follicles. Number of observations in E, F, G and H: 153 growing ++ follicles; 105 growing I+B+ follicles; 15 PPOV I+B+ follicles and 38 PPOV I+B+ follicles). Statistically significant differences between follicular stage are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001), while genotype differences are depicted by non-consecutive letters (P < 0.05).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Relative mRNA expression levels in cumulus cell masses from ++ and I+B+ ewes (A, B, C and D) and in growing and PPOV follicles (E, F, G and H). Data represent the geometric mean and 95% confidence limit of the linearized CT values (Number of observations in A, B, C and D: 256 growing follicles; and 53 PPOV follicles. Number of observations in E, F, G and H: 153 growing ++ follicles; 105 growing I+B+ follicles; 15 PPOV I+B+ follicles and 38 PPOV I+B+ follicles). Statistically significant differences between follicular stage are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001), while genotype differences are depicted by non-consecutive letters (P < 0.05).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Relative mRNA expression levels in cumulus cell masses from ++ and I+B+ ewes (A, B, C and D) and in growing and PPOV follicles (E, F, G and H). Data represent the geometric mean and 95% confidence limit of the linearized CT values (Number of observations in A, B, C and D: 256 growing follicles; and 53 PPOV follicles. Number of observations in E, F, G and H: 153 growing ++ follicles; 105 growing I+B+ follicles; 15 PPOV I+B+ follicles and 38 PPOV I+B+ follicles). Statistically significant differences between follicular stage are denoted by asterisk (*P < 0.05, **P < 0.01, ***P < 0.001), while genotype differences are depicted by non-consecutive letters (P < 0.05).
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
There were no genotype differences in the expression levels of HAS2, HSP90B1, FSHR or GJA1 mRNA and data from both genotypes were pooled (Fig. 2A, B, C and D). There were main effects of follicular stage with HAS2 mRNA levels being higher (P < 0.001) and HS90B1, FSHR and GJA1 being lower (P < 0.01; P < 0.001; P < 0.001, respectively) in PPOV, compared to growing, follicles.
In both ++ and I+B+ ewes, mRNA levels of VCAN, PGR, EREG and BMPR2 were higher in PPOV, compared to growing, follicles (Fig. 2E, F, G and H). Genotype differences were evident in growing follicles for VCAN (P < 0.0005) and BMPR2 (P = 0.001), with levels being higher in I+B+ follicles. Mean EREG levels were up-regulated in PPOV, compared to growing, follicles (P < 0.001), and within each follicular stage the EREG levels were lower (P = 0.035) in I+B+ follicles. No genotype differences were noted for PGR.
Experiment 2: Oocyte-derived gene expression
The expression levels of key oocyte-derived genes involved in candidate pathways for the attainment of oocyte maturation are depicted in Fig. 3. There were no effects of genotype or follicular stage, or any interaction, for any genes measured. However, there was a strong trend towards lower (P = 0.051) BMP15 expression levels in oocytes from I+B+, compared to ++, ewes.
Relative mRNA expression levels in oocytes in growing and PPOV follicles from ++ and I+B+ ewes. Data represent the mean ± s.e.m. or as geometric mean and 95% confidence limit, where data were non-normally distributed, of the linearized CT values (Number of observations: 176 growing ++ follicles; 111 growing I+B+ follicles; 12 PPOV I+B+ follicles and 32 PPOV I+B+ follicles). Where there was a trend towards significance for the main effect of genotype, the P-value has been reported beside the appropriate graph.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Relative mRNA expression levels in oocytes in growing and PPOV follicles from ++ and I+B+ ewes. Data represent the mean ± s.e.m. or as geometric mean and 95% confidence limit, where data were non-normally distributed, of the linearized CT values (Number of observations: 176 growing ++ follicles; 111 growing I+B+ follicles; 12 PPOV I+B+ follicles and 32 PPOV I+B+ follicles). Where there was a trend towards significance for the main effect of genotype, the P-value has been reported beside the appropriate graph.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Relative mRNA expression levels in oocytes in growing and PPOV follicles from ++ and I+B+ ewes. Data represent the mean ± s.e.m. or as geometric mean and 95% confidence limit, where data were non-normally distributed, of the linearized CT values (Number of observations: 176 growing ++ follicles; 111 growing I+B+ follicles; 12 PPOV I+B+ follicles and 32 PPOV I+B+ follicles). Where there was a trend towards significance for the main effect of genotype, the P-value has been reported beside the appropriate graph.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Discussion
Mutations in GDF9, BMP15, BMPR1B or combinations thereof result in smaller preovulatory follicles containing fewer GC. These GC cells appear to mature earlier than those of wild-type follicles, becoming more responsive to LH at a smaller follicular diameter. These radical changes in ovarian follicular development, resulting in an altered biochemical and molecular microenvironment, have raised the question of whether all oocytes matured in these I+B+ follicles gain full developmental competency. This uncertainty was amplified by the observations that the addition of BMP15 to in vitro maturation cultures increased oocyte developmental competency (Hussein et al. 2006, Gilchrist et al. 2008). Our study has confirmed that developmental competency of oocytes developed and matured in I+B+ follicles is successfully attained, as evidenced by similar embryo/fetal survival rate in animals heterozygous for these mutations, compared to wild-type equivalents (Supplementary data 1).
Changes in the microenvironment during follicular maturation
To better understand how oocyte quality is maintained in maturing ovarian follicles of I+B+ ewes, it is first necessary to understand what changes normally occur during follicular maturation. Within the follicular microenvironment, maturation of the follicle was associated with changes in concentrations of many amino acids, energy substrates and steroid hormones. When assessing these changes, it is important to consider changes in follicle diameter. Growing (non-responsive and FSH-responsive) follicles were smaller than PPOV (LH-responsive) follicles. Thus, concentration differences due to follicular type may not be entirely attributed to differences in intra-follicular metabolic rates but also to differences in surface area:volume ratio. The latter would affect both transfer rates of substrates into and dilution factors within follicles. One piece of evidence that suggests that surface area:volume ratio may not impact amino acid concentrations in follicular fluid is that threonine concentrations remain constant between the two follicular sizes. Therefore, we suggest that the differences in concentrations of all other amino acids between growing and PPOV follicles are most likely related to GC numbers and rates of utilisation and not increased volume.
Concentrations of the majority of amino acids tested, as well as glucose and cholesterol, were lower in PPOV follicles compared with growing follicles, when assessed as total concentration in follicular fluid. This is consistent with an increased requirement for substrates by the higher number of metabolically active cells synthesising proteins and steroids as the follicle matures. Even when expressed as availability per million GC, many amino acids, as well as cholesterol and glucose, were lower in PPOV follicles.
Glucose concentrations were greatly depleted in PPOV follicles, supporting data that utilisation of glucose is minimal in less mature follicles and increases during the final stages of maturation (Boland et al. 1994, Harris et al. 2007). Glutamine was also greatly depleted in PPOV follicles, which is consistent with its role in glutathione (GSH) synthesis under increased glucose utilization (Sutton-McDowall et al. 2010). Utilisation of asparagine, glycine, phenylalanine and serine is also likely important for GSH homeostasis within the follicle, which relies on the adequate provision of sulfur-containing amino acids as well as glutamate (glutamine or BCAAs), glycine or serine and asparagine as stimulators of a GSH co-factor. Thus, these amino acids are involved not only in the protection of follicular cells from reactive oxygen species (ROS), but also in the inhibition of apoptosis (Li & Wu 2018). The production of GSH, an antioxidant, is critical in high metabolic tissues to prevent cellular damage by ROS.
Alanine and ornithine are unique in that they are produced, rather than utilised, during follicular maturation to aid in the removal of excess ammonium. Alanine is produced from the transamination of pyruvate and glutamic acid and is associated with sequestration of the potentially toxic effects of ammonia. The increased surplus of alanine may occur as a result of increased transamination of pyruvate and glutamate to alanine and α-ketoglutarate.
As expected, a significant effect of follicular stage was identified for steroid hormone concentrations in follicular fluid. Concentrations of androstenedione and testosterone decreased with maturity, while the reverse was true of oestradiol and progesterone. These results are verified by data on expression of steroidogenic enzymes in ovine follicles (Moor et al. 1975, McNatty et al. 1979, 1984, Logan et al. 2002). Likewise, our data reflects with the increased oestradiol synthesis from GC that express LH receptors (i.e. those of the PPOV follicles) and coincides with an increase in the rate of androstenedione and testosterone utilisation (McNatty et al. 1984).
The expression levels of many of the cumulus cell-derived genes (i.e. HAS2, VCAN, PGR, EREG and BMPR2) differed significantly between growing and PPOV follicles. This was not unexpected, given these genes are either mediated by LH- (Park & Mayo 1991, Lydon et al. 1995, Robker et al. 2000, Russell et al. 2003, Park et al. 2004, Sugiura et al. 2009, Cotterill et al. 2012) or EGF-like peptide-signaling cascades (Sugimura et al. 2015) or are positively associated with follicular size and thus follicular stage (Feary et al. 2007, Chen et al. 2009, Paradis et al. 2009). In contrast, expression levels of FSHR, GJA1 and HSP90B1 decreased as follicles matured. This was also expected as FSH-responsiveness diminishes in GC that acquire LH receptors, and the preovulatory LH surge disrupts gap junctions within the COC (Calder et al. 2003, Jeppesen et al. 2012, Li et al. 2015). The role of the HSP90B1 gene in cumulus cells is not fully understood, but in other tissues, it plays an integral role in the regulation of protein folding and secretion and has been associated with a wide-range of situations that lead to endoplasmic reticulum stress (Yang & Li 2005). Higher levels present in growing, compared to PPOV follicles may reflect an increased protein synthesis in less mature follicles and are also consistent with the increased levels of methionine reported herein.
The oocyte genes investigated in this study are reported to play key roles in cell cycle regulation, DNA damage and replication repairs during meiosis (PTTG1 and ATR) (Chu et al. 2012), polyadenylation of mRNA (PAPOLA) (Dutta et al. 2016), facilitating GVBD and fertilization (ZP3) (Gao et al. 2017), regulation of intraoocyte cAMP concentrations (PDE3A) (Richard et al. 2001) and the regulation of stress responses involving heat shock proteins critical for early embryonic development (HSF1) (Christians et al. 2000, Bierkamp et al. 2010). Surprisingly, some of these genes are involved in oocyte maturation pathways, but their expression levels did not differ between growing and PPOV follicles indicating constitutive expression.
Differences in the follicular microenvironment of I+B+ ewes
A summary of the key differences in the follicular microenvironment of I+B+ ewes, compared to ++ ewes, is illustrated in Fig. 4. In particular, genotype differences were observed in amino acid concentrations with most being higher in I+B+ follicles when calculated as concentrations in follicular fluid on a per follicle basis and when normalised against GC numbers. The later suggests that the GC in I+B+ ewes are less metabolically active, which is supported by a previous study of follicular fluid of B+ ewes (Guo et al. 2018). The decreased metabolism of several of these amino acids in I+B+ follicles that have known follicular functions may provide insight into mechanistic differences between genotypes.
Summary of genes (in italics), gonadotrophin-responsiveness and biochemical factors that were different in I+B+ growing (top) and preovulatory (PPOV; bottom) follicles, calculated on a per follicle (left) and per granulosa cell (right; with the exception that genes were per cumulus cell) basis. Note that only factors that were different in follicles of I+B+, compared to wild-type, ewes are displayed. The higher concentrations of biochemical factors indicate a lower utilisation and thus metabolism rate. EREG, epiregulin gene; VCAN, versican gene; BMPR2, bone morphogenetic protein receptor 2; A4, androstenedione; capital letters denote amino acids; Cit, citrulline; Hyp, hydroxyproline; Tau, taurine.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Summary of genes (in italics), gonadotrophin-responsiveness and biochemical factors that were different in I+B+ growing (top) and preovulatory (PPOV; bottom) follicles, calculated on a per follicle (left) and per granulosa cell (right; with the exception that genes were per cumulus cell) basis. Note that only factors that were different in follicles of I+B+, compared to wild-type, ewes are displayed. The higher concentrations of biochemical factors indicate a lower utilisation and thus metabolism rate. EREG, epiregulin gene; VCAN, versican gene; BMPR2, bone morphogenetic protein receptor 2; A4, androstenedione; capital letters denote amino acids; Cit, citrulline; Hyp, hydroxyproline; Tau, taurine.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Summary of genes (in italics), gonadotrophin-responsiveness and biochemical factors that were different in I+B+ growing (top) and preovulatory (PPOV; bottom) follicles, calculated on a per follicle (left) and per granulosa cell (right; with the exception that genes were per cumulus cell) basis. Note that only factors that were different in follicles of I+B+, compared to wild-type, ewes are displayed. The higher concentrations of biochemical factors indicate a lower utilisation and thus metabolism rate. EREG, epiregulin gene; VCAN, versican gene; BMPR2, bone morphogenetic protein receptor 2; A4, androstenedione; capital letters denote amino acids; Cit, citrulline; Hyp, hydroxyproline; Tau, taurine.
Citation: Reproduction 159, 5; 10.1530/REP-19-0613
Proline is an important signalling molecule in the mTOR pathway in embryos (Washington et al. 2010). The mTOR pathway is important for integration of nutrients, growth factors, energy status, and stress and regulates cellular processes such as proliferation, protein synthesis, glucose homeostasis and metabolism. This pathway is highly active during follicular development (Telfer & Zelinski 2013) as is observed by its depletion in PPOV follicles, but is not as heavily utilised by GC in I+B+ follicles.
The oxidation of citrulline to arginine by nitric oxide synthase generates nitric oxide (Kwon et al. 1990), which in turn activates the guanylate cyclase enzyme and thereby the synthesis of cGMP (Bilodeau-Goeseels 2007). The production of cGMP within follicles is essential as it outcompetes cAMP for phosphodiesterase-induced catabolism, thereby maintaining intra-oocyte cAMP levels and meiotic arrest (reviewed by Gilchrist et al. 2016). Isoleucine and leucine are precursors for the derivation of NADH and FADH, which are utilised by the COC to generate ATP. ATP is generated mainly via glycolysis and mitochondrial oxidative phosphorylation. The fact that key genes within the glycolytic pathway are expressed poorly in GCs (Eppig 2001, Sugiura et al. 2005, Su et al. 2008), compared to cumulus cells (Downs & Utecht 1999, Harris et al. 2007, Scaramuzzi et al. 2010, Sutton-McDowall et al. 2010) suggests that GC proliferation is reliant upon mitochondrial ATP production. Similarly, methionine, under the activation of ATP, is converted to S-adenosylmethionine, a critical donor of the methyl group involved in protein methylation (Bottiglieri 2002) and DNA repair (Mattson & Shea 2003). As expected, the depletion of arginine, isoleucine, leucine and methionine in PPOV follicles are correlated to higher GC number, but these amino acids are not as heavily utilised by GC in I+B+ follicles.
Evidence of increased antioxidant capacity has been observed in follicular fluid of other high OR genotypes, including BB and B+ ewes (Guo et al. 2018). As discussed earlier, glutamine, glycine, serine, asparagine and phenylalanine have roles in the production of GSH which functions as an antioxidant. Interestingly, the concentrations of glycine and serine were similar between genotypes in growing follicles, indicating that these amino acids are independent of GC numbers at this development stage. Increased utilisation of these amino acids was evident in PPOV follicles, but these amino acids were less utilised by GC in I+B+ follicles, perhaps leading to better protection from ROS.
Interestingly, androstenedione concentrations were higher in growing follicles from I+B+ ewes, while no differences were observed in the PPOV follicles. Similarly, a study in cultured theca cells from small antral follicles of BB ewes revealed an increased basal rate of androstenedione production as well as the production of more androstenedione in response to low doses of LH (Campbell et al. 2006). This study also revealed an absence in genotype differences in regards to androstenedione production of theca cells exposed to higher doses of LH, which may explain the lack of genotype differences in androstenedione concentrations in PPOV follicles in our study. Furthermore, LH-dependent androstenedione production was detected in small antral follicles (<5 mm) in a bovine model and occured prior to measurable oestradiol synthesis (McNatty et al. 1984). Thus, higher concentrations of androstenedione in growing follicles of I+B+ may be attributable to precocious LH-responsiveness. This is supported by higher VCAN and BMPR2 mRNA expression levels in growing I+B+ follicles under low levels of LH signalling.
If GC of I+B+ follicles are indeed more responsive to FSH signaling, as indicated by cAMP production per GC, CYP19A1 mRNA expression and activity may be increased. Recently, CYP19A1 mRNA levels were reported to be higher in small (1–3 mm) and medium (3–<4.5 mm) sized follicles in I+B+ ewes (Juengel et al. 2017). In combination, higher androstenedione concentrations would allow for increased synthesis of testosterone as well as increased cytochrome P450 aromatase activity and may explain why similar concentrations of oestradiol were measured in PPOV follicles from I+B+ and ++ ewes, despite the former having fewer GC.
Through their acquisition of LH receptors, the ability of GC to respond to LH (or hCG) is unique to follicles capable of ovulation (Webb & England 1982). The attainment of LH receptors on GC of comparatively smaller follicles of I+B+ ewes observed in this study was consistent with previous studies of B+, BB, I+ and I+B+ ewes (McNatty et al. 1986, 2009, 2016, Henderson et al. 1987, Shackell et al. 1993, Crawford et al. 2011). Our study has identified distinct differences in cumulus cell function that may accommodate oocyte maturation in spite of fewer GC in follicles of I+B+ ewes. In particular, expression levels of VCAN and BMPR2 genes were higher in growing, but not PPOV, follicles of I+B+, compared to ++, ewes. The VCAN gene regulates cumulus cell expansion (LeBaron et al. 1992, Fülöp et al. 1997), while BMPR2 gene encodes a serine-threonine kinase type II receptor involved in the signalling of specific transforming growth factor β superfamily members secreted from the oocyte (Vitt et al. 2002, Moore et al. 2003). The transcription of both genes are regulated by LH (Park & Mayo 1991, Lydon et al. 1995, Robker et al. 2000, Russell et al. 2003, Park et al. 2004, Sugiura et al. 2009, Cotterill et al. 2012), thus the genotype differences observed in growing follicles are likely due to the early acquisition of LH receptors in I+B+ follicles. This may compensate for lower GC numbers, relative to maturational stage, while the associated oocyte continues to achieve full developmental competency. Additionally, higher BMPR2 expression in growing follicles of I+B+ ewes may also indicate an increased sensitivity to oocyte-secreted growth factors, perhaps to counterbalance the lower levels of BMP15 secreted from the oocytes of these ewes carrying an I+ mutation.
Mean EREG mRNA levels were lower at both follicular stages in I+B+, compared to ++, ewes. The EREG gene produces an EGF-like peptide that is important for propagating the downstream effects of the preovulatory LH surge (Park et al. 2004). Moreover, the expression of BMPR2 mRNA in GC in vitro is reported to be upregulated by oestradiol (Jayawardana et al. 2006), but downregulated by hCG and LH under both in vivo and in vitro conditions (Jayawardana et al. 2006, Brannian et al. 2010, Nio-Kobayashi et al. 2015). This may explain our results, where lower expression of EREG mRNA was associated with higher BMPR2 mRNA in non-estrogenic growing follicles of I+B+, compared to ++, ewes. However, despite lower EREG expression in PPOV follicles of I+B+ ewes, follicular fluid levels of oestradiol and BMPR2 expression levels were similar between genotypes in these mature follicles. While EGF-like factors including epiregulin facilitate LH-signaling in the cumulus cells, EREG expression was also reported to be modulated by BMP15 in cumulus cells in mice (Yoshino et al. 2006). Therefore, it is plausible that lower BMP15 protein levels in follicles of I+B+ ewes decreased EREG expression in cumulus cells. The importance of the EGF-like peptide pathway in oocyte developmental competency was demonstrated following the discovery that cAMP, GDF9 and BMP15 modulated the functionality of the EGFR (Sugimura et al. 2015). Thus, the lower EREG mRNA levels in I+B+ follicles are intriguing, considering the concomitant lower amounts of intra-follicular cAMP and presumably BMP15. Particularly, the associated COC from I+B+ follicles successfully underwent final maturational events as evidenced by cumulus cell expansion, and HAS2 and VCAN mRNA levels were equivalent to PPOV follicles of ++ ewes.
Quantification of cAMP production by GC in response to either FSH or hCG treatment was used to classify follicles into developmental stages as has been used previously (McNatty et al. 1986, 2009, 2016, Henderson et al. 1987, Jolly et al. 1997, Crawford et al. 2011). When cAMP levels were calculated on a per follicle basis in this study, less cAMP was produced in I+B+ follicles due to fewer GC. Interestingly though, when normalized for GC number, I+B+ follicles that were unresponsive to gonadotropins appeared to have a higher basal cAMP production rate than equivalent follicles from ++ ewes. Moreover, this higher cAMP response was also observed in FSH-responsive and LH-responsive follicles of I+B+ ewes following FSH and hCG treatment, respectively. Initially, GC from I+ and BB ewes were reported to have higher FSH-induced cAMP synthesis compared to that from similar-sized follicles in ++ ewes (McNatty et al. 1990, Shackell et al. 1993). However, subsequent studies using a highly purified FSH preparation could not corroborate these findings, indicating these earlier results were due contamination with LH (McNatty et al. 2009, Crawford et al. 2011). While this current study also used a highly purified ovine FSH preparation, the timing of ovary recovery was later, resulting in follicles being classified based on gonadotropin-responsiveness rather than follicular diameter. In fact, BMP15 has been reported to inhibit FSH-responsiveness by reducing FSHR expression in in vitro-cultured rat GC (Otsuka et al. 2001). In support, GC from BB follicles exhibited increased surface FSHR, LHR and BMPR1B densities compared to ++ equivalents (Regan et al. 2016). While FSHR gene expression levels in cumulus cells in the current study and in whole follicles in a previous study were not higher in I+B+ ewes (Juengel et al. 2017), gene expression does not always corroborate with protein production. Moreover, the total number of FSH- and LH-responsive GC per ewe were not different between I+, BB and B+ genotypes (Henderson et al. 1985, Niswender et al. 1990, Shackell et al. 1993). Collectively, this implies a similar overall endocrine output from GC, despite major morphological and molecular differences (Niswender et al. 1990, Juengel et al. 2004, 2013, Fabre et al. 2006).
With the exception of the BMP15 gene, the oocyte-derived genes did not differ between genotypes. We have shown previously that one mutated copy of BMPRIB, as is present in these I+B+ ewes, resulted in lower BMP15 expression levels (Crawford et al. 2011). What is interesting is that BMP15 mRNA levels do not appear to be lowered further when inactivating single-copy mutations for BMP15 and BMPRIB are combined. While it might be assumed that lower BMP15 activity might reduce oocyte quality, particularly as GDF9 and BMP15 have both been shown to increase blastocyst and offspring rates (Juengel 2018), lowering concentrations of BMP15 or GDF9 through naturally occurring inactivating mutations (e.g. I+ ewes) or active immunizations did not affect embryo or foetal survival (Juengel 2018). This may suggest that there is considerable plasticity in the amount of BMP15 required for normal oocyte maturation.
Conclusions
This study offers new insights into the novel physiological processes within the ovarian follicle that enables a low OR species to develop a greater than normal number of developmental competency oocytes in one reproductive cycle. The GC of growing I+B+ follicles have a higher basal cAMP rate and an increased responsiveness to gonadotrophins. The higher intracellular cAMP levels in GC will likely lead to increased protein kinase A activation and steroidogenic enzyme expression, such as CYP19A1 and cytochrome P450 aromatase. In concert, elevated VCAN and BMPR2 genes in cumulus cells of growing follicles of I+B+ ewes reveal that key maturation processes are initiated earlier, enabling the earlier acquisition of LH receptors. The GC of I+B+ ewes are also less metabolically active. This culminates in a unique follicular microenvironment demonstrated by higher concentrations of amino acids and metabolic substrates that may serve to protect the maturing I+B+ oocyte from ROS. These differences enable the oocyte to develop normally despite marked changes in GC number induced by I+B+ mutations.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-19-0613.
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
The work was supported by funding by Royal Society of New Zealand Marsden grant (13-VUW-153).
Author contribution statement
Z Clark performed tissue collection, laboratory work and prepared the majority of first draft of the manuscript and contributed to subsequent revisions. D Heath performed follicle collections and GC cultures. A O’Connell planned, supervised and assisted with data analysis from Experiment 1. J Juengel supervised and participated in the animal work, was involved in experimental design planning and contributed to all revised manuscript drafts. K McNatty performed follicle collections and GC cultures, was involved in experimental design planning and contributed to all revised manuscript drafts. J Pitman oversaw all experimental procedures, performed tissue collection, was involved in experimental design planning and contributed to all manuscript drafts.
Acknowledgments
The authors would also like to acknowledge the contribution of Eric Thorstensen (University of Auckland, New Zealand) for the measurement of plasma and follicular fluid constituents, Laurel Quirke for her help with the embryo transfer studies, and Alexia Kauff and Kieran Hyslop for their assistance on Experiment 2 and the Invermay reproduction and farm staff for their help with animal handling and care.
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