Rémi Labrecque

FSH withdrawal improves developmental competence of oocytes in the bovine model

Combinations of genetic, environmental, and management factors are suspected to explain the loss in fertility observed for over 20 years in dairy cows. In some cases, IVF is used. When compared with in vivo embryo production, IVF resulted in low success rates until the FSH coasting process (FSH starvation after superstimulation) was introduced in 2002. Increased competence associated with FSH withdrawal of aspirated oocyte for in vitro maturation and IVF has not been optimized nor explained yet. The goal here was to determine and characterize the optimal oocyte competence acquisition window during the coasting period by determining blastocyst rates and follicular cohort development. Commercial milking cycling cows (n=6) were stimulated with 3 days of FSH (6×40 mg NIH Folltropin-V given at 12 h intervals) followed by a coasting period of 20, 44, 68, or 92 h. Each animal was exposed to the four conditions and served as its own control. At the scheduled time, transvaginal aspirations of immature oocytes were performed followed by IVF of half the oocytes. The outcomes were as follows: i) FSH coasting was optimal at a defined period: between 44 and 68 h of coasting; ii) The best estimated coasting duration was ∼54±7 h; iii) Under these conditions, the best statistical blastocyst rate estimation was ∼70%; iv) Between 44 and 68 h of coasting, follicle size group proportions were similar; v) Follicle diameter was not linearly associated with competence. In conclusion, coasting duration is critical to harvest the oocytes at the right moment of follicular differentiation.

The study of mammalian oocyte competence by transcriptome analysis: progress and challenges

Various morphological and cytological traits of oocytes and their surrounding cumulus cells may be used to select oocytes for assisted reproduction. However, even with careful selection, successful IVF and subsequent embryo development remain uncertain. The factors that ensure oocyte competence are unclear and other approaches to assessing developmental potential must be explored. With the constant development of the molecular toolbox, genomic/transcriptomic analysis is becoming a more and more interesting approach to understand oocyte quality on the basis of RNA composition. Using bovine and mouse models as well as human oocytes of known developmental potential, various efforts are underway to characterize the mRNA profile of the competent oocyte using microarray technology. The proliferation of gene expression data sets raises new opportunities to identify the mechanisms involved in this complex phenotype, which should lead to improved techniques of assisted reproduction. Although several molecular markers of oocyte quality are known, translating these into cellular functions remains challenging, largely due to the poor correlation between mRNA level and protein synthesis. Unlike most somatic cells, the oocyte can store mRNA for days, with transcriptional activity remaining at a halt during the 4-5 days beginning before ovulation and ending with embryonic genome activation. This review provides an overview of the transcriptomic data obtained from oocytes of different quality as well as interesting avenues to explore in order to improve our understanding of oocyte competence.

Gene expression analysis of bovine oocytes with high developmental competence obtained from FSH-stimulated animals.

Recent progress in the ovarian stimulation protocol used for bovine in vitro maturation and fertilization, especially through optimization of the follicle‐stimulating hormone (FSH) withdrawal period (“coasting”) after ovarian pre‐treatment with FSH, has significantly improved blastocyst outcome. Despite this important success, the underlying factors leading to improved oocyte quality have not yet been identified. The aim of this project was to compare the transcriptome of germinal vesicle‐stage oocytes collected from FSH‐stimulated cows after various coasting periods (20, 44, 68, and 92 hr) to determine which transcripts were accumulated or depleted during the rise and fall of competence. Oocytes from each coasting period were compared to the three other times (optimal conditions, 44 and 68 hr; under‐matured, 20 hr; and over‐matured, 92 hr) per animal, allowing each cow to be its own control (24 collections). Microarray analysis revealed that between 5 and 338 transcripts were significantly different across the six comparisons, with an important longitudinal modulation in terms of gene expression profile. Not surprisingly, as the transcriptional activity decreased in these oocytes, several transcripts that are significantly modulated during coasting are related to RNA processing functions, as shown by functional analysis. Ingenuity Pathway Analysis also highlighted another important function: the control of chromosome segregation. The results presented here indicate that the quality gained with the optimal coasting time does not last, and also suggests a possible mechanism of control by transcript degradation that could be implicated if the oocyte is not ovulated at the right time. Mol. Reprod. Dev. 80: 428–440, 2013.

Chromatin remodelling and histone mRNA accumulation in bovine germinal vesicle oocytes

Major remodelling of the chromatin enclosed within the germinal vesicle occurs towards the end of oocyte growth in mammals, but the mechanisms involved in this process are not completely understood. In bovine, four distinct stages of chromatin compaction—ranging from a diffused state (GV0) to a fully compacted configuration (GV3)—are linked to the gradual acquisition of developmental potential. To better understand the molecular events and to identify mRNA modulations occurring in the oocyte during the GV0‐to‐GV3 transition, transcriptomic analysis was performed with the EmbryoGENE microarray platform. The mRNA abundance of several genes decreased as chromatin compaction increased, which correlates with progressive transcriptional silencing that is characteristic of the end of oocyte growth. On the other hand, the abundance of some transcripts increased during the same period, particularly several histone gene transcripts from the H2A, H2B, H3, H4, and linker H1 family. In silico analysis predicted RNA‐protein interactions between specific histone transcripts and the bovine stem‐loop binding protein 2 (SLBP2), which helps regulate the translation of histone mRNA during oogenesis. These results suggest that some histone‐encoding transcripts are actively stored, possibly to sustain the needs of the embryo before genome activation. This dataset offers a unique opportunity to survey which histone mRNAs are needed to complete chromatin compaction during oocyte maturation and which are stockpiled for the first three cell cycles following fertilization. Mol. Reprod. Dev. 82: 450–462, 2015.

Gene expression analysis of bovine oocytes at optimal coasting time combined with GnRH antagonist during the no-FSH period

Ovarian stimulation with FSH combined with an appropriate period of FSH withdrawal (coasting) before ovum pick-up now appears to be a successful way to obtain oocytes with high developmental competence in bovine. Recent results showed that extending follicular growth by only 24 hours has a detrimental effect on oocyte quality as shown by the reduced blastocyst formation rate. Although these treatments are initiated during the luteal phase with low LH level, the small LH pulsatility present at that time could potentially impact follicular development as well as oocyte quality. In this study, a GnRH antagonist (Cetrotide) was used to suppress LH secretion during follicular differentiation to get a better insight into the physiological importance of the LH support during that period. Oocytes were collected by ovum pick-up, and quality was assessed by measuring the blastocyst formation rate obtained after IVM-IVF. The oocyte transcriptome from GnRH antagonist-treated animals was also compared with that from a control group (coasting duration of 68 hours) to detect possible alterations at the messenger RNA (mRNA) level. The oocyte quality was not statistically affected by the treatment as shown by the blastocyst formation rate obtained. However, microarray analysis showed that a total of 226 genes had a significant difference (fold change > 2; P < 0.05) at the mRNA level, with the majority being in overabundance in the treated group. Many genes related to RNA posttranscriptional modifications presented different abundance at the mRNA level significant differences in the control group (68 hours), whereas translation function appeared to be affected, with many genes related to structural constituents of the ribosome presenting a overabundance in the GnRH antagonist-treated group. Specific mRNAs with crucial roles in chromosome segregation control also showed significant difference at the mRNA level after Cetrotide treatment. The results presented here indicated that the suppression of the LH secretion in an optimal stimulated context would have an impact on the oocyte, with the possible alteration of critical functions related to translation capacity and chromosome segregation control.

Evolutionary conservation of the oocyte transcriptome among vertebrates and its implications for understanding human reproductive function

Cross-phylum and cross-species comparative transcriptomic analyses provide an evolutionary perspective on how specific tissues use genomic information. A significant mRNA subset present in the oocytes of most vertebrates is stabilized or stored for post-LH surge use. Since transcription is arrested in the oocyte before ovulation, this RNA is important for completing maturation and sustaining embryo development until zygotic genome activation. We compared the human oocyte transcriptome with an oocyte-enriched subset of mouse, bovine and frog (Xenopus laevis) genes in order to evaluate similarities between species. Graded temperature stringency hybridization on a multi-species oocyte cDNA array was used to measure the similarity of preferentially expressed sequences to the human oocyte library. Identity analysis of 679 human orthologs compared with each identified official gene symbol found in the subtractive (somatic-oocyte) libraries comprising our array revealed that bovine/human similarity was greater than mouse/human or frog/human similarity. However, based on protein sequence, mouse/human similarity was greater than bovine/human similarity. Among the genes over-expressed in oocytes relative to somatic tissue in Xenopus, Mus and Bos, a high level of conservation was found relative to humans, especially for genes involved in early embryonic development.

Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis.

Follicle size is recognized as a predictor of the potential for the enclosed oocyte to yield an embryo following in vitro maturation and in vitro fertilization. Oocytes from larger follicles are more likely to reach the blastocyst stage than those from smaller follicles. A growing oocyte accumulates all the transcripts needed to ensure development until the maternal embryonic transition, and this accumulation must be completed before the period of transcriptional arrest. Accordingly, the transcriptomes of bovine germinal‐vesicle‐stage oocytes collected from follicles of increasing sizes (<3, 3–5, >5–8, and >8 mm) were evaluated, using the EmbryoGENE bovine transcriptomic platform (custom Agilent 4 × 44 K), to better understand transcriptional modulation in the oocyte as the follicle becomes larger. Microarray analyses revealed very few differences between oocytes from small follicles (<3 vs. 3–5 mm), whereas an important number of differences were detected at the mRNA level between oocytes from larger follicles. Weighted gene correlation network analysis allowed for the identification of several hub genes involved in crucial functions such as transcriptional regulation (TAF2), chromatin remodeling (PPP1CB), energy production (SLC25A31), as well as transport of key molecules within the cell (NAGPA, CYHR1, and SLC3A12). The results presented here thus reinforce the hypothesis that developmental competence acquisition cannot be seen as a simple one‐step process, especially in regards to the modulation of mRNA. Mol. Reprod. Dev. 83: 558–569, 2016.

Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation

The processes underlying the very first moments of embryonic development are still not well characterised in mammals. To better define the kinetics of events taking place following fertilisation, it would be best to have perfect synchronisation of sperm entry. With fertilisation occurring during a time interval of 6 to 12h in the same group of fertilised oocytes, this causes a major variation in the time of activation of embryonic development. Bovine parthenogenesis could potentially result in better synchronisation and, if so, would offer a better model for studying developmental competence. In the present study, bovine oocytes were either parthenogenetically activated or fertilised and cultured in vitro for 7 days. Gene expression analysis for those two groups of embryos at early and expanded stages was performed with BlueChip, a customised 2000-cDNA array developed in our laboratory and enriched in clones from various stages of bovine embryo development. The microarray data analysis revealed that only a few genes were differentially expressed, showing the relative similarity between those two kinds of embryos. Nevertheless, the fact that we obtained a similar diversity of developmental stages with parthenotes suggests that synchronisation is more oocyte-specific than sperm entry-time related. We then analysed our data with Ingenuity pathway analysis. Networks of genes involved in blastocyst implantation but also previous stages of embryo development, like maternal-to-embryonic transition, were identified. This new information allows us to better understand the regulatory mechanisms of embryonic development associated with embryo status.

ASAS-SSR Triennial Reproduction Symposium: The use of natural cycle’s follicular dynamic to improve oocyte quality in dairy cows and heifers1,2

The selection of the best dairy heifers is mainly driven by the genetic value of their parents. The phenotype analysis of cows and of the daughters of bulls has been used to identify the best genetic value for decades before being replaced by genomic selection of individuals that are not yet parents. Because it is possible to predict the future value of an individual by its genetic makeup, it becomes feasible to do it as early as the blastocyst stage and to decide which should be transferred or not. Because we know the genotype of an animal at birth, or even before, it is becoming desirable to reproduce this animal as soon as possible to reduce generation interval and improve selection speed. Nature provides constraints that can be overcome: a single oocyte per cycle and age at puberty. Indeed, it is now possible to super-stimulate the ovary at any age and to start collecting oocytes at 6 mo by trans-vaginal ultrasonography. The challenge becomes the production of good eggs and embryos capable of implanting and developing into healthy calves. Our understanding of ovarian follicular physiology has been instrumental in designing stimulation protocols that may be adjusted to any physiological context including age, and even the individual animal, to obtain a good response. Therefore, the combination of procedures developed in cows to optimize oocyte quality, for example, FSH coasting, in association with in vitro fertilization and optimal culture conditions can now result in the production of several female embryos twice a month from animals 6 to12 mo of age. The transcriptomic and epigenetic analyses of embryos produced from the same females at different ages were compared and few differences were noted in particular in relation to embryo metabolism. These embryos are as good as the ones obtained from adult animals and can be produced with sexed sperm of bulls 12 mo of age. This combination of these technical optimizations with blastocyst genotyping allows the selection of a second generation within a year.

Effect of heifer age on the granulosa cell transcriptome after ovarian stimulation

Genomic selection is accelerating genetic gain in dairy cattle. Decreasing generation time by using younger gamete donors would further accelerate breed improvement programs. Although ovarian stimulation of peripubertal animals is possible and embryos produced in vitro from the resulting oocytes are viable, developmental competence is lower than when sexually mature cows are used. The aim of the present study was to shed light on how oocyte developmental competence is acquired as a heifer ages. Ten peripubertal Bos taurus Holstein heifers underwent ovarian stimulation cycles at the ages of 8, 11 (mean 10.8) and 14 (mean 13.7) months. Collected oocytes were fertilised in vitro with spermatozoa from the same adult male. Each heifer served as its own control. The transcriptomes of granulosa cells recovered with the oocytes were analysed using microarrays. Differential expression of certain genes was measured using polymerase chain reaction. Principal component analysis of microarray data revealed that the younger the animal, the more distinctive the gene expression pattern. Using ingenuity pathway analysis (IPA) and NetworkAnalyst (www.networkanalyst.ca), the main biological functions affected in younger donors were identified. The results suggest that cell differentiation, inflammation and apoptosis signalling are less apparent in peripubertal donors. Such physiological traits have been associated with a lower basal concentration of LH.