Acquisition Of Developmental Competence Research Articles

Identification of novel cattle (Bos taurus) genes and biological insights of their function in pre-implantation embryo development

BackgroundAppropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes.ResultsWe identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10–8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10–7).ConclusionsOur results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

Maternal regulation of the vertebrate oocyte-to-embryo transition.

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.

Metabolic imaging of human cumulus cells reveals associations with pregnancy and live birth.

Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos? FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not. CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART. A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied. This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant. A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not. Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection. Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART. National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1. N/A.

P-219 Single-follicle spatial proteomics of the whole ovary by MALDI mass spectrometry imaging: towards the identification of protein-markers of the follicle's quality

Abstract Study question Can we improve our knowledge of the single-follicle proteome signature by MALDI mass spectrometry imaging? Summary answer We identified follicle-type proteins that function during follicle growth (NUMA1, TPM2), GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3). What is known already The acquisition of oocytes developmental competence occurs through a bidirectional exchange of molecular information between the oocyte, the companion follicle cells, the stroma, and the vascular network. This reciprocal relationship is still poorly understood and particularly scarce is our knowledge of the proteins that are playing a regulative role during folliculogenesis. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool that allows the study of the proteomic landscape of small morphological structures inside a tissue, but in the field of reproduction has been only used to analyse rodent spermatogenesis. Study design, size, duration Nano-scale liquid chromatography-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS) was combined with MALDI-MSI to identify the proteomic landscape and to map the changes occurring throughout folliculogenesis. We performed single-follicle spatial proteomics on all the follicles, from the secondary to the fully-grown preovulatory, present in histological serial sections of an entire prepubertal 25-day-old mouse ovary. Cross-referencing our results with those obtained by previous proteomics and transcriptomics analyses allowed to strengthen their significance. Participants/materials, setting, methods A single 25-day-old mouse ovary was fixed in 10% formalin, dehydrated, embedded in paraffin, and sectioned to obtain 133 6-μm serial sections. After paraffin removal, trypsin digestion and matrix deposition, mass spectra were acquired using a rapifleX MALDI Tissuetyper™ (Bruker) with a raster sampling of 20 μm in both x and y axes. Then, tryptic peptides were extracted from sections and analysed with nLC-ESI-MS/MS using a Dionex-UltiMate-3000 LC-nano-system coupled with an Impact HD™ UHR-QqToF (Bruker). Main results and the role of chance This study proposes a spatial proteomics workflow to investigate the proteome of a whole prepubertal 25-day-old mouse ovary, preserving the spatial relationship of the peptides in this complex histological context. A total of 401 proteins were identified by nLC-ESI-MS/MS, 69 with a known function in ovary biology. Enrichment analysis highlighted significant KEGG and Reactome pathways, with apoptosis, developmental biology, PI3K-Akt, epigenetic regulation of gene expression, and extracellular matrix organisation being well represented. Then, correlating these data with the spatial information provided by MALDI-MSI on 276 follicles highlighted 94 proteins that were detected throughout the secondary to the pre-ovulatory transition. Of these, 37 proteins showed a gradual quantitative change during follicle differentiation, comprising 10 with a known role in follicle growth (NUMA1, TPM2), oocyte GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3). The proteome landscape identified includes molecules of known function in the ovary, but also those whose specific role is emerging. Altogether, this work demonstrates the utility of performing spatial proteomics in the context of the ovary and offers sound bases for more in-depth investigations that aim to further unravel its spatial proteome. Limitations, reasons for caution A limitation of MALDI-MSI is its 20 µm/pixel resolution which 1) leads to the acquisition of spectra comprising follicular and extra-follicular tissue, particularly with small follicles (15-30µm in diameter); and 2) does not distinguish among the different follicular cell types, thus precluding its application for single-cell in situ proteomics. Wider implications of the findings MALDI-MSI is a potent technology to study the proteome of ovary specimens, but it could also be used for the analysis of their lipidic or metabolic profiles. Our pipelline aim to identify protein-markers of the follicle's quality and, in turn, of the oocyte's developmental competence. Trial registration number not appplicable

Effects of cortisol on granulosa cell function during follicle maturation in the horse

Follicle maturation and acquisition of developmental competence by the oocyte require an interplay between granulosa cells and the oocyte, involving Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) mediated modulation of follicular steroidogenesis. In the horse, dynamic intrafollicular concentrations of cortisol were associated with follicle growth and oocyte maturation, suggesting a functional relationship between cortisol and oocyte-derived factors. The aim of this study was to analyze effects of cortisol on steroidogenic activity, apoptosis and proliferative capacity of equine granulosa cells, as well as the relationship with the oocyte-derived factor GDF9. For this purpose, granulosa cells were collected three times in four mares each during the breeding season by transvaginal aspiration in the absence of a dominant follicle. Cells collected on the same day were pooled and then seeded on 12-well plates to grow to 70-80% confluence before being stimulated with 100 ng/ml IGF1 for 24h, in the presence or absence of cortisol in increasing concentrations: 50, 100 and 150 ng/ml. These concentrations were previously observed in antral follicles before deviation (50 ng/ml) and after deviation (120 ng/ml). In blocking experiments, cells were preincubated with 20 uM H89 (protein kinase A inhibitor). All experiments were repeated three times. GraphPad software was used for statistical analysis with one-way analysis of variance (ANOVA), followed by non-parametric multiple comparison post-hoc test.Expression of Star was similar in all groups (P>0.05), independent of cortisol concentration and was decreased (P<0.05) after preincubation with H89. Transcriptional levels of HSD3B2 were higher (P<0.001) after adding IGF1 and cortisol simultaneously, but not separately. The rise in HSD3B2 levels was independent of cortisol dose used (P>0.05). Blocking of Star with H89 increased GDF9 expression (P<0.01), both in the presence and absence of cortisol. Interestingly, addition of cortisol alone in all analyzed dosages raised BCL2 levels (P<0.05) and this effect was inhibited by H89 (P<0.05). However, there was no effect on CASP3 levels (P>0.05), regardless of treatment used. A higher proliferating capability of equine granulosa cells, as assessed by PCNA levels, was observed in response to IGF1 treatment (P<0.05), but only in the presence of 150 ng/ml cortisol. Despite apparently elevated, the expression of Ki67 marker in response to IGF1 in the presence of cortisol proved to be not significant (P>0.05). Taken together, our results demonstrate enhanced steroidogenesis as well as reduced apoptosis in equine granulosa cells in response to cortisol. Moreover, reduced steroidogenic activity (i.e. decreased expression of genes involved in progesterone synthesis) of granulosa cells increased GDF9 expression, which has previously been shown to be detrimental for the acquisition of developmental competence by the oocyte.

Review: The putative role of Progesterone Receptor membrane Component 1 in bovine oocyte development and competence.

Acquisition of developmental competence is a complex process in which many cell types cooperate to support oocyte maturation, fertilisation, and preimplantation embryonic development. In recent years, compelling evidence has shown that Progesterone Receptor Membra Component 1 (PGRMC1) is expressed in many cell types of the mammalian reproductive system where it exerts diverse functions. In the ovary, PGRMC1 affects follicular growth by controlling cell viability and proliferation of granulosa cells. PGRMC1 has also a direct role in promoting a proper completion of bovine oocyte maturation, as altering its function leads to defective chromosome segregation and polar body extrusion. Strikingly, the mechanism by which PGRMC1 controls mitotic and meiotic cell division seems to be conserved, involving an association with the spindle apparatus and the chromosomal passenger complex through Aurora kinase B. Conclusive data on a possible role of PGRMC1 in the preimplantation embryo are lacking and further research is needed to test whether the mechanisms that are set in place in mitotic cells also govern blastomere cleavage and subsequent differentiation. Finally, PGRMC1 is also expressed in oviductal cells and, as such, it might also impact fertilisation and early embryonic development, although this issue is completely unexplored. However, the study of PGRMC1 function in the mammalian reproductive system remains a complex matter, due to its pleiotropic function.

Review: Roles of follicle-stimulating hormone in preantral folliculogenesis of domestic animals: what can we learn from model species and where do we go from here?

The pituitary gonadotropin FSH is a glycoprotein critical for the development of ovarian follicles. Upon binding to its G protein-coupled membrane receptor located on the granulosa cells of ovarian follicles, FSH elicits a cascade of downstream intracellular responses to promote follicle growth, maturation and steroidogenic activity, leading to the acquisition of meiotic and developmental competence of the enclosed oocyte. The essential role of FSH for proper antral follicle development and fertility is indisputable; over the decades, increasing evidence has also pointed toward survival and growth-promoting effects elicited by FSH in earlier-stage preantral follicles, deeming these follicles FSH-responsive as opposed to the FSH-dependent antral follicles. Transgenic mouse models lacking GnRH1, Fshβ or Fshr clearly demonstrate this difference by showing that, morphologically, preantral follicles develop to the secondary stage without FSH signaling; however, exogenous expression or administration of FSH to hormone-deficient mice promotes preantral follicle development, with more pronounced effects seen in earlier stages (i.e., primary follicles). In hypophysectomized sheep, FSH administration also promotes the growth of primary-stage preantral follicles. However, in vivo studies in this area are more challenging to perform in domestic animals compared to rodents, and therefore most of the research to date has been done in vitro. Here, we present the existing evidence for a role of FSH in regulating the growth and survival of preantral follicles from data generated in rodents and domestic animals. We provide an overview of the process of folliculogenesis, FSH synthesis and cellular signaling, and the response to FSH by preantral follicles in vivo and in vitro, as well as interactions between FSH and other molecules to regulate preantral folliculogenesis. The widespread use of FSH in ovarian stimulation programs for assisted reproduction creates a real need for a better understanding of the effects of FSH beyond stimulation of antral follicle growth, and more research in this area could lead to the development of more effective fertility programs. In addition to its importance as an agricultural species, the cow provides a desirable model for humans regarding ovarian stimulation due to similar timing of folliculogenesis and follicle size, as well as similar ovarian architecture. The refinement of minimally invasive methods to allow the study of preantral folliculogenesis in live animals will be critical to understand the short- and long-term effects of FSH in ovarian folliculogenesis.

Restoration of Developmental Competence in Low-Quality Porcine Cumulus–Oocyte Complexes through the Supplementation of Sonic Hedgehog Protein during In Vitro Maturation

The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus-oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB- (low-quality), (ii) BCB- + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

A refined culture system of oocytes from early antral follicles promotes oocyte maturation and embryo development in cattle.

The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency. The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus-oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development

To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. Laboratory study. Private fertility clinic. Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. Transcriptomic profiles comparison between different stages of early development in AGs and PGs. The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.

Cumulus cells secrete micro-vesicles carrying miRNAs: their role in the acquisition of mouse and human oocyte's developmental competence

Background and Aim: During folliculogenesis oocytes acquire competence also thanks to a bidirectional communication with companion cumulus cells (CCs). The nature of the CC-determinants contributing to this remains unclear. We recently showed that when mouse CCs-free antral oocytes are cultured to MII with a feeder layer (FL) of CCs isolated from developmentally-competent surrounded-nucleolus (FL-SN-CCs) or -incompetent not-surrounded-nucleolus (FL-NSN-CCs) oocytes, the former acquire developmental competence to blastocyst, whereas the latter arrest.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

Bovine In Vitro Oocyte Maturation and Embryo Production Used as a Model for Testing Endocrine Disrupting Chemicals Eliciting Female Reproductive Toxicity With Diethylstilbestrol as a Showcase Compound.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10–9 M, 10–7 M, 10–5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10–9 M or 10–7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10–5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Human oocyte meiotic maturation is associated with a specific profile of alternatively spliced transcript isoforms.

The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is required for the acquisition of oocyte developmental competence. We hypothesize that the expression of specific genes at the in vivo matured (MII) stage could be modulated by posttranscriptional mechanisms, particularly regulation of alternative splicing (AS). In this study, we examined the transcriptional activity of GV oocytes after ovarian stimulation followed by oocyte pick-up and the landscape of alternatively spliced isoforms in human MII oocytes. Individual oocytes were processed and analyzed for transcriptional activity (GV), gene expression (GV and MII), and AS signatures (GV and MII) on HTA 2.0 microarrays. Samples were grouped according to maturation stage, and then subgrouped according to women's age and antral follicular count (AFC); array results were validated by quantitative polymerase chain reaction. Differentially expressed genes between GV and MII oocytes clustered mainly in biological processes related to mitochondrial metabolism. Interestingly, 16 genes that were related to the regulation of transcription and mitochondrial translation showed differences in alternatively spliced isoform profiles despite not being differentially expressed between groups. Altogether, our results contribute to our understanding of the role of AS in oocyte developmental competence acquisition.

150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P&amp;lt;0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P&amp;lt;0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P&amp;lt;0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.

Potential of aquaporins and connexins in dogs and their relation to the reproductive tract

The complexity of processes in the female reproductive system of mammals is extremely sophisticated. The overall relationship between the processes during the oestrus cycle in animals is quite well understood, but the molecular background of these processes still requires an in-depth analysis. Bitches are distinguished by a specific course of sexual cycle during which the oocyte matures after ovulation in the oviduct. Other species of mammals are characterized by maturation of the oocyte within the ovary. Acquisition of developmental competence by cumulus – oocyte complexes seems to be a process with a complex molecular background, and the key to understanding it may be the analysis of intercellular channels. Aquaporins and connexins are structural proteins that are built into the cell membrane. Their location is widespread in many body tissues. Recent years have shown that they exhibit significant expression in different parts of the mammalian reproductive system, although the number of studies on dogs is still negligible. This review paper presents the current state of knowledge of water channels and gap junction connections in different animal species, with particular focus on dogs, and also explores the role of aquaporins and connexins in the acquisition of reproductive competences.

In Vitro Maturation of Fully Grown Mouse Antral Follicles in the Presence of 1\xa0nM 2-Hydroxyestradiol Improves Oocytes\u2019 Developmental Competence

Cathecolestrogens are estradiol metabolites produced during folliculogenesis in the mammalian ovary. 2-Hydroxyestradiol (2-OHE2) is one of the most abundant although its role remains unknown. The aim of this study is to investigate whether the presence of 2-OHE2 during the germinal vesicle-to-metaphase II transition affects oocyte meiotic and preimplantation developmental competence. Mouse cumulus-oocyte complexes (COCs), isolated from fully grown antral follicles, were in vitro–matured (IVM) in the presence of 2-OHE2 (0.1, 1, 10 or 100 nM) for 6 or 15 h; then, their meiotic and developmental competence was evaluated using a number of cytological quality markers. With the exception of the highest dose (100 nM), the addition of 2-OHE2 to the IVM medium, did not alter, compared with untreated control, the frequency of oocytes that reached the MII stage. Instead, IVM in the presence of 1 nM 2-OHE2 highly increased the rate of preimplantation development and blastocyst quality. To understand whether this positive effect could be attributed to the events occurring during meiosis resumption, we analysed a number of specific cytological quality markers of the asymmetric division, such as PB-I volume and position, presence and extension of the cortical F-actin cap, meiotic spindle shape and area, and microtubule organisation centre localisation. The results highlighted how the presence of 1 nM 2-OHE2 significantly improved the overall cytological organisation required for a correct asymmetric division. Our results contribute a first step to acknowledge a potential role of this estradiol metabolite during the GV-to-MII transition, contributing to the acquisition of oocytes developmental competence.

The blueprint of RNA storages relative to oocyte developmental competence in cattle (Bos taurus).

From the time oocytes leave quiescence, there are constant microenvironmental influences contributing to development, thus acquiring developmental competence is not a simple, linear phenomenon. During folliculogenesis, oocytes experience many morphological and cytological changes that contribute toward the acquisition of developmental competence, a process defined by an oocyte's ability to progress through folliculogenesis, be fertilized, undergo cleavage, and develop into an embryo. Many factors, such as ovarian follicle size, cow age, and the morphology of the cumulus-oocyte complex, have been extensively investigated to understand this process. In parallel to aiding in the understanding of oocyte biology, these features have been used to characterize an oocyte's ability to achieve competence. In addition, oocytes undergo intense gene transcription and protein translation to accumulate the maternal stores. When the oocyte is fully grown, most genes are transcriptionally inactive, and the chromatin is densely compacted. More recently, RNA profiling has been used to further define the transcriptional parameters that are associated with oocyte development. Here, focusing on cattle, we provide an overview of the experimental models commonly used to understand the underlying biology related to oocyte developmental competence. We compiled public data and showed that cattle oocytes can express over 15000 protein-coding genes, suggesting a complex transcriptome landscape. Surprisingly, less than 2% of the expressed genes have been linked to developmental competence. The identification of the gene products that contribute to oocyte development, and understanding their biological function, are a vital component of our quest toward defining oocyte developmental competence at the molecular level.