Age-dependent high-yield isolation of primordial, primary, and early secondary follicles from the bovine ovarian cortex.

In brief: Preantral follicles constitute the largest follicle reserve in the mammalian ovary. This study assesses a mechanical isolation method to maximize the number of follicles retrieved from a defined cortex volume. Abstract: Primordial, primary, and secondary follicles (collectively defined as preantral follicles) constitute the most abundant source of gametes inside the mammalian ovarian cortex. The massive isolation of preantral follicles and the refinement of stage-specific protocols for in vitro follicle growth would provide a powerful tool to boost the rescue and restoration of fertility in assisted reproduction interventions in human medicine, animal breeding, and vulnerable species preservation. Nevertheless, together with an efficient culture system, the most significant limitation to implementing in vitro follicle growth is the lack of an efficient method to isolate viable and homogeneous subpopulations of primordial, primary, and secondary follicles suitable for in vitro culture. Our study provides a strategy for high-yielding mechanical isolation of primordial, primary, and early secondary follicles from a limited portion of the ovarian cortex in the bovine animal model. In the first part of the study, we refined a mechanical isolation protocol of preantral follicles, adopting specific methodological strategies to separate viable and distinct subpopulations of primordial (oblate and prolate forms), primary, and early secondary follicles from 0.16 cm3 of the ovarian cortex. In the second part of the study, we tested the effectiveness of the isolation protocol, considering the individual's age as a critical factor, bearing in mind the progressive decrease in the ovarian reserve that naturally accompanies the reproductive life span. Our study provides a way for designing quantitative and conservative fertility preservation approaches to preserve organ function and minimize the invasiveness of the interventions, also considering age-related differences.

Meiotic maturation failure in primary ovarian insufficiency: insights from a bovine model.

PURPOSE: Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. METHODS: Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. RESULTS: In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. CONCLUSION: Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

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

In brief: 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. Abstract: 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.

Culture of vitrified bovine ovarian tissue on agarose gel inserts maintains follicle integrity.

In brief: Ovarian tissue cryopreservation and culture provide an option for fertility preservation without tissue grafting, but need optimization. This study reveals that vitrified bovine ovarian tissue can be cultured on agarose gel and maintain follicle morphology, low activation, and low apoptosis. Abstract: Ovarian tissue preservation is hitherto a promising fertility insurance option for precious animals. Ovarian tissue vitrification and culture combined approach would eliminate the need of transplanting ovarian tissue to obtain mature oocytes. We aimed at optimizing vitrification and in vitro culture conditions for improved bovine ovarian tissue viability. Ovaries obtained from the slaughterhouse were punched into fragments and divided into three groups. Group 1 (fresh) was divided into two and immediately placed in two-culture systems (culture inserts and agarose inserts). Group 2 was vitrified, warmed, and placed in the two-culture systems, while group 3 was only equilibrated and then placed in the two-culture systems. All cultures were maintained for 6 days and spent media were collected on alternate days for cytokine (interleukin 1ß and interleukin 6) evaluation. Fragments were fixed for morphology assessment and immunohistochemistry. Higher percentages (P < 0.05) of grade 1 (morphologically intact) follicles were observed in fragments on agarose compared to those on culture inserts on days 2 and 4 of the culture. Conversely, we found higher (P < 0.05) shifts of primordial follicles to transitional follicles in fragments on culture inserts vis-à-vis agarose inserts which was consistent with a higher proportion of Ki-67 and MCM-7 and activated caspase-3-positive follicles. In conclusion, in vitro culture of bovine ovarian tissue on agarose inserts maintained follicle morphology, low follicle activation, and low apoptosis compared to culture inserts.

A century of programmed cell death in the ovary: a commentary.

Programmed cell death (PCD) has been discussed and categorized endlessly over the years. Herein, we comment on the chaotic classification of PCD in the mammalian ovary and the compartmentalized ovarian follicle based on an unfiltered overview of the extent of publication about some of the different modes of PCD on PubMed.

Physiological parameters related to oocyte nuclear differentiation for the improvement of IVM/IVF outcomes in women and cattle.

In vitro maturation (IVM) has been applied in numerous different contexts and strategies in humans and animals, but in both cases it represents a challenge still far from being overcome. Despite the large dataset produced over the last two decades on the mechanisms that govern antral follicular development and oocyte metabolism and differentiation, IVM outcomes are still unsatisfactory. This review specifically focuses on data concerning the potential consequences of using supraphysiological levels of FSH during IVM, as well as on the regulation of oocyte chromatin dynamics and its utility as a potential marker of oocyte developmental competence. Taken together, the data revisited herein indicate that a significant improvement in IVM efficacy may be provided by the integration of pre-OPU patient-specific protocols preparing the oocyte population for IVM and more physiological culture systems mimicking more precisely the follicular environment that would be experienced by the recovered oocytes until completion of metaphase II.

Zinc supports transcription and improves meiotic competence of growing bovine oocytes.

In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

Successful in vitro maturation of oocytes: a matter of follicular differentiation.

Folliculogenesis is remarkably similar in cattle and humans. In this review, we consider the known differences and provide a possible explanation for the greater success of oocyte in vitro maturation in cattle. Two different parallel processes that are critical for oocyte competence acquisition are examined. The first occurs in the follicle and in turn influences the oocyte, the second occurs within the oocyte itself and involves the gradual cessation of the transcription machinery with additional changes observable in the chromatin structure. We expect this insight to contribute to the improvement of human fertility programs based on in vitro fertilization, and particularly to the development of controlled ovarian stimulation protocols that yield more high-quality oocytes and thereby improve the clinical performance of treatments for infertility.

PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes.

Progesterone receptor membrane component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1's nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi)-mediated gene silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H2O2) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1's ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1's well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress.

Method of Isolation and In Vitro Culture of Primordial Follicles in Bovine Animal Model.

The mammalian ovary is a substantial source of oocytes arranged into follicles at various stages of folliculogenesis, from the primordial to the ovulatory ones. Primordial follicles constitute the most abundant source of gametes inside the mammalian ovary at any given time.The isolation of a high number of primordial follicles, together with the development of protocols for in vitro follicle growth, would provide a powerful tool to fully exploit the female reproductive potential and boost the rescue and restoration of fertility in assisted reproduction technologies in human medicine, animal breeding, and preservation of threatened species. However, the most significant limitation is the lack of efficient methods for isolating a healthy and homogeneous population of viable primordial follicles suitable for in vitro culture. Here, we provide a fast and high-yield strategy for the mechanical isolation of primordial follicles from limited portions of the ovarian cortex in the bovine animal model.

In vitro production of meiotically competent oocytes from early antral follicles in sheep.

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350-450 µm) and cultured in TCM199 medium supplemented with 0.15 µg/mL Zn sulfate, 10-4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B.

Oocytes isolated from cows of reproductive age with reduced antral follicle counts (AFC) have a diminished capacity of embryonic development, which may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Because we demonstrated that progesterone receptor membrane component 1 (PGRMC1) is involved in chromosome congression and metaphase II (MII) plate formation, the present study was designed to determine 1) if the decrease in oocyte developmental competence observed in dairy cows with a reduced AFC is due to a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. Oocytes from ovaries with reduced AFC and age-matched controls were matured in vitro and the occurrence of aneuploidy determined as well as the mRNA level and localization of PGRMC1. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3-fold increase in aneuploidy compared to oocytes isolated from control ovaries (P < 0.05). Although Pgrmc1 mRNA levels were not altered, PGRMC1 and aurora kinase B (AURKB) failed to localize to precise focal points on MII chromosomes of oocytes from ovaries with reduced AFC. Furthermore, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity, their MII plate was disrupted and PGRMC1 was not properly localized to the chromosomes. These results suggest that alterations in PGRMC1 and/or AURKB localization account in part for the increased aneuploidy and low development competence of oocytes from ovaries with reduced AFC.

Neuregulin 1 (NRG1) modulates oocyte nuclear maturation during IVM and improves post-IVF embryo development.

Oocyte in vitro maturation (IVM) is still a major challenge in human and animal assisted reproduction. Gradual instead of abrupt activation of the ovulatory cascade during IVM has been proposed to enhance nuclear-cytoplasmic synchrony and cumulus-oocyte communication, thus favoring oocyte developmental competence. Herein, we assessed the effects of neuregulin 1 (NRG1), an EGF-like factor that modulates EGFR signaling, on oocyte nuclear maturation dynamics, cumulus expansion and expression of mRNAs regulating these processes during IVM, as well as on post-IVF embryo development following AREG-stimulated IVM in cattle. In experiment 1, cumulus-oocyte complexes (COCs) were subjected to IVM with graded doses of NRG1 (1, 10 or 100 ng/mL) for 6, 9, 12, 20, and 24 h, after which oocyte nuclear status and cumulus mRNA expression were assessed. At 6 h of IVM, NRG1 at 1 ng/mL significantly decreased the percentage of GVBD (germinal vesicle breakdown) oocytes without altering later meiotic dynamics or the percentage of oocytes achieving meiosis II. In experiment 2, adding NRG1 (1 ng/mL) to the IVM medium did not affect cumulus expansion but increased the percentage of expanded and hatched blastocysts, and blastocyst total cell number following IVF/IVC. NRG1 decreased EGFR mRNA abundance while increasing NPR2 and PTX3 mRNA levels at 9 h, and TNFAIP6 mRNA abundance at 20 h of IVM. This is the first study that reports the modulatory effect of NGR1 during oocyte maturation in a mono-ovulatory species and demonstrates that this action may be applied during IVM to improve post-IVF embryo development.

Canine amniotic fluid at birth: From a discarded sample to a potential diagnostic of neonatal maturity.

The definition of new reliable markers for neonatal maturity evaluation is crucial in canine clinical practice. Concerns about the safety of amniotic sampling in pregnant dogs have prevented its collection for diagnostic purposes. Moreover, amniotic fluid had been considered waste material until the latest studies reported amniocentesis as a reliable and safe procedure, even in the canine species. In our study, amniotic fluid (n = 63) collected at birth from ten dogs undergoing elective Caesarean sections at term was analysed to discover new potential indices of canine neonatal maturity. Based on gestational age, mothers and puppies were divided into two groups: the early group (≤65 days from luteinizing hormone (LH) surge, n = 5) and the late group (>65 days from LH surge, n = 5). Amniotic parameters of the lightest and heaviest puppy in individual/each litter, with a birth weight difference of at least 20% among littermates, were also compared. In particular, the content of lecithin, sphingomyelin, surfactant protein A (SP-A), cortisol, and pentraxin 3 (PTX3) in amniotic fluid, which is considered predictive of foetal development in humans, were investigated. Maternal serum SP-A and cortisol were also measured simultaneously. All amniotic parameters were detectable in canine amniotic fluid. Interestingly, the concentrations of different amniotic parameters correlated with each other. Lecithin was positively correlated with sphingomyelin (p < 0.0001), maternal SP-A (p < 0.0005), and the ratio of amniotic and maternal cortisol (p < 0.004). Amniotic SP-A was inversely correlated to maternal SP-A (p < 0.05), lecithin (p < 0.005), and lecithin-sphingomyelin ratio (p < 0.05). A positive correlation was also recorded between amniotic and maternal cortisol (p < 0.008). Considering that all puppies were born alive and mature, these data could provide a potential range of expected amniotic values in full-term new-born dogs. Furthermore, since gestational age was positively correlated with both maternal and amniotic cortisol (p < 0.0001) and amniotic PTX3 (p < 0.05), amniotic fluid seems to be an attractive, innovative, and minimally invasive matrix with potential diagnostic and prognostic utility for the investigation of canine maturity.

Progress toward species-tailored prematuration approaches in carnivores.

In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies. Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans. An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.

Phylogenomic evidence for the presence of a flagellum and cbb(3) oxidase in the free-living mitochondrial ancestor.

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Insights on the Role of PGRMC1 in Mitotic and Meiotic Cell Division.

During mitosis, chromosome missegregation and cytokinesis defects have been recognized as hallmarks of cancer cells. Cytoskeletal elements composing the spindle and the contractile ring and their associated proteins play crucial roles in the faithful progression of mitotic cell division. The hypothesis that PGRMC1, most likely as a part of a yet-to-be-defined complex, is involved in the regulation of spindle function and, more broadly, the cytoskeletal machinery driving cell division is particularly appealing. Nevertheless, more than ten years after the preliminary observation that PGRMC1 changes its localization dynamically during meiotic and mitotic cell division, this field of research has remained a niche and needs to be fully explored. To encourage research in this fascinating field, in this review, we will recap the current knowledge on PGRMC1 function during mitotic and meiotic cell division, critically highlighting the strengths and limitations of the experimental approaches used so far. We will focus on known interacting partners as well as new putative associated proteins that have recently arisen in the literature and that might support current as well as new hypotheses of a role for PGRMC1 in specific spindle subcompartments, such as the centrosome, kinetochores, and the midzone/midbody.

A Nuclear and Cytoplasmic Characterization of Bovine Oocytes Reveals That Cysteamine Partially Rescues the Embryo Development in a Model of Low Ovarian Reserve.

Decreased oocyte quality is a major determinant of age-associated fertility decline. Similarly, individuals affected by early ovarian aging carry low-quality oocytes. Using an established bovine model of early ovarian aging, we investigated key features of 'quality' oocyte maturation, associated with the onset of egg aneuploidy and reproductive aging, such as histone modifications, mitochondria distribution and activity, reduced glutathione (GSH) content, and gap junction functionality. Bovine ovaries were classified according to the antral follicle count (AFC), and the retrieved oocytes were processed immediately or matured in vitro. We observed alterations in several cellular processes, suggesting a multifactorial etiology of the reduced oocyte quality. Furthermore, we performed a rescue experiment for one of the parameters considered. By adding cysteamine to the maturation medium, we experimentally increased the free radical scavenger ability of the 'low competence' oocytes and obtained a higher embryo development. Our findings show that adopting culture conditions that counteract the free radicals has a positive impact on the quality of 'compromised' oocytes. Specifically, cysteamine treatment seems to be a promising option for treating aging-related deficiencies in embryo development.

Recreating the Follicular Environment: A Customized Approach for In Vitro Culture of Bovine Oocytes Based on the Origin and Differentiation State.

The mammalian ovary is a large source of oocytes organized into follicles at various stages of folliculogenesis. However, only a limited number of them can be used for in vitro embryo production (IVEP), while most have yet to complete growth and development to attain full meiotic and embryonic developmental competence. While the in vitro growth of primordial follicles in the ovarian cortex has the potential to produce mature oocytes, it is still at an experimental stage. The population of early antral follicles (EAFs), instead, may represent a reserve of oocytes close to completing the growth phase, which might be more easily exploited in vitro and could increase the number of female gametes dedicated to IVEP.Here we present in vitro culture strategies that have been developed utilizing physiological parameters to support the specific needs of oocytes at distinct stages of differentiation, in order to expand the source of female gametes for IVEP by maximizing the attainment of fertilizable oocytes. Furthermore, these culture systems provide powerful tools to dissect the molecular processes that direct the final differentiation of the mammalian oocyte.

Involvement of E-cadherin in early in vitro development of adult and juvenile sheep embryos.

The oocyte-to-embryo transition in mammals depends on maternal proteins and transcripts, which accumulate during oocyte differentiation. The aim of the present study was to examine the role of the junctional proteins beta-catenin and E-cadherin during preimplantation in vitro embryo development in sheep, comparing the competence of adult and prepubertal oocytes. We analysed the concentration of beta-catenin and E-cadherin in immature and in vitro-matured oocytes. There was a significant increase in E-cadherin concentration after 24 h of in vitro maturation and this was lower in prepubertal oocytes than in adult ones. We therefore studied the expression and distribution of E-cadherin during the major transition from maternal to embryonic genome. E-cadherin distribution and localisation in sheep was age- and developmental-stage dependent and was related to developmental kinetics. In fact, in adults, the majority of embryos showed the proper distribution of E-cadherin just beneath the membrane surfaces of all blastomeres and the percentage of embryos with this distribution increased with the increase in cell number during development. On the contrary, and regardless of their developmental stage, the majority of prepubertal embryos showed an uneven distribution of the protein, often associated with the occurrence of cellular fragmentation. In conclusion, our results suggest that E-cadherin plays a pivotal role during preimplantation embryo growth in sheep and may be one of the possible cytoplasmic factors involved in the reduced developmental competence of prepubertal female gametes.