Preantral Stage Research Articles

In vitro development of mechanically and enzymatically isolated cat ovarian follicles.

Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts.

Current views on the molecular mechanisms of the initial stages of folliculogenesis

The ovary is a unique structure of the female body, which simultaneously presents various morphohistological units-from primordial to dominant follicles. Over the past decades, scientists have focused on studying the mechanisms of folliculogenesis at the gonadotropin-dependent stage. While more complex and lengthy processes that determine the fate of the follicle occur from the moment of their recruitment to the preantral stage of maturity (about 290 days), another 60 days pass before the dominant maturity. Currently, it has been proved that intercellular communication is established within the follicle, which involves a bidirectional exchange of information between the oocyte and its companions granulose and Teka cells through auto-and paracrine interactions using various genes, growth factors and cytokines. The purpose of this review was to study intrafollicular factors that control the early stages of folliculogenesis and other disorders that may ultimately lead to the development of premature ovarian failure.

Reproductive Performance of Dairy Cows Fed a Diet Supplemented with n-3 Polyunsaturated Fatty Acids – A Review

Abstract The aim of this review is to summarize the current state of knowledge of reproductive performance of high-yielding dairy cows fed a diet supplemented with n-3 fatty acids (FA s), and to recommend a feeding schedule that can increase the success rate in reproduction. Dietary supplementation with fat, especially FA, has become an accepted strategy for supporting the fertility of high-yielding dairy cows. The two main categories of FA s, unsaturated fatty acids (UFA s) and saturated fatty acids (SFA s), exert distinct effects on reproductive functions, with UFA having a conclusively beneficial impact. Polyunsaturated fatty acids (PUFAs) are of particular importance on account of their biological properties. Standard feedstuffs (such as soybean) are rich in n-6 FA s, whereas few feedstuffs contain n-3 FA s. Neither the n-3 nor n-6 PUFA s are synthetized by the animal. Several field experiments have indicated that the recommended schedule of n-3 supplementation should last 160–170 days, from the eighth week before calving to 100 or 120 days postpartum. Supplementation of the diet thus covers the period from the late preantral stage of the follicle, the entire development of the antrum, oocyte ovulation, and fertilization, as well as the development of the preattachment embryo and its implantation. The following effects are among the important beneficial results of supplementation with n-3 FA s: a higher number of antral follicles suitable for ovum pick-up (OPU), larger dominant follicles and corresponding CL, better quality oocytes and embryos, and higher implantation rates resulting from improved uterine environment (e.g., reduced synthesis of luteolytic PGF2α). We conclude that dietary supplementation for high-yielding dairy cows with n-3 FA s supports ovarian functions and helps the embryo to survive in the adverse environment of the reproductive tract of the postpartum cow.

Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†.

Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.

Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit

Breast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits.

Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

Gene and protein expression of connexins 37 and 43 in cumulus-oocytes complexes throughout the canine oestrous cycle.

The aim of this study was to evaluate the expression of connexin (Cx) 37 and Cx43 in canine cumulus-oocyte complexes (COCs) during the oestrous cycle. Cx localisation was analysed by immunohistochemistry and immunofluorescence, whereas protein and gene expression was evaluated by western blotting and quantitative polymerase chain reaction respectively; comparisons were made using analysis of variance. Both Cx37 and Cx43 were expressed in all follicular stages; Cx43 was identified in cumulus cells and Cx37 was identified in cumulus cells, zonae pellucida and oocytes. Immunofluorescence analyses showed that Cx37 remained unchanged during the preovulatory stage but decreased after ovulation, whereas Cx43 remained unchanged before and after ovulation. Cx43 transcripts increased (P<0.05) during anoestrus and dioestrus in medium-sized follicles but remained unaltered during the pro-oestrus and antral stages during oestrus, before and after ovulation. Cx37 mRNA levels decreased in ovulated COCs (P<0.05). The highest levels of Cx37 protein (P<0.05) were detected in the preantral stage during anoestrus. In contrast, strong Cx43 signals were detected in oestrus and in medium-sized antral follicles in dioestrus (P<0.05). Overall, we demonstrated that Cx37 and Cx43 exhibit different expression patterns, suggesting specific roles throughout growth. Maintenance of Cx expression before ovulation indicates the involvement of Cx37 and Cx43 in the prolonged meiotic arrest.

Periovulatory insulin signaling is essential for ovulation, granulosa cell differentiation, and female fertility.

Recent studies have demonstrated an essential role for insulin signaling in folliculogenesis as conditional ablation of Igf1r in primary follicles elicits defective follicle-stimulating hormone responsiveness blocking development at the preantral stage. Thus the potential role of insulin action in the periovulatory window and in the corpus luteum is unknown. To examine this, we generated conditional Insr,Igf1r, and double receptor knockout mice driven by Pgr-Cre. These models escape the preantral follicle block and in response to superovulatory gonadotropins exhibit normal distribution of ovarian follicles and corpora lutea. However, single ablation of Igf1r leads to subfertility and mice lacking both receptors are infertile. Double knockout mice have impaired oocyte development and ovulation. While some oocytes are released and fertilized, subsequent embryo development is retarded, and the embryos potentially fail to thrive due to lack of luteal support. In support of this, we found reduced expression of key enzymes in the steroid synthesis pathway and reduced serum progesterone. In addition to metabolic and steroidogenic pathways, RNA-sequencing analysis revealed transcription factor-3 as an important transcription factor downstream of insulin signaling. Collectively, these results highlight the importance of growth factors of the insulin family during two distinct windows of follicular development, ovulation, and luteinization.

Initial steps in reconstruction of the human ovary: survival of pre-antral stage follicles in a decellularized human ovarian scaffold.

Can a reconstructed ovary using decellularized human ovarian tissue (DCT) support survival of pre-antral stage follicles? We have demonstrated an effective protocol for decellularization of human ovarian tissues and successful recellularization with isolated human ovarian cells and pre-antral follicles. Survivors of leukemia or ovarian cancer run a risk of reintroducing malignancy when cryopreserved ovarian tissue is transplanted to restore fertility. A reconstructed ovary free of malignant cells could provide a safe alternative. Decellularization of ovarian tissue removes all cells from the extracellular matrix (ECM) including possible malignancies and leaves behind a physiological scaffold. The ECM offers the complex milieu that facilitates the necessary interaction between ovarian follicles and their surroundings to ensure their growth and development. Previous studies have shown that decellularized bovine ovarian scaffolds supported murine follicle growth and restoration of ovarian function in ovariectomized mice. Optimizing a decellularization protocol for human ovarian tissues and testing biofunctionality of the decellularized scaffolds in vitro and in vivo by reseeding with both murine and human pre-antral follicles and ovarian cells. Donated human ovarian tissue and isolated pre-antral follicles were obtained from women undergoing ovarian tissue cryopreservation for fertility preservation. Ovarian cortical and medullary tissues were decellularized using 0.1% sodium dodecyl sulfate (SDS) for 3, 6, 18 and 24hours followed by 24hours of 1mg/mL DNase treatment and washing. Decellularization of ovarian tissues and preservation of ECM were characterized by morphological evaluation using Periodic Acid-Schiff (PAS) staining, DNA quantification, histochemical quantification of collagen content and immunofluorescence analysis for collagen IA, laminin, fibronectin and DNA. Human ovarian stromal cells and isolated human pre-antral follicles were reseeded on the DCT and cultured in vitro. Isolated murine (N= 241) and human (N= 20) pre-antral follicles were reseeded on decellularized scaffolds and grafted subcutaneously to immunodeficient mice for 3 weeks. Incubation in 0.1% SDS for 18-24hours adequately decellularized both human ovarian medullary and cortical tissue by eliminating all cells and leaving the ECM intact. DNA content in DCT was decreased by >90% compared to native tissue samples. Histological examination using PAS staining confirmed that the cortical and medullary tissues were completely decellularized, and no visible nuclear material was found within the decellularized sections. DCT also stained positive for collagen I and collagen quantities in DCT constituted 88-98% of the individual baselines for native samples. Human ovarian stroma cells were able to recellularize the DCT and isolated human pre-antral follicles remained viable in co-culture. Xenotransplantation of DCT reseeded with human or murine pre-antral follicles showed, that the DCT was able to support survival of human follicles and growth of murine follicles, of which 39% grew to antral stages. The follicular recovery rates after three weeks grafting were low but similar for both human (25%) and murine follicles (21%). N/A. Further studies are needed to increase recovery and survival of the reseeded follicles. Longer grafting periods should be evaluated to determine the developmental potential of human follicles. Survival of the follicles might be impaired by the lack of stroma cells. This is the first time that isolated human follicles have survived in a decellularized human scaffold. Therefore, this proof-of-concept could be a potential new strategy to eliminate the risk of malignant cell re-occurrence in former cancer patients having cryopreserved ovarian tissue transplanted for fertility restoration. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. Furthermore, Project ITN REP-BIOTECH 675526 funded by the European Union, European Joint Doctorate in Biology and Technology of the Reproductive Health, the Research Pools of Rigshospitalet, the Danish Cancer Foundation and Dagmar Marshalls Foundation are thanked for having funded this study. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

Leptin induced in vitro development of ovarian follicles in sheep is related to the expression of P450 aromatase and steroidogenesis

The objective of this study was to test the hypothesis that Leptin induced in vitro growth in preantral follicles in sheep involves modulation of P450 aromatase expression and steroidogenesis. Accordingly, the expression of P450 aromatase gene was studied in the cumulus cells and oocytes isolated from different stages of preantral follicles (PFs') grown in vivo, cultured in TCM 199B, TCM 199B + Leptin (10 ng/ml) (TCM199BL) or a standard PF culture medium supplemented with Leptin (10 ng/ml) (SML). Ovarian follicles grown in vivo or in SML expressed P450 aromatase both in cumulus cells and oocytes at all the development stages. In the oocytes from PFs' grown in vitro, P450 expression was consistently lower than in those from in vivo grown follicles at all except the preantral stage. The patterns of expression of aromatase gene in the cumulus cells from in vivo grown and the PFs' cultured in TCM 199BL were similar. Significantly higher levels of progesterone production were supported by SML at all the development stages than the other two media. Oestradiol concentration in the spent TCM 199B and SML showed a significant increase as the development progressed from preantral to large antral stage. However, such increase was not sustained beyond early antral stage in the PFs’ cultured in TCM199BL. It is concluded that Leptin modulates the expression P450 aromatase while supporting the in vitro development of the ovarian follicles in sheep.

Control of ovarian follicular development to the gonadotrophin-dependent phase: a 2006 perspective

In sheep, as in other mammals, ovarian follicular growth is regulated mainly by intraovarian growth factors during early development with pituitary hormones increasingly important during the final phases to ovulation. Most follicles are present as primordial structures and these express many hundreds of genes that fulfil an array of housekeeping and signalling functions. Once growth has been initiated, at least two oocyte-derived growth factors, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), are critical for ongoing development to ovulation, most likely by regulating the proliferative and differentiative functions of adjacent follicular cells. In sheep, the granulosa cell populations double some 12–14 times and a well-defined thecal layer differentiates before antrum formation and the time taken to complete this process varies between 50–150 days with very little follicular atresia. During preantral growth, FSH and LH receptors coupled to the cyclic AMP second messenger system develop in granulosa and thecal cells respectively. From the late preantral stage, GDF9, BMP15 and perhaps other factors are thought to regulate gene expression in cumulus cells to enhance metabolic cooperativity with the oocyte and in mural granulosa cells to regulate their responses to pituitary hormones. In sheep, artral follicular development is characterized by a much faster rate of growth, additional increases in the numbers of granulosa (4–5 more doublings) and thecal cells, an increased level of steroid and inhibin secretion in response to FSH and LH, but also by most follicles undergoing atresia. The final number of follicles that go on to ovulate is dependent upon FSH as well as the intrafollicular concentrations of GDF9 and BMP15.

Quantitative mass spectrometric analysis to unravel glycoproteomic signature of follicular fluid in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy affecting women of reproductive age, and whose etiology is not well understood yet. In these women, the follicular growth is arrested at preantral stage leading to cyst formation, consequently resulting in anovulatory infertility in these women. As the follicular fluid provides the conducive microenvironment for the growth of oocytes, molecular profiling of the fluid may provide unique information about pathophysiology associated with follicular development in PCOS. Post-translational addition of oligosaccharide residues is one of the many modifications of secreted proteins influencing their functions. These glycoproteins play a significant role in disease pathology. Despite glycoproteins having such essential functions, very limited information is available on their profiling in human reproductive system, and glycoproteomic profile of follicular fluid of women with PCOS is yet unexplored. In the present study, we performed a comparative glycoproteomic analysis of follicular fluid between women with PCOS and controls undergoing in vitro fertilization, by enrichment of glycoproteins using three different lectins viz. concanavalin A, wheat germ agglutinin and Jacalin. Peptides generated by trypsin digestion were labeled with isobaric tags for relative and absolute quantification reagents and analyzed by liquid chromatography tandem mass spectrometry. We identified 10 differentially expressed glycoproteins, in the follicular fluid of women with PCOS compared to controls. Two important differentially expressed proteins- SERPINA1 and ITIH4, were consistently upregulated and downregulated respectively, upon validation by immunoblotting in follicular fluid and real-time polymerase chain reaction in granulosa cells. These proteins play a role in angiogenesis and extracellular matrix stabilization, vital for follicle maturation. In conclusion, a comparative glycoproteomic profiling of follicular fluid from women with PCOS and controls revealed an altered expression of proteins which may contribute to the defects in follicle development in PCOS pathophysiology.

What Changed on the Folliculogenesis in the Process of Mouse Ovarian Aging?

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.

Reconstruction of the ovary microenvironment utilizing macroporous scaffold with affinity-bound growth factors

Implementing ovarian tissue engineering for the maturation of primordial follicles, the most abundant follicle population in the ovary, holds great potential for women fertility preservation. Here, we evaluated whether macroporous alginate scaffolds with affinity-bound bone morphogenetic protein-4 (BMP-4) could mimic the ovary microenvironment and support the culture and growth of primordial follicles seeded with supporting ovarian cells. Porcine primordial follicles developed in the alginate scaffolds up to the pre-antral stage within 21 days. Affinity-bound BMP-4 significantly contributed to follicular maturation, as evident by the 5-fold increase in the number of developing follicles and enhanced estradiol secretion in these cultures compared to when BMP-4 was added to cultures with no affinity binding. After 21 days in culture, an increase in GDF-9/AMH gene expression, which is correlated with follicular development, was statistically significant when BMP-4 was affinity bound, compared to all other scaffold groups. When developed in-vivo, after xeno-transplantation of the follicle devices supplemented with additional angiogenic factors, the follicles reached antral size and secreted hormones at levels leading to restoration of ovarian function in ovariectomized severe combined immunodeficiency (SCID) mice. Altogether, our results provide first affirmation for the applicability of macroporous alginate scaffolds as a suitable platform for promoting follicle maturation in-vitro and in-vivo, and lay the foundations for the advantageous use of affinity binding presentation of growth factors to cultured follicles.

Individual 17-Hydroxyprogesterone Responses to hCG Are Not Correlated With Follicle Size in Polycystic Ovary Syndrome.

ContextIn women with polycystic ovary syndrome (PCOS), 17-hydroxyprogesterone (17-OHP) responses to gonadotropin stimulation vary from increased to indistinguishable compared with normal controls.ObjectiveTo determine whether 17-OHP responses to recombinant-human chorionic gonadotropin (r-hCG) are individually correlated to the size of antral follicles among women with PCOS.Design, Setting, and ParticipantsA prospective study conducted in 19 women with PCOS and 20 normal controls at an academic medical center.InterventionsBlood samples were obtained before and 24 hours after administration of 25 μg of r-hCG. Ovarian imaging was conducted with three-dimensional pelvic ultrasonography. Each subject underwent a 2-hour oral glucose tolerance test.Main Outcome MeasuresBasal and stimulated levels of 17-OHP, androgens, estradiol, progesterone, anti-Mullerian hormone (AMH), insulin, glucose, follicle number, and size.ResultsIn women with PCOS, mean antral follicle count (AFC) was greater than that of controls, although the size of cohort follicles within individual subjects was not correlated to 17-OHP responses. The numbers of 2- to 3-mm and 3- to 4-mm follicles in PCOS were significantly greater than in controls, whereas differences between larger follicles were not observed. Increased AMH in PCOS was correlated to AFC, but not 17-OHP responses. Insulin sensitivity did not correlate to r-hCG‒stimulated 17-OHP after adjustment for body mass index.Conclusions17-OHP responses to hCG in individuals with PCOS were not correlated to the distribution of antral follicles. Greater numbers of small antral follicles in women with PCOS than in controls suggest an extension of accelerated growth from the preantral stage.

Stage-specific modulation of antimüllerian hormone promotes primate follicular development and oocyte maturation in the matrix-free three-dimensional culture

To study whether follicular growth and oocyte maturation can be improved by antimüllerian hormone (AMH) modulation at specific stages of follicular development. Primary and secondary follicles were cultured in a matrix-free system and were assigned to the control group and the group with AMH supplementation during the preantral stage and neutralizing AMH antibody addition during the antral stage. National primate research center. Adult, female rhesus macaques (Macaca mulatta). None. Follicle survival, growth, steroid and paracrine factor production, and oocyte competence were evaluated. Follicles were assessed for expression of genes that are critical for gonadotropin signaling, cumulus cell glycolysis, and oocyte quality. Primary follicles formed "organoids" and developed to the antral stage in group culture. AMH exposure during the preantral stage increased organoid diameters. Oocytes from the AMH-treated organoids had greater diameters and matured to the metaphase II (MII) stage. Secondary follicles developed to the antral stage during individual culture. The AMH exposure during the preantral stage and AMH antibody treatment during the antral stage increased follicle diameters, vascular endothelial growth factor and follistatin production, differentiation factor 9 expression, and oocyte diameters. The MII oocytes from the AMH-modulated group developed to the morula stage after IVF, with one to the blastocyst stage. AMH supplementation at the preantral stage and depletion at the antral stage enhanced primate follicular development and oocyte competence invitro. The improved embryonic development supports invitro follicle maturation as a potential approach for fertility preservation.

Evaluation of an ovary-on-a-chip in large mammalian models: Species specificity and influence of follicle isolation status.

The ability to grow oocytes from immature ovarian follicles in vitro has significant potential for fertility preservation; yet, it has proved challenging in large mammalian species due to the complex metabolic needs and long-term culture requirements. Currently, follicular incubations are based on a "static" system with manual exchange of medium. Despite the numerous advantages of conventional culturing approaches, recapitulating the native microenvironment and supporting the survival of ovarian follicles from large mammalian species still represent challenges. In this study, we utilized an innovative, dynamic microfluidic system to support the in vitro survival of domestic cat and dog follicles enclosed within the ovarian cortex or isolated from ovarian cortex. Results indicate both species-specific and tissue type-specific differences in response to microfluidic culture. Domestic cat but not dog ovarian cortical tissues maintained viability under flow similar to conventional agarose gel controls. Preantral stage isolated follicles from both species that grew most favourably in conventional alginate bead culture, but overall, there was no influence of culture system on expression of follicle development or oocyte health markers. This system represents an important exploration toward the development of an improved ovarian in vitro culture system of large mammalian species (e.g., cats and dogs), which has potential applications for fertility preservation, reproductive toxicology, and endangered mammal conservation efforts.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector+recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Platelet-rich plasma promotes the development of isolated human primordial and primary follicles to the preantral stage

This study aimed to assess the effects of platelet-rich plasma (PRP) on growth and survival of isolated early human follicles in a three-dimensional culture system. After fresh and vitrified-warmed ovarian tissue was digested, isolated early preantral follicles and ovarian cells were separately encapsulated in 1% alginate (w/v). The encapsulated follicles and ovarian cells were cultured together in a medium supplemented with foetal bovine serum (FBS), PRP, PRP + FBS, or human serum albumin (HSA) for 10 days. Growth and survival of the follicles were assessed by measurement of diameter and staining with trypan blue. Follicular integrity was assessed by histological analysis. After culturing, all follicles increased in size, but growth rate was greater in follicles isolated from fresh samples than those from vitrified-warmed ones (P < 0.001). Similarly, follicular viability of fresh samples after culturing was higher than that of vitrified-warmed ones. The growth and survival rates of follicles from both fresh and vitrified groups cultured in PRP supplemented media were significantly higher than those of other groups (growth P < 0.001 and survival P < 0.05, in both groups). In conclusion, media supplementation with PRP can better support viability and growth of isolated human early preantral follicles in vitro.

Embryonic poly(A)-binding protein is required at the preantral stage of mouse folliculogenesis for oocyte-somatic communication.

Embryonic poly(A)-binding protein (EPAB)-deficient mice are infertile due to defects in both the oocyte and the somatic cells of the ovary. Since EPAB is oocyte specific, the abnormalities in the somatic compartment of Epab−/− mice are likely due to factors inherent to the oocyte. Herein, we investigated whether oocyte–somatic communication is disrupted as a result of EPAB deficiency. We found that gap junctions are disrupted at the late preantral stage of folliculogenesis in Epab−/– mice and remain disrupted in cumulus-enclosed oocytes (COCs) from antral follicles. Consistent with the timing of gap junction dysfunction, F-actin staining of transzonal processes (TZPs) is lower in Epab−/− follicles at the late preantral stage and completely absent in Epab−/− COCs. Epab−/− oocytes express significantly lower levels of the junction protein E-cadherin, which is likely to be a contributing factor leading to premature TZP retraction. Overall, these results demonstrate that EPAB is important for oocyte–somatic communication by maintaining TZPs and gap junctions at the preantral stage of folliculogenesis.