Agarose-based 3D culture improved the developmental competence of oocyte-granulosa complex isolated from porcine preantral follicle.

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established. The present study indicated that the use of a modified medium with Ascorbic Acid (50 µM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3-0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac-H3-K9) and methylation of histone H3 at lysine 4 (Me-H3-K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.

Expression of luteinizing hormone receptor during development of bovine fetal ovary.

Steroids and gonadotrophins are essential for the regulation of late stages of preantral development and antral follicular development. Although the luteinizing hormone receptor (LHCGR) has been detected in the preantral follicles of rats, rabbits, and pigs, its expression, in bovine fetal ovary, has not been demonstrated. Based on this, we aimed to investigate the expression of the LHCGR and LHCGR mRNA binding protein (LRBP), as well as, to quantify bta-miR-222 (a regulatory microRNA of the LHCGR gene) during the development of bovine fetal ovary. In summary, LHCGR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. The LHCGR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHCGR. In conclusion, we suggest the involvement of LHCGR/LRBP/bta-mir222 with mechanisms related to the development of preantral follicles in cattle.

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.

Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

BACKGROUND: The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. MATERIALS AND METHODS: In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. RESULTS: The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. CONCLUSION: The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

The effect of Coenzyme Q10 on mitochondrial biogenesis in mouse ovarian follicles during in vitro culture.

The aim of this research was to investigate the effect of Coenzyme Q10 (CoQ10) on the expression of the Transcription Factor A Mitochondrial (Tfam) gene and mtDNA copy number in preantral follicles (PFs) of mice during in vitro culture. To conduct this experimental study, PFs were isolated from 14-day-old National Medical Research Institute mice and cultured in the presence of 50 µm CoQ10 for 12 days. On the 12th day, human chorionic gonadotropin was added to stimulate ovulation. The fundamental parameters, including preantral follicle developmental rate and oocyte maturation, were evaluated. Additionally, the Tfam gene expression and mtDNA copy number of granulosa cells and oocytes were assessed using the real-time polymerase chain reaction. The results revealed that CoQ10 significantly increased the diameter of PFs, survival rate, antrum formation, and metaphase II (MII) oocytes (P < 0.05). Moreover, in the CoQ10-treated groups, the Tfam gene expression in granulosa cells and oocytes increased considerably compared with the control group. The mtDNA copy number of granulosa cells and oocytes cultured in the presence of CoQ10 was substantially higher compared with the control groups (P < 0.05). The addition of CoQ10 to the culture medium enhances the developmental competence of PFs during in vitro culture by upregulating Tfam gene expression and increasing mtDNA copy number in oocyte and granulosa cells.

Effects of different cryopreservation methods on canine isolated preantral follicles.

The aim of the present study was to compare the survival and developmental rate of canine isolated preantral follicles (PAFs) after cryopreservation with different methods (closed vs open vitrification). Follicles were isolated from ovaries randomly divided into three groups: fresh control, OPS (open pulled straw) vitrified and cryotube (CT) vitrified. Post-thaw viability of follicles and oocytes was assessed. Fresh and vitrified/thawed PAFs were cultured in 20 µl drops of FSH-supplemented medium for 10 days. Follicular growth, survival rate, estradiol production and ovulation rate were examined. CT method resulted in lower rate of live cells (58.7%) and oocytes (38.8%) than that of fresh ones (83.6% and 64%, respectively) and OPS (80.3% and 79.3%, respectively). Survival rate was similar to fresh follicles in OPS group (98.5% and 95.4%, respectively), while CT decreased the survival to 81.2%. Fresh follicles showed continuous growth, while CT follicles stopped to increase their size after 2 day. In the OPS vitrified follicles, this halting occurred between Day5 and Day10. Fresh follicles showed the highest estradiol production (range: 26.9 - 266.2 pg/ml). Comparing the two vitrified groups, lower estradiol concentration range was measured in the CT group (7.8-48.7 pg/ml vs. 15.4-89.6 pg/ml). Ovulation rate in each group was lowest in the OPS group (1.7% vs 7% and 8.9% in fesh and CT, respectively). Our data show that OPS vitrification provides superior survival rate, in vitro growth and hormonal production to CT. To our knowledge, these are the first results on comparing different cryopreservation protocols on canine isolated preantral follicles.

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

A self-gelling hydrogel based on thiolated hyaluronic acid for three-dimensional culture of ovine preantral follicles.

Three-dimensional (3D) ovarian follicle culture offers a promising option for fertility preservation in patients who cannot receive ovarian tissue transplantation. Our research evaluated the potential of a hydrogel composed of thiolated hyaluronic acid (HA-SH) for ovine preantral follicle development compared to routinely used alginate hydrogel (ALG). Synthesized via a carbodiimide reaction, HA-SH facilitated a self-crosslinking hydrogel through disulfide bond formation. Ovine preantral follicles (200-300 µm) retrieved through mechanical and enzymatic methods were encapsulated individually in either ALG or HA-SH hydrogels. Although both hydrogels adequately supported follicle survival, 3D integrity, and antrum formation over a 17-day in vitro culture, follicle growth was significantly higher within the HA-SH hydrogel. Gene expression analysis underscored that some folliculogenesis-related genes (ZP3, BMP7, and GJA1) and a steroidogenic gene (CYP19A1) demonstrated higher expression levels in HA-SH encapsulated follicles versus ALG. Collectively, our findings advocate for HA-SH hydrogel as a potent biomaterial for in vitro follicle cultures, attributing its efficacy to facile gelation, bio-responsiveness, and superior support for follicle growth.

Cystic endometrial hyperplasia-pyometra syndrome impairs the preantral follicle reserve in domestic bitches (Canis familiaris).

Cystic endometrial hyperplasia (CEH)-pyometra syndrome is the most common uterine pathological condition reported in breeding bitches, however, their described effects on fertility are limited to uterine disorders and conception rates. As the preantral follicle population represents the available reserve of gametes recruited during the lifespan, the aim of this study was to evaluate the effects of CEH-pyometra syndrome on the: (i) preantral follicle morphology, (ii) developing follicle rates, and (iii) preantral follicle and stromal cell densities. Ovarian fragments from bitches subjected to elective or therapeutic ovariohysterectomy were allocated according to uterine diagnosis as follows: control (n = 7, clinically healthy), CEH-mucometra (n = 8, uterine lumen filled with a sterile mucus), and pyometra (n = 17, presence of a purulent mucus) groups. Overall, the control group had 3.4 and 4.1-fold higher probability (P < 0.0001) of the presence of normal preantral follicles compared with CEH-mucometra and pyometra groups, respectively. Moreover, ovarian fragments from the pyometra group showed an increase in the percentage of developing follicles (P < 0.05) compared to the control. Both CEH-mucometra and pyometra groups showed lower (P < 0.05) preantral follicle and stromal cell densities (P < 0.05) compared to the control. In summary, the CEH-pyometra syndrome decreased the percentage of morphologically normal follicles and enhanced the developing follicle rates. Additionally, a reduction of preantral follicle and stromal cell densities suggests that the inappropriate uterine environment induced by CEH-pyometra syndrome can lead to premature depletion of ovarian reserve.

Kisspeptin promotes follicular development through its effects on modulation of P450 aromatase expression and steroidogenesis in sheep ovarian follicles.

The present study was conducted to ascertain whether the role of kisspeptin in promoting in vitro development of preantral follicles was through the regulation of P450 aromatase gene expression and steroidogenesis in sheep. Accordingly, the cumulus cells and oocytes were collected from different development stages of preantral follicles grown in vivo and cultured in vitro in TCM199B (Group I), TCM199B + KP (10 µg/mL) (Group II) and Standard medium + KP (10 µg/mL). To measure the steroid (Estradiol-17ß; E2 and Progesterone; P4 ) synthesis through ELISA, spent culture medium was collected separately from the same in vitro groups. E2 synthesis in the spent medium collected from all the three groups showed an increasing trend from PFs' exposed to respective culture media for 3 min to 2-day culture stage but decreased thereafter till 6-day culture stage. This is followed by a sharp increase in E2 concentration in the spent medium collected after in vitro maturation. However, P4 synthesis in group III followed increased pattern as the development progressed from PFs' exposed to culture medium for 3 min to in vitro maturation stage. The steroid production was observed at all stages of in vitro development in altered supplemented conditions. The steroid synthesis in the spent medium was highest in the 6 day cultured PFs' in Standard medium + KP matured in vitro for 24 h. Therefore, supplementation of kisspeptin along with other growth factors promoted steroid production in cultured preantral follicles far better than in other media.

BPA interferes with granulosa cell development and oocyte meiosis in mouse preantral follicles.

Bisphenol A (BPA) is an established environmental endocrine disruptor and can interfere with the development of female germ cells. However, the underlying mechanisms are still unclear. We investigated the effects of BPA on granulosa cell development and meiosis of oocytes using in vitro culture system of mouse preantral follicles. Preantral follicles from D14 mouse ovary were treated with 10 µg/mL BPA in vitro for 11 days. The adherent area of follicles was measured. On D11, cumulus cell expansion was observed. The meiosis recovery rate was calculated. Western blot detected P53, proliferating cell nuclear antigen (PCNA), estrogen receptor α (ERα), and cyclin B1. ELISA measured estrogen and progesterone levels. Immunofluorescence detected Cx37 on oocyte membrane. Gap junction communication was assessed. We found that BPA significantly promoted the expressions of PCNA and ERα in granulosa cells and the secretion of estrogen and progesterone by granulosa cells on D10 and significantly increased the attachment area of the follicles on D8 and D10. However, it reduced the expansion of cumulus cells, Cx37 expression, and the gap junction communication between cumulus cells and oocytes on D11. BPA promoted the recovery of oocytes from meiosis, interrupted the expression of cyclin B1 protein in arrested germinal vesicle breakdown (GVBD) oocytes, and reduced the in vitro maturation rate of oocytes. These GVBD oocytes were live without apoptosis or death. Conclusively, BPA disturbs the development of granulosa cells and the meiosis progression of oocytes by decreasing gap junction communication between oocytes and the granulosa cells as well as regulating cyclin B1 expression in GVBD oocytes.

The mechanisms that control the preantral to early antral follicle transition and the strategies to have efficient culture systems to promote their growth in vitro.

Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.

Bone Morphogenetic Protein 15 (BMP-15) Improves In Vitro Mouse Folliculogenesis.

Multilayered secondary follicles were encapsulated in a 0.5% alginate matrix and cultured in a 3D culture system supplemented with bone morphogenetic protein 15 (BMP-15; 15 ng/mL) for 12 days. The in vitro development of ovarian follicles was evaluated. On day 12, the follicle diameter, follicle survival rate, and antrum formation rate were significantly higher for follicles cultured in BMP-15-supplemented medium than those cultured in regular medium. The percentage of ovulated metaphase II oocytes retrieved from follicles cultured in BMP-15-supplemented medium was greater than that of oocytes retrieved from follicles cultured in regular medium. The secretion of P4 was significantly higher on days 6, 8, and 10 in follicles cultured in BMP-15-supplemented medium. The result for E2 tended toward significance on day 12. Intracellular reactive oxygen species levels were higher and glutathione levels were lower in mature oocytes from the in vitro culture than in mature oocytes from an in vivo control. A 3D culture system using an alginate matrix and supplemented with BMP-15 effectively improves the outcomes of in vitro ovarian follicle culture.

Granulocyte colony-stimulating factor priming improves embryos and pregnancy rate in patients with poor ovarian reserve: a randomized controlled trial.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) administration increased ovarian preantral follicles and anti-Müllerian hormone (AMH) in animal models with diminished ovarian reserve. We investigated whether G-CSF priming before treatment with assisted reproductive technology (ART) improved embryo development and pregnancy rate while increasing serum AMH in patients with poor ovarian reserve. METHODS: In this prospective randomized open-label controlled trial, 100 patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of in vitro fertilization/intracytoplasmic sperm injection and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming. RESULTS: Fertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts. The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04-7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. No adverse effects of priming were observed. CONCLUSION: G-CSF priming improved embryonic development and pregnancy rate during ART treatment and increased AMH in patients with poor ovarian reserve. Enhanced preantral follicle growth likely was responsible. TRIAL REGISTRATION: UMIN registration in Japan (UMIN000013956) on May 14, 2014.  https://www.umin.ac.jp/ctr/index.htm .

Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum-free medium containing FSH, EGF and IGF-I.

Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum-free medium containing FSH, EGF and IGF-I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G-0%), 0.5% (G-0.5%) or 1% (G-1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF-I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48 h and samples were stored at -20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24 h. G-0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 µm; p < .05) than G-0.5% and G-1% follicles (157.89 ± 8.47 µm and 95.23 ± 1.67 µm, respectively) which maintained three-dimensional organization, being larger in G-0.5% than in G-1% (p < .05). G-0.5% follicles attained the multi-layer preantral follicle stage on day 7 of culture, whereas G-1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p < .05) in G-0% than in G-1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G-0.5% were comprised between those of G-0% and G-1% (p > .05). In conclusion, two-layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF-I can develop up to the multi-layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three-dimensional organization, and experienced regression with compromised steroidogenesis, respectively.

Anti-Müllerian hormone-mediated preantral follicle atresia is a key determinant of antral follicle count in mice.

STUDY QUESTION: Does anti-Müllerian hormone (AMH) induce preantral follicle atresia in mice? SUMMARY ANSWER: The present findings suggest that AMH-mediated follicle atresia only occurs in early follicles before they become sensitive to FSH. WHAT IS KNOWN ALREADY: Most prior studies have investigated the ability of AMH to inhibit primordial follicle activation. Our previous study showed that AMH-overexpressing mice had fewer preantral follicles than expected after accounting for primordial follicle inhibition but the reason for this was not determined. STUDY DESIGN, SIZE, DURATION: Cross-sectional-control versus transgenic/knockout mouse studies were carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Studies were conducted on female wild-type (Amh+/+), AMH-knockout (Amh-/-) and AMH overexpressing (Thy1.2-AMHTg/0) mice on a C57Bl/6J background (age: 42-120 days). The follicle counts were conducted for primordial, transitioning, primary, secondary and antral follicles in Amh-/- and Amh+/+ mice. After confirming that follicle development speeds were identical (proliferating cell nuclear antigen immunohistochemistry), the ratio of follicles surviving beyond each stage of folliculogenesis was determined in both genotypes. Evidence for increased rates of preantral follicle atresia was assessed by active caspase-3 immunohistochemistry in wild-type and Thy1.2-AMHTg/0 mice. MAIN RESULTS AND THE ROLE OF CHANCE: Amh -/- mice at 100-120 days of age had lower primordial follicle counts but higher primordial follicle activation rates compared to Amh+/+ mice. These counteracting effects led to equivalent numbers of primordial follicles transitioning to the primary stage in Amh+/+ and Amh-/- mice. Despite this, Amh+/+ mice had fewer primary, secondary, small antral and medium antral follicles than Amh-/- mice indicating differing rates of developing follicle atresia between genotypes. Cleaved caspase-3 immunohistochemistry in Thy1.2-AMHTg/0 ovaries revealed high rates of granulosa cell and oocyte apoptosis in late primary/early secondary follicles of Thy1.2-AMHTg/0 mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The findings were shown only in one species and additional research will be required to determine generalizability to other species. WIDER IMPLICATIONS OF THE FINDINGS: This study is consistent with prior studies showing that Amh-/- mice have increased primordial follicle activation but these new findings demonstrate that AMH-mediated preantral follicle atresia is a predominant cause of the increased small antral follicle counts in Amh-/- mice. This suggests that the role of AMH is not to conserve the ovarian reserve to prolong fertility, but instead to prevent the antral follicle pool from becoming too large. While this study may demonstrate a new function for AMH, the biological purpose of this function requires further investigation, particularly in mono-ovulatory species. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Health Research Council of New Zealand and the University of Otago. No competing interests to declare.

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential medium alone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a (p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

When Electrospun Fiber Support Matters: In Vitro Ovine Long-Term Folliculogenesis on Poly (Epsilon Caprolactone) (PCL)-Patterned Fibers.

Current assisted reproduction technologies (ART) are insufficient to cover the slice of the population needing to restore fertility, as well as to amplify the reproductive performance of domestic animals or endangered species. The design of dedicated reproductive scaffolds has opened the possibility to better recapitulate the reproductive 3D ovarian environment, thus potentially innovating in vitro folliculogenesis (ivF) techniques. To this aim, the present research has been designed to compare ovine preantral follicles in vitro culture on poly(epsilon-caprolactone) (PCL)-based electrospun scaffolds designed with different topology (Random vs. Patterned fibers) with a previously validated system. The ivF performances were assessed after 14 days under 3D-oil, Two-Step (7 days in 3D-oil and on scaffold), or One-Step PCL protocols (14 days on PCL-scaffold) by assessing morphological and functional outcomes. The results show that Two- and One-Step PCL ivF protocols, when performed on patterned scaffolds, were both able to support follicle growth, antrum formation, and the upregulation of follicle marker genes leading to a greater oocyte meiotic competence than in the 3D-oil system. In conclusion, the One-Step approach could be proposed as a practical and valid strategy to support a synergic follicle-oocyte in vitro development, providing an innovative tool to enhance the availability of matured gametes on an individual basis for ART purposes.

Exposure to non-esterified fatty acids in vitro results in changes in the ovarian and follicular environment in cattle.

Negative energy balance (NEB) in the postpartum period of dairy cows is associated with reduced fertility to insemination later in lactation. We hypothesized that elevated non-esterified fatty acids (NEFA) levels that occur during NEB result in accumulation of fatty acids within the ovarian tissue and preantral follicles, causing changes in ovarian gene expression that would indicate a response to injury. We performed ovarian cortex culture and oocyte maturation in medium containing a combination of palmitic, oleic and stearic acid (NEFA). Ovarian cortex was subjected to RNA sequencing and lipid content analysis via Nile Red staining and gas chromatography; oocytes were analyzed for maturation rate and mitochondrial mass and localization following in vitro maturation (IVM). Accumulation of lipids associated with the plasma membrane was increased in granulosa cells of preantral follicles exposed to NEFA in vitro; RNA sequencing revealed changes in biological functions associated with metabolic disease, stimulation of an inflammatory response, and reduction in glucose uptake. Oocyte maturation under high NEFA compromised nuclear, but not cytoplasmic maturation. These data demonstrate that exposure to NEFA in vitro affects the ovary, preantral follicles and cumulus-oocyte complexes, and provides further insight into the potential links between metabolic imbalance and infertility.

Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue.

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.