Ti3C2 nanosheet-induced autophagy derails ovarian functions.

BACKGROUND: Two-dimensional ultrathin Ti3C2 (MXene) nanosheets have gained significant attention in various biomedical applications. Although previous studies have described the accumulation and associated damage of Ti3C2 nanosheets in the testes and placenta. However, it is currently unclear whether Ti3C2 nanosheets can be translocated to the ovaries and cause ovarian damage, thereby impairing ovarian functions. RESULTS: We established a mouse model with different doses (1.25, 2.5, and 5 mg/kg bw/d) of Ti3C2 nanosheets injected intravenously for three days. We demonstrated that Ti3C2 nanosheets can enter the ovaries and were internalized by granulosa cells, leading to a decrease in the number of primary, secondary and antral follicles. Furthermore, the decrease in follicles is closely associated with higher levels of FSH and LH, as well as increased level of E2 and P4, and decreased level of T in mouse ovary. In further studies, we found that exposure toTi3C2 nanosheets increased the levels of Beclin1, ATG5, and the ratio of LC3II/Ι, leading to autophagy activation. Additionally, the level of P62 increased, resulting in autophagic flux blockade. Ti3C2 nanosheets can activate autophagy through the PI3K/AKT/mTOR signaling pathway, with oxidative stress playing an important role in this process. Therefore, we chose the ovarian granulosa cell line (KGN cells) for in vitro validation of the impact of autophagy on the hormone secretion capability. The inhibition of autophagy initiation by 3-Methyladenine (3-MA) promoted smooth autophagic flow, thereby partially reduced the secretion of estradiol and progesterone by KGN cells; Whereas blocking autophagic flux by Rapamycin (RAPA) further exacerbated the secretion of estradiol and progesterone in cells. CONCLUSION: Ti3C2 nanosheet-induced increased secretion of hormones in the ovary is mediated through the activation of autophagy and impairment of autophagic flux, which disrupts normal follicular development. These results imply that autophagy dysfunction may be one of the underlying mechanisms of Ti3C2-induced damage to ovarian granulosa cells. Our findings further reveal the mechanism of female reproductive toxicity induced by Ti3C2 nanosheets.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

HDAC6-dependent deacetylation of NGF dictates its ubiquitination and maintains primordial follicle dormancy.

Rationale: Primordial follicles are limited in number and cannot be regenerated, dormant primordial follicles cannot be reversed once they enter a growth state. Therefore, the length of the female reproductive lifespan depends on the orderly progression and selective activation of primordial follicles, the mechanism of which remains unclear. Methods: We used human ovarian cortical biopsy specimens, granulosa cells from diminished ovarian reserve (DOR) patients, Hdac6-overexpressing transgenic mouse model, and RNA sequencing to analyze the crucial roles of histone deacetylase 6 (HDAC6) in fertility preservation and primordial follicle activation. Results: In the present study, we found that HDAC6 was highly expressed in most dormant primordial follicles. The HDAC6 expression was reduced accompanying reproductive senescence in human and mouse ovaries. Overexpression of Hdac6 delayed the rate of primordial follicle activation, thereby prolonging the mouse reproductive lifespan. Short-term inhibition of HDAC6 promoted primordial follicle activation and follicular development in humans and mice. Mechanism studies revealed that HDAC6 directly interacted with NGF, reducing acetylation modification of NGF and thereby accelerating its ubiquitination degradation. Consequently, the reduced NGF protein level maintained the dormancy of primordial follicles. Conclusions: The physiological significance of the high expression of HDAC6 in most primordial follicles is to reduce NGF expression and prevent primordial follicle activation to maintain female fertility. Reduced HDAC6 expression increases NGF expression in primordial follicles, activating their development and contributing to reproduction. Our study provides a clinical reference value for fertility preservation.

Oocyte Competence of Prepubertal Sheep and Goat Oocytes: An Assessment of Large-Scale Chromatin Configuration and Epidermal Growth Factor Receptor Expression in Oocytes and Cumulus Cells.

The oocyte competence of prepubertal females is lower compared to that of adults, mainly because they originate from small follicles. In adult females, the germinal vesicle (GV) and epidermal growth factor receptor (EGFR) have been associated with oocyte competence. This study aimed to analyze GV chromatin configuration and EGFR expression in prepubertal goat and sheep oocytes obtained from small (<3 mm) and large (≥3 mm) follicles and compare them with those from adults. GV chromatin was classified from diffuse to condensed as GV1, GVn, and GVc for goats and NSN, SN, and SNE for sheep. EGFR was quantified in cumulus cells (CCs) by Western blotting and in oocytes by immunofluorescence. Oocytes from prepubertal large follicles and adults exhibited highly condensed chromatin in goats (71% and 69% in GVc, respectively) and sheep (59% and 75% in SNE, respectively). In both species, EGFR expression in CCs and oocytes was higher in prepubertal large follicles than in small ones. In adult females, EGFR expression in oocytes was higher than in prepubertal large follicles. In conclusion, GV configuration and EGFR expression in CCs and oocytes were higher in the large than small follicles of prepubertal females.

Mesenchymal stem cells promote ovarian reconstruction in mice.

BACKGROUND: Studies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem. METHODS: In the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay. RESULTS: Our study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary. CONCLUSIONS: The presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn't proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

Exposure to nicotine regulates prostaglandin E2 secretion and autophagy of granulosa cells to retard follicular maturation in mammals.

Exposure to nicotine by cigarette smoking have shown strongly defectives on the physiological function of ovaries, which in turn leads to disorders of fertility in women. However, the potential molecular mechanisms remain to be elucidated. In this study, we notably found that nicotine was likely to specifically raise the expression of histone deacetylase 3 (HDAC3) to promote the apoptosis and autophagy of granulosa cells (GCs) and block follicular maturation. Moreover, prostaglandin E2 (PGE2) inhibited the apoptosis of GCs and facilitated follicular maturation, and nicotine appeared to inhibit PGE2 secretion by freezing the expression of cyclooxygenase 1 (COX1), which was the rate-limiting and essential enzyme for PGE2 synthesis. Epigenetically, the nicotine was observed to diminish the histone H3 lysine 9 acetylation (H3K9ac) level and compact the chromatin accessibility in -1776/-1499 bp region of COX1 by evoking the expression of HDAC3, with the deactivated Cas9-HDAC3/sgRNA system. Mechanistically, the COX1 protein was found to pick up and degrade the autophagy related protein beclin 1 (BECN1) to control the autophagy of GCs. These results provided a potential new molecular therapy to recover the damage of female fertility induced by nicotine from cigarette smoking.

ERß Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles.

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

BACKGROUND: Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. METHODS: Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. CONCLUSIONS: GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary.

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

Transcriptome analysis during 4-vinylcyclohexene diepoxide exposure-induced premature ovarian insufficiency in mice.

The occupational chemical 4-Vinylcyclohexene diepoxide (VCD) is a reproductively toxic environmental pollutant that causes follicular failure, leading to premature ovarian insufficiency (POI), which significantly impacts a woman's physical health and fertility. Investigating VCD's pathogenic mechanisms can offer insights for the prevention of ovarian impairment and the treatment of POI. This study established a mouse model of POI through intraperitoneal injection of VCD into female C57BL/6 mice for 15 days. The results were then compared with those of the control group, including a comparison of phenotypic characteristics and transcriptome differences, at two time points: day 15 and day 30. Through a comprehensive analysis of differentially expressed genes (DEGs), key genes were identified and validated some using RT-PCR. The results revealed significant impacts on sex hormone levels, follicle number, and the estrous cycle in VCD-induced POI mice on both day 15 and day 30. The DEGs and enrichment results obtained on day 15 were not as significant as those obtained on day 30. The results of this study provide a preliminary indication that steroid hormone synthesis, DNA damage repair, and impaired oocyte mitosis are pivotal in VCD-mediated ovarian dysfunction. This dysfunction may have been caused by VCD damage to the primordial follicular pool, impairing follicular development and aggravating ovarian damage over time, making it gradually difficult for the ovaries to perform their normal functions.

Effect of breeding season and age on follicular dynamics and hemodynamics in embryo donor mares subjected to luteolysis after embryo flushing.

Background: Mares are the only companion animals simulating women in the large diameter of their follicles. Horses start reproduction at the age of three years, and some of them live for >30 years, so aging influences their reproductive capacity. Mares are sensitive to summer heat stress as they can sweat like humans. Aim: The current work aimed to study the effects of age (young versus senile), season (cold versus hot), and the hormonal treatments during embryo collection on the dominant and subordinate follicular dynamics and hemodynamics and circulating ovarian hormones in embryo donor mares ovulated twice spontaneously before inducing ovulation for flushing embryos. Methods: Spontaneous oestrous cycles were studied for young mares (<10 years; N = 6) or senile (>20 years; N = 5) during months of the cold season (November to April) and hot season (May to August). In young embryo donor mares, oestrous cycles after inducing ovulation and luteolysis were studied using Doppler ultrasound. Estradiol (E2), progesterone (P4), nitric oxide (NO), total cholesterol, and lactate dehydrogenase (LDH) were measured in blood serum. Results: A decrease in the dominant follicle antrum diameter (p > 0.05) and LDH (p = 0.016) was observed after inducing luteolysis in young embryo donor mares. Both human chorionic gonadotropin (hCG) and PGF2α treatments increased dominant follicle area (p = 0.0001), antrum area (p = 0.001), perimeter (p = 0.001), granulosa area (p = 0.0001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001). The dominant follicle area, antrum area, perimeter, color area, granulosa area, LDH, cholesterol, NO, and E2 increased (p = 0.0001) during the oestrous cycles of the hot season, but the circulatory % (p = 0.0001) declined. Senile mares had lower dominant follicle area (p = 0.002), antrum area (p = 0.0001), granulosa area (p > 0.05), LDH (p = 0.001), cholesterol (p = 0.0001), NO (p = 0.0001), and E2 (p = 0.0001) but higher circulatory % (p = 0.0001) and color area % (p = 0.023). The dominant follicle possesses the largest diameter, area, perimeter, granulosa area, and color area but the lowest circulatory % during spontaneous oestrous cycles, after inducing ovulation, or luteolysis with significant effects of the day of the spontaneous oestrous cycles on their dynamics and hemodynamics. Conclusion: During hot months, mares treated with hCG ovulated 24 hours later and prostaglandin-induced luteolysis was followed by new ovulation five days later. Follicles ovulated during the hot months were larger than those ovulated during the cold months and both had nearly the same color area %. Senile mares ovulated follicles with a lower area and antrum area but a higher color area %, so senile mares can be used as embryo or oocyte donors during the hot season.

The value of low-intensity pulsed ultrasound in reducing ovarian injury caused by chemotherapy in mice.

BACKGROUND: Ovarian damage and follicle loss are major side effects of chemotherapy in young female patients with cancer. However, effective strategies to prevent these injuries are still lacking. The purpose of this study was to verify low-intensity pulsed ultrasound (LIPUS) can reduce ovarian injury caused by chemotherapy and to explore its underlying mechanisms in mice model. METHODS: The mice were randomly divided into the Control group, Cisplatin group, and Cisplatin + LIPUS group. The Cisplatin group and Cisplatin + LIPUS group were intraperitoneally injected with cisplatin every other day for a total of 10 injections, and the Control group was injected with saline. On the second day of each injection, the Cisplatin + LIPUS group received irradiation, whereas the other two groups received sham irradiation. We used a variety of biotechnologies to detect the differences in follicle count, granulosa cell apoptosis, fibrosis, transcriptome level, oxidative damage, and inflammation in differently treated mice. RESULT: LIPUS was able to reduce primordial follicle pool depletion induced by cisplatin and inhibit the apoptosis of granulosa cells. Transcriptomic results confirmed that LIPUS can reduce ovarian tissue injury. We demonstrated that LIPUS can relieve ovarian fibrosis by inhibiting TGF-ß1/Smads pathway. Meanwhile, it can reduce the oxidative damage and reduced the mRNA levels of proinflammatory cytokines caused by chemotherapy. CONCLUSION: LIPUS can reduce the toxic effects of chemotherapy drugs on ovaries, inhibit ovarian fibrosis, reduce the inflammatory response, and redcue the oxidative damage, reduce follicle depletion and to maintain the number of follicle pools.

Ovarian Follicle Development in Ascidians.

Ovarian follicle development is an essential process for continuation of sexually reproductive animals, and is controlled by a wide variety of regulatory factors such as neuropeptides and peptide hormones in the endocrine, neuroendocrine, and nervous systems. Moreover, while some molecular mechanisms underlying follicle development are conserved, others vary among species. Consequently, follicle development processes are closely related to the evolution and diversity of species. Ciona intestinalis type A (Ciona rubusta) is a cosmopolitan species of ascidians, which are the closest relative of vertebrates. However, unlike vertebrates, ascidians are not endowed with the hypothalamus-pituitary-gonadal axis involving pituitary gonadotropins and sexual steroids. Combined with the phylogenetic position of ascidians as the closest relative of vertebrates, such morphological and endocrine features suggest that ascidians possess both common and species-specific regulatory mechanisms in follicle development. To date, several neuropeptides have been shown to participate in the growth of vitellogenic follicles, oocyte maturation of postvitellogenic follicles, and ovulation of fully mature follicles in a developmental stage-specific fashion. Furthermore, recent studies have shed light on the evolutionary processes of follicle development throughout chordates. In this review, we provide an overview of the neuropeptidergic molecular mechanism in the premature follicle growth, oocyte maturation, and ovulation in Ciona, and comparative views of the follicle development processes of mammals and teleosts.

Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth.

BACKGROUND: Mammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. RESULTS: Single-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 µm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 µm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100-109 versus 110-119 µm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. CONCLUSIONS: The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

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.

The pFSH dose affects the efficiency of in vivo embryo production in Santa Inês ewes.

This study compared the follicular growth, superovulatory response, and in vivo embryo production after administering two doses of porcine follicle-stimulating hormone (pFSH) in Santa Inês ewes. The estrous cycle of 36 multiparous ewes was synchronized with the Day 0 protocol and superovulated with 133 mg (G133, n=18) or 200 mg (G200, n=18) of pFSH. Ultrasonographic evaluations of the ovaries were performed, ewes were mated and submitted to non-surgical embryo recovery. Viable blastocysts were stained with Nile Red and Hoechst. The G200 had a greater number of medium and large follicles, as well as a larger size of the third largest follicle. A total of 97.2% (35/36) of the ewes came into estrus and it was possible to transpose cervix in 80.6% (29/36). There were no effects of treatments in the response to superovulation, the proportion of ewes in which was possible to transpose the cervix, the number of corpora lutea, the number of anovulatory follicles, the proportion of ewes flushed with at least one recovered structure, number of recovered structures, number of viable embryos, viability rate, and recovery rate. The G200 ewes were in estrus for a longer period of time than the G133 ewes (54.0 ± 4.5 h vs. 40.3 ± 3.6 h) and produced more freezable embryos (6.5 ± 1.6 vs. 2.3 ± 0.7) than G133. Both doses promoted an efficient superovulatory response and did not affect embryonic lipid accumulation. The dose of 200 mg of pFSH showed greater potential to increase the superovulatory response, as it increased follicular recruitment and the recovery of freezable embryos.

Integrated transcriptomic analysis on chicken ovary reveals CYP21A1 affects follicle granulosa cell development and steroid hormone synthesis.

Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.

FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

The therapeutic effect of anti-CD19 antibody on DHEA-induced PCOS mice.

Immune dysregulation has been summarized as a critical factor in the occurrence and development of Polycystic ovary syndrome (PCOS), but potential mediators and mechanisms remain unclear. Our previous study showed that CD19+ B cells were involved in the pathogenesis of dehydroepiandrosterone (DHEA)-induced PCOS mice. Here, we studied the therapeutic potential of anti-CD19 antibody (aCD19 Ab) on DHEA-induced PCOS mice. The results showed that aCD19 Ab treatment improved ovarian pathological structure and function of PCOS mice, manifested by an increased number of corpus luteum, a decreased number of cystic follicles and atretic follicles, and regular estrus cycles. The aCD19 Ab treatment reduced the proportion of splenic CD21+ CD23low marginal zone B cells as well as the level of serum IgM and decreased the percentage of peripheral blood and splenic neutrophils. In particular, aCD19 Ab treatment reduced the apoptosis of granulosa cells and macrophage infiltration in ovarian secondary follicles of PCOS mice, as well as the expression of TNF-α in ovarian tissue and serum TNF-α levels. Moreover, we confirmed that TNF-α induced the apoptosis of human ovarian granulosa tumor cell line cells in vitro. Thus, our work demonstrates that aCD19 Ab treatment improves ovarian pathological phenotype and function by reducing local and systemic inflammation in PCOS mice, which may provide a novel insight into PCOS therapy.