Gap junctions in polycystic ovary syndrome: Implications for follicular arrest.

Gap junctions are specialized intercellular conduits that provide a direct pathway between neighboring cells, which are involved in numerous physiological processes, such as cellular differentiation, cell growth, and metabolic coordination. The effect of gap junctional hemichannels in folliculogenesis is particularly obvious, and the down-regulation of connexins is related to abnormal follicle growth. Polycystic ovary syndrome (PCOS) is a ubiquitous endocrine disorder of the reproductive system, affecting the fertility of adult women due to anovulation. Exciting evidence shows that gap junction is involved in the pathological process related to PCOS and affects the development of follicles in women with PCOS. In this review, we examine the expression of connexins in follicular cells of PCOS and figure out whether such communication could have consequences for PCOS women. While along with results from clinical and related animal studies, we summarize the mechanism of connexins involved in the pathogenesis of PCOS.

ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice.

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

Identification of functional circRNAs regulating ovarian follicle development in goats.

BARKGROUND: Circular RNAs (circRNAs) play important regulatory roles in a variety of biological processes in mammals. Multiple birth-traits in goats are affected by several factors, but the expression and function of circRNAs in follicular development of goats are not clear. In this study, we aimed to investigate the possible regulatory mechanisms of circRNA and collected five groups of large follicles (Follicle diameter > 6 mm) and small follicles (1 mm < Follicle diameter < 3 mm) from Leizhou goats in estrus for RNA sequencing. RESULTS: RNA sequencing showed that 152 circRNAs were differentially expressed in small and large follicles. Among them, 101 circRNAs were up-regulated in large follicles and 51 circRNAs were up-regulated in small follicles. GO and KEGG enrichment analyses showed that parental genes of the differential circRNAs were significantly enriched in important pathways, such as ovarian steroidogenesis, GnRH signaling pathway, animal autophagy and oxytocin signalling pathway. BioSignal analysis revealed that 152 differentially expressed circRNAs could target 91 differential miRNAs including miR-101 family (chi-miR-101-3p, chi-miR-101-5p), miR-202 family (chi-miR-202-5p, chi-miR-202-3p),60 circRNAs with translation potential. Based on the predicted sequencing results, the ceRNA networks chicirc_008762/chi-miR-338-3p/ARHGAP18 and chicirc_040444/chi-miR-338-3p/STAR were constructed in this study. Importantly, the new gene circCFAP20DC was first discovered in goats. The EDU assay and flow cytometry results indicated that circCFAP20DC enhanced the proliferation of follicular granulosa cells(GCs). Real-time quantitative PCR and western blotting assays showed that circCFAP20DC activated the Retinoblastoma(RB) pathway and promoted the progression of granulosa cells from G1 to S phase. CONCLUSION: Differential circRNAs in goat size follicles may have important biological functions for follicular development. The novel gene circCFAP20DC activates the RB pathway, promoting the progression of GCs from G1 to S phase. This, in turn, enhances the proliferation of follicular GCs in goats.

Long non-coding RNA PWRN1 affects ovarian follicular development by regulating the function of granulosa cells.

RESEARCH QUESTION: What is the role of Prader-Willi region non-protein coding RNA 1 (PWRN1) in ovarian follicular development and its molecular mechanism? DESIGN: The expression and localization of PWRN1 were detected in granulosa cells from patients with different ovarian functions, and the effect of interfering with PWRN1 expression on cell function was detected by culturing granulosa cells in vitro. Furthermore, the effects of interfering with PWRN1 expression on ovarian function of female mice were explored through in-vitro and in-vivo experiments. RESULTS: The expression of PWRN1 was significantly lower in granulosa cells derived from patients with diminished ovarian reserve (DOR) compared with patients with normal ovarian function. By in-vitro culturing of primary granulosa cells or the KGN cell line, the results showed that the downregulation of PWRN1 promoted granulosa cell apoptosis, caused cell cycle arrested in S-phase, generated high levels of autophagy and led to significant decrease in steroidogenic capacity, including inhibition of oestradiol and progesterone production. In addition, SIRT1 overexpression could partially reverse the inhibitory effect of PWRN1 downregulation on cell proliferation. The results of in-vitro culturing of newborn mouse ovary showed that the downregulation of PWRN1 could slow down the early follicular development. Further, by injecting AAV-sh-PWRN1 in mouse ovarian bursa, the oestrous cycle of mouse was affected, and the number of oocytes retrieved after ovulation induction and embryos implanted after mating was significantly reduced. CONCLUSION: This study systematically elucidated the novel mechanism by which lncRNA PWRN1 participates in the regulation of granulosa cell function and follicular development.

Multi-Omics Reveals the Role of Arachidonic Acid Metabolism in the Gut-Follicle Axis for the Antral Follicular Development of Holstein Cows.

In vitro embryonic technology is crucial for improving farm animal reproduction but is hampered by the poor quality of oocytes and insufficient development potential. This study investigated the relationships among changes in the gut microbiota and metabolism, serum features, and the follicular fluid metabolome atlas. Correlation network maps were constructed to reveal how the metabolites affect follicular development by regulating gene expression in granulosa cells. The superovulation synchronization results showed that the number of follicle diameters from 4 to 8 mm, qualified oocyte number, cleavage, and blastocyst rates were improved in the dairy heifers (DH) compared with the non-lactating multiparous dairy cows (NDC) groups. The gut microbiota was decreased in Rikenellaceae_RC9_gut_group, Alistipes, and Bifidobacterium, but increased in Firmicutes, Cyanobacteria, Fibrobacterota, Desulfobacterota, and Verrucomicrobiota in the NDC group, which was highly associated with phospholipid-related metabolites of gut microbiota and serum. Metabolomic profiling of the gut microbiota, serum, and follicular fluid further demonstrated that the co-metabolites were phosphocholine and linoleic acid. Moreover, the expression of genes related to arachidonic acid metabolism in granulosa cells was significantly correlated with phosphocholine and linoleic acid. The results in granulosa cells showed that the levels of PLCB1 and COX2, participating in arachidonic acid metabolism, were increased in the DH group, which improved the concentrations of PGD2 and PGF2α in the follicular fluid. Finally, the expression levels of apoptosis-related proteins, cytokines, and steroidogenesis-related genes in granulosa cells and the concentrations of steroid hormones in follicular fluid were determinants of follicular development. According to our results, gut microbiota-related phosphocholine and linoleic acid participate in arachidonic acid metabolism in granulosa cells through the gut-follicle axis, which regulates follicular development. These findings hold promise for enhancing follicular development and optimizing oocyte quality in subfertile dairy cows.

YTHDF2 as a Mediator in BDNF-Induced Proliferation of Porcine Follicular Granulosa Cells.

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) are critical in determining the fate of follicles and are influenced by various factors, including brain-derived neurotrophic factor (BDNF). Previous research has shown that BDNF primarily regulates GC proliferation through the PI3K/AKT, NF-kB, and CREB tumour pathways; however, the role of other molecular mechanisms in mediating BDNF-induced GC proliferation remains unclear. In this study, we investigated the involvement of the m6A reader YTH domain-containing family member 2 (YTHDF2) in BDNF-stimulated GC proliferation and its underlying mechanism. GCs were cultured in DMEM medium supplemented with varying BDNF concentrations (0, 10, 30, 75, and 150 ng/mL) for 24 h. The viability, number, and cell cycle of GCs were assessed using the CCK-8 assay, cell counting, and flow cytometry, respectively. Further exploration into YTHDF2's role in BDNF-stimulated GC proliferation was conducted using RT-qPCR, Western blotting, and sequencing. Our findings indicate that YTHDF2 mediates the effect of BDNF on GC proliferation. Additionally, this study suggests for the first time that BDNF promotes YTHDF2 expression by increasing the phosphorylation level of the ERK1/2 signalling pathway. This study offers a new perspective and foundation for further elucidating the mechanism by which BDNF regulates GC proliferation.

miR-128-3p Regulates Follicular Granulosa Cell Proliferation and Apoptosis by Targeting the Growth Hormone Secretagogue Receptor.

The proliferation and apoptosis of granulosa cells (GCs) affect follicle development and reproductive disorders, with microRNAs playing a crucial regulatory role. Previous studies have shown the differential expression of miR-128-3p at different stages of goat follicle development, which suggests its potential regulatory role in follicle development. In this study, through the Cell Counting Kit-8 assay, the EDU assay, flow cytometry, quantitative real-time polymerase chain reaction, Western blot, and the dual-luciferase reporter assay, we used immortal human ovarian granulosa tumor cell line (KGN) cells as materials to investigate the effects of miR-128-3p and its predicted target gene growth hormone secretagogue receptor (GHSR) on GC proliferation and apoptosis. The results show that overexpression of miR-128-3p inhibited the proliferation of KGN cells, promoted cell apoptosis, and suppressed the expression of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2 (BCL2) while promoting that of Bcl-2 associated X protein (BAX). The dual-luciferase reporter assay revealed that miR-128-3p bound to the 3' untranslated region sequence of GHSR, which resulted in the inhibited expression of GHSR protein. Investigation of the effects of GHSR on GC proliferation and apoptosis revealed that GHSR overexpression promoted the expression of PCNA and BCL2, enhanced GC proliferation, and inhibited cell apoptosis, whereas the opposite effects were observed when GHSR expression was inhibited. In addition, miR-128-3p and GHSR can influence the expression of extracellular signal-regulated kinase 1/2 protein. In conclusion, miR-128-3p inhibits KGN cell proliferation and promotes cell apoptosis by downregulating the expression of the GHSR gene.

Follicular development and ovary aging: single-cell studies.

Follicular development is a critical process in reproductive biology that determines the number of oocytes and interacts with various cells within the follicle (such as oocytes, granulosa cells, cumulus cells and theca cells, etc.), and plays a vital role in fertility and reproductive health due to the dogma of a limited number of oogonia. Dysregulation of follicular development can lead to infertility problems and other reproductive disorders. To explore the physiological and pathological mechanisms of follicular development, immunology-based methods, microarrays, and next-generation sequencing have traditionally been used for characterization at the tissue level. However, with the proliferation of single-cell sequencing techniques, research has uncovered unique molecular mechanisms in individual cells that have been masked by previous holistic analyses. In this review, we briefly summarize the achievements and limitations of traditional methods in the study of follicular development. Simultaneously, we focus on how to understand the physiological process of follicular development at the single-cell level and reveal the relevant mechanisms leading to the pathology of follicular development and intervention targets. Moreover, we also summarize the limitations and application prospects of single cell sequencing in follicular development research.

Maternal tamoxifen exposure leads to abnormal primordial follicle assembly.

Tamoxifen (TAM) is an accredited drug used for treatment and prevention of breast cancer. Due to the long-term taking and the trend for women to delay childbearing, inadvertent conception occasionally occurs during TAM treatment. To explore the effects of TAM on a fetus, pregnant mice at gestation day 16.5 were orally administrated with different concentrations of TAM. Molecular biology techniques were used to analyze the effects of TAM on primordial follicle assembly of female offspring and the mechanism. It was found that maternal TAM exposure affected primordial follicle assembly and damaged the ovarian reserve in 3 dpp offspring. Up to 21 dpp, the follicular development had not recovered, with significantly decreased antral follicles and decreased total follicle number after maternal TAM exposure. Cell proliferation was significantly inhibited; however, the cell apoptosis was induced by maternal TAM exposure. Epigenetic regulation was also involved in the process of TAM induced abnormal primordial follicle assembly. The changed levels of H3K4me3, H3K9me3, and H3K27me3 presented the function of histone methylation in the regulation of the effects of maternal TAM exposure on the reproduction of female offspring. Moreover, the changed level of RNA m6A modification and the changed expression of genes related to transmethylation and demethylation proved the role of m6A in the process. Maternal TAM exposure led to abnormal primordial follicle assembly and follicular development by affecting cell proliferation, cell apoptosis, and epigenetics.

The effect of large follicle puncture and aspiration on the outcomes of IVF-ET in patients with asynchronized follicles under the long GnRH-a protocol: a retrospective cohort study.

OBJECTIVE: This retrospective study aimed to explore whether puncturing and aspirating asynchronized large follicles during long GnRH-a protocol COH impacted IVF-ET outcomes. METHODS: A total of 180 patients with asynchronized follicles during long GnRH-a protocol COH were retrospectively analyzed. They were divided into a puncture group, Group 1 (n = 81), and a non-puncture group, Group 2 (n = 99), according to whether puncture and aspiration were performed on the prematurely developing large follicles. The data of the selected patients were statistically analyzed to assess the effect of large follicle puncture and aspiration during ovulation induction on the final pregnancy results. In addition, we tentatively divided these 180 patients into either Group A (DF ≤ 14 mm) or Group B (DF > 14 mm) according to whether the diameter of the dominant large follicles (DF) exceeded 14 mm at the time of appearance. These two groups were then further divided into four subgroups: Subgroup A1 (DF ≤ 14 mm, patients underwent large follicle puncture), Subgroup A2 (DF ≤ 14 mm, patients did not undergo large follicle puncture), Subgroup B1 (DF > 14 mm, patients underwent large follicle puncture), and Subgroup B2 (DF > 14 mm, patients did not undergo large follicle puncture) based on whether large follicle puncture and aspiration were performed or not, aiming to compare the effects of large follicle puncture and aspiration on the clinical outcomes of patients with dominant large follicles at different time points. RESULTS: Group 1 exhibited significantly higher oocyte maturation rate (92.3% vs. 88.9%, P = 0.009) and high-quality embryo rate (75.2% vs. 65.7%, P = 0.007) compared with Group 2. No differences were observed in the number of oocytes retrieved, 2PN fertilization rate, clinical pregnancy rate, abortion rate, and live birth rate between the two groups (P > 0.05). When the dominant large follicles' diameter was ≤ 14 mm, the final oocyte maturation rate (92.7% vs. 88.1%, P = 0.023), high-quality embryo rate (72.9% vs. 61.8%, P = 0.047) and live birth rate (54.5% vs. 31.9%, P = 0.043) of Subgroup A1 were significantly higher than those of Subgroup A2. In contrast, when the dominant large follicles' diameter was > 14 mm, no statistical difference was observed in all data. CONCLUSIONS: Large follicle puncture and aspiration in long GnRH-a protocol COH could improve the oocyte maturation rate and high-quality embryo rate in patients with asynchronized follicles. However, clinical pregnancy and live birth rates were not significantly improved. In addition, when the dominant follicles' diameter did not exceed 14 mm, large follicles puncture and aspiration significantly improved the patient's oocyte maturation rate, high-quality embryo rate and live birth rate.

Role and mechanism of the p-JAK2/p-STAT3 signaling pathway in follicular development in PCOS rats.

OBJECTIVE: To identify the association between the phosphorylated Janus kinase 2/phosphorylated signal transducer and activator of transcription (p-JAK2/p-STAT3) signaling pathway and follicular development in polycystic ovary syndrome (PCOS) rats, and explore the underlying mechanism. To evaluate the role of exogenous JAK2 inhibitor AG490 in the model and the associations among luteinizing hormone/choriogonadotropin receptor (LHCGR), follicle-stimulating hormone receptor (FSHR), cytochrome P450 17α (CYP17a), cytochrome P450 19 (CYP19), and PCOS. RESULTS: Rat models of PCOS was established. PCOS rats were intraperitoneally treated with double-distilled water (ddH2O)/DMSO/AG490. The rate of ovarian morphological recovery in the AG490 group was significantly higher compared with the DMSO group (83.3 % vs 9.1 %, X2 = 12.68, P < 0.001). Moreover, the short in the time the estrous cycle was resumed in the AG490 group (hazard ratio = 16.32, P < 0.001) compared with the DMSO group. Compared with the controls, p-JAK2, p-STAT3, LHCGR, and CYP17a expression levels were increased whereas that of FSHR and CYP19 were decreased in the ovaries of PCOS rats. However, an opposite trend was observed after treatment with AG490. Software prediction revealed that the p-STAT3 bound to the promoter regions of LHCGR, FSHR, CYP17a, and CYP19 genes. This finding was confirmed by results of correlation analysis (R = 0.834, -0.836, 0.875 and -0.712, respectively, all P < 0.001). CONCLUSION: This study demonstrated that the p-JAK2/p-STAT3 signaling pathway was involved in follicular development in PCOS rats by upregulating LHCGR and CYP17a expression, and downregulating that of FSHR and CYP19. AG490 treatment exerted beneficial effects. LHCGR, FSHR, CYP17a, and CYP19 are candidate genes associated with follicular development in PCOS rats.

Protective effect of wuzibushen recipe on follicular development via regulating androgen receptor in polycystic ovary syndrome model rats.

OBJECTIVES: The study aimed to explore the role and mechanism of WZBS recipe on PCOS. METHODS: PCOS model was established. After modeling, PCOS rats were intragastrically administered with Diane-35 or WZBS recipe (6.93 g/kg/d). Then, the ovarian and uterine morphology were observed, the estrous cycle was assessed. HE and oil red O staining were conducted for ovarian morphological analysis and counting ovarian follicle and corpora lutea number. Furthermore, the serum content of testosterone (T) and sex-hormone-binding globulin (SHBG) were assessed by ELISA kits. The androgen receptor (AR), CX43 mRNA and protein expression were measured by q-PCR and Western blot. RESULTS: WZBS recipe increased uterine implanted blastocysts, reduced cystic dilated follicles, and normalized estrous cycle in PCOS rats. Meanwhile, WZBS recipe alleviated ovarian injury, increased mature follicles and corpora lutea number in PCOS rats. Moreover, WZBS recipe decreased serum T content, AR expression and increased serum SHBG content, CX43 expression in PCOS rats. CONCLUSIONS: This study reveals that WZBS recipe may attenuate PCOS by protecting follicular development via down-regulating AR.

Obesity-induced inflammatory miR-133a mediates apoptosis of granulosa cells and causes abnormal folliculogenesis.

Obesity has been reported to promote disordered folliculogenesis, but the exact molecular mechanisms are still not fully understood. In this study, we find that miR-133a is involved in obesity-induced follicular development disorder. After feeding with a high-fat diet (HFD) and fructose water for nine weeks, the mouse body weight is significantly increased, accompanied by an inflammatory state and increased expression of miR-133a in the adipose tissues and ovaries as well as accelerated follicle depletion. Although miR-133a is increased in the fat and ovaries of HFD mice, the increased miR-133a in the HFD ovaries is not derived from exosome transferred from obese adipose tissues but is synthesized by ovarian follicular cells in response to HFD-induced inflammation. In vivo experiments show that intrabursal injection of miR-133a agomir induces a decrease in primordial follicles and an increase in antral follicles and atretic follicles, which is similar to HFD-induced abnormal folliculogenesis. Overexpression of miR-133a modestly promotes granulosa cell apoptosis by balancing the expression of anti-apoptotic proteins such as C1QL1 and XIAP and pro-apoptotic proteins such as PTEN. Overall, this study reveals the function of miR-133a in obesity-induced ovarian folliculogenesis dysfunction and sheds light on the etiology of female reproductive disorders.

Dynamic changes in gene expression during follicle development and the efficacy of four timed AI protocols in non-suckling female yaks (Bos grunniens).

The objectives of the study were to investigate changes in the mRNA expression levels of five genes during antral follicle development and to assess the efficacy of four timed-artificial insemination (TAI) protocols in female yaks (Bos grunniens). RT-qPCR analysis revealed that expression levels were greater for follicle-stimulating hormone receptor and bone morphogenic protein 15 in the small follicle, luteinizing hormone receptor, and kit ligand in the large follicle, and growth differentiation factor 9 in the medium follicle (p < 0.05). Non-suckling yaks were treated as a 7-d CIDR, and PGF2α + eCG at CIDR withdrawal and TAI with frozen yak semen at 56-58 h after PGF2α (PPe-7d); either a 7-d CIDR (PPG-7d) or a 5-d CIDR (PPG-5d), and PGF2α at CIDR withdrawal and TAI + GnRH at 70-72 h after PGF2α; and GnRH treatment on Day 0, followed by PGF2α on Day 7 and TAI + GnRH on Day 9 (GPG-7d). The results showed that the pregnancy rate (P/AI) was greater in PPG-5d than in GPG-7d (p < 0.05), but the P/AI was not different among the other TAI protocols. In conclusion, the expression levels of these genes in follicles are dynamically changed during antral follicle development in yaks. The PPG-5d protocol achieved a greater P/AI.

Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus.

Estradiol and progesterone have been recognized as important mediators of reproductive events in the female mainly via binding to their receptors. This study aimed to characterize the immunolocalization of the estrogen receptor alfa (ERα), estrogen receptor beta (ERß) and progesterone receptor (PR) in the ovarian follicles of the lizard Sceloporus torquatus. The localization of steroid receptors has a spatio-temporal pattern that depends on the stage of follicular development. The immunostaining intensity of the three receptors was high in the pyriform cells and the cortex of the oocyte of previtellogenic follicles. During the vitellogenic phase, the granulosa and theca immunostaining was intense even with the modification of the follicular layer. In the preovulatory follicles, the receptors were found in yolk and additionally, ERα was also located in the theca. These observations suggest a role for sex steroids in regulating follicular development in lizards, like other vertebrates.

Impacts of dietary betaine on rectal temperature, laying performance, metabolism, intestinal morphology, and follicular development in heat-exposed laying hens.

This experiment assessed the influences of betaine (BET; 2000 mg/kg) on rectal temperature (Tr), laying performance, metabolism, intestinal morphology, and follicular development in heat-stressed hens. One-hundred and twenty-eight Hisex white hens (42wks) were housed in 4 battery cages (8 pens/cage; 4 hens/pen) and divided into 4 treatments: 1) thermoneutral (TN) environments and a control diet (TNCON), 2) TN and a diet accompanied with BET (TNBET), 3) heat stress (HS) environments and a control diet (HSCON), or 4) HS and a diet accompanied with BET (HSBET). Following acclimation (15d), hens of TNCON and TNBET remained in TN, while HSCON and HSBET hens were subjected to cyclical HS (5d; 16.9-37.5 °C). Cyclical HS increased Tr compared with TN hens (1.6 °C; P < 0.01), but supplemental BET decreased Tr (0.4 °C; P < 0.01). Relative to TN treatments, HS declined egg production, weight, and mass (18, 4.2, and 26%, respectively; P < 0.01), but BET ameliorated the egg production and mass (13.1 and 16.2%, respectively; P < 0.01). Compared with HSCON, feed conversion ratio and survival rate were improved in HSBET hens (12.3 and 6.25%, respectively; P ≥ 0.03). Relative to TN hens, HS elevated glucose and blood urea nitrogen (BUN) levels (15 and 4%, respectively; P ≤ 0.04). Supplemental BET decreased BUN levels (6.6%; P < 0.01) relative to HSCON hens. Furthermore, HS diminished jejunal villus height and villus surface area (∼27 and 35%, respectively; P < 0.01) relative to TN hens but were unaltered by BET supplementation. Relative to TN hens, HS decreased oviduct's weight, ovary's length, and ovarian primordial and primary follicles count (18, 23, 34 and 44%, respectively; P < 0.01) and caused fibrosis in shell gland (3-fold; P = 0.05). Collectively, HS impaired productivity, metabolism, intestinal architecture, and reproductive efficiency. Feeding BET reduced Tr, improved laying performance, and slightly altered metabolism but did not affect intestinal and follicular measurements in heat-stressed hens.

Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells.

In poultry, prolactin (PRL) plays a key role in the regulation of incubation behavior, hormone secretion, and reproductive activities. However, previous in vitro studies have focused on the actions of PRL in ovarian follicles of poultry, relying on the use of exogenous or recombinant PRL, and the true role of PRL in regulating ovarian granulosa cell (GC) functions in poultry awaits a further investigation using endogenous native PRL. Therefore, in this study, we first isolated and purified recombinant goose PRL protein (rPRL) and native goose PRL protein (nPRL) using Ni-affinity chromatography and rabbit anti-rPRL antibodies-filled immunoaffinity chromatography, respectively. Then, we analyzed and compared the effects of rPRL and nPRL at different concentrations (0, 3, 30, or 300 ng/mL) on the proliferation and apoptosis of both GCs isolated from goose ovarian pre-hierarchical follicles (phGCs) and from hierarchical follicles (hGCs). Our results show that rPRL at lower concentrations increased the viability and proliferation of both phGCs and hGCs, while it exerted anti-apoptotic effects in phGCs by upregulating the expression of Bcl-2. On the other hand, nPRL increased the apoptosis of phGCs in a concentration-dependent manner by upregulating the expressions of caspase-3 and Fas and downregulating the expressions of Bcl-2 and Becn-1. In conclusion, this study not only obtained a highly pure nPRL for the first time, but also suggested a dual role of PRL in regulating the proliferation and apoptosis of goose GCs, depending on its concentration and the stage of follicle development. The data presented here can be helpful in purifying native proteins of poultry and enabling a better understanding of the roles of PRL during the ovarian follicle development in poultry.

Lovastatin, an Up-Regulator of Low-Density Lipoprotein Receptor, Enhances Follicular Development in Mouse Ovaries.

Ovarian aging hampers in vitro fertilization in assisted reproductive medicine and has no cure. Lipoprotein metabolism is associated with ovarian aging. It remains unclear how to overcome poor follicular development with aging. Upregulation of the low-density lipoprotein receptor (LDLR) enhances oogenesis and follicular development in mouse ovaries. This study investigated whether upregulation of LDLR expression using lovastatin enhances ovarian activity in mice. We performed superovulation using a hormone and used lovastatin to upregulate LDLR. We histologically analyzed the functional activity of lovastatin-treated ovaries and investigated gene and protein expression of follicular development markers, using RT-qPCR and Western blotting. Histological analysis showed that lovastatin significantly increased the numbers of antral follicles and ovulated oocytes per ovary. The in vitro maturation rate was 10% higher for lovastatin-treated ovaries than for control ovaries. Relative LDLR expression was 40% higher in lovastatin-treated ovaries than in control ovaries. Lovastatin significantly increased steroidogenesis in ovaries and promoted the expression of follicular development marker genes such as anti-Mullerian hormone, Oct3/4, Nanog, and Sox2. In conclusion, lovastatin enhanced ovarian activity throughout follicular development. Therefore, we suggest that upregulation of LDLR may help to improve follicular development in clinical settings. Modulation of lipoprotein metabolism can be used with assisted reproductive technologies to overcome ovarian aging.

[Advances in the Regulation of Follicular Development by Extracellular Vesicles and Non-Coding RNAs].

Extracellular vesicles (EV),nanoscale vesicles encapsulated by phospholipid bilayers,are rich in biological molecules such as nucleic acids,metabolites,proteins,and lipids derived from parental cells.They are mainly involved in intercellular communication,signal transmission,and material transport and affect the functions of target cells.Ovulation disorders account for a higher proportion in the factors causing infertility which demonstrates increasing incidence year by year.Non-coding RNAs participate in a series of physiological and pathological processes of follicular development,playing a key role in female infertility.This review systematically introduces the types and biological roles of EV and elaborates on the regulation of follicular development from the effects of EV and non-coding RNAs on granulosa cell function,oocyte maturation,ovulation,luteal formation,and steroid hormone synthesis,providing a new idea and a breakthrough point for the diagnosis and treatment of infertility.

Role of autophagy in follicular development and maintenance of primordial follicular pool in the ovary.

The reproductive life span of the organism mainly depends on follicular development that maintains the primordial follicle pool in the cohort of follicles within the ovary. The total count of primordial follicles decreases with age due to ovulation and follicular atresia. Follicular atresia, a process of ovarian follicles degradation, mainly occurs via apoptosis, but recent studies also favor autophagy existence. Autophagy is a cellular and energy homeostatic response that helps to maintain the number of healthy primordial follicles, germ cell survival, and removal of corpus luteum remnants. But the excessive autophagic cell death changes both the quality and quantity of oocytes that ultimately affect female reproductive health. Autophagy regulation occurs by various autophagy-regulated genes like BECN1 and LC3-II (autophagy marker genes). Their abnormal regulation or mutation highly influences follicular development by alteration of primordial follicles formation, the decline in oocytes count, and germ cell loss. Various classical signaling pathways such as PI3K/AKT/mTOR, MAPK/ERK1/2, AMPK, and IRE1 are involved in granulosa and oocytes autophagy, while mTOR signaling is the primary mechanism. Along with basal level autophagy, chemical/hormone/stress-mediated autophagy also affects follicular development and female reproduction. In this review, we have primarily focused on granulosa cell and oocytes' autophagy, mechanism, and the role of autophagy determining marker genes in follicular development.