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PAFAH1B1 governs follicular development by modulating the protein complex of CCNE1-CDK2-CDK1 to induce cell cycle arrest.

BACKGROUND: Ovarian follicle development plays a crucial role in mammalian fertility, which is primarily regulated by granulosa cell (GC) proliferation and cell cycle. Cell cycle dysregulation collectively might drive follicular atresia through GC dysfunction. However, the underlying molecular mechanisms remain largely unexplored. METHODS: The scRNA-seq and integrative analysis revealed that PAFAH1B1 was involved in cell cycle. Functional assays, including overexpression/knockdown, flow cytometry, EdU, HE, and TUNEL, confirmed that PAFAH1B1 regulated cell cycle and follicular development in vitro and in vivo. CoIP showed that PAFAH1B1 bound CCNE1-CDK2-CDK1 to arrest G2/M phase. Chromatin accessibility and CRISPR/dCas9-TET1 demonstrated that DNA methylation modulated PAFAH1B1 transcription. RESULTS: A novel regulator of cell cycle, PAFAH1B1, was identified in Pig Genotype-Tissue Expression (PigGTEx). During GC proliferation, we found that PAFAH1B1 transcription was correlated with the distribution rate of G1 phase in GCs. PAFAH1B1 protein was confirmed to specifically bind to CCNE1-CDK2-CDK1 to arrest G2/M phase. Notably, PAFAH1B1 appeared to hinder the development of follicles. Furthermore, the demethylation significantly promoted the transcription activity and chromatin accessibility of CpG island (-7 bp to +170 bp) of PAFAH1B1. Taken together, PAFAH1B1 physically interacted with the CCNE1-CDK2-CDK1 complex to arrest G2/M phase and inhibit the GCs proliferation and follicular development. Additionally, demethylation of CpG island significantly promoted the transcription of PAFAH1B1. CONCLUSION: These findings not only advance understanding of cell proliferation and cycle regulation but also identify PAFAH1B1 as a candidate gene for further investigation in follicular development.

CCNE1-CDK2-CDK1 complex

Estradiol receptor content in rat granulosa cells during follicular development: modification by estradiol and gonadotropins.

To examine the relationships between gonadotropic hormones, ovarian follicular development, and estradiol receptor content in rat granulosa cells, follicular growth was stimulated in intact immature rats and hypophysectomized immature rats. In intact rats, both PMSG and oFSH given for 2 days stimulated follicular growth and endogenous estrogen production but caused estradiol receptor content in granulosa cells to decrease markedly. In hypophysectomized rats estradiol (2 mg/day X 4) was capable of increasing the content of its own receptor, stimulated preantral follicular development, and potentiated the responses of granulosa cells to highly purified hFSH. Thus, subsequent treatment with hFSH stimulated antral formation and maintained high levels of estradiol receptor content in granulosa cells. LH, on the other hand, caused a rapid decline in the content of estradiol receptor in nuclei of granulosa cells. In hypophysectomized rats primed with estradiol alone, a high dose of oLH caused follicles to undergo atresia. In hypophysectomized rats primed with hFSH in addition to estradiol, oLH stimulated luteinization. Thus, induction of atresia or luteinization by oLH in these rats appears to be associated (cause or effect?) with a loss of estradiol receptor in granulosa cells. Whether follicles of estrogen-treated, hypophysectomized rats luteinize or undergo atresia in response to LH appears to be determined by their stage of differentiation and the nature of the prior stimulation by FSH.

Aging

Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone.

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.

Animals

Celastrol modulates IRS1 expression to alleviate ovarian aging and to enhance follicular development.

Ovarian aging significantly contributes to the decline of the female reproductive system, adversely affecting fertility and endocrine homeostasis. To address the challenges posed by reproductive aging, natural products have shown promising preventive and therapeutic effects. Here, we investigated the beneficial effects of natural compound celastrol on ovarian development and aging, together with its underlying mechanisms. We found that celastrol administration at a concentration of 3 mg/kg promoted follicle development in young mice and enhanced porcine oocyte maturation, while regulating granulosa cell proliferation and apoptosis. In 12-month-old mice (equivalent to middle-aged adults), celastrol exhibited similar beneficial effects. Transcriptomic analysis revealed that differentially expressed genes post-celastrol treatment were associated with steroid biosynthesis, estrogen signaling pathways, type 2 diabetes, insulin secretion, meiosis, and apoptosis. Additionally, insulin receptor substrate 1 (IRS1), an adapter protein in insulin signaling, was shown to advance puberty in young mice and to facilitate oocyte maturation. Overexpression of IRS1 in oocytes promoted follicular development and oocyte maturation, resulting in enhanced steroid hormone levels, whereas IRS1 knockdown inhibited these processes. Our findings indicate that celastrol may regulate ovarian development and aging by modulating IRS1 expression and its related pathways, suggesting celastrol as a novel small-molecule compound targeting IRS1, and offering new perspectives for potential therapeutic strategies against reproductive aging and infertility.

Animals

Follicular development during late human pregnancy.

In contrast to the accepted view of ovarian quiescence during pregnancy, the ovaries of preparturient women are covered with a dense population of small superficial follicles. In this study we have measured the follicular size and status of the oocytes and cumulus oophorus in 298 follicles from the ovaries of 30 women. The samples were taken at cesarean section and were examined with Nomarski differential interference microscopy. No oocyte was recovered from 50% of the follicles; 79% of the recovered oocytes and 78% of their cumuli were degenerative. Degeneration was correlated with appearance of phagocytes in the follicular fluid. These findings suggest that the endocrine status of gestation does not prevent early follicular development, but induces premature atresia.

Female

Ovarian H3K27ac remodeling is associated with impaired follicular development in laying hens with fatty liver hemorrhagic syndrome.

Fatty liver hemorrhagic syndrome (FLHS) is a metabolic disease of laying hens that reduces egg production and is accompanied by reproductive impairment, but the ovarian regulatory mechanisms that connect nutritional stress to follicular dysfunction remain unclear. This study examined whether active chromatin remodeling in the ovary is associated with FLHS induced by a high-energy, low-protein (HELP) diet. Hy-Line Brown hens were assigned to a basal diet or HELP diet, and ovarian tissue was collected for histone H3 lysine 27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing and RNA sequencing. The HELP diet reduced laying performance and the numbers of small yellow and hierarchical follicles, indicating compromised follicular development. Genome-wide H3K27ac profiling identified 2,111 regions with lower acetylation and 1,707 regions with higher acetylation in FLHS ovaries. Genes linked to differential H3K27ac regions were enriched in pathways related to oocyte meiosis, cell cycle control, FoxO signaling, gonadotropin-releasing hormone signaling, and steroid hormone biosynthesis. RNA sequencing identified 341 differentially expressed genes, with a predominance of downregulated genes. Integration of chromatin and transcriptome data highlighted folliculogenesis-related genes, including FGF1, FGF9, and MMP10, that showed reduced H3K27ac enrichment together with decreased expression. Super-enhancer analysis further identified 131 regions with reduced H3K27ac signal in FLHS ovaries, including regions located near PCNA and RAP1A, two genes involved in cellular proliferation and survival signaling. Motif enrichment of differential H3K27ac regions implicated Fos, SF-1/NR5A1, and GATA-4 as candidate transcriptional regulators. These findings indicate that HELP diet-induced FLHS is associated with broad attenuation of active ovarian regulatory elements and reduced expression of genes required for follicle growth, tissue remodeling, and steroidogenic function. The study provides an ovarian epigenomic framework for understanding reproductive decline in FLHS-affected laying hens.

Fatty liver hemorrhagic syndrome

Inhibitory effect of charcoal-treated porcine follicular fluid upon serum FSH levels and follicular development in the rhesus monkey.

Injection of charcoal treated porcine follicular fluid into 4 intact, 4 long term castrate and 2 postmenopausal rhesus monkeys brought about a selective decline in serum FSH levels. Luteinizing hormone levels were not significantly decreased. Intraperitoneal injection of 4 ml porcine follicular fluid every 8 hours for the first 4 days of the menstrual cycle led to a decrease in follicle and granulosa cell growth observed at that midcycle. Treatment with porcine serum did not exert this effect.

Animals

The morphology of follicular development and ovulation in non-human primates.

A colony of Macaca fascicularis have been under continuous observation for the past 4 years to ascertain the follicular morphological changes that occur prior to ovulation and during the development of the corpus luteum. Of 609 experimental cycles, laparoscopy was performed at least once in 44-5% of the cycles. Of the 104 cycles where the ovulatory status was definitely known, 89-4% were deemed ovulatory and 10-6% anovulatory. The presence or absence of ovulation in the previous cycle did not have an effect on the cycle length either for the total cycles or when analysing only cycles over 28 days. Similarly, the occurrence of two consecutive ovulations (in consecutive cycles) on the same vs. opposite ovaries did not have a significant effect on the cycle length. Neither laparoscopic stress or anaesthesia effected the normal cyclicity of the animals. The characteristic changes in follicular morphology are most clearly defined in M. fascicularis. In this species the 24 of 36 hr prior to ovulation are accompanied by discrete changes which occur in a fixed sequence, allowing one to predict the time of ovulation with reasonable accuracy. In S. sciureus ovulation is preceded by extensive bulging at the follicular apex and haemorrhaging at the base of the follicle. Due to this haemorrhaging post-ovulatory follicles in S. sciureus are generally more easily discernible than in fascicularis. The formation of clear areas (stigma) is not as evident in either S. sciureus or G. senegalensis as in the macaque. Actual ovulation has been observed four times in M. fascicularis and twice in S. sciureus.

Animals

Follicular development in ovaries of children with Down's syndrome.

Ovaries of children with Down's syndrome were examined histologically in order to determine in what way the ovarian development differed from the normal. Twenty-six specimens obtained at autopsy at various ages between birth and 14 1/2 years were available and compared with ovaries from normal children of similar ages. All ovaries from normal children were characterized by active follicle growth. The ovaries of the children with Down's syndrome, however, showed absence or retardation of follicle growth. Furthermore the number as well as the size of the antral follicles differed from those in the normal ovary. The decrease of the number of small follicles occurs earlier in life in the ovaries of children with Down's syndrome than in the control. The possible relationship between an abnormal ovarian growth pattern and hormonal imbalance is discussed.

Adolescent

[Development of follicular and parafollicular adenomas in the thyroid of rats treated with thiamazole (author's transl)].

Hypothyroidism is induced in rats treated with thiamazole, an antithyroid drug. If this phase lasts long enough, the follicular cells develop adenomas. Within the same period, the number of parafollicular or C cells increases threefold on an average, but without producing corresponding adenomas, the formation of which is inhibited. When treatment is stopped, a second phase appears during which thyroid function reverts to normal. The previously observed inhibition disappears, and, after a period of latency, the hyperplastic C cells develop parafollicular adenomas. The type of tumors to be found in treated rats (either of them, or both simultaneously) is determined by respective durations of the two phases. The endocrine mechanisms of these phenomena are discussed.

Adenoma