SOX4 regulates proliferation and apoptosis of human ovarian granulosa-like tumor cell line KGN through the Hippo pathway.

The proliferation and apoptosis of ovarian granulosa cells are important for folliculogenesis. As a transcription factor, SRY-box transcription factor 4 (SOX4) has important roles in regulating cellular proliferation and apoptosis. Nonetheless, the regulatory mechanisms of SOX4 on proliferation and apoptosis of granulosa cells remain elusive. Therefore, a stably overexpressed SOX4 ovarian granulosa cell line KGN was generated by lentivirus encapsulation. We observed that overexpression of SOX4 inhibits apoptosis, promotes proliferation and migration of KGN cells. Comparative analysis of the transcriptome revealed 868 upregulated and 696 downregulated DEGs in LV-SOX4 in comparison with LV-CON KGN cell lines. Afterward, further assessments were performed to explore the possible functions about these DEGs. The data showed their involvement in many biological processes, particularly the Hippo signaling pathway. Moreover, the expression levels of YAP1, WWTR1, WTIP, DLG3, CCN2, and AMOT, which were associated with the Hippo signaling pathway, were further validated by qRT-PCR. In addition, the protein expression levels of YAP1 were markedly elevated, while p-YAP1 were notably reduced after overexpression of SOX4 in KGN cells. Thus, these results suggested that SOX4 regulates apoptosis, proliferation and migration of KGN cells, at least partly, through activation of the Hippo signaling pathway, which might be implicated in mammalian follicle development.

Comparative transcriptome analysis of ovaries and testes reveals sex-biased genes and pathways in zebrafish.

Zebrafish (Danio rerio) is a widely recognized and extensively studied model organism in scientific research. The regulatory mechanism of gonadal development and differentiation of this species has aroused considerable attention. Nonetheless, the major sex-biased genes and pathways associated with gonadal development remain elusive. Therefore, to comprehend this intricate process, gonadal transcriptome sequencing was carried out to identify differentially expressed genes (DEGs) between the testes and ovaries of adult zebrafish. The preliminary assessment yielded a total of 23,529,272 and 23,521,368 clean reads from the cDNA libraries of ovaries and testes. Afterward, a comparative analysis of the transcriptome revealed 3,604 upregulated and 11,371 downregulated DEGs in the ovaries compared to the testes. Of these genes, 428 were exclusively expressed in females, while 3,516 were exclusively expressed in males. Additionally, further assessments were conducted to explore the functions associated with these DEGs in various biological processes. The data revealed their involvement in sex-biased pathways, such as progesterone-mediated oocyte maturation, oocyte meiosis, cytokine-cytokine receptor interaction, and cardiac muscle contraction. Finally, the expression levels of 14 sex-biased DEGs (cdc20, ccnb1, ypel3, chn1, bmp15, rspo1, tnfsf10, egfra, acta2, cox8a, gsdf, dmrt1, star, and cyp17a1) associated with the enriched pathways were subjected to further validation through qRT-PCR. The data acquired from these investigations offer valuable resources to support further exploration of the mechanisms governing sexual dimorphism and gonadal development in zebrafish.

Difluoromethylation-Carboxylation and -Deuteration of Alkenes Triggered by Electroreduction of Difluoromethyltriphenylphosphonium Bromide.

It is significant to develop novel difluoromethylation methods because of the important roles of difluoromethyl groups in the medicinal chemistry and material industries. Here, we developed a novel difluoromethylation-carboxylation and difluoromethylation-deuteration method triggered by a difluoromethyl radical generated by electroreduction of stable and easily available difluoromethyltriphenylphosphonium bromide. Various molecules containing difluoromethyl and carboxyl or deuterium groups can be synthesized through this method. The establishment of this method will provide an alternative to radical difluoromethylation reactions.

Magnesium-Mediated Umpolung Carboxylation of p-Quinone Methides with CO2.

Magnesium-mediated reductive carboxylation of p-QMs with CO2 via an Umpolung strategy has been developed, which can be used for the preparation of various aryl acetic acids. This protocol featured high atom economy, mild conditions, and operational simplicity. The creation of this Umpolung carboxylation of p-QMs will unprecedentedly extend the application of p-QMs to nucleophilic reagents.

Role of EGFR expressed on the granulosa cells in the pathogenesis of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is one of the most common endocrinological disorders affecting between 6 to 20% of reproductive aged women. However, the etiology of PCOS is still unclear. Epidermal growth factor receptor (EGFR) plays a critical role in the growth and development of ovarian follicles. In our previous study, we showed that the expression level of EGFR was significantly higher in the cumulus granulosa cells from women with PCOS than that of normal women, suggesting that EGFR may play a potential role in the pathogenesis of PCOS. The present study further evaluated the association between EGFR and PCOS through both in clinical observation and animal experiments. We firstly validated the differential expression of EGFR in cumulus granulosa cells between PCOS patients and normal subjects by qRT-PCR and immunofluorescence staining. Then we generated a mouse model (n=20) of PCOS by injecting dehydroepiandrosterone (DHEA). The PCOS mice were then injected with an E corpus GFR inhibitor (AG1478) (n=10), which significantly improved the sex hormone levels in the estrous cycle stage, and the serum levels of LH, FSH and testosterone were compared with the PCOS mice without EGFR inhibitor treatment (n=10). Decreasing the expression level of EGFR in the PCOS mice also improved the ovulatory function of their ovaries which was indicated by the multifarious follicle stage in these mice as compared with the PCOS mice without EGFR inhibitor treatment. Also, the number of corpopa lutea were higher in the control group and the EGFR inhibitor treated group than in the PCOS group. The sex hormone levels and reproductive function were not significantly different between the control mice and the PCOS mice treated with the EGFR inhibitor. Our results demonstrated that EGF/EGFR signaling affected the proliferation of cumulus granulosa cells, oocyte maturation and meiosis, and played a potential role in the pathogenesis of PCOS. Therefore, the selective inhibition of EGFR may serve as a novel strategy for the clinical management of PCOS.

Perceptions and attitudes towards elective egg freezing of Chinese college students: a survey from eastern China.

PURPOSE: Few options are available for preserving female fertility to postpone childbirth. Although egg freezing with successful thawing is now possible, women' attitudes towards its use or the circumstances under which this technique may be considered remain unclear. METHODS: This study is a cross-sectional online survey. From November 2020 to January 2021, 848 questionnaires were collected through the Questionnaire Star Network platform, and a total of 750 valid answers were obtained. RESULTS: For more than 40% of the interviewees, the level of knowledge about egg freezing was only 0-25%; 36.9% of the interviewees supported elective egg freezing, and the main factor affecting their approval was major; approximately 60% of interviewees believed that being married should not be a condition for freezing eggs; and 56.7% of the interviewees supported the establishment of an egg bank in China, and the main factor affecting their acceptance was the place of residence. CONCLUSION: College students generally have a high level of recognition regarding elective egg freezing and the establishment of an egg bank, but their level of knowledge about egg freezing is low. Relevant knowledge must be strengthened to help college students achieve a correct understanding of elective egg freezing and egg bank establishment and then guide college students in developing a scientific dialectical attitude towards this technology.

Identification of Serotonin as a Predictive Marker for Breast Cancer Patients.

PURPOSE: Cumulative evidence has demonstrated that breast cancer was the most commonly diagnosed cancer in women. Despite growing evidence for a link between serotonin and tumorigenesis, research on the expression of serotoninergic systems in the human breast cancer cell and tissue has only rarely been reported. METHODS: First, immunofluorescence staining, ELISA and Western blotting were used to detect serotonin and melatoninergic systems in various breast cancer cell types. Then, serotonin expression was evaluated in the cultures of TPBC cell line BT-474 and TNBC cell line MDA-MB-231 using immunofluorescence assay. To further explore the diagnostic role of serotonin in breast cancer, serotonin expression was conducted in the TPBC and TNBC tumor sections by immunostaining analysis. RESULTS: Our results suggested that both human breast cancer cells and human breast epithelial cell line could synthesize serotonin and melatonin. Unlike melatonin, serotonin levels varied significantly between human breast cancer and breast epithelial cell line (p<0.01). In addition, serotonin N-acetyltransferase (NAT) and acetylserotonin methyltransferase (ASMT), the key enzymes in the pathway of melatonin synthesis from serotonin, were also detectable. In agreement with these findings of human breast cancer cell and human breast epithelial cell line, serotonin expression was also much higher in triple-negative (PR-, ER-, HER-2-) breast cancer (TNBC) and triple-positive breast cancer (TPBC) compared to para-carcinoma tissues (PCTs). CONCLUSION: Here, we provided evidence that the human breast cancer cell (MCF-7, Bcap-37) and human breast epithelial cell (MCF-10A) could synthesize intrinsic serotonin and melatonin, and serotonin expression was higher in the breast cancer tissue compared with PCT. The findings suggested that serotonin might be used as a predictive marker for breast cancer patients.

Melatonin ameliorates ovarian dysfunction by regulating autophagy in PCOS via the PI3K-Akt pathway.

Emerging evidence has demonstrated that melatonin (MT) plays a crucial role in regulating mammalian reproductive functions. It has been reported that MT has a protective effect on polycystic ovary syndrome (PCOS). However, the protective mechanisms of MT remain poorly understood. This study aims to explore the effect of MT on ovarian function in PCOS and to elucidate the relevant molecular mechanisms in vivo and in vitro. We first analysed MT expression levels in the follicular fluid of PCOS patients. A significant reduction in MT expression levels was noted in PCOS patients. Intriguingly, reduced MT levels correlated with serum testosterone and inflammatory cytokine levels in follicular fluid. Moreover, we confirmed the protective function of MT through regulating autophagy in a DHEA-induced PCOS rat model. Autophagy was activated in the ovarian tissue of the PCOS rat model, whereas additional MT inhibited autophagy by increasing PI3K--Akt pathway expression. In addition, serum-free testosterone, inflammatory and apoptosis indexes were reduced after MT supplementation. Furthermore, we also found that MT suppressed autophagy and apoptosis by activating the PI3K-Akt pathway in the DHEA-exposed human granulosa cell line KGN. Our study showed that MT ameliorated ovarian dysfunction by regulating autophagy in DHEA-induced PCOS via the PI3K-Akt pathway, revealing a potential therapeutic drug target for PCOS.

ASMT Regulates Tumor Metastasis Through the Circadian Clock System in Triple-Negative Breast Cancer.

OBJECTIVE: Triple-negative (PR-, ER-, HER-2-) breast cancer (TNBC) is regarded as more aggressive and more likely to recur after medical care. Emerging evidence has demonstrated that the circadian clock system regulates cell-signaling pathways critical to cancer cell proliferation, survival and metastasis, meaning that it could be a good candidate for TNBC treatment. As such, the aim of the current study was to examine the molecular mechanism by which the circadian clock system contributes to cancer progression in TNBC. METHODS: Cancer cells and primary breast cancer tissues were immunostained for the measurement of circadian clock proteins (CLOCK, BMAL1 and PER1) and acetylserotonin methyltransferase (ASMT). The association between ASMT and clock proteins was assessed using siRNA and Western blot. Transwell assays were used to detect cancer cell migration and invasion while MTT assays were utilized to evaluate cell proliferation. RESULTS: Circadian clock proteins (CLOCK, BMAL1, and PER1) and ASMT expression were higher in TNBC and triple positive breast cancer (TPBC) compared with para-carcinoma tissues (PCTs). Intriguingly, there was an obvious correlation between circadian clock proteins and ASMT expression in both TPBC and TNBC. Similarly, circadian clock proteins and ASMT were expressed to a greater extent in BT-474 (triple-positive) cells than in MDA-MB-231 (triple-negative) cells. The inhibition of ASMT reduced circadian clock protein levels in both breast cancer cell lines. Further analysis showed that the expression levels of ASMT and circadian clock proteins did not correlate with clinical parameters such as age, tumor size, histologic grade and CK5/6, but increased significantly with lymphatic invasion in TNBC. In agreement with this finding, knockdown of ASMT significantly leads to reductions in migration and invasion in MDA-MB-231 cells. However, over-expression of CLOCK reversed the decreases seen in ASMT inhibited cells. CONCLUSION: Our study suggests that ASMT regulates the circadian clock system in breast cancer and inhibition of ASMT reduces the invasiveness of triple-negative breast cancer cells by downregulating clock protein in a certain extent, indicating the potential value of ASMT as a drug target for TNBC treatment.

Melatonin maintains mitochondrial membrane potential and decreases excessive intracellular Ca2+ levels in immature human oocytes.

AIMS: Previous reports have demonstrated that melatonin exists in multiple extrapineal sites, and higher amounts of melatonin are present in human follicular fluid than in serum, which indicates that it might play key roles in human oocyte maturation and subsequent embryonic development. Melatonin has been shown to be a potent antioxidant and might be beneficial to human oocytes during in vitro maturation (IVM). However, the underlying mechanisms of melatonin action during IVM have not been thoroughly investigated. MAIN METHODS: Immunofluorescence staining, western blotting, and ELISA were applied to investigate whether melatoninergic components are expressed in the cultured human ovarian cumulus cells. TMRE staining and Fluo-4 AM staining were performed to detect the mitochondrial membrane potential and intracellular Ca2+ levels of immature human oocytes respectively. KEY FINDINGS: First, cultured human ovary cumulus cells synthesized melatonin in vitro, and it expressed serotonin (the precursor of melatonin) and the two key enzymes, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Additionally, the results suggest that melatonin maintains the mitochondrial membrane potential and decrease excessive Ca2+ levels in immature human oocytes during IVM. SIGNIFICANCE: In conclusion, we provide evidence that the melatoninergic components were expressed in cultured human ovarian cumulus cells, and melatonin might reduce oxidative stress of human oocytes by ameliorating mitochondrial function. In view of the significant clinical value that immature human oocytes have in assisted reproductive technology (ART), our findings highlight a potential treatment strategy of using melatonin to improve mitochondrial function and to enhance the quality of human oocytes during IVM.

The BAF45D Protein Is Preferentially Expressed in Adult Neurogenic Zones and in Neurons and May Be Required for Retinoid Acid Induced PAX6 Expression.

Adult neurogenesis is important for the development of regenerative therapies for human diseases of the central nervous system (CNS) through the recruitment of adult neural stem cells (NSCs). NSCs are characterized by the capacity to generate neurons, astrocytes, and oligodendrocytes. To identify key factors involved in manipulating the adult NSC neurogenic fate thus has crucial implications for the clinical application. Here, we report that BAF45D is expressed in the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ) of the lateral ventricle, and the central canal (CC) of the adult spinal cord. Coexpression of BAF45D with glial fibrillary acidic protein (GFAP), a radial glial like cell marker protein, was identified in the SGZ, the SVZ and the adult spinal cord CC. Quantitative analysis data indicate that BAF45D is preferentially expressed in the neurogenic zone of the LV and the neurons of the adult CNS. Furthermore, during the neuroectoderm differentiation of H9 cells, BAF45D is required for the expression of PAX6, a neuroectoderm determinant that is also known to regulate the self-renewal and neuronal fate specification of adult neural stem/progenitor cells. Together, our results may shed new light on the expression of BAF45D in the adult neurogenic zones and the contribution of BAF45D to early neural development.