Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

The corpus luteum (CL) is a transient ovarian endocrine structure that maintains pregnancy in primates during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL lifespan, growth, and differentiation are tightly regulated by survival and cell death signals through luteotrophic and luteolytic factors, including the epidermal growth factor (EGF)-like factor family. Neuregulin 1 (NRG1), a member of the EGF family, mediates its effect through ErbB2/3 receptors. However, the functional role of NRG1 in luteal cells (LCs) is unknown. Thus, this study investigated the role of NRG1 and its molecular mechanism of action in rat LC. Our experimental results suggest a strong positive correlation between steroidogenic acute regulatory protein (StAR) and NRG1 expression in mid-CL and serum P4 and estrogen (E2) production. In contrast, there was a decrease in StAR and NRG1 expression and P4 and E2 production with an increase in tumor necrosis factor α (TNFα) expression in regressing CL. Further in vitro studies in LCs showed that the knockdown of endogenous Nrg1 promoted the expression of proinflammatory and proapoptotic factors and decreased prosurvival factor expression. Subsequently, treatment with exogenous TNFα under these experimental conditions profoundly elevated proinflammatory and proapoptotic factors. Further analysis demonstrated that the phosphorylation status of ErbB2/3, PI3K, Ak strain transforming or protein kinase B (Akt), and ErK1/2 was significantly inhibited under these experimental conditions, whereas the treatment of TNFα further inhibited the phosphorylation of ErbB2/3, PI3K, Akt, and ErK1/2. Collectively, these studies provide new insights into the NRG1-mediated immunomodulatory and prosurvival role in LCs, which may maintain the function of CL.

Cordycepin suppresses steroidogenic acute regulatory protein expression by reducing SP1 in human granulosa-lutein cells.

In brief: Cordycepin (COR), a compound derived from Cordyceps, is recognized as an adenosine analog with numerous beneficial effects on human health. However, its impact on steroidogenic acute regulatory protein (STAR) expression in ovarian granulosa cells is not well understood. This study demonstrates that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. Abstract: Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders.

The effects of the activation of TLR2/TLR1 on in vitro angiogenesis in an immortalized ovine luteal endothelial cell line.

In brief: Activation of TLR2/TLR1 alters in vitro formation of capillary-like structures and induces inflammatory processes in ovine luteal endothelial (OLENDO) cells. Abstract: Postpartum bacterial infections of the uterus affect uterine physiology and ovarian activity, causing fertility problems. The outer membrane component of Gram-negative bacteria, lipopolysaccharide, is involved in the initiation of the local inflammatory processes, and other bacterial toxins, particularly lipopeptides, have also been shown to be potent cytokine inducers, acting via Toll-like receptor-2 (TLR2). However, the possible adverse effects of TLR2 on ovarian and luteal activities have not yet been investigated in depth. The strong expression of TLR2 in the blood vessels of the corpus luteum led us to hypothesize that TLR2 activation might participate in the disruption of luteal vascular functionality. Therefore, we analyzed the effects of Pam3CSK4 (Pam3CysSerLys4), a synthetic triacylated lipopeptide and TLR2/TLR1 ligand, on the functionality of gap junctional intercellular communication (GJIC), endothelial cell invasion, and in vitro capillary-like network formation in an immortalized ovine luteal endothelial (OLENDO) cell line. Pam3CSK4 treatment of OLENDO cells disrupted in vitro tube formation but had no effect on GJIC or migration of OLENDO cells. Furthermore, Pam3CSK4 induced the expression of NFKB, IL6, and IL8 in OLENDO cells. Additionally, the basal availability of TLRs (TLR1-10) and TLR co-receptors (MYD88, LY96/MD2, and CD14) in OLENDO cells was confirmed by conventional PCR. Finally, the activation of TLR2/TLR1 appears to alter in vitro formation of capillary-like structures and induce inflammatory processes in OLENDO cells.

Visfatin impact on the proteome of porcine luteal cells during implantation.

Visfatin (VIS) is a hormone belonging to the adipokines' group secreted mainly by the adipose tissue. VIS plays a crucial role in the control of energy homeostasis, inflammation, cell differentiation, and angiogenesis. VIS expression was confirmed in the hypothalamic-pituitary-gonadal (HPG) axis structures, as well as in the uterus, placenta, and conceptuses. We hypothesised that VIS may affect the abundance of proteins involved in the regulation of key processes occurring in the corpus luteum (CL) during the implantation process in pigs. In the present study, we performed the high-throughput proteomic analysis (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the in vitro influence of VIS (100 ng/mL) on differentially regulated proteins (DRPs) in the porcine luteal cells (LCs) on days 15-16 of pregnancy (implantation period). We have identified 511 DRPs, 276 of them were up-regulated, and 235 down-regulated in the presence of VIS. Revealed DRPs were assigned to 162 gene ontology terms. Western blot analysis of five chosen DRPs, ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), lanosterol 14-α demethylase (CYP51A1), inhibin subunit beta A (INHBA), notch receptor 3 (NOTCH3), and prostaglandin E synthase 2 (mPGES2) confirmed the veracity and accuracy of LC-MS/MS method. We indicated that VIS modulates the expression of proteins connected with the regulation of lipogenesis and cholesterologenesis, and, in consequence, may be involved in the synthesis of steroid hormones, as well as prostaglandins' metabolism. Moreover, we revealed that VIS affects the abundance of protein associated with ovarian cell proliferation, differentiation, and apoptosis, as well as CL new vessel formation and tissue remodelling. Our results suggest important roles for VIS in the regulation of ovarian functions during the peri-implantation period.

NR4A1-mediated regulation of lipid droplets in progesterone synthesis in goat luteal cells†.

Nuclear receptor NR4A1 is a key factor in glycolipid metabolism and steroidogenesis, while lipid droplets serve as crucial dynamic organelles for lipid metabolism in luteal cells. To investigate the effects of NR4A1 on lipid droplet metabolism and progesterone (P4) synthesis in goat corpus luteum in vitro, luteal cells from the middle-cyclic corpus luteum were isolated and treated with Cytosporone B (CSNB, an agonist) or siRNA of NR4A1. Results showed that both low (1 µM) and high (50 µM) concentrations of CSNB promoted lipid droplet accumulation, while NR4A1 knockdown reduced lipid droplet content. CSNB increased while siNR4A1 decreased total cholesterol content; however, CSNB and siNR4A1 did not change triglyceride content. CSNB increased the expression of perilipins at mRNA and protein levels, also increased LDLR, SCARB1, SREBFs, and HMGCR mRNA abundance. Treatment with siNR4A1 revealed opposite results of CSNB, except for HMCGR and SREBF2. For steroidogenesis, 1 µM CSNB increased, but 50 µM CSNB inhibited P4 synthesis, NR4A1 knockdown also reduced the P4 level. Further analysis demonstrated that 1 µM CSNB increased the protein levels of StAR, HSD3B, and P-HSL, while 50 µM CSNB decreased StAR, HSD3B, and CYP11A1 protein levels. Moreover, 50 µM CSNB impaired active mitochondria, reduced the BCL2, and increased DRP1, Caspase 3, and cleaved-Caspase 3 protein levels. siNR4A1 consistently downregulated the P-HSL/HSL ratio and the steroidogenic protein levels. In conclusion, NR4A1-mediated lipid droplets are involved in the regulation of progesterone synthesis in goat luteal cells.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

The in vitro mechanism of Vaspinregulates the proliferation and steroidogenesis of rat lutein cells.

OBJECTIVE: Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism. METHODS: Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway. RESULTS: Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels. CONCLUSION: Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Molecular characterization and expression profile of the ALDH1A1 gene and its functions in yak luteal cells.

The ALDH1A1 gene encodes a cytoplasmic member of the aldehyde dehydrogenase 1 family, which plays an important role in regulating animal reproductive performance, including estrus cycle and embryonic development. The aim of this study was to characterize ALDH1A1 activity in ovaries of 3-5 year-old yaks and to determine its effects on cell proliferation, apoptosis, and progesterone secretion in luteal cells (LCs). The coding sequence (CDS) of the ALDH1A1 gene was cloned by reverse transcription-PCR and immunohistochemical analysis was used to confirm localization of the ALDH1A1 protein in the ovary. To assess the activity of ALDH1A1 in regulating progesterone secretion, si-ALDH1A1 was transfected into LCs in vitro and progesterone levels in LC supernatants were measured by ELISA. The interference efficiency was assessed by real-time quantitative PCR (RT-qPCR) and immunofluorescence staining, and cell proliferation and apoptosis were evaluated by EdU and TUNEL staining, respectively. The cloned ALDH1A1 sequence contained 1462 bp, encoding 487 amino acids. Immunohistochemical analysis showed that ALDH1A1 protein expression, which was significantly higher in LCs, was mainly found in antral follicles and the corpus luteum (CL). The expression of ALDH1A1 mRNA in LCs was effectively inhibited by si-ALDH1A1transfection, and progesterone secretion was markedly decreased along with the significant down-regulation of progesterone pathway-related genes, STAR, CYP11A1, CYP19A1, CYP17A1, 3ß-HSD, and HSD17B1. Knockdown of ALDH1A1 mRNA expression decreased cell proliferation and increased apoptosis in LCs. The mRNA expression of the proliferation-related genes, PCNA, CCND1, CCNB1 and CDC25A, was significantly down-regulated, while expression of the apoptosis-promoting CASP3 gene was significantly increased. In summary, we characterized the yak ALDH1A1 gene and revealed that ALDH1A1 knockdown promoted apoptosis, repressed cell proliferation, and decreased progesterone secretion by yak LCs, potentially by regulating the mRNA expression of genes related to proliferation, apoptosis, and progesterone synthesis and secretion.

Interleukin-1 increases SERPINE1 expression in human granulosa-lutein cell via P50/P52 signaling pathways.

It has been reported that immune factors are associated with the occurrence of polycystic ovary syndrome (PCOS). Interleukin-1 (IL-1) is a member of the interleukin family that widely participates in the regulation of the inflammatory response in the immune system. In addition, it has been reported that aberrant IL-1 accumulation in serum is associated with the occurrence of PCOS. However, little is known about how IL-1 participates in the pathogenesis of PCOS. In the present study, we demonstrated that the immune microenvironment was altered in follicular fluid from PCOS patients and that the expression levels of two IL-1 cytokines, IL-1α and IL-1ß were increased. Transcriptome analysis revealed that IL-1α and IL-1ß treatment induced primary human granulosa-lutein (hGL) cell inflammatory response and increased the expression of serpin family E member 1 (SERPINE1). Mechanistically, we demonstrated that IL-1α and IL-1ß upregulated SERPINE1 expression through IL-1R1-mediated activation of downstream P50 and P52 signaling pathways in human granulosa cells. Our study highlighted the role of immune state changes in the occurrence of PCOS and provided new insight into the treatment of patients with IL-1-induced ovarian function disorders.

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

PURPOSE: To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. METHODS: hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2 mU/L). Cells were cultured at 37 °C in DMEM, supplemented with 5% FBS. The cells were treated with 1 nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. RESULTS: The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. CONCLUSIONS: Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000 nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells.

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-ß) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

Transcriptomic profile of GLCs of PCOS women highlights metabolic dysregulation as a plausible contributor to PCOS pathophysiology.

Polycystic ovary syndrome (PCOS) is a complex heterogeneous disorder with reproductive and metabolic consequences whose aetiology is still elusive. To understand the cellular mechanisms that potentially govern follicular defect in women with PCOS, we performed transcriptomic profiles of granulosa-lutein cells (GLCs) by RNA-Seq analysis. We found differential expression of 876 genes in GLCs between PCOS and controls that belonged to various processes such as cell cycle, extracellular matrix organization, angiogenesis, oxidative stress, metabolism, etc. that support folliculogenesis, oocyte development, and maturation. The cross-talk between oocyte and GLCs is a fundamental cornerstone in determining oocyte quality and highly interlinked pathways of metabolism and redox homeostasis may influence this. We found several genes involved in the metabolism of carbohydrates, nucleotides, cholesterol, and lipids were dysregulated, which may impair the supply of metabolites to the growing oocyte, affecting oocyte development and competence. Additionally, high metabolic activity during folliculogenesis may augment oxidative damage to cells and macromolecules if not counter-balanced. We observed dysregulation of redox homeostasis and AGE-RAGE signalling in the follicular environment. Among the validated genes, prokineticin-1 and growth differentiation factor-15 were found to be negatively regulated, while, S100, calcium-binding protein A9 and angiomotin-like-2 were positively regulated in GLCs of women with PCOS. Comparing our data with previously published relevant transcriptomic studies showed metabolic, cytokine-cytokine receptor interaction, IL-17, and chemokine signalling pathways were most commonly affected in PCOS. Overall, this data can provide insights into mechanisms contributing to PCOS pathophysiology and can be explored as potential indicators for oocyte/embryo quality in IVF settings.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells.

BACKGROUND: The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined. METHODS: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. RESULTS: The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells. CONCLUSIONS: BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

miR-665 overexpression inhibits the apoptosis of luteal cells in small ruminants suppressing HPGDS.

Evidence has shown that microRNA-665 (miR-665) is highly expressed in the mid-luteal phase compared with the early and end-luteal phase of the corpus luteum (CL) life cycle. However, whether miR-665 is a positive regulator of the life span of the CL is still unknown. The objective of this study is to explore the effect of miR-665 on the structural luteolysis in the ovarian CL. In this study, the targeting relationship between miR-665 and hematopoietic prostaglandin synthase (HPGDS) was firstly verified by dual luciferase reporter assay. Then, quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-665 and HPGDS in luteal cells. Following miR-665 overexpression, the apoptosis rate of the luteal cells was determined using flow cytometry; B-cell lymphoma-2 (BCL-2) and caspase-3 mRNA and protein were measured using qRT-PCR and Western blot (WB) analysis. Finally, the DP1 and CRTH2 receptors of PGD2, a synthetic product of HPGDS, were localized using immunofluorescence. Results confirmed that HPGDS was a direct target gene of miR-665, and miR-665 expression was negatively correlated with HPGDS mRNA expression in luteal cells. Meanwhile, after miR-665 was overexpressed, the apoptotic rate of the luteal cells showed a significant decrease (P < 0.05) and this was accompanied by elevated expression levels of anti-apoptotic factor BCL-2 mRNA and protein and decreased expression levels of apoptotic factor caspase-3 mRNA and protein (P < 0.01). Moreover, the immune fluorescence staining results showed that the DP1 receptor was also significantly decreased (P < 0.05), but the CRTH2 receptor was significantly increased (P < 0.05) in luteal cells. Overall, these results indicate that miR-665 reduces the apoptosis of luteal cells via inhibiting caspase-3 expression and promoting BCL-2 expression, and the biological function of miR-665 may be attributed to its target gene HPGDS which regulates the balance of DP1 and CRTH2 receptors expression in luteal cells. As a consequence, this study suggests that miR-665 might be a positive regulator of the life span of the CL rather than destroy the integrity of CL in small ruminants.

Resveratrol Attenuates Hydrogen Peroxide-induced Injury of Rat Ovarian Granulosa-lutein Cells by Resisting Oxidative Stress via the SIRT1/Nrf2/ARE Signaling Pathway.

INTRODUCTION: This paper aims to reveal the molecular mechanism of resveratrol against oxidative stress and cell injury. The ovarian granulosa-lutein cell injury and apoptosis induced by oxidative stress may be responsible for female luteal phase deficiency. The antioxidant function of resveratrol has been confirmed; however, its effect on the expression of antioxidant enzymes and regulatory mechanisms in ovarian granulosa-lutein cells remains unclear. OBJECTIVE: This study aimed to investigate the role of the SIRT1/Nrf2/ARE signaling pathway in the effect of resveratrol on the hydrogen peroxide-induced injury of rat ovarian granulosa-lutein cells. METHODS: In this study, ovarian granulosa-lutein cells extracted from 3-week female SD rats were treated with 200 µM H2O2 in the presence or absence of 20 µM resveratrol. siRNA-SIRT1 and siRNA-Nrf2 were used to inhibit the expression of SIRT1 and Nrf2, respectively. Cell counting kit 8 (CCK-8), cellular morphology, progesterone secretion, and estradiol were used to evaluate cell injury. Hoechst 33258 staining was used to measure cell apoptosis. DHE staining, DCFH-DA staining, malondialdehyde content, protein carbonyl content, total antioxidant capacity and SOD viability were used to estimate the levels of oxidative stress. Western blot analysis was used to detect the levels of apoptosis-related proteins, and SIRT1/Nrf2/ARE signaling pathway-related proteins. RESULTS: The H2O2 treatment-induced rat ovarian granulosa-lutein cells injury was shown as decreased cell viability, impaired cellular morphology, and decreased levels of progesterone and estradiol. The H2O2 treatment also exacerbated cell apoptosis demonstrated as more apoptotic cells stained by Hoechst staining, decreased level of anti-apoptosis protein Bcl-2 and increased level of pro-apoptosis protein Bax. These effects of cell injury and apoptosis induced by H2O2 can be ameliorated by resveratrol. Resveratrol also alleviated oxidative stress induced by H2O2, supported by decreased superoxide anion and cellular total ROS, decreased malondialdehyde and protein carbonyl levels, and increased total antioxidant capacity and SOD viability. Western blot results demonstrated resveratrol reversed the H2O2-induced decrease in levels of antioxidant enzymes containing ARE sequences and activated SIRT1/Nrf2 pathway. Further treatment by siRNA-Nrf2 suggested resveratrol could not activate the expression of antioxidant enzymes under a condition of inhibition of Nrf2. CONCLUSION: This study demonstrates that resveratrol attenuated oxidative stress to protect H2O2-induced rat ovarian granulosa-lutein cell injury and apoptosis via SIRT1/Nrf2/ARE signaling pathway.

Prokineticin 1 is a novel factor regulating porcine corpus luteum function.

Prokineticin 1 (PROK1) is a pleiotropic factor secreted by endocrine glands; however, its role has not been studied in the corpus luteum (CL) during pregnancy in any species. The present study aimed to investigate the contribution of PROK1 in regulating processes related to porcine CL function and regression: steroidogenesis, luteal cell apoptosis and viability, and angiogenesis. The luteal expression of PROK1 was greater on Days 12 and 14 of pregnancy compared to Day 9. PROK1 protein expression during pregnancy increased gradually and peaked on Day 14, when it was also significantly higher than that on Day 14 of the estrous cycle. Prokineticin receptor 1 (PROKR1) mRNA abundance increased on Days 12 and 14 of pregnancy, whereas PROKR2 elevated on Day 14 of the estrous cycle. PROK1, acting via PROKR1, stimulated the expression of genes involved in progesterone synthesis, as well as progesterone secretion by luteal tissue. PROK1-PROKR1 signaling reduced apoptosis and increased the viability of luteal cells. PROK1 acting through PROKR1 stimulated angiogenesis by increasing capillary-like structure formation by luteal endothelial cells and elevating angiogenin gene expression and VEGFA secretion by luteal tissue. Our results indicate that PROK1 regulates processes vital for maintaining luteal function during early pregnancy and the mid-luteal phase.

Divergent roles of sirtuin 1 in human granulosa-lutein cells: similarities to human chorionic gonadotropin.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that modifies gene expression through histone deacetylation. It also deacetylates nonhistone substrates, e.g., tumor suppressor p53, NOS3, HIF1A, NFKB, FOXO3a, PGC-1α, and PPARγ. Consequently, it regulates a wide range of physiological functions including cell cycle control, energy expenditure, oxidative stress response, apoptosis, and aging. SIRT1 is expressed in ovarian granulosa cells (GCs) of various species including humans at different stages of the reproductive cycle. The importance of SIRT1 in female reproduction is supported by the findings that SIRT1-knockout mice exhibit defects in reproductive tissue development. These mice were found to have a thin-walled uterus, small ovaries, with follicles present but no corpora lutea. This review aims to provide state-of-the-art information on SIRT1's mode of action and its roles in human granulosa-lutein cells and GCs from other species where data are available. It also discusses the overlapping actions of SIRT1 and human chorionic gonadotropin on the production of critical GC-borne factors.

Interleukin-1 increases cyclooxygenase-2 expression and prostaglandin E2 production in human granulosa-lutein cell via nuclear factor kappa B/P65 and extracellular signal-regulated kinase 1/2 signaling pathways.

A multitude of cytokines have been reported to participate in the folliculogenesis process in female. Interleukin-1 (IL-1), belonging to interleukin family, is originally identified as an important immune factor involved in inflammation response. Besides the immunity system, IL-1 is also expressed in reproductive system. However, the role of IL-1 in regulating ovarian follicle function remains to be elucidated. In the current study, using the primary human granulosa-lutein (hGL) and immortalized human granulosa-like tumor cell line (KGN) models, we demonstrated that both IL-1α and IL-1ß increased prostaglandin E2 (PGE2) production via upregulating its cyclooxygenase (COX) enzyme COX-2 expression in human granulosa cells. Mechanistically, IL-1α and IL-1ß treatment activated nuclear factor kappa B (NF-κB) signaling pathway. Using the specific siRNA to knock down endogenous gene expression, we found that the inhibition of p65 expression abolished IL-1α and IL-1ß-induced upregulation of COX-2 expression whereas knockdown of p50 and p52 had no effect. Moreover, our results also showed that IL-1α and IL-1ß promoted the nuclear translocation of p65. ChIP assay demonstrated the transcriptional regulation of p65 on COX-2 expression. Additionally, we also found that IL-1α and IL-1ß could activate the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The inhibition of ERK1/2 signaling pathway activation reversed IL-1α and IL-1ß-induced upregulation of COX-2 expression. Our findings shed light on the cellular and molecular mechanisms by which IL-1 modulates the COX-2 expression through NF-κB/P65 and ERK1/2 signaling pathways in human granulosa cells.