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.

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.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

BACKGROUND: The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. METHOD AND RESULTS: The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. CONCLUSION: The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

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.

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.

Bone morphogenetic protein 6 induces downregulation of pentraxin 3 expression in human granulosa lutein cells in women with polycystic ovary syndrome.

PURPOSE: To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells. METHODS: The expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-ß type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells. RESULTS: The levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells. CONCLUSIONS: The suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.

Visfatin Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy.

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation.

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.

LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro.

STUDY QUESTION: Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER: A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT IS KNOWN ALREADY: Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION: hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE OF CHANCE: hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION: Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS OF THE FINDINGS: This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

TGF-ß1 upregulates secreted protein acidic and rich in cysteine expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a serious complication during in vitro fertilization (IVF) treatment. The upregulation of ovarian transforming growth factor-beta 1 (TGF-ß1) is involved in the development of OHSS. The secreted protein acidic and rich in cysteine (SPARC) is a secreted multifunctional matricellular glycoprotein. Although the regulatory effects of TGF-ß1 on SPARC expression have been reported, whether TGF-ß1 regulates SPARC expression in the human ovary remains unknown. In addition, the role of SPARC in the pathogenesis of OHSS is unclear. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing IVF treatment were used as experimental models. OHSS was induced in rats, and ovaries were collected. Follicular fluid samples were collected from 39 OHSS and 35 non-OHSS patients during oocyte retrieval. The underlying molecular mechanisms mediating the effect of TGF-ß1 on SPARC expression were explored by a series of in vitro experiments. RESULTS: TGF-ß1 upregulated SPARC expression in both KGN and hGL cells. The stimulatory effect of TGF-ß1 on SPARC expression was mediated by SMAD3 but not SMAD2. The transcription factors, Snail and Slug, were induced in response to the TGF-ß1 treatment. However, only Slug was required for the TGF-ß1-induced SPARC expression. Conversely, we found that the knockdown of SPARC decreased Slug expression. Our results also revealed that SPARC was upregulated in the OHSS rat ovaries and in the follicular fluid of OHSS patients. Knockdown of SPARC attenuated the TGF-ß1-stimulated expression of vascular endothelial growth factor (VEGF) and aromatase, two markers of OHSS. Moreover, the knockdown of SPARC reduced TGF-ß1 signaling by downregulating SMAD4 expression. CONCLUSIONS: By illustrating the potential physiological and pathological roles of TGF-ß1 in the regulation of SPARC in hGL cells, our results may serve to improve current strategies used to treat clinical infertility and OHSS. Video Abstract.

Androgen receptor gene deficiency results in the reduction of steroidogenic potential in porcine luteal cells.

Luteal steroidogenesis is critical to implantation and pregnancy maintenance in mammals. The role of androgen receptors (AR) in the progesterone (P4) producing luteal cells of porcine corpus luteum (CL) remains unexplored. The aim of the present study was to establish AR gene knock out (KO) porcine luteal cell culture system model by CRISPR/Cas9 genome editing technology and to study the downstream effects of AR gene deficiency on steroidogenic potential and viability of luteal cells. For this purpose, genomic cleavage detection assay, microscopy, RT-qPCR, ELISA, annexin, MTT, and viability assay complemented by bioinformatics analysis were employed. There was significant downregulation (p < 0.05) in the relative mRNA expression of steroidogenic marker genes STAR, CYP11A1, HSD3B1 in AR KO luteal cells as compared to the control group, which was further validated by the significant (p < 0.05) decrease in the P4 production. Significant decrease (p < 0.05) in relative viability on third passage were also observed. The relative mRNA expression of hypoxia related gene HIF1A was significantly (p < 0.05) downregulated in AR KO luteal cells. Protein-protein interaction analysis mapped AR to signaling pathways associated with luteal cell functionality. These findings suggests that AR gene functionality is critical to luteal cell steroidogenesis in porcine.

The interleukin-6 trans-signaling promotes progesterone production in human granulosa-lutein cells†.

As a critical paracrine regulator of multiple reproductive functions, the cytokine interleukin-6 (IL-6) is expressed in human granulosa cells and can be detected in follicular fluid. At present, the functional role of IL-6 in the regulation of ovarian steroidogenesis is controversial. Moreover, the detailed molecular mechanisms by which IL-6 regulates the production of progesterone in human granulosa cells remain to be elucidated. In the present study, we used primary and immortalized human granulosa-lutein (hGL) cells to investigate the effects of IL-6 on progesterone synthesis and the underlying molecular mechanisms. We found that IL-6 trans-signaling by the combined addition of IL-6 and soluble IL-6 receptor (sIL-6Rα)-induced steroidogenic acute regulatory expression and progesterone production in hGL cells. Additionally, IL-6/sIL-6Rα activated the phosphorylation of Janus activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), and the cellular effects were abolished by AG490 (JAK2 inhibitor), C188-9 (STAT3 inhibitor), or siRNA-mediated knockdown of STAT3. IL-6 trans-signaling-induced activation of JAK2/STAT3 also upregulated the expression of suppressor of cytokine signaling 3, which, in turn, negatively regulated the JAK2/STAT3 pathway by suppressing STAT3 activation and its downstream effects. Our findings provide insight into the molecular mechanisms by which IL-6 trans-signaling modulates steroidogenesis in hGL cells.

Expression of arylalkylamine n-acetyltransferase (AANAT) and acetylserotonin o-methyltransferase (ASMT) in the corpus luteum of pregnant sows and synthesis of melatonin in luteal cells.

In vertebrates, melatonin is mainly synthesized from serotonin in the pineal gland. Many reports have documented that melatonin is also synthesized in the extra-pineal tissues, but the synthesis of melatonin in the corpus luteum (CL) of pregnant sows has never been studied. The objectives of this study were to evaluate the expression of melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), in the CL of sows during pregnancy and to investigate the synthesis of melatonin in luteal cells. Results showed that AANAT and ASMT were both expressed in the CL of sows during pregnancy, higher levels were observed in the early- and mid-stage CL, and the lowest abundance was found in the regressing CL (later-stage). The immunostaining for AANAT and ASMT was predominantly localized in the large luteal cells of porcine CL during pregnancy. Furthermore, melatonin was synthesized in luteal cells from serotonin in a dose- and time-dependent manner. And the expressions of AANAT and ASMT were upregulated by serotonin in luteal cells. In addition, progesterone (P4) secretion and cell viability were promoted in luteal cells treated with serotonin, and the stimulatory effects were blocked by luzindole (a non-selective MT1 and MT2 antagonist). Finally, the expressions of MT1 and MT2 were augmented by serotonin in luteal cells. In conclusion, this study demonstrates for the first time the developmental expression of AANAT and ASMT in the CL and a local synthesis of melatonin in luteal cells of pregnant sows, and suggests a paracrine and/or autocrine role for melatonin in luteal function.

Growth differentiation factor-11 downregulates steroidogenic acute regulatory protein expression through ALK5-mediated SMAD3 signaling pathway in human granulosa-lutein cells.

BACKGROUND: Growth differentiation factor-11 (GDF-11) belongs to the transforming growth factor-ß (TGF-ß) superfamily. To date, the expression of GDF-11 in the ovary and its role in regulating ovarian function are completely unknown. Ovarian granulosa cell-mediated steroidogenesis plays a pivotal role in maintaining normal female reproductive function. GDF-11 and GDF-8 share high sequence similarity and exhibit many similar features and functions. Steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis and its expression can be downregulated by GDF-8. Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. The expression levels of GDF-8 are upregulated in the human follicular fluid and granulosa-lutein (hGL) cells of PCOS patients. However, whether similar results can be observed for the GDF-11 needs to be determined. METHODS: The effect of GDF-11 on StAR expression and the underlying molecular mechanisms were explored by a series of in vitro experiments in a primary culture of hGL cells obtained from patients undergoing in vitro fertilization (IVF) treatment. Human follicular fluid samples were obtained from 36 non-PCOS patients and 36 PCOS patients. GDF-11 levels in follicular fluid were measured by ELISA. RESULTS: GDF-11 downregulates StAR expression, whereas the expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) are not affected by GDF-11 in hGL cells. Using pharmacological inhibitors and a siRNA-mediated approach, we reveal that ALK5 but not ALK4 mediates the suppressive effect of GDF-11 on StAR expression. Although GDF-11 activates both SMAD2 and SMAD3 signaling pathways, only SMAD3 is involved in the GDF-11-induced downregulation of StAR expression. In addition, we show that SMAD1/5/8, ERK1/2, and PI3K/AKT signaling pathways are not activated by GDF-11 in hGL cells. RT-qPCR and ELISA detect GDF-11 mRNA expression in hGL cells and GDF-11 protein expression in human follicular fluid, respectively. Interestingly, unlike GDF-8, the expression levels of GDF-11 are not varied in hGL cells and follicular fluid between non-PCOS and PCOS patients. CONCLUSIONS: This study increases the understanding of the biological function of GDF-11 and provides important insights into the regulation of ovarian steroidogenesis.

Bone morphogenetic protein 2 upregulates SERPINE2 expression through noncanonical SMAD2/3 and p38 MAPK signaling pathways in human granulosa-lutein cells.

Serine protease inhibitor-E2 (SERPINE2) is highly expressed in the granulosa cells of growing follicles and the dynamic changes in SERPINE2 expression are correlated with follicular development and ovulation in several mammals, including mice, cattle, sheep, and humans. Bone morphogenetic proteins (BMPs) and their functional receptors are extensively expressed in the ovary and play critical roles in the regulation of ovarian folliculogenesis and luteal function. To date, whether BMPs regulate the expression of SERPINE2 during human follicular development remains to be elucidated. The aim of this study was to investigate the effects of BMPs on the regulation of SERPINE2 expression (a major regulator of plasminogen activators [PA]) and the underlying mechanisms using primary and immortalized human granulosa-lutein (hGL) cells. Our results demonstrated that these BMPs (BMP2, BMP4, BMP6, BMP7, and BMP15) induced differential upregulation of SERPINE2 expression. In this regard, BMP2 is the major modulator that has the best cellular activity, which further decreased the production of urokinase PA and tissue PA in hGL cells. In addition to canonical SMAD1/5/8 signaling, BMP2 also activates noncanonical SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) signaling. Using two inhibition approaches (kinase receptor inhibitors and siRNA-mediated knockdown), we found that SMAD2/3-SMAD4 and p38 MAPK, but not SMAD1/5/8 signaling, was involved in the BMP2-induced upregulation of SERPINE2 expression via activin receptor-like kinase 3. These findings deepen our understanding of the differential effect of BMPs in regulating follicular function and provide new insights of the molecular mechanisms by which BMP2 regulates the expression of SERPINE2 in human granulosa cells.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. RESULTS: Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. CONCLUSION: This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract.

MicroRNA let-7i inhibits granulosa-luteal cell proliferation and oestradiol biosynthesis by directly targeting IMP2.

RESEARCH QUESTION: Increased granulosa cell division is associated with abnormal folliculogenesis in polycystic ovary syndrome (PCOS). Lethal-7i microRNA (let-7i) may play an important role in the follicular development and granulosa cell growth; therefore is let-7i involved in PCOS pathogenesis? DESIGN: The expression of let-7i was measured in granulosa-luteal cells (GLC) from women with or without PCOS. A human granulosa cell line, KGN, was used for the functional study. Mimics and inhibitors of let-7i, lentiviruses expressing insulin-like growth factor 2 mRNA binding protein (IMP2), and small-interfering RNAs were transfected into KGN cells. KGN cell proliferation was determined by 5-ethynyl-2'-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays. The cell cycle and apoptosis were assessed by propidium iodide-annexin V (PI-A) staining and fluorescence-activated cell sorting. Oestradiol concentration was determined by enzyme-linked immunoassay. Bioinformatics analysis and luciferase reporter assay were applied to confirm the let-7i target genes. RESULTS: The study showed that let-7i was down-regulated in PCOS GLC (P = 0.001). Mimics of let-7i inhibited KGN proliferation (P = 0.001), and decreased aromatase expression (P = 0.030) and oestradiol production (P = 0.029), whereas let-7i inhibitors had the opposite effect. Bioinformatics analysis and quantitative real-time (qRT) PCR identified IMP2 as a target of let-7i (P = 0.021). qRT-PCR and western blot analysis indicated that IMP2 was up-regulated in GLC in women with PCOS (P = 0.001 and P = 0.044), and IMP2 expression was suppressed by let-7i in KGN cells (P < 0.001). Luciferase reporter assay results (P = 0.002), combined with the rescue assay, confirmed that let-7i inhibited KGN cell proliferation and reduced oestradiol concentration by directly targeting IMP2. CONCLUSIONS: let-7i was down-regulated in PCOS GLC. Overexpression of let-7i inhibited KGN cell proliferation and decreased oestradiol production in an IMP2-dependent manner, providing a new molecular mechanism for PCOS.

TGF-ß1 stimulates aromatase expression and estradiol production through SMAD2 and ERK1/2 signaling pathways in human granulosa-lutein cells.

Estradiol (E2), one of the main steroid hormones secreted by the ovaries, plays an important role in maintaining normal female reproductive function. Ovarian granulosa cells are the main source of E2 production because these cells express aromatase, which is encoded by the CYP19A1 gene and catalyzes the final step in E2 biosynthesis from androgens. Transforming growth factor-beta 1 (TGF-ß1) and its receptors are expressed in human granulosa cells, and TGF-ß1 expression can be detected in human follicular fluid. To date, TGF-ß1 has been shown to regulate various ovarian functions. However, whether aromatase can be regulated by TGF-ß1 in human granulosa cells has not been determined. In the present study, we demonstrate that TGF-ß1 stimulates aromatase expression in primary human granulosa-lutein cells and in the human ovarian granulose-like tumor cell line, KGN. We used pharmacological inhibitors and small interfering RNA-mediated knockdown approaches to reveal that the SMAD2 and ERK1/2 signaling pathways are involved in TGF-ß1-induced aromatase expression and E2 production. These results not only provide important insights into the molecular mechanisms that mediate TGF-ß1-induced aromatase expression and E2 production in human granulosa cells but also increase the understanding of the normal physiological roles of TGF-ß1 in the ovary.

Melatonergic systems of AANAT, melatonin, and its receptor MT2 in the corpus luteum are essential for reproductive success in mammals†.

Corpus luteum (CL) plays a critical role in mammalian reproductive physiology. Its dysfunction will lead to infertility or habitual abortion. In the current study, by use of melatonin specific membrane receptor 2 (MT2) knocking out (KO) mice model combined with RNA-Seq, immunohistochemistry, and immunofluorescence analyses, the genes of melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT) and MT2 were identified to strongly express in the CL of sows and mice. KO MT2 significantly impaired the reproductive performance in mice indicated by the reduced litter sizes. Melatonin treatment elevated the progesterone production in sows suggesting the improved CL function. Mechanistic analysis showed that melatonin upregulated a set of progesterone synthesis-related genes including cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), aldo-keto reductase family 1, member C18 (Akr1c18), isopentenyl-diphosphate delta isomerase 1 (Idi1), and luteinizing hormone/choriogonadotropin receptor (Lhcgr). The upregulation of these genes directly related to the increased progesterone production. The regulatory effects of melatonin on these gene expressions were mediated by MT2 and MT2KO diminished the effects of melatonin in this respect. Thus, the presence of melatonergic system of AANAT, melatonin, and its receptor MT2 in CL is essential for reproductive success in mammals.

Cloprostenol, a synthetic analog of prostaglandin F2α induces functional regression in cultured luteal cells of felids†.

In the present study, we investigated the effect of the synthetic analog of prostaglandin F2α (PGF2α)-cloprostenol-on cultured steroidogenic luteal cells of selected felid species over a 2-day culture period. The changes induced by cloprostenol were measured based on progesterone concentration and mRNA expression analysis of selected genes. Cloprostenol significantly reduced concentration of progesterone in cell culture medium of small luteal cells isolated from domestic cat corpora lutea (CL) at the development/maintenance stage (P < 0.05), but did not influence progesterone production in cultured cells from the regression stage. A decrease or complete silencing of progesterone production was also measured in cultured luteal cells of African lion (formation stage) and Javan leopard (development/maintenance stage). Gene-expression analysis by real-time PCR revealed that treatment with cloprostenol did not have an influence on expression of selected genes coding for enzymes of steroidogenesis (StAR, HSD3B, CYP11A1) or prostaglandin synthesis (PTGS2, PGES), nor did it effect hormone receptors (AR, ESR1, PGR, PTGER2), an anti-oxidative enzyme (SOD1) or factors of cell apoptosis (FAS, CASP3, TNFRSF1B, BCL2) over the studied period. Significant changes were measured only for expressions of luteinizing hormone (P < 0.05), prolactin (P < 0.05) and PGF2α receptors (P < 0.005) (LHCGR, PRLR, and PTGFR). The obtained results confirm that PGF2α/cloprostenol is a luteolytic agent in CL of felids and its impact on progesterone production depends on the developmental stage of the CL. Cloprostenol short-term treatment on luteal cells was associated only with functional but not structural changes related to luteal regression.