Granulosa Lutein Research Articles

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.

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

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

PurposeTo 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.MethodsThe 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-β 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.ResultsThe 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.ConclusionsThe 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.

Insights into dysregulation of luteinizing hormone receptor (LHR) signaling in granulosa lutein (GL) cells from women with polycystic ovary syndrome (PCOS)

Insights into dysregulation of luteinizing hormone receptor (LHR) signaling in granulosa lutein (GL) cells from women with polycystic ovary syndrome (PCOS)

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH

To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

TGF-β1 suppresses de novo cholesterol biosynthesis in granulosa-lutein cells by down-regulating DHCR24 expression via the GSK-3β/EZH2/H3K27me3 signaling pathway

Cholesterol is a precursor to steroid hormones and can be obtained from serum LDL or de novo synthesis in steroidogenic cells. Before luteinizing hormone (LH) surge-induced ovulation, follicles remain avascular, and cholesterol required for progesterone production in granulosa cells (GCs) is derived from de novo biosynthesis. Previous studies have verified that the intrafollicular TGF-β1 plays inhibitory roles in GCs luteinization, vascularization, and progesterone production. Nevertheless, the regulatory function of TGF-β1 on de novo cholesterol synthesis in granulosa-lutein (GL) cells remains largely unknown. We aim to investigate this aspect in this study using in vivo cultured human GL cells. Our results suggested that TGF-β1 significantly suppresses intracellular cholesterol levels and down-regulates the expression of the final step enzyme, DHCR24, that catalyzes de novo cholesterol synthesis. We used specific inhibitors and siRNA-mediated knockdown approaches demonstrate that TGF-β1 suppression of DHCR24 expression in GL cells is mediated by the GSK-3β/EZH2/H3K27me3 signaling pathway. Further ChIP assays revealed that elevated H3K27me3 levels in the promoter region of DHCR24 play a vital role in TGF-β1-induced DHCR24 down-regulation, and RNA-sequencing results confirmed these findings. Notably, our study provides a novel insight into the molecular mechanisms by which TGF-β1 suppresses de novo cholesterol biosynthesis in GL cells.

P-207 Molecular mechanisms of melatonin action in human reproductive system – implications for assisted reproduction

Abstract Study question What is the potential role of melatonin and melatonin receptors activation on the reproductive function in humans? Summary answer The presence of membrane melatonin receptors in male gametes and female hGLCs suggests a role for melatonin on motility performance of mature spermatozoa and oogenesis. What is known already The interest in the influence of the external environment on the reproductive performance in humans facilitates reevaluation of its main mediators in the body. One of the most important among them is melatonin circadian secretion by the pineal gland, a major synchronizer of the diurnal rest-activity cycle. Activation of melatonin-linked GPCR receptors by melatonin is coupled to inhibition of cAMP accumulation in target cells, which makes research on membrane receptor-mediated signaling in human reproductive system very attractive. Study design, size, duration Pilot study on the presence of MT1 and MT2 receptors in human spermatozoa and human granulosa luteinized cells (hGLCs) and the presence and localization of α- and ß-tubulin, melatonin receptors (MT1, MT2) and P450 aromatase in hGLCs. Participants/materials, setting, methods Human donor samples (spermatozoa and granulosa lutein cells (hGLC) were collected after informed consent signed from patients recruited at IVF clinics in Sofia. Two-step quantitative real time RT-PCR (qRT-PCR) was used to compare individual variations between donors. Selected proteins in samples and derived cell cultures like α- and ß-tubulin, melatonin receptors (MT1, MT2) and P450 aromatase were studied by Western blotting and localization visualized by confocal immune fluorescence. Main results and the role of chance PCR amplification of cDNA in the samples resulted in products of the expected size of 516 bp corresponding to the MTNR1A gene. In purified spermatozoa, intense expression of a MT1 receptor was found in the post-acrosomal region and the neck. In hGLC cultures up-regulation of melatonin receptors after in-vitro melatonin treatment was observed both by protein expression and densitometry of immune fuorescence. Our results demonstrate the presence in situ of membrane melatonin receptor in competent human male gametes and female hGLCs. The observed localization of the receptor protein suggests a role for melatonin on motility performance of mature spermatozoa, which may have several important implications to assisted reproductive techniques. Limitations, reasons for caution The discrete functional responses and the transduction pathways linked to specific receptor activation by melatonin in human reproductive system have further to be elucidated. Wider implications of the findings Future studies could reveal the potential therapeutic role of Melatonin in infertility patients. Trial registration number Not applicable

In vitro effects of dihydrotestosterone (DHT) on gonadotropin receptor function and steroidogenesis in human granulosa lutein cells

<i>In vitro</i> effects of dihydrotestosterone (DHT) on gonadotropin receptor function and steroidogenesis in human granulosa lutein cells

THE POSSIBLE EFFECT OF FLAXSEED EXTRACT ON LETROZOLE-INDUCED POLYCYSTIC OVARY RAT MODEL: CORRELATIVE HISTOLOGICAL AND FUNCTIONAL STUDY

Background: Polycystic Ovary Syndrome (PCOS) is one of the most common metabolic disorders affecting women in the reproductive age leading to infertility/subfertility. It may occur as a complication of Letrozole (LTZ) therapy in breast cancer patients. Objective: This was a preliminary study to assess the possible protective and/or curative effect of flaxseed extract in a letrozole-induced PCO rat model. Material and Methods: Thirty-six adult female albino rats were divided into five main groups: Group I kept as the control group, Group II (Letrozole-induced PCO group), received letrozole solution (3 mg/kg) orally once daily by gastric tube for 21 days. Group III (combined flaxseed and letrozole group), received aqueous flaxseed extract (500 mg /kg/day) orally one hour before letrozole in the same dose and duration as group II, Group IV (recovery group), received letrozole in the same dose and duration as group II and then left without treatment for the next 4 weeks after PCO induction, Group V (flaxseed-treated group), treated with aqueous flaxseed extract orally once daily for the next 4 weeks after PCO induction. At the end of the experiment, final body weight, ovarian weight, blood glucose, serum antioxidants assay, and sex hormones levels were measured. Also, specimens of ovaries were processed for light and electron microscopic studies. Histomorphometric and statistical analyses were done. Results: letrozole-induced PCO resulted in a significant increase in final body weight, ovarian weight as well as abnormal blood glucose, sex steroids hormone levels, and antioxidant assays. Moreover, numerous ovarian cystic follicles, widespread cytoplasmic vacuolation in the granulosa lutein cells, congested blood capillaries, and increased fibrosis within the ovarian medulla were evident in the PCO group in concomitant with a significant increase in area percentage of collagen fibers. The ultrastructural assessment confirmed these distortions. The protective and curative potential of flaxseed is mostly comparable revealed improvement in the final body weight, biochemical markers and histological changes, with the protective effect was more pronounced than the curative one. Conclusion and Recommendation: Flaxseed succeeded in ameliorating letrozole-induced polycystic ovary owing to its phytochemicals, antioxidant and anti-inflammatory properties with folliculogenesis potentials. So, it is recommended to use flaxseed as a co-therapy with letrozole to reduce its hyperandrogenic effects.

The interleukin 6 trans-signaling increases prostaglandin E2 production in human granulosa cells†.

As a potent autocrine regulator, the proinflammatory cytokine interleukin 6 (IL6) is expressed in granulosa cells and is involved in the modulation of various follicular functions, including follicular development and ovulation. At present, the detailed molecular mechanisms by which IL6 regulates the event of ovulation remain to be elucidated. In the present study, primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used to investigate the effects of IL6 on the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and the subsequent synthesis of prostaglandin E2 (PGE2) and to investigate the underlying molecular mechanisms. We found that instead of classic signaling, IL6/soluble form of the IL6 receptor (sIL-6Ralpha) trans-signaling induced the expression of PTGS2 and production of PGE2 in both SVOG cells and primary hGL cells. Moreover, IL6/sIL-6Ralpha activated the phosphorylation of Janus-activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), which in turn induced STAT3 nuclear translocation. In addition, these effects were suppressed by the addition of inhibitors (AG490 for JAK2 and C188-9 for STAT3) and by the small interfering RNA-mediated knockdown of STAT3. In addition, suppressor of cytokine signaling 3 (SOCS3) acts as a negative-feedback regulator in IL6/sIL-6Ralpha-induced cellular activities, including the activation and nuclear translocation of STAT3, upregulation of PTGS2 expression, and increase in PGE2 production in SVOG cells. In conclusion, IL6 trans-signaling upregulates the expression of PTGS2 and increases the production of PGE2 via the JAK2/STAT3/SOCS3 signaling pathway in hGL cells. Our findings provide insights into the molecular mechanisms by which IL6 trans-signaling may potentially modulate the event of ovulation in human ovaries.

Quantification of hormone membrane receptor FSHR, GPER and LHCGR transcripts in human primary granulosa lutein cells by real-time quantitative PCR and digital droplet PCR

BackgroundQuantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology. ObjectivesHerein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes (FSHR, GPER and LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction. MethodsTotal RNA was extracted from human primary granulosa lutein cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature. Immunostaining for protein localization in cell membranes was also performed. ResultsBoth techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 40- to 260-fold lower amount than LHCGR transcripts. qPCR and ddPCR data are convergent with immunofluorescence analysis of membrane receptor expression in human primary granulosa lutein cells. ConclusionThese results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.

HISTOLOGY AND HISTOMORPHOMETRY OF GAYO MARE OVARIES

This study aims to determine the histology, the number and diameter of follicles, and the corpus albican of the Gayo mare ovary. The micro technical process was applied to 3 pairs of ovarian samples for further hematoxylin-eosin (HE) staining. Observation of ovarian structure was carried out microscopically and the data were analyzed statistically. The results showed that the Gayo mare ovary consists of the medulla on the outside and the cortex on the inside. Medulla consists of small follicles, blood vessels, nerves, and connective tissue. Meanwhile, the cortex consists of de Graff's follicle, corpus luteum, corpus albican, and ovulatory fossa. Follicles are composed of oocytes, granulosa cells, internal and external theca cells, oophorous cumulus, follicular antrum, and follicular fluid. Atretic follicles contain lutein cells that have been damaged. The corpus luteum is composed of granulosa lutein tissue, internal theca and external. The corpus albican is composed of scar tissue and lutein cells. The measurement results showed that the primordial follicle diameter was 26.60±2.37 µm, primary follicle 54.33±6.70 µm, secondary follicle 119.32±25.55 µm, tertiary follicle 250.86±49.46 µm, atretic follicle 49.03±45.47 µm, the corpus albican 511.10±132.41 µm. Thus, it can be concluded that the Gayo mare ovary has a histological structure that is not different from the ovary of other mares. Follicular growth occurs in the medulla of the ovary, and de Graff's follicles are present in the cortex of the ovaries.

Sphingosine-1 phosphate induces cAMP/PKA-independent phosphorylation of the cAMP response element-binding protein (CREB) in granulosa cells

Background and aimsSphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. ConclusionsThis study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

Twohuman menopausal gonadotrophin (hMG) preparations display different early signaling in vitro.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

Effect of Social Isolation on Rat Ovary and the Possible Protective Role of Melatonin: Light, Electron Microscopic, Biochemical and Molecular Study

Abstract Background: Social Isolation (SI) is an influential stressor in animals and humans, which has a negative impact on mental and physical health. It is associated with increased oxidative stress and decreased antioxidant capacity. Melatonin (MT) is well known by its antioxidant properties that help in prevention of tissue damage. Aim of Study: The present study aimed to investigate the harmful effect of SI on rat ovary and the possible protective role of MT. Material and Methods: Twenty adult female albino rats were divided into four groups; five rats in each (control, MT, SI and SI + MT). Histological examination was done using hematoxylin and eosin, semithin sections were stained with toluidine blue and ultrathin sections were performed. Meas-urements of Malondialdehyde (MDA) and Superoxide Dis-mutase (SOD) activity in ovarian tissue were carried out. Quantitative analysis of SIRT1 gene expression was done by real-time Polymerase Chain Reaction (PCR). Morphometric measurements were also performed. Results: Light microscopic examination of ovarian follicles from SI group showed shrunken granulosa cells with pyknotic nuclei and irregular oocytes with rarified cytoplasm. Cyto-plasmic vacuolation was observed in granulosa cells, theca cells and granulosa lutein cells. Ultrastructural examination of granulocsa cells showed swollen mitochondria and abundant lipid droplets. The oocytes exhibited disrupted microvilli. There was significant decrease in the mean follicular diameter, granulosa and theca layer thickness in SI group compared to control group. These morphological changes were associated with significant increase in MDA and decrease in SOD activity, in addition to increased SIRT1 gene expression. Concomitant administration of MT improved the previously mentioned alterations. Conclusion: Social isolation induced histological, ul-trastructural and morphometric changes in the ovary of rats. These changes were associated with oxidative stress, decreased antioxidant capacity and increased SIRT1 gene expression. Melatonin administration ameliorated these harmful effects, possibly due to its antioxidant property.

ALK4-SMAD3/4 mediates the effects of activin A on the upregulation of PAI-1 in human granulosa lutein cells

In the mammalian ovary, the proteolysis of the extracellular matrix is dynamically regulated by plasminogen activator and plasminogen activator inhibitor (PAI), and it is a critical event that influences various physiological and pathological processes. Activin A is a member of the transforming growth factor-β superfamily and is expressed at a high level in human luteal cells that play an essential role in the regulation of the luteal function. At present, it is not known whether activin A can regulate the expression and production of PAI in human granulosa lutein (hGL) cells. The present study aimed to examine the effects of activin A on the expression and production of intraovarian PAI-1 and the underlying molecular mechanisms. Using primary and immortalized hGL cells as the cell model, we demonstrated that activin A upregulated the expression of PAI-1 and increased the production of PAI-1 in an autocrine/paracrine manner. Additionally, using a dual inhibition approach (molecular inhibitors and siRNA-mediated knockdown), we showed that this biological function is mediated by the ALK4-mediated SMAD3-SMAD4-dependent signaling pathway. Our findings suggest that activin A may be involved in the regulation of luteal function via the induction of PAI-1 expression and an increase in PAI-1 production.

The expression of angiotensin II receptors mRNA in granulosa-lutein cells in endometriosis patients who underwent ovarian surgery before in vitro fertilization

Angiogenesis is important to folliculogenesis and oocyte maturation. Vascular endothelial growth factor (VEGF) in follicular fluid (FF) induces folliculogenesis. Angiotensin II is involved in angiogenesis through angiotensin II receptors. To elucidate the mechanisms of oocyte maturation disorders in endometriotic patients, the authors investigated the concentration of VEGF, angiotensin II, and soluble receptor of VEGF (sFlt-1) in FF and the expression of angiotensin II receptors mRNA in granulosa-lutein (GL) cells in endometriosis patients with controlled ovarian stimulation. FF samples were obtained from 56 IVF cycles (control = 47, endometriosis = 9). GL cells were obtained from 15 IVF cycles (control = 10, endometriosis = 5). The authors examined the concentration of VEGF, sFlt-1, and angiotensin II in FF by ELISA, and the expression of angiotensin II receptors mRNA by quantitative RT-PCR. Informed consents were obtained from all patients and IRB approval was obtained. The expression levels of angiotensin II type 1 and type 2 receptor mRNA in endometriosis was significantly lower than the control. The number of immature oocytes was significantly higher in endometriosis than in control. Lower expression of angiotensin II receptors may affect folliculogenesis and oocyte maturation.

Differential expression in granulosa-lutein (GL) cells from polycystic ovaries of genes implicated in assembly and modification of extracellular matrix (ECM)

Differential expression in granulosa-lutein (GL) cells from polycystic ovaries of genes implicated in assembly and modification of extracellular matrix (ECM)