Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Non-esterified Fatty Acid-Induced Reactive Oxygen Species Mediated Granulosa Cells Apoptosis Is Regulated by Nrf2/p53 Signaling Pathway

Antioxidants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 523
Author(s):  
Yiru Wang ◽  
Chengmin Li ◽  
Julang Li ◽  
Genlin Wang ◽  
Lian Li
Keyword(s):  
Reactive Oxygen Species ◽  
Fatty Acid ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Reactive Oxygen ◽  
Follicular Development ◽  
Oxygen Species ◽  
Related Protein ◽  
Induced Apoptosis

Negative energy balance (NEB) during the perinatal period can affect dairy cow follicular development and reduce the fecundity. Non-esterified fatty acid (NEFA) concentration is elevated during NEB, and is known to be toxic for multiple cell types. In the ovary, NEB increased NEFA, and may influences follicular growth and development. However, the effect and mechanism of NEFA on granulosa cells (GCs) in vitro remains unknown. In this study, we found that NEFA dose-dependently induced apoptosis in primary cultured granulosa cells. Mechanistically, our data showed that NEFA significantly increased reactive oxygen species (ROS) levels, resulting in the activation of endoplasmic reticulum stress (ERS) and eventually cell apoptosis in GCs. Moreover, NEFA also increased the phosphorylation levels of ERK1/2 and p38MAPK pathways, upregulated the expression of p53 and potentially promoted its translocation to the nuclear, thus transcriptionally activated Bax, a downstream gene of this pathway. NEFA also promoted nuclear factor E2 (Nrf2) expression and its level in the nuclear. To elucidate the mechanism of NEFA action, N-acetyl-l-cysteine (NAC), a ROS scavenger was used to verify the role of ROS in NEFA induced apoptosis of GCs. NAC pretreatment reversed the NEFA-induced ERS-related protein and apoptosis-related protein levels. Meanwhile, NAC pretreatment also blocked the phosphorylation of ERK1/2 and p38 induced by NEFA, and the nucleation of Nrf2 and p53, suggesting that ROS plays a crucial role in regulating the NEFA-induced apoptosis of GCs. Together, these findings provide an improved understanding of the mechanisms underlying GCs apoptosis, which could potentially be useful for improving ovarian function.

scholarly journals rno-miR-128-3p promotes apoptosis in rat granulosa cells (GCs) induced by norepinephrine through Wilms tumor 1 (WT1)

2021 ◽  
Author(s):  
Ming Li ◽  
Ling Xue ◽  
Weibin Xu ◽  
Pingping Liu ◽  
Feng Li
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Wilms Tumor ◽  
Sympathetic Innervation ◽  
Follicular Development ◽  
Wilms Tumor 1 ◽  
Ovarian Follicular Development ◽  
Induced Apoptosis

AbstractThe mechanism related to ovarian follicular is complex, which has not been fully elucidated. Abundant reports have confirmed that the ovarian function development is closely related to sympathetic innervation. As one of the major neurotransmitters, norepinephrine (NE) is considered an effective regulator of ovarian functions like granulosa cell (GC) apoptosis. However, the mechanism between NE and GC apoptosis in rat is still unclear. In our study, GCs were isolated and cultured in vitro with NE treatment. The apoptosis of GCs was facilitated by NE. Wilms tumor 1 (WT1) was found to be significantly downregulated in GCs after NE treatment, and overexpression of WT1 repressed apoptosis in rat GCs induced by NE. rno-miR-128-3p was found to be significantly enhanced by NE in GCs, and inhibition of rno-miR-128-3p repressed apoptosis in rat GCs induced by NE. Mechanistically, rno-miR-128-3p interacted with WT1 and repressed its expression. In summary, inhibition of rno-miR-128-3p may enhance WT1 expression, and then repress NE-induced apoptosis in rat GCs. Our research may provide a new insight for the improvement of ovarian follicular development.

scholarly journals A rapid and robust method for the cryopreservation of human granulosa cells

2021 ◽  
Author(s):  
Sarah Beschta ◽  
Katja Eubler ◽  
Nancy Bohne ◽  
Ignasi Forne ◽  
Dieter Berg ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Large Scale ◽  
Ovarian Function ◽  
Oocyte Retrieval ◽  
Cell Contact ◽  
Cellular Model ◽  
Preovulatory Follicle ◽  
Human Granulosa Cells ◽  
Ovarian Cells

AbstractHuman primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

scholarly journals AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽  
2014 ◽  
Vol 147 (1) ◽  
pp. 73-80 ◽  
Author(s):  
JongYeob Choi ◽  
MinWha Jo ◽  
EunYoung Lee ◽  
DooSeok Choi
Keyword(s):  
Cell Death ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Apoptotic Cell ◽  
Follicular Development ◽  
Negative Regulator ◽  
Metabolic Clearance ◽  
Akt Inhibitor ◽  
Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

scholarly journals Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

2021 ◽  
Author(s):  
C. Chetan Kumar ◽  
B. Rambabu Naik ◽  
A.V.N. Siva Kumar ◽  
A. Ravi ◽  
L.S.S. Varaprasad Reddy ◽  
...  
Keyword(s):  
Growth Factors ◽  
Ovarian Function ◽  
Follicular Development ◽  
Oocyte Quality ◽  
Free Radical Scavenger ◽  
Radical Scavenger ◽  
Optimum Level ◽  
Preantral Follicles ◽  
Positive Effects

Background: Melatonin, a powerful free radical scavenger and broad-spectrum antioxidant may directly affect ovarian function by regulating folliculogenesis, maintenance of follicular integrity, oocyte quality and maturation capacity. Therefore, we aimed to study effects of melatonin and its interaction with growth factors in sheep preantral follicles. Methods: The influence of different concentrations of Melatonin (5-500 pM) on in vitro culture of preantral follicles (PFs’) isolated from sheep ovaries was studied. Experiments I and II were conducted to standardize the optimum concentration of Melatonin that supports better development of preantral follicles. Experiment III was conducted with the optimum level of Melatonin derived in the Experiments I and II to evaluate the effect of melatonin at 100pM in combination with various growth factors. Result: Overall follicular development was found to be the best in the PFs’ cultured in medium supplemented with 100pM of Melatonin. Melatonin supplementation showed positive effects on the preantral follicular development in combination with different growth factors.

scholarly journals Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†

2019 ◽  
Vol 102 (2) ◽  
pp. 511-520
Author(s):  
Yanrong Kuai ◽  
Xiaobo Gao ◽  
Huixia Yang ◽  
Haiyan Luo ◽  
Yang Xu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Crop Production ◽  
Follicular Development ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Serum Progesterone ◽  
Primordial Follicles ◽  
Progesterone Production

Abstract Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300 mg/kg by daily gavage for 7 days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

Relationships between concentrations of steroids, igf-1 and igf binding proteins during follicular development in the ewe

1995 ◽  
Vol 1995 ◽  
pp. 56-56
Author(s):  
M. Khalid ◽  
W. Haresign
Keyword(s):  
Follicular Fluid ◽  
Biochemical Markers ◽  
Binding Proteins ◽  
Ovarian Function ◽  
Follicular Development ◽  
Physiological Status ◽  
Igf Binding Proteins ◽  
Ovarian Cells ◽  
Igf Binding

Insulin-like growth factor-1 (IGF-1) is one of the potential autocrine/paracrine regulators of ovarian function. Not only do relationships exist between follicular fluid concentrations of IGF-1 and various biochemical markers of follicular differentiation, but IGF-1 has also been shown to stimulate both proliferation and steroidogenesis in ovarian cells in vitro (Adashi et al., 1985). The actions of IGF-1 are thought to be modulated by IGF-binding proteins (IGFBPs). Indeed, follicular growth and atresia in the ewe have been reported to be determined more by changes in IGFBPs than by changes in IGF-1 (Monget et al., 1993). However, in mat particular study, stage of follicular development was determined by follicle size and by microscopic examination of the granulosa cells of individual follicles rather than by biochemical markers of follicle status. The objective of the present study was, therefore, to investigate changes in IGF-1 and IGFBPs levels in follicular fluid and to relate these to the physiological status as determined by steroidogenic content of follicular fluid.

scholarly journals Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells

2002 ◽  
Vol 172 (1) ◽  
pp. 45-59 ◽  
Author(s):  
F Le Bellego ◽  
C Pisselet ◽  
C Huet ◽  
P Monget ◽  
D Monniaux
Keyword(s):  
Estrous Cycle ◽  
Granulosa Cells ◽  
Physiological Role ◽  
Follicular Development ◽  
Antral Follicles ◽  
Final Development ◽  
Beta 1 Integrin ◽  
Arginine Glycine Aspartic Acid

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.

scholarly journals Gonadotropin-Induced Expression of Messenger Ribonucleic Acid for Cyclooxygenase-2 and Production of Prostaglandins E and F2α in Bovine Preovulatory Follicles Are Regulated by the Progesterone Receptor

Endocrinology ◽  
2006 ◽  
Vol 147 (10) ◽  
pp. 4713-4722 ◽  
Author(s):  
P. J. Bridges ◽  
C. M. Komar ◽  
J. E. Fortune
Keyword(s):  
Granulosa Cells ◽  
Medroxyprogesterone Acetate ◽  
Cyclooxygenase 2 ◽  
Induced Expression ◽  
Messenger Ribonucleic Acid ◽  
Preovulatory Follicles ◽  
Gonadotropin Surge ◽  
Induced Changes

Follicular production of prostaglandins (PGs) is essential for ovulation, but the factors mediating gonadotropin-induced secretion of PGE and PGF2α remain largely unknown. We tested the hypothesis that gonadotropin-induced changes in progesterone and its receptor (PR) mediate the increase in periovulatory PGs. Heifers were treated with PGF2α and GnRH to induce luteolysis and the LH/FSH surge (ovulation occurs ∼30 h after GnRH). Because there are two increases in intrafollicular progesterone/PR mRNA during the bovine periovulatory period, we first examined the temporal pattern of PG production by follicles collected at 0, 3.5, 6, 12, 18, and 24 h after GnRH. Although PGs did not increase in the follicular fluid until 24 h after GnRH, acute secretion of PGs by follicle wall (theca + granulosa cells) was initiated by 18 h and had increased manyfold by 24 h after GnRH. In vitro, FSH and LH induced dramatic transient increases in PG production by follicle wall and granulosa, but not theca, cells isolated from preovulatory follicles (0 h after GnRH). PG accumulation peaked on d 2 of culture, mimicking the secretion pattern after a gonadotropin surge in vivo. In cultures of follicle wall and granulosa cells, the PR antagonist mifepristone (MIFE, 1 μm) inhibited LH-induced PG secretion and the progestin medroxyprogesterone acetate (1 or 10 μm), but not the glucocorticoid dexamethasone (1 or 10 μm), overcame the effect of MIFE on PGs. Semiquantitative RT-PCR revealed that MIFE inhibited LH-induced expression of cyclooxygenase-2 mRNA in granulosa cells in vitro. Again, treatment with medroxyprogesterone acetate overcame the effect of MIFE. Together these results provide strong evidence that periovulatory increases in cyclooxygenase-2 mRNA, PGE, and PGF2α are mediated by gonadotropin-induced increases in progesterone/PR, indicating that in some species there is an important functional relationship between these pathways in the ovulatory cascade.

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