Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†

Abstract Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

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Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells

Reproduction ◽

10.1530/rep-15-0021 ◽

2015 ◽

Vol 150 (1) ◽

pp. 53-63 ◽

Cited By ~ 17

Author(s):

Mélodie Diot ◽

Maxime Reverchon ◽

Christelle Ramé ◽

Yannick Baumard ◽

Joëlle Dupont

Keyword(s):

Granulosa Cells ◽

Specific Inhibitor ◽

Follicular Development ◽

Rt Pcr ◽

Nicotinamide Phosphoribosyltransferase ◽

Theca Cells ◽

Protein Levels ◽

Progesterone Secretion ◽

Ovulatory Follicles

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

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Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry

Reproduction ◽

10.1530/rep.0.1230363 ◽

2002 ◽

pp. 363-369 ◽

Cited By ~ 83

Author(s):

CJ Souza ◽

BK Campbell ◽

AS McNeilly ◽

DT Baird

Keyword(s):

Granulosa Cells ◽

Polyclonal Antibodies ◽

Follicular Development ◽

Ovarian Surface Epithelium ◽

Bone Morphogenetic Protein 2 ◽

Antigen Retrieval ◽

Surface Epithelium ◽

Inhibin A ◽

Bmp Receptors

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.

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Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture

Reproduction ◽

10.1530/rep.1.00868 ◽

2006 ◽

Vol 131 (4) ◽

pp. 641-649 ◽

Cited By ~ 62

Author(s):

Inger B Carlsson ◽

Mika P E Laitinen ◽

Jennifer E Scott ◽

Henna Louhio ◽

Louiza Velentzis ◽

...

Keyword(s):

Granulosa Cells ◽

Developmental Stages ◽

Culture Media ◽

Follicular Development ◽

Ovarian Follicles ◽

Kit Ligand ◽

Kit Receptor ◽

Ovarian Follicular Development

The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit usingin situhybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian folliclesin vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development.

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Dynamic characteristics of lipid metabolism in cultured granulosa cells from geese follicles at different developmental stages

Bioscience Reports ◽

10.1042/bsr20192188 ◽

2019 ◽

Vol 39 (12) ◽

Cited By ~ 4

Author(s):

Shanyan Gao ◽

Xiang Gan ◽

Hua He ◽

Shenqiang Hu ◽

Yan Deng ◽

...

Keyword(s):

Lipid Metabolism ◽

Granulosa Cells ◽

Dynamic Characteristics ◽

Developmental Stages ◽

Lipid Synthesis ◽

Follicular Development ◽

Vital Role ◽

Intracellular Lipids ◽

Expression Of Genes

Abstract Previous studies have shown that lipid metabolism in granulosa cells (GCs) plays a vital role during mammalian ovarian follicular development. However, little research has been done on lipid metabolism in avian follicular GCs. The goal of the present study was to investigate the dynamic characteristics of lipid metabolism in GCs from geese pre-hierarchical (6–10 mm) and hierarchical (F4-F2 and F1) follicles during a 6-day period of in vitro culture. Oil red O staining showed that with the increasing incubation time, the amount of lipids accumulated in three cohorts of GCs increased gradually, reached the maxima after 96 h of culture, and then decreased. Moreover, the lipid content varied among these three cohorts, with the highest in F1 GCs. The qPCR results showed genes related to lipid synthesis and oxidation were highest expressed in pre-hierarchical GCs, while those related to lipid transport and deposition were highest expressed in hierarchical GCs. These results suggested that the amount of intracellular lipids in GCs increases with both the follicular diameter and culture time, which is accompanied by significant changes in expression of genes related to lipid metabolism. Therefore, it is postulated that the lipid accumulation capacity of geese GCs depends on the stage of follicle development and is finely regulated by the differential expression of genes related to lipid metabolism.

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Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽

10.1017/s0967199419000832 ◽

2020 ◽

Vol 28 (2) ◽

pp. 154-159

Author(s):

Juliana I. Candelaria ◽

Anna C. Denicol

Keyword(s):

Granulosa Cells ◽

Developmental Stages ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Cell Number ◽

Culture Conditions ◽

Preantral Follicles ◽

Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

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

Reproduction ◽

10.1530/rep-13-0386 ◽

2014 ◽

Vol 147 (1) ◽

pp. 73-80 ◽

Cited By ~ 43

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.

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

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0180027 ◽

1997 ◽

Vol 18 (1) ◽

pp. 27-35 ◽

Cited By ~ 2

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.

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FSH Promotes Rat Follicle Development Through Inhibition of the Hippo Signalling Pathway

10.21203/rs.3.rs-784763/v1 ◽

2021 ◽

Author(s):

Tairen Chen ◽

Mengjing Wu ◽

Yuting Dong ◽

Bin Kong ◽

Yufang Cai ◽

...

Keyword(s):

Western Blot ◽

In Vitro Culture ◽

Granulosa Cells ◽

Signalling Pathway ◽

Control Group ◽

Follicle Development ◽

Follicle Growth ◽

Expression Levels ◽

Hippo Signalling

Abstract Purpose: Whether FSH promotes follicle growth by inhibiting the Hippo signalling pathway.METHODS: Ovaries were cultured in vitro into a control group (no intervention), an FSH group (0.3 IU/mL FSH), and a VP group (10 µg/mL vetiporfin). HE staining and follicle counts were performed at each stage after 3 hours of in vitro culture. Immunohistochemistry was performed to study the expression levels of LATS2, YAP, PLATS2, and PYAP, and their expression levels in each group were also analysed by Western blot.The number of secondary follicles was significantly increased in the FSH group, the arrangement of granulosa cells was neater, the nuclear fixation was reduced, and the number of atretic follicles was decreased in the VP group. The number of secondary follicles was significantly increased, the number of atretic follicles was reduced, and granulosa cell nuclear consolidation was reduced in the VP+FSH group. Immunohistochemistry showed that LATS2 and YAP expression levels were significantly increased and PLATS2 and PYAP expression levels were relatively decreased in the FSH group, PYAP and PLATS2 expression levels were significantly increased and YAP expression was significantly decreased in the VP group, and YAP and LATS2 expression levels were significantly increased and PYAP and PLATS2 expression levels were significantly decreased in the VP+FSH group. By Western blot, LATS2 and YAP were elevated and PYAP and PLAT2 were decreased in the FSH group, LATS2 and YAP were decreased and PYAP and PLATS were significantly elevated in the VP group, and LATS2 and YAP were elevated and PYAP and PLATS2 were decreased in the VP+FSH group.CONCLUSION: FSH promotes follicle development by inhibiting the Hippo signalling pathway.

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

Biology of Reproduction ◽

10.1093/biolre/ioz195 ◽

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.

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

Journal of Endocrinology ◽

10.1677/joe.0.1720045 ◽

2002 ◽

Vol 172 (1) ◽

pp. 45-59 ◽

Cited By ~ 30

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.

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