Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation

in Journal of Molecular Endocrinology
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  • 1 Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, Georgia, USA
  • | 2 Reproductive Science Research Program, Morehouse School of Medicine, Atlanta, Georgia, USA
  • | 3 Department of Neurobiology, Morehouse School of Medicine, Atlanta, Georgia, USA
  • | 4 Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  • | 5 Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia, USA

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Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

Abstract

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

Introduction

Mammalian granulosa cells (GCs) are multilayered somatic cells residing within the ovarian follicle that support oogenesis through proliferation, differentiation, and interactive communications. These events are under the control of endocrine factors, including gonadotrophins (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) (Havelock et al. 2004). During these processes, GCs produce and secrete multiple autocrine and paracrine factors, and steroid hormones that play important roles as regulators of oogenesis and folliculogenesis. The coordinated biosynthesis of steroids in the ovary is critical for progression of the reproductive cycle, successful ovulation, and eventually pregnancy (Jamnongjit & Hammes 2006). Gonadotrophin-induced synthesis and secretions of steroids, progesterone (P4) and estradiol (E2), involve multiple steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (cyp19a1) (Miller & Bose 2011). All steroidogenic pathways begin in the mitochondria, where regulation of the cellular steroidogenic capacity occurs through key enzymatic rate-limiting steps (notably p450scc and StAR) (Duarte et al. 2014, Prasad et al. 2015). Both p450scc and StAR are localized in a complex multicomponent ‘transduceosome’ on the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) (Duarte et al. 2014, Prasad et al. 2015).

Prohibitin (PHB) is a member of the highly conserved ubiquitous protein family and plays a pleiotropic role in cell cycle control, differentiation, and senescence, in addition to having antiproliferative and anti-apoptotic roles (Chowdhury et al. 2016). A growing body of experimental evidence implicates PHB in the maintenance of integrity of mitochondrial structure, functions, inheritance, and cellular homeostasis (Thompson et al. 1999, 2001, 2003, Sutovsky et al. 2000, Kawashima et al. 2006, 2008, Artal-Sanz & Tavernarakis 2009, Chowdhury et al. 2016). Recently, PHB has been identified as a substrate for Ras-Raf and Akt, and is essential for activation of the Ras-Raf-MEK1-ERK1/2 signaling pathway (Rajalingam et al. 2005, Han et al. 2008). Previous studies have shown that PHB is widely expressed in rat ovary and its expression is regulated by age and follicular stage (Thompson et al. 1999, 2004). Moreover, recent studies in pre-antral GCs isolated from diethylstilbestrol (DES)-treated rats and antral GCs isolated from equine chorionic gonadotrophin (eCG)-treated rats have shown that PHB is regulated by FSH in a follicular stage-dependent manner in vitro and that the role PHB plays in regulating steroidogenesis is dependent on the differentiation status of GCs (Wang et al. 2013a,b). However, the specific roles and contribution of mitochondrial PHB and phospho-PHB (pPHB) in mediating differentiation of normal undifferentiated GCs (mainly steroidogenesis) is not known.

The experimental studies presented here were designed to decipher the physiological effects of knocking down endogenous PHB on steroid (E2 and P4) synthesis, secretions, and the activation of MEK1-ERK1/2 pathway in FSH-induced rat ovarian undifferentiated GC model. Immunoblot studies were carried out under these experimental conditions to analyze the expression levels of selective steroidogenic enzymes including StAR, p450scc, 3β-HSD, and Cyp19a1, which are commonly regulated in the steroidogenesis pathway at the protein level. Parallel studies were carried out to examine expression of the acidic (phosphorylated) isoform of PHB, phosphorylated-PHB (pPHB), and to decipher its relationship with activation of the MEK1-ERK1/2 pathway in FSH-treated GCs. In addition, we analyzed the role of PHB in FSH-induced mitochondrial morphological changes using assays that examined changes in the mitochondrial fusion protein optic atrophy 1 (OPA1). OPA1 is a nuclear-encoded large dynamin-like GTPase that is found in the mitochondrial intermembrane space and regulates both mitochondrial fusion and cristae morphogenesis during steroidogenesis (Wasilewski et al. 2012).

Materials and methods

Animals

Sprague–Dawley (SD) rats (female, 21 days old) were purchased from Charles River Laboratories. Animals were given food and water ad libitum, and kept under a regular day/night (12h light:12h darkness) cycle. All animal care handling procedures in this study were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health (NIH) and the US Department of Agriculture, and approved by the Morehouse School of Medicine Animal Care and Use Committee.

Primary GC cultures

Primary GC cultures were isolated from immature (23–25 days old) SD rat ovaries as described previously (Chowdhury et al. 2007). GCs from sexually immature 23- to 25-day-old rats are referred to as undifferentiated because they lack the presence of functional LH receptor and do not produce E2 or P4 under basal conditions, and these GCs have not been exposed to pubertal cyclic gonadotrophins. However, these cells respond to FSH with respect to the production of cAMP and induction of LH receptor activation of the E2 and P4 biosynthetic pathways (Bebia et al. 2001).

Adenoviral infection of GCs and treatments

Undifferentiated GCs were grown on a 6-well culture dish (~2×106cells/well; 2 ovaries/rat/plate) in M199 media supplemented with 10% fetal bovine serum (FBS). Subsequently, the medium was removed and cells were washed twice with M199 (antibiotics-free) and infected with or without adenoviral (Ad) vectors (Ad-eGFP-scrambled: adenovirus with scrambled sequence RNA with green fluorescent protein (GFP); Ad-eGFP-shPhb: adenovirus with siRNA designed for knockdown of PHB with GFP) at a multiplicity of infection (MOI) of 5, 10, and 20 plaque-forming units per cell (pfu/cell) with occasional rocking as described previously by Chowdhury et al. (2007, 2011, 2013). After 2h of incubation, the media was replaced with fresh M199 media without FBS and incubated for 24h. Infected GCs showed 95–100% GFP expressions. Twenty-four hours after exposure to adenoviruses, the media was replaced with fresh M199 media without FBS with testosterone (30ng/mL) in presence or absence of FSH (100ng/mL) for 48h.

For inhibitor studies, after 24h culture of GCs, the media was replaced with fresh M199 media without FBS in the presence or absence of MEK inhibitor (PD98059; PD 20μM), and DMSO as parallel control for 1h. Followed by MEK inhibitor pretreated GCs were treated with testosterone in presence or absence of FSH for 6h, since half-life of the MEK inhibitor is less than 8h. When reagents were dissolved in DMSO, the same concentrations of DMSO were added to the control cells medium. The final concentration of DMSO was less than 0.1%. The dose and time for inhibitor studies were based on Supplementary Table 1 (see section on supplementary data given at the end of this article) and previous publication (Chowdhury et al. 2013).

After completion of each experimental group, total proteins were isolated for further analysis by Western blot analysis as described below.

Assessment of survival state of GCs after completion of treatments

A detailed analysis of the morphological changes occurring in GCs after 48h treatment with testosterone or FSH+testosterone was carried out using live cell photographs taken under an inverted epifluorescence microscope to image the green fluorescence signals for the eGFP-shPhb or the control eGFP-scrambled construct alone. Phase-contrast pictures were taken to assess the survival status. The percentage of apoptosis (Hoechst 33248 staining) and caspase-3 activity was determined as described previously (Chowdhury et al. 2007, 2011, 2013).

Assessment of ultrastructural changes in GCs using transmission electron microscopy

Transmission electron microscopy (TEM) for GCs was carried out as described by Thompson et al. (1997). Mitochondrial and lipid droplets cross-sectional areas were determined using ImageJ software (NIH, Bethesda, MD, USA).

Immunofluoresence microscopy

Immunofluorescence microscopy for GCs was carried out by methods previously described by Chowdhury et al. (2007).

Isolation of S-100 fraction and mitochondria

S-100 (cytosolic) fractions and mitochondria were prepared as described previously (Chowdhury et al. 2007, 2011). Protein levels of cellular fractions were analyzed by Western blot analysis.

Generation of PHB anti-phospho-Tyr249 and anti-phospho-Thr258/Tyr259 antibodies

PHB contains multiple residues that could serve as phosphorylation sites in FSH stimulation of GCs (Rajalingam et al. 2005, Han et al. 2008, Ande et al. 2009, Chiu et al. 2013). It has been reported that insulin phosphorylates PHB at Y114 and Y259 (Ande et al. 2009), whereas Akt phosphorylates PHB at T258 (Han et al. 2008). In addition, PHB phosphorylation sites also occur within the Raf1 binding domain of PHB (residues 243–272). The MEK1-ERK1/2-signaling pathway is known to be involved in GC survival (Moore et al. 2001, Dewi et al. 2002, Amsterdam et al. 2003, Su et al. 2006, Woods & Johnson 2007, Duarte et al. 2014, Chowdhury et al. 2016). Thus, based on our unpublished phosphosite bioinformatic studies using complete PHB amino acid sequences, we developed our own custom-made pPHB antibodies. The anti-phospho-Tyr249 (anti-pTyr249) and anti-phospho-Thr258/Tyr259 (anti-pThr258/Tyr259) rabbit phospho-specific antibody against PHB peptide Ac-EDIA[pY]QLSRSRN-amide phosphorylated at tyrosine 249 residue and Ac-LSRSRNI[pT][pY]LPAGQS-amide phosphorylated at threonine 258 and tyrosine 259 residues were custom prepared and affinity purified by Thermo Fisher Scientific. In brief, two rabbits were immunized with the PHB phosphopeptide. The terminal bleeds were adsorbed over a nonphosphopeptide column and then subjected to two successive purifications over a phosphopeptide column. ELISA analysis showed a greater than 1000-fold preference for phosphorylated peptide over nonphosphopeptide. The specificity of pPHB antibodies were analyzed by Thermo Fisher Scientific.

Phosphatase treatment

Phosphatase treatment was carried out as described by Pon et al. (1986) and Thompson et al. (1997).

Western blot analysis

GC protein extracts obtained from different treatment conditions were subjected to 1D or 2D gel electrophoresis and Western blot analysis, as described previously (Thompson et al. 2001, Chowdhury et al. 2007, 2011, 2013; given in Table 1). For 1D gel electrophoresis, equal amounts of protein (25μg) were applied to each lane. For 2D gel electrophoresis, 80μg of protein purified from mitochondrial fractions isolated from cultured GCs after treatment were focused in the first dimension immobilized pH gradient (IPG) 3–10 or 4–7 strips for 60kV-h using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blot analysis. PHB antibody was used to detect protein spots corresponding to 30kDa marker.

Table 1

List of antibodies used for Western blot analysis.

Primary antibodyOriginDilution used
Prohibitin (PHB)Rabbit polyclonal, Neomarks, Fremont, CA, USA1:1000
Steroidogenic acute regulatory protein (StAR) Rabbit polyclonal, Santa Cruz Biotechnology1:1000
p450 cholesterol side-chain cleavage enzyme (P450scc) Rabbit polyclonal, Santa Cruz Biotechnology1:1000
Hydroxysteroid dehydrogenase-3β (3B-HSD)Rabbit polyclonal, Santa Cruz Biotechnology1:1000
Aromatase (cyp19a1)Rabbit polyclonal, Abcam1:1000
Total ERK1/2 and pERK1/2Rabbit polyclonal, Cell Signaling1:1000
Anti-OPA1Rabbit polyclonal, Abcam1:1000
PorinRabbit polyclonal, Cell Signaling1:1000
TubulinMouse monoclonal, Sigma-Aldrich1:1000

Quantification of the PHB phosphorylation and steroidogenic protein ratios

Quantitative analysis of the PHB phosphorylation and steroidogenic protein expression was carried out using a scanning densitometer and Multianalyst Software version 1.0.2 (Bio-Rad) as described previously (Chowdhury et al. 2013).

Steroids secretion analysis

Spent medium from GC cultures was collected, centrifuged (900g, 5min) and kept at −80°C for hormone analysis. 17β-estradiol (E2) and progesterone (P4) concentrations in spent medium were measured using RIA (El-Hefnawy & Zeleznik 2001, Zeleznik et al. 2003, Parakh et al. 2006, Escamilla-Hernandez et al. 2008). The detection limitation of E2 was 0.05pg/mL, and the intra-assay and inter-assay coefficients of variation were 6 and 4%, respectively. The detection limitation of P4 was 0.31pg/mL, and the intra-assay and inter-assay coefficients of variation were 5 and 4%, respectively.

Statistical analysis

All experimental data are expressed as mean±s.e.m. of three separate experiments, each carried out in replicate. Statistical analysis was carried out by one-way ANOVA using SPSS version 11.0. Multiple comparisons were done by Newman–Keuls test. Differences were considered to be significant at P≤0.05.

Results

Ultrastructural changes in GCs in response to FSH and testosterone

To delineate the role of PHB in GCs differentiation, we first examined the impact of gonadotrophin-induced steroidogenesis by using our established rat primary GC culture system in vitro (Supplementary Fig. 1).

TEM was used to analyze the effects of testosterone, FSH, or testosterone+FSH on mediating ultrastructural changes in undifferentiated and differentiated GCs. In this ultrastructure analysis, particular attention was paid to the oil droplet numbers and mitochondrial morphology of GCs under the respective culture condition (control, testosterone, FSH, and testosterone+FSH; Fig. 1A (parts a–d), B, and C). The cytoplasmic matrix of GCs cultured in control medium contained many ribosomes, no lipid droplets, few mitochondria with transversely oriented lamellar cristae, and rough endoplasmic reticulum (ER) (Fig. 1A (part a)). In GCs cultured with either testosterone or FSH alone, many cisternae of the rough ER and smooth ER with large number of elongated mitochondria were observed with few lipid droplets (Fig. 1A (b and c)). By contrast, GCs cultured with testosterone+FSH had many cisternae of the rough and smooth ER. It also had larger elongated mitochondria closely associated with some cisternae of the smooth ER with dense lipid droplets present (Fig. 1A (part d)) compared with GCs cultured with testosterone alone or FSH alone.

Figure 1
Figure 1

Ultrastructural changes in rat GC in response to FSH and testosterone. GCs were treated with or without FSH (100 ng/mL) and testosterone (30 ng/mL) or with FSH+testosterone for 48 h. Parallel control (C) GCs were maintained without any treatments. After completing the various treatments, GCs were fixed, mounted, and micrographs were taken with a TEM. (A) represents electron micrographs as control (a), testosterone (b), FSH (c), and testosterone+FSH (d). N, nucleus; M, mitochondria; GC, Golgi complex; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; C, centriole; LD, lipid droplets. (B and C) are the cross-sectional areas of mitochondria and lipid droplets, respectively represented as percentage fold change (arbitrary unit). The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All groups are significantly different (P≤0.05).

Citation: Journal of Molecular Endocrinology 56, 4; 10.1530/JME-15-0278

Based on results from Fig. 1 and Supplementary Fig. 1, testosterone with or without FSH was used in all subsequent studies that were carried out for the induction of steroidogenesis in GCs, where testosterone served as a substrate of aromatase (cyp19a1) as well as a paracrine regulator of FSH action (Hsueh et al. 1983, Thompson et al. 1997, El-Hefnawy & Zeleznik 2001, Zeleznik et al. 2003, Parakh et al. 2006, Wayne et al. 2007, Escamilla-Hernandez et al. 2008). In addition, in our subsequent studies, GCs were lysed and fractionated into mitochondrial and cytosolic fractions, and equal amounts of protein extracts were analyzed by 2D gel electrophoresis and Western blot analysis.

Phb-knockdown inhibits steroidogenesis in gonadotrophin-induced GCs

As PHB is an important survival factor, knockdown of Phb sensitized the GCs (Chowdhury et al. 2016). Therefore, before delineating the effects of FSH in Phb-knockdown GCs, survival studies were carried out under these experimental conditions. The Supplementary Fig. 2 suggests that lower levels of knockdown of Phb do not affect the survivability of GCs. However, higher levels of knockdown of Phb in GCs tilt the balance of survivability toward cell death in a time-dependent manner.

To delineate the gonadotrophin effects on differentiation state after Phb knockdown in the GCs, we first examined whether infection with empty vector (Ad-scrambled) alone showed any changes in testosterone+FSH-induced P4 and E2 secretions in GCs. FSH+testosterone significantly stimulated P4 and E2 secretion (P<0.05; Newman–Keuls test) in Ad-scrambled-infected GCs (Fig. 2A). Similar results were observed in parallel controls without infected GCs treated with testosterone+FSH. Western blot analyses show (Fig. 2B and C) that testosterone+FSH significantly stimulated the expression of total PHB and key steroidogenic enzymes (StAR, p450scc, 3β-HSD, and aromatase (cyp19a1)) in Ad-scrambled infected and parallel controls (without infected) GCs treated with testosterone+FSH (P<0.05; Newman–Keuls test). These studies suggest that the Ad-scrambled construct has no adverse effect on the expression of PHB and steroidogenic enzymes, and P4 and E2 synthesis and secretions.

Figure 2
Figure 2

PHB knockdown inhibits steroidogenesis in gonadotrophin-induced rat GCs. GCs were transiently infected with Ad-shPhb (MOI 5, 10, and 20) or Ad-scrambled (MOI 5, 10, and 20) for 2 h and maintained in culture for 24 h in serum-free media and treated with testosterone (30 ng/mL) in presence or absence of FSH (100 ng/mL) for 48 h in serum-free media. (A) Bar diagram represents estradiol (E2) and progesterone (P4) secretions into the medium as measured by RIA. (B) Representative Western blot analyses of protein expression levels of PHB, StAR, p450scc, 3β-HSD, and aromatase in GCs induced by FSH under PHB knockdown experimental conditions. Equal amounts of protein (25 μg) were applied to each lane. Western blot analyses were analyzed for PHB, StAR, p450scc, 3β-HSD, and aromatase. Tubulin was used as an internal control for cytosol. (C) The bar graphs represent the relative percentage change in expression of PHB, StAR, p450scc, 3β-HSD, and aromatase as a ratio of the protein levels normalized by tubulin. The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All are significantly different (P≤0.05) except with same alphabets (a, b, c, and d). C, parallel control group.

Citation: Journal of Molecular Endocrinology 56, 4; 10.1530/JME-15-0278

We then examined the effects of testosterone+FSH on secretions of P4 and E2 and key steroidogenic enzymes expression in the Phb-knockdown GCs. A significant dose-dependent knockdown of Phb expression occurred in Ad-shPhb-infected GCs treated with or without testosterone+FSH (P<0.05; Newman–Keuls test) (Fig. 2B). Interestingly, under dose-dependent knockdown of Phb, GCs when treated with testosterone+FSH showed a significant dose-dependent decrease in P4 and E2 secretions (Fig. 2A) with suppression of the key steroidogenic enzymes (StAR, p450scc, 3β-HSD, and aromatase) content (Fig. 2B and C) (P<0.05; Newman–Keuls test).

Gonadotrophin stimulates phosphorylation of mitochondrial PHB in GCs

Co-localization studies shown in Supplementary Fig. 1D suggest that FSH+testosterone-dependent upregulated PHB is mainly localized to mitochondria, and confirmed that the mitochondria is the key organelle for steroidogenesis (Poderoso et al. 2008, Miller & Bose 2011, Wasilewski et al. 2012, Duarte et al. 2014, Prasad et al. 2015). Moreover, previous studies have shown that PHB is a substrate for MEK-ERK pathway (Rajalingam et al. 2005, Han et al. 2008, Chowdhury et al. 2013, 2016). Therefore, we designed further studies that aimed at (i) determining whether mitochondrial PHB is phosphorylated (pPHB) during either FSH or testosterone or testosterone+FSH treatments and (ii) determining whether MEK-ERK signaling pathway was involved in FSH-induced mitochondrial PHB expression in GCs. After completion of each treatment, mitochondrial protein fractions were isolated and 2D Western blot analysis was carried out to analyze for PHB expression. As shown in Fig. 3A, PHB within the mitochondrial fraction in the controls (without treatment) and testosterone-treated GCs both have single spots, whereas a shift in the mobility of the PHB protein was observed toward the acidic region in 2D Western blot analysis with two distinct spots seen in the FSH and testosterone+FSH-treated groups. The level of PHB increased when GCs were cultured in the presence of FSH alone and FSH+testosterone, and the GCs were actively producing P4 and E2 (Fig. 2A). These results suggest that under these experimental conditions, the PHB protein has an acidic isoform that is most likely due to phosphorylation. To further determine whether these results are correct, mitochondrial pPHB in testosterone+FSH-treated GCs protein fractions were purified from isolated mitochondria and incubated with alkaline phosphatase. This phosphatase treatment resulted in disappearance of the acidic mitochondrial isoform appearing at isoelectric point 5.6 (Fig. 3B). These experimental results suggest that PHB within the mitochondrial fraction is phosphorylated with stimulation of FSH+testosterone.

Figure 3
Figure 3

Gonadotrophin stimulates phosphorylation of mitochondrial PHB in GCs. (A) GCs were treated with or without FSH (100 ng/mL) and testosterone (30 ng/mL) or in combination (FSH+testosterone) for 48 h. Parallel controls (C) of GCs were maintained without any treatments. After 48 h, 80 μg of protein purified from the respective mitochondrial fractions were focused in the first dimension on IPG 4–7 strips for 60 kV-h using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blot analysis. PHB antibody was used to detect protein spots corresponding to PHB. (B) Alkaline phosphatase-treated GCs. GCs were cultured in the presence of FSH+testosterone for 48 h. Mitochondrial fractions were collected and treated with and without alkaline phosphatase followed by separation on 2D gel electrophoresis, and PHB spots were identified by Western blot analysis. (C) Representative Western blot analyses of mitochondrial protein levels PHB, pPHB (T258/259 and Y249), and OPA1 in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial fractions were isolated, and pPHB and OPA1 were analyzed by Western blot analyses. Equal amounts of protein (25μg) were applied to each lane, and Western blot analyses were analyzed for PHB, pPHB, and OPA1. Porin was used as an internal control for mitochondria. (D) The bar graphs represent the relative percentage change in expression levels of PHB, pPHB, and OPA1 as a ratio of the protein levels normalized by porin from three independent experiments as mean±s.e.m. All are significantly different (P≤0.05) except with same alphabets (a, b, c, d, e, and f). (E) The bar graphs represent the relative percentage change in expression of mitochondrial PHB/porin and pERK/ERK in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial protein was isolated and analyzed by Western blot analyses for PHB, pERK, and ERK. Porin was used as an internal control for mitochondria. The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All are significantly different (P≤0.05) except with same alphabets (a and b).

Citation: Journal of Molecular Endocrinology 56, 4; 10.1530/JME-15-0278

We further explored whether mitochondrial pPHB and OPA1 expression were affected by testosterone+FSH treatment after knockdown of Phb expression in the GCs. We found that mitochondrial PHB was phosphorylated at residues Y249, T258, and Y259 in response to FSH+testosterone stimulation (Fig. 3C). In addition, the levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly higher in FSH+testosterone treated GCs than was observed in the parallel controls GCs (P<0.05; Newman–Keuls test). Interestingly, the levels of PHB phosphorylation at Y249 and T258/Y259 were significantly low in GCs without FSH treatment (P<0.05; Newman–Keuls test). Moreover, there was a significant increase in mitochondrial pPHB (Y249 and T258/Y259), PHB, and OPA1 expression in Ad-scrambled-infected GCs treated with testosterone+FSH, which is similar to that observed in control GCs (uninfected Ad-virus) treated with FSH+testosterone (P<0.05; Newman–Keuls test; Fig. 3C and D). By contrast, a significant dose-dependent knockdown of mitochondrial Phb expression occurred in Ad-shPhb-infected GCs with a concomitant decrease in OPA1 expression (P<0.05; Newman–Keuls test; Fig. 3C and D). Furthermore, Ad-shPhb-infected GCs treated with testosterone+FSH showed significantly (P<0.05; Newman–Keuls test) lower concentrations of pPHB (Y249 and T258/Y259) and OPA1 compared with Ad-scrambled-infected GCs treated with testosterone+FSH.

We further examined whether FSH stimulates MEK-ERK pathway in Phb knockdown GCs alone or in combination with MEK inhibitor (PD98059). As shown in Fig. 3E, under FSH stimulation ERK1/2 is phosphorylated at significantly higher levels compared with the parallel control groups (P<0.05; Newman–Keuls test). Interestingly, Phb knockdown in GCs with FSH stimulation resulted in a marked decrease in the levels of ERK1/2 phosphorylation compared with the parallel control groups (P<0.05; Newman–Keuls test). Moreover, the presence of MEK inhibitor PD98059 with stimulation by FSH+testosterone resulted in drastically inhibited ERK1/2 phosphorylation (Fig. 3E). Consistently, we observed that depletion of PHB had a negative impact on FSH-induced phosphorylation of ERK1/2 without changes in its total protein expression levels.

Discussion

This study demonstrates for the first time that PHB/pPHB plays a key differentiation role in activating the steroidogenic pathway and regulating the expression of the steroidogenic proteins in gonadotrophin-induced differentiation model of rat ovarian undifferentiated GCs. In our experimental culture system, we have shown that GCs have a strong steroidogenic response to FSH+testosterone, which is in agreement with several published observations that FSH+testosterone stimulates StAR, 3β-HSD, p450scc, and aromatase (Cyp19a1) expression, and E2 and P4 secretions (Hsueh et al. 1983, El-Hefnawy & Zeleznik 2001, Eimerl & Orly 2002, Zeleznik et al. 2003, Parakh et al. 2006, Wayne et al. 2007, Escamilla-Hernandez et al. 2008). Moreover, the TEM studies showed that the steroidogenic activity of GCs is supported morphologically by the appearance of smooth and rough ER, an apparent increase in the size and number of the mitochondria, and an increase in the number of oil droplets present in both the FSH or FSH+testosterone-treated GCs when compared with untreated parallel control GC (Thompson et al. 1997). Both mitochondria and ER play a critical role in steroidogenesis (Walther & Farese 2009, Miller & Bose 2011). Under these experimental conditions, the expression level of PHB is upregulated in presence of testosterone, FSH, and testosterone+FSH, suggesting that PHB plays a role in the steroidogenic pathway in undifferentiated GCs (Thompson et al. 2004). Therefore, in subsequent studies, dose-dependent knockdown of Phb was carried out in GCs using Ad-shPhb construct followed by treatment with FSH. The decrease in E2 and P4 secretion occurring in Phb-knockdown GCs treated with FSH is due to loss of StAR, 3β-HSD, p450scc, and aromatase (Cyp19a1) expression, which are known to govern steroidogenic pathways (Miller & Bose 2011, Duarte et al. 2014). The StAR is the acute steroidogenic response to tropic stimuli and facilitates cholesterol transport from the OMM to the IMM for steroidogenesis (Pescador et al. 1997, Wayne et al. 2007, Miller & Auchus 2011).

Our studies have also confirmed that phosphorylation is a fundamental post-translational modification that regulates PHB function, localization, and binding specificity of the protein (Rajalingam et al. 2005, Han et al. 2008, Ande et al. 2009, Chowdhury et al. 2016). Interestingly, all functional phosphorylation sites (Y249 and T258/Y259) of PHB are within the Raf1 binding region of PHB residues (residues 243–272) (Rajalingam & Rudel 2005, Chiu et al. 2013). However, whether there are direct interactions between these PHB phosphorylation sites with Raf have not been clarified in the current studies. The functional role that PHB/pPHB in the mitochondria plays in GCs differentiation may be explained by altered processing of OPA1 (Merkwirth et al. 2008). The reduced PHB/pPHB content in mitochondria resulting from Phb-knockdown GCs also resulted in inhibition of OPA1 expression in the mitochondria and is likely to be one of the mechanisms that explains the role of PHB in stabilizing the mitochondrial integrity and membrane potential and its subsequent involvement in regulating steroidogenesis in mitochondria (Artal-Sanz et al. 2003, Chowdhury & Bhat 2010, Chowdhury et al. 2016). Previous studies have strongly supported our results that OPA1 is absolutely required for maintaining mitochondrial morphology and ultrastructure for efficient support of steroidogenesis (Wasilewski et al. 2012). The detailed mechanisms by which PHB/pPHB affects OPA1 processing are currently unknown.

In addition, our current studies have demonstrated a role for PHB in MEK-ERK signaling pathway during gonadotrophin-stimulated steroidogenesis in GCs. The role for Ras-Raf-MEK-ERK signaling in regulating gonadotrophin-induced steroidogenesis in mammalian and hen GCs has been well studied (Bygrave & Roberts 1995, Moore et al. 2001, Richards 2001, Baines et al. 2002, Dewi et al. 2002, Cottom et al. 2003, Alonso et al. 2004, Hunzicker-Dunn & Maizels 2006, Su et al. 2006, Woods & Johnson 2007, Wayne et al. 2007, Duarte et al. 2014). Based on our studies, it appears that pPHB/PHB in the GC plays an indispensable role in MEK1-ERK1/2 activation. Furthermore, we have also observed that a decrease in PHB levels in the GCs inhibited MEK1-ERK1/2-signaling cascades and resulted in inhibition of GCs differentiation. Moreover, mitochondrial fusion is associated with the efficient localization of StAR and ERK activity in mitochondria (Poderoso et al. 2008, Duarte et al. 2014). Our data in this study further suggests that PHB is required for the phosphorylation of ERK in FSH-stimulated GCs.

Upon induction of steroidogenesis by FSH, mito­chondrial PHB through pPHB mediates the activation of pMEK-pERK expression that subsequently enhances steroidogenic pathway activity and stimulates E2 and P4 synthesis and secretions. An alternative mechanism that can explain the inhibition of steroidogenesis after mitochondrial Phb knockdown is by inhibition of steroidogenic enzymes in the mitochondria, thus protecting the mitochondria from undergoing functional incapacitation. Of note, however, our in vitro experimental results were not in agreement with previously published work that gonadotrophin treatment had an inhibitory effect on the mRNA/protein expression levels of PHB in GCs (Wang et al. 2013a,b). This apparent discrepancy could be explained by differences in the gonadotrophin dosages that were used in the respective studies, and the fact that the isolated DES and eCG primed pre-antral and antral GCs were then further treated with gonadotrophin in vitro (Boone et al. 1995, Karakji & Tsang 1995a,b, Minegishi et al. 1997, Simoni et al. 1997, Pellatt et al. 2011, Scheetz et al. 2012).

In summary, this study indicates that gonadotroph­in-dependent steroidogenesis in undifferentiated GCs are influenced not only by the expression of the key steroidogenic enzymes, but also by the 30-kDa PHB/pPHB, which acts as an obligatory mitochondrial protein that regulates mitochondrial function and, thus plays a central role as a key modulator of GC differentiation.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/JME-15-0278.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported in part by NIH Grants 1RO1HD057235, HD41749, 1SC3 GM113751, and G12-RR03034. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #bib6 RR18386 from NIH/National Center for Research Resources (NCRR). Part of this work was presented at the 43rd Annual Meeting of the Society for the Study of Reproduction in Milwaukee, WI, USA, 30 July–3 August 2010; the 46th Annual Meeting of the Society for the Study of Reproduction in Montreal, Canada, 22–26 July 2013; the 47th Annual Meeting of the Society for the Study of Reproduction, Grand Rapids, MI, USA, 19–23 July 2014; and 14th Research Centers in Minority Institutions (RCMI) International Symposium (Women’s Health), Washington, DC, USA, 1–3 December 2014.

References

  • Alonso M, Melani M, Converso D, Jaitovich A, Paz C, Carreras MC, Medina JH & Poderoso JJ 2004 Mitochondrial extracellular signal-regulated kinases 1/2 (ERK1/2) are modulated during brain development. Journal of Neurochemistry 89 248256. (doi:10.1111/j.1471-4159.2004.02323.x)

    • Search Google Scholar
    • Export Citation
  • Amsterdam A, Tajima K, Frajese V & Seger R 2003 Analysis of signal transduction stimulated by gonadotropins in granulosa cells. Molecular and Cellular Endocrinology 202 7780. (doi:10.1016/S0303-7207(03)00066-2)

    • Search Google Scholar
    • Export Citation
  • Ande SR, Gu Y, Nyomba BL & Mishra S 2009 Insulin induced phosphorylation of prohibitin at tyrosine 114 recruits Shp1. Biochimica et Biophysica Acta 1793 13721378. (doi:10.1016/j.bbamcr.2009.05.008)

    • Search Google Scholar
    • Export Citation
  • Artal-Sanz M & Tavernarakis N 2009 Prohibitin and mitochondrial biology. Trends in Endocrinology and Metabolism 20 394401. (doi:10.1016/j.tem.2009.04.004)

    • Search Google Scholar
    • Export Citation
  • Artal-Sanz M, Tsang WY, Willems EM, Grivell LA, Lemire BD, van der Spek H & Nijtmans LG 2003 The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans. Journal of Biological Chemistry 278 3209132099. (doi:10.1074/jbc.M304877200)

    • Search Google Scholar
    • Export Citation
  • Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R & Ping P 2002 Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAP K activation in PKCepsilon-induced cardioprotection. Circulation Research 90 390397. (doi:10.1161/01.RES.0000012702.90501.8D)

    • Search Google Scholar
    • Export Citation
  • Bebia Z, Somers JP, Liu G, Ihrig L, Shenker A & Zeleznik AJ 2001 Adenovirus-directed expression of functional luteinizing hormone (LH) receptors in undifferentiated rat granulosa cells: evidence for differential signaling through follicle-stimulating hormone and LH receptors. Endocrinology 142 22522259.

    • Search Google Scholar
    • Export Citation
  • Boone DL, Yan W & Tsang BK 1995 Identification of a deoxyribonuclease I-like endonuclease in rat granulosa and luteal cell nuclei. Biology of Reproduction 53 10571065. (doi:10.1095/biolreprod53.5.1057)

    • Search Google Scholar
    • Export Citation
  • Bygrave FL & Roberts HR 1995 Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers. FASEB Journal 9 12971303.

    • Search Google Scholar
    • Export Citation
  • Chiu CF, Ho MY, Peng JM, Hung SW, Lee WH, Liang CM & Liang SM 2013 Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane. Oncogene 32 777787. (doi:10.1038/onc.2012.86)

    • Search Google Scholar
    • Export Citation
  • Chowdhury I & Bhat GK 2010 Mitochondria – in cellular life and death. In Mitochondria Structure, Functions and Dysfunctions, pp 247376. Ed Svensson OL. New York, NY, USA: NOVA Publishers.

    • Search Google Scholar
    • Export Citation
  • Chowdhury I, Xu W, Stiles JK, Zeleznik A, Yao X, Matthews R, Thomas K & Thompson WE 2007 Apoptosis of rat granulosa cells after staurosporine and serum withdrawal is suppressed by adenovirus-directed overexpression of prohibitin. Endocrinology 148 206217. (doi:10.1210/en.2006-0187)

    • Search Google Scholar
    • Export Citation
  • Chowdhury I, Branch A, Olatinwo M, Thomas K, Matthews R & Thompson WE 2011 Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells. Life Sciences 89 295303. (doi:10.1016/j.lfs.2011.06.022)

    • Search Google Scholar
    • Export Citation
  • Chowdhury I, Thompson WE, Welch C, Thomas K & Matthews R 2013 Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins. Apoptosis 18 15131525. (doi:10.1007/s10495-013-0901-z)

    • Search Google Scholar
    • Export Citation
  • Chowdhury I, Thompson WE & Thomas K 2016 Prohibitin (PHB) roles in granulosa cell physiology. Cell and Tissue Research 363 1929. (doi:10.1007/s00441-015-2302-9)

    • Search Google Scholar
    • Export Citation
  • Cottom J, Salvador LM, Maizels ET, Reierstad S, Park Y, Carr DW, Davare MA, Hell JW, Palmer SS, Dent P et al., . 2003 Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. Journal of Biological Chemistry 278 71677179. (doi:10.1074/jbc.M203901200)

    • Search Google Scholar
    • Export Citation
  • Dewi DA, Abayasekara DR & Wheeler-Jones CP 2002 Requirement for ERK1/2 activation in the regulation of progesterone production in human granulosa-lutein cells is stimulus specific. Endocrinology 143 877888. (doi:10.1210/endo.143.3.8677)

    • Search Google Scholar
    • Export Citation
  • Duarte A, Castillo AF, Podestá EJ & Poderoso C 2014 Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria. PLoS ONE 9 e100387.

    • Search Google Scholar
    • Export Citation
  • Eimerl S & Orly J 2002 Regulation of steroidogenic genes by insulin-like growth factor-1 and follicle-stimulating hormone: differential responses of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and 3beta-hydroxysteroid dehydrogenase/isomerase in rat granulosa cells. Biology of Reproduction 67 900910. (doi:10.1095/biolreprod.101.002170)

    • Search Google Scholar
    • Export Citation
  • El-Hefnawy T & Zeleznik AJ 2001 Synergism between FSH and activin in the regulation of proliferating cell nuclear antigen (PCNA) and cyclin D2 expression in rat granulosa cells. Endocrinology 142 43574362. (doi:10.1210/endo.142.10.8438)

    • Search Google Scholar
    • Export Citation
  • Escamilla-Hernandez R, Little-Ihrig L, Orwig KE, Yue J, Chandran U & Zeleznik AJ 2008 Constitutively active protein kinase A qualitatively mimics the effects of follicle-stimulating hormone on granulosa cell differentiation. Molecular Endocrinology 22 18421852. (doi:10.1210/me.2008-0103)

    • Search Google Scholar
    • Export Citation
  • Han EK, Mcgonigal T, Butler C, Giranda VL & Luo Y 2008 Characterization of Akt overexpression in MiaPaCa-2 cells: prohibitin is an Akt substrate both in vitro and in cells. Anticancer Research 28 957963.

    • Search Google Scholar
    • Export Citation
  • Havelock JC, Rainey WE & Carr BR 2004 Ovarian granulosa cell lines. Molecular and Cellular Endocrinology 228 6778. (doi:10.1016/j.mce.2004.04.018)

    • Search Google Scholar
    • Export Citation
  • Hsueh AJ, Jones PB, Adashi EY, Wang C, Zhuang LZ & Welsh TH Jr 1983 Intraovarian mechanisms in the hormonal control of granulosa cell differentiation in rats. Journal of Reproduction and Fertility 69 325342. (doi:10.1530/jrf.0.0690325)

    • Search Google Scholar
    • Export Citation
  • Hunzicker-Dunn M & Maizels ET 2006 FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A. Cell Signaling 18 13511359. (doi:10.1016/j.cellsig.2006.02.011)

    • Search Google Scholar
    • Export Citation
  • Jamnongjit M & Hammes SR 2006 Ovarian steroids: the good, the bad, and the signals that raise them. Cell Cycle 5 11781183. (doi:10.4161/cc.5.11.2803)

    • Search Google Scholar
    • Export Citation
  • Karakji EG & Tsang BK 1995a Regulation of rat granulosa cell plasminogen activator system: influence of interleukin-1 beta and ovarian follicular development. Biology of Reproduction 53 13021310.

    • Search Google Scholar
    • Export Citation
  • Karakji EG & Tsang BK 1995b Growth hormone releasing factor and vasoactive intestinal peptide stimulate rat granulosa cell plasminogen activator activity in vitro during follicular development. Molecular and Cellular Endocrinology 107 105112.

    • Search Google Scholar
    • Export Citation
  • Kasashima K, Ohta E, Kagawa Y & Endo H 2006 Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. Journal of Biological Chemistry 281 3640136410. (doi:10.1074/jbc.M605260200)

    • Search Google Scholar
    • Export Citation
  • Kasashima K, Sumitani M, Satoh M & Endo H 2008 Human prohibitin 1 maintains the organization and stability of the mitochondrial nucleoids. Experimental Cell Research 314 988996. (doi:10.1016/j.yexcr.2008.01.005)

    • Search Google Scholar
    • Export Citation
  • Merkwirth C, Dargazanli S, Tatsuta T, Geimer S, Löwer B, Wunderlich FT, von Kleist-Retzow JC, Waisman A, Westermann B & Langer T 2008 Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria. Genes and Development 22 476488. (doi:10.1101/gad.460708)

    • Search Google Scholar
    • Export Citation
  • Miller WL & Auchus RJ 2011 The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews 32 81151. (doi:10.1210/er.2010-0013)

    • Search Google Scholar
    • Export Citation
  • Miller WL & Bose HS 2011 Early steps in steroidogenesis: intracellular cholesterol trafficking. Journal of Lipid Research 52 21112135. (doi:10.1194/jlr.R016675)

    • Search Google Scholar
    • Export Citation
  • Minegishi T, Tano M, Kishi H, Kameda T & Miyamoto K 1997 Follicle-stimulating hormone regulation on its receptor messenger ribonucleic acid levels in cultured rat granulosa cells. Biochimica et Biophysica Acta 1359 165173. (doi:10.1016/S0167-4889(97)00095-5)

    • Search Google Scholar
    • Export Citation
  • Moore RK, Otsuka F & Shimasaki S 2001 Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells. Biochemical and Biophysical Research Communincations 289 796800. (doi:10.1006/bbrc.2001.6052)

    • Search Google Scholar
    • Export Citation
  • Parakh TN, Hernandez JA, Grammer JC, Weck J, Hunzicker-Dunn M, Zeleznik AJ & Nilson JH 2006 Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin. PNAS 103 1243512440. (doi:10.1073/pnas.0603006103)

    • Search Google Scholar
    • Export Citation
  • Pellatt L, Rice S, Dilaver N, Heshri A, Galea R, Brincat M, Brown K, Simpson ER & Mason HD 2011 Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells. Fertility and Sterility 96 12461251.e1. (doi:10.1016/j.fertnstert.2011.08.015)

    • Search Google Scholar
    • Export Citation
  • Pescador N, Houde A, Stocco DM & Murphy BD 1997 Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. Biology of Reproduction 57 660668. (doi:10.1095/biolreprod57.3.660)

    • Search Google Scholar
    • Export Citation
  • Poderoso C, Converso DP, Maloberti P, Duarte A, Neuman I, Galli S, Cornejo Maciel F, Paz C, Carreras MC, Poderoso JJ et al., . 2008 A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis. PLoS ONE 3 e1443.

    • Search Google Scholar
    • Export Citation
  • Pon LA, Hartigan JA & Orme-Johnson NR 1986 Acute ACTH regulation of adrenal corticosteroid biosynthesis. Journal of Biological Chemistry 261 1330913316.

    • Search Google Scholar
    • Export Citation
  • Prasad M, Kaur J, Pawlak KJ, Bose M, Whittal RM & Bose HS 2015 Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction. Journal of Biological Chemistry 290 26042616. (doi:10.1074/jbc.M114.605808)

    • Search Google Scholar
    • Export Citation
  • Rajalingam K & Rudel T 2005 Ras-Raf signaling needs prohibitin. Cell Cycle 4 15031505. (doi:10.4161/cc.4.11.2142)

  • Rajalingam K, Wunder C, Brinkmann V, Churin Y, Hekman M, Sievers C, Rapp UR & Rudel T 2005 Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nature Cell Biology 7 837843. (doi:10.1038/ncb1283)

    • Search Google Scholar
    • Export Citation
  • Richards JS 2001 Perspective: the ovarian follicle – a perspective in 2001. Endocrinology 142 21842193.

  • Scheetz D, Folger JK, Smith GW & Ireland JJ 2012 Granulosa cells are refractory to FSH action in individuals with a low antral follicle count. Reproduction, Fertility and Development 24 327336. (doi:10.1071/RD11020)

    • Search Google Scholar
    • Export Citation
  • Simoni M, Gromoll J & Nieschlag E 1997 The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocrine Reviews 18 739773.

    • Search Google Scholar
    • Export Citation
  • Su YQ, Nyegaard M, Overgaard MT, Qiao J & Giudice LC 2006 Participation of mitogen-activated protein kinase in luteinizing hormone-induced differential regulation of steroidogenesis and steroidogenic gene expression in mural and cumulus granulosa cells of mouse preovulatory follicles. Biology of Reproduction 75 859867. (doi:10.1095/biolreprod.106.052613)

    • Search Google Scholar
    • Export Citation
  • Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C & Schatten G 2000 Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biology of Reproduction 63 582590. (doi:10.1095/biolreprod63.2.582)

    • Search Google Scholar
    • Export Citation
  • Thompson WE, Sanbuissho A, Lee GY & Anderson E 1997 Steroidogenic acute regulatory (StAR) protein (p25) and prohibitin (p28) from cultured rat ovarian granulosa cells. Journal of Reproduction and Fertility 109 337348. (doi:10.1530/jrf.0.1090337)

    • Search Google Scholar
    • Export Citation
  • Thompson WE, Powell JM, Whittaker JA, Sridaran R & Thomas KH 1999 Immunolocalization and expression of prohibitin, a mitochondrial associated protein within the rat ovaries. Anatomical Record 256 4048. (doi:10.1002/(sici)1097-0185(19990901)256:1<40::aid-ar6>3.0.co;2-x)

    • Search Google Scholar
    • Export Citation
  • Thompson WE, Branch A, Whittaker JA, Lyn D, Zilberstein M, Mayo KE & Thomas K 2001 Characterization of prohibitin in a newly established rat ovarian granulosa cell line. Endocrinology 142 40764085. (doi:10.1210/endo.142.9.8354)

    • Search Google Scholar
    • Export Citation
  • Thompson WE, Ramalho-Santos J & Sutovsky P 2003 Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control. Biology of Reproduction 69 254260. (doi:10.1095/biolreprod.102.010975)

    • Search Google Scholar
    • Export Citation
  • Thompson WE, Asselin E, Branch A, Stiles JK, Sutovsky P, Lai L, Im G-S, Prather RS, Isom SC, Rucker E III et al., . 2004 Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary. Biology of Reproduction 71 282290. (doi:10.1095/biolreprod.103.024125)

    • Search Google Scholar
    • Export Citation
  • Walther TC & Farese RV Jr 2009 The life of lipid droplets. Biochimica et Biophysica Acta 1791 459466. (doi:10.1016/j.bbalip.2008.10.009)

  • Wang Q, Leader A & Tsang BK 2013a Follicular stage-dependent regulation of apoptosis and steroidogenesis by prohibitin in rat granulosa cells. Journal of Ovarian Research 6 23.

    • Search Google Scholar
    • Export Citation
  • Wang Q, Leader A & Tsang BK 2013b Inhibitory roles of prohibitin and chemerin in FSH-induced rat granulosa cell steroidogenesis. Endocrinology 154 956967.

    • Search Google Scholar
    • Export Citation
  • Wasilewski M, Semenzato M, Rafelski SM, Robbins J, Bakardjiev AI & Scorrano L 2012 Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts. Current Biology 22 12281234. (doi:10.1016/j.cub.2012.04.054)

    • Search Google Scholar
    • Export Citation
  • Wayne CM, Fan HY, Cheng X & Richards JS 2007 Follicle-stimulating hormone induces multiple signaling cascades: evidence that activation of Rous sarcoma oncogene, RAS, and the epidermal growth factor receptor are critical for granulosa cell differentiation. Molecular Endocrinology 21 19401957. (doi:10.1210/me.2007-0020)

    • Search Google Scholar
    • Export Citation
  • Woods DC & Johnson AL 2007 Protein kinase C activity mediates LH-induced ErbB/Erk signaling in differentiated hen granulosa cells. Reproduction 133 733741. (doi:10.1530/REP-06-0261)

    • Search Google Scholar
    • Export Citation
  • Zeleznik AJ, Saxena D & Little-Ihrig L 2003 Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 144 39853994. (doi:10.1210/en.2003-0293)

    • Search Google Scholar
    • Export Citation

Supplementary Materials

 

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    Ultrastructural changes in rat GC in response to FSH and testosterone. GCs were treated with or without FSH (100 ng/mL) and testosterone (30 ng/mL) or with FSH+testosterone for 48 h. Parallel control (C) GCs were maintained without any treatments. After completing the various treatments, GCs were fixed, mounted, and micrographs were taken with a TEM. (A) represents electron micrographs as control (a), testosterone (b), FSH (c), and testosterone+FSH (d). N, nucleus; M, mitochondria; GC, Golgi complex; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; C, centriole; LD, lipid droplets. (B and C) are the cross-sectional areas of mitochondria and lipid droplets, respectively represented as percentage fold change (arbitrary unit). The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All groups are significantly different (P≤0.05).

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    PHB knockdown inhibits steroidogenesis in gonadotrophin-induced rat GCs. GCs were transiently infected with Ad-shPhb (MOI 5, 10, and 20) or Ad-scrambled (MOI 5, 10, and 20) for 2 h and maintained in culture for 24 h in serum-free media and treated with testosterone (30 ng/mL) in presence or absence of FSH (100 ng/mL) for 48 h in serum-free media. (A) Bar diagram represents estradiol (E2) and progesterone (P4) secretions into the medium as measured by RIA. (B) Representative Western blot analyses of protein expression levels of PHB, StAR, p450scc, 3β-HSD, and aromatase in GCs induced by FSH under PHB knockdown experimental conditions. Equal amounts of protein (25 μg) were applied to each lane. Western blot analyses were analyzed for PHB, StAR, p450scc, 3β-HSD, and aromatase. Tubulin was used as an internal control for cytosol. (C) The bar graphs represent the relative percentage change in expression of PHB, StAR, p450scc, 3β-HSD, and aromatase as a ratio of the protein levels normalized by tubulin. The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All are significantly different (P≤0.05) except with same alphabets (a, b, c, and d). C, parallel control group.

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    Gonadotrophin stimulates phosphorylation of mitochondrial PHB in GCs. (A) GCs were treated with or without FSH (100 ng/mL) and testosterone (30 ng/mL) or in combination (FSH+testosterone) for 48 h. Parallel controls (C) of GCs were maintained without any treatments. After 48 h, 80 μg of protein purified from the respective mitochondrial fractions were focused in the first dimension on IPG 4–7 strips for 60 kV-h using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blot analysis. PHB antibody was used to detect protein spots corresponding to PHB. (B) Alkaline phosphatase-treated GCs. GCs were cultured in the presence of FSH+testosterone for 48 h. Mitochondrial fractions were collected and treated with and without alkaline phosphatase followed by separation on 2D gel electrophoresis, and PHB spots were identified by Western blot analysis. (C) Representative Western blot analyses of mitochondrial protein levels PHB, pPHB (T258/259 and Y249), and OPA1 in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial fractions were isolated, and pPHB and OPA1 were analyzed by Western blot analyses. Equal amounts of protein (25μg) were applied to each lane, and Western blot analyses were analyzed for PHB, pPHB, and OPA1. Porin was used as an internal control for mitochondria. (D) The bar graphs represent the relative percentage change in expression levels of PHB, pPHB, and OPA1 as a ratio of the protein levels normalized by porin from three independent experiments as mean±s.e.m. All are significantly different (P≤0.05) except with same alphabets (a, b, c, d, e, and f). (E) The bar graphs represent the relative percentage change in expression of mitochondrial PHB/porin and pERK/ERK in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial protein was isolated and analyzed by Western blot analyses for PHB, pERK, and ERK. Porin was used as an internal control for mitochondria. The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All are significantly different (P≤0.05) except with same alphabets (a and b).