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. Author manuscript; available in PMC: 2023 Jan 15.
Published in final edited form as: J Endocrinol. 2022 Aug 17;254(3):187–199. doi: 10.1530/JOE-22-0043

Exogenous cholesterol acquisition signaling in LH-responsive MA-10 Leydig cells and in adult mice

Prasanthi P Koganti 1,†, Amy H Zhao 1,†, Vimal Selvaraj 1,*
PMCID: PMC9840751  NIHMSID: NIHMS1856174  PMID: 35900012

Abstract

MA-10 cells, established four decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), use of a cell permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro, and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to regulation of endocytic traffic, is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that use of serum could confound or interfere with trophic stimulation, and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.

Keywords: Leydig cells, luteinizing hormone, steroidogenesis, mitochondria, cholesterol

Introduction

Steroid hormone biosynthesis starts with cholesterol as a substrate. Bioconversion of cholesterol to the first steroid precursor, pregnenolone (Lynn Jr. et al. 1955; Saba & Hechter 1955; Staple et al. 1956), by CYP11A1 within the mitochondria is the first step in steroidogenesis (Constantopoulos & Tchen 1961; Halkerston et al. 1961; Simpson & Boyd 1966, 1967; Greengard et al. 1967). Subsequent enzymatic conversions of pregnenolone to generate different steroids are regulated on the basis of tissue-specific hormonal outputs (Miller 1988; Miller & Auchus 2011). Studies on steroidogenic cell function over the past six decades have uncovered cholesterol regulatory and transport systems (Kraemer 2007; Miller & Bose 2011; Kraemer et al. 2013), and signaling pathways associated with triggering a steroidogenic response in granulosa, luteal, adrenocortical and Leydig cells (Haynes Jr. et al. 1959; Matthews & Saffran 1973; Hamberger et al. 1978; Fortune & Vincent 1986; Fortune et al. 1986; Chaudhary & Stocco 1988; Stocco & Kilgore 1988; Clark et al. 1994; Lin et al. 1995). Across all steroidogenic model systems that have been pivotal to elucidating many of the mechanisms, in vitro studies continue to present contextual limitations to refining specific functional descriptions.

One such example can be found in the progression of in vitro studies performed to define regulation of cholesterol homeostasis in steroidogenic cells. As steroids are only synthesized on demand, there is sudden escalation in need for acquiring extracellular cholesterol and/or mobilizing cholesterol from internal sources in steroidogenic cells (Aoki & Massa 1975; Zoller & Malamed 1975; Freeman 1987, 1989). Nevertheless, stimulation of steroidogenic cells in culture has been recommended and always performed under serum-free conditions that provide no extracellular cholesterol (Hornsby & McAllister 1991). Although this had no adverse ramifications in studies that examined mitochondrial cholesterol import predominant in the 1990s (Stocco et al. 2017), it distorts physiological relevance in studies investigating upstream signaling events and intricate mechanisms that determine cholesterol sourcing and mitochondrial delivery.

Another example lies in the choice of stimulation provided to elicit responses in steroidogenesis studies. When first derived from murine Leydig cell tumors (Ascoli 1981), the MA-10 cell line shared many features of in vivo Leydig cells which included the ability to respond to luteinizing hormone (LH). It has since served as a valuable model for steroidogenesis studies (Freeman & Ascoli 1981; Ascoli 2007), although they produced progesterone rather than testosterone due to loss of 17alpha-hydroxylase/C17–20 lyase activity. In the early 2000s, it was noted that MA-10 cells became almost devoid of cell surface LH receptor (LHR) activity, with low LH binding density and LH-induced steroidogenic response. Although the precise reason remains elusive, this loss-of-function could be reversed when exogenous LHR was expressed in transient transfections (Hirakawa et al. 2002). Nevertheless, deficiency of endogenous LHR signaling had little impact on MA-10 cell applications as the cell permeable cAMP analog (dibutyryl-cAMP/Bt2cAMP) was already being routinely used to stimulate steroidogenesis in studies that not only dissected mitochondrial cholesterol import (Clark et al. 1994), but also upstream signaling mechanisms (Manna et al. 2009, 2011), and transcriptional regulation (Manna et al. 2002; Martin et al. 2008).

Upstream signaling in LH-induced steroidogenesis is not only via cAMP and activation of protein kinase A (PKA) signaling, but includes other independent and crosscutting mechanisms and regulation involving protein kinase C (PKC), protein kinase G (PKG), extracellular signal-regulated kinase (ERK), arachidonic acid, calcium ions and chloride ions (Choi & Cooke 1990; Cooke et al. 1992; Gyles et al. 2001; Stocco et al. 2005; Manna et al. 2006; Andric et al. 2007). Therefore, use of Bt2cAMP for stimulating steroidogenesis in MA-10 cells, together with providing medium devoid of serum, could have seriously limited the scope of cellular LH-mediated responses pertinent to physiological interpretations.

Based on heterogeneity observed in the MA-10 parent cell line from our previous selection screens (Tu et al. 2015; Selvaraj & Stocco 2018), we evaluated and identified subclones that robustly respond to physiological stimulation via LHR. Use of these LH-responsive MA-10Slip clones allowed documenting the effects of serum, and testing proteomic data-driven systems biology uncovering signaling that directs cholesterol sourcing for steroidogenesis. Our results demonstrate that physiological stimulation via LHR and culture medium that incorporates serum might be important to accurately deduce the cellular and molecular basis of steroidogenesis.

Materials and methods

MA-10 cell cultures and stimulation

MA-10 Leydig tumor cells (Ascoli 1981), were cultured in DMEM containing 10% fetal bovine serum (FBS), 1% non-essential amino acids and 1% penicillin-streptomycin as previously described (Morohaku et al. 2013; Zhao et al. 2016). Clones from single cells were derived by plating MA-10 cells at a dilution corresponding to 1 cells/well in a 96 well plate. The wells were observed using a light microscope for the colony forming units (CFU) and those with single CFUs were allowed to grow for two weeks, and the colonies that formed in some of the wells (~25%) were further expanded to obtain subclones. For testing the ability of single-cell clones in producing progesterone, 5 × 104 cells from each subclone were plated in a 96-well plate and their progesterone producing capability was tested when stimulated with 1.5 IU/mL hCG or 0.5mM Bt2cAMP for 3 or 6 hours in DMEM supplemented with 10% FBS. For testing the hCG-induced increases to progesterone levels over time, samples were collected after 30 min, 1-, 2-, 3- and 6-hour time periods. To measure progesterone, cell culture medium was collected and held at −20°C. Total protein content in each well was measured immediately thereafter; cells were lysed with SDS lysis buffer (1% w/v in water) and protein concentration was measured using the bicinchoninic acid assay (Pierce™, BCA protein assay kit, ThermoFisher Scientific).

Effect of serum lipids on steroidogenesis

To test the influence of serum lipids on hormone production, MA-10 cells (parent line and slip clones) were tested for progesterone synthesis using three different conditions: (a) serum free medium (DMEM and 1% non-essential amino acids), (b) complete medium with serum (DMEM and 10% FBS and 1% non-essential amino acids), (c) lipoprotein depleted serum medium (DMEM and 10% lipoprotein depleted serum and 1% non-essential amino acids). Each condition was stimulated with either 1.5 IU/ml hCG (Sigma) or 0.5mM Bt2cAMP (Sigma), or maintained without any treatment (controls) for 6 hours. Plating cells and collection of supernatants for the estimation of progesterone is as described in the previous section.

Measurement of progesterone

Progesterone levels in cell culture supernatants were measured as previously described (Tu et al. 2015). In brief, cell culture supernatants were incubated overnight with I125-labeled progesterone (MP Bio) and anti-progesterone antibody (Staigmiller et al. 1979) at 4°C for competitive binding. A charcoal-dextran suspension was then added and incubated for 10 minutes at 4°C to absorb the free fraction. Samples were then centrifuged at 1700 × g for 10 minutes and the supernatant was collected. Radioactivity in each collected sample was measured using a scintillation counter (Clinigamma Automatic, Wallac). Progesterone levels were estimated relative to detection of an all-encompassing range of progesterone standards/standard curve. The progesterone levels were subsequently normalized to the total protein content of each well.

Immunoblots for LHR and STAR expression

General procedures for performing immunoblot are as previously described (Morohaku et al. 2013, 2014). For detecting LHR, a commercially available affinity purified polyclonal rabbit primary antibody against hLHCGR (peptide YLSICNTGIRKFPDVTK; Boster Bio, #PA1552) was tested and used. For detecting STAR (STARD1), a polyclonal primary antibody was custom generated against the murine peptide epitope EGWKKESQQENGD in rabbits and affinity purified (EZBiolab). Primary antiserum was validated for detection of STAR (Supplementary Figure S1). For the tissue blot, C57BL/6 mice were euthanized, and brain, thymus, heart, lung, liver spleen and adrenal were dissected and processed for protein preparation. Mice were maintained in accordance to the NIH Guidelines for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Cornell University approved their use in this study. For MA-10 cell blots, cultures were used with their respective treatment and stimulation conditions. For the immunoblot procedures, proteins were separated by SDS-PAGE and immunoblotted using rabbit primary antibodies, multiplexed with the control for protein loading, mouse monoclonal β-actin (Li-Cor; #926–42212). Simultaneous detection was performed using species directed IRDye 700 and 800 conjugated secondary antibodies. Images were acquired using a laser fluorescence scanner (Li-Cor Odyssey) and processed using ImageJ for visualization as described previously (Morohaku et al. 2014).

Shotgun proteomics

MA-10 cell pellets (6 × 106 cells) were solubilized in 6 M urea in 50mM ammonium bicarbonate. Dithiothreitol (DTT) was added to a final concentration of 5 mM and samples were incubated for 30 min at 37°C. Subsequently, 20 mM iodoacetamide (IAA) was added to a final concentration of 15 mM and incubated for 30 min at room temperature, followed by the addition of 20 μL DTT to quench the IAA reaction. Lys-C/trypsin (Promega) was used at a 1:25 ratio (enzyme:protein) and incubated at 37°C for four hours. Samples were then diluted to <1 M urea by the addition of 50 mM ammonium bicarbonate and digested overnight at 37°C. The following day, samples were desalted using C18 Macro Spin columns (Waters™ Corp) and dried down by vacuum centrifugation.

LC separation was done on a Proxeon Easy-nLC II HPLC (Thermo Scientific) with a Proxeon nanospray source and mass spectra were collected on an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) as previously described (Pillai et al. 2017). Tandem mass spectra were extracted by Proteome Discoverer v1.2. (Thermo Scientific). All MS/MS samples were analyzed using X! Tandem (The GPM, www.thegpm.org; CYCLONE 2013.02.01.1). X! Tandem was set up to search the Uniprot murine proteome (55,398 entries) and 116 common laboratory contaminants (www.thegpm.org/crap) with an equal number of reverse decoy sequences assuming the digestion enzyme trypsin. Scaffold (version 4.2.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 79.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability to achieve an FDR less than 5.0% and contained at least 2 unique peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al. 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

Bioinformatics and enrichment analysis

Identified proteins were first compared against all mouse genes using the PANTHER (protein analysis through evolutionary relationships) database (Mi et al. 2021), and the diversity of proteins identified were delineated under the protein class terms to visualize the detection/expression landscape. Using the full dataset, functional pathways detection and statistical enrichment analysis were performed using Reactome (Jassal et al. 2020). Results were visually examined using graphing tools (chord diagrams) in RStudio (version 1.3).

Effects of modulating Rho signaling in steroidogenesis

Treating LH-responsive MA-10Slip cells to either activate Rho-signaling (Rho Activator II/Rho-a, Cytoskeleton Inc.), or inhibit Rho-associated coiled-coil containing kinase/Rock (Y-27632/Rock-i, MCE®) prior to steroidogenic stimulation was performed to determine their impacts on progesterone production in both serum containing and serum-free medium conditions. Pretreatments were performed with either 20 μM of Rho-a or 5 μM of Rock-i for 1 hour. Induction of steroidogenesis, and all steps towards the measurement of progesterone levels were as previously described.

Measuring in vivo hCG-induced testosterone production

Male mice (Mus musculus; C57BL/6J, 8 weeks of age) were used to evaluate the effect of Rock-i on hCG-induced testosterone production. For this experiment, mice first received intraperitoneal injections of Rock-i (Y-27632, 30 mg/kg in 1% DMSO) or vehicle (control). After 1 hour of Rock-i exposure, testosterone production was induced using hCG (7.5 IU, intraperitoneal). Mice were then euthanized after 1 hour of hCG stimulation, and blood was collected in heparinized tubes. Plasma was separated by centrifugation (2000 × g for 10 mins), and stored at −80°C. All animal procedures were approved by The Institutional Animal Care and Use Committee of Cornell University. Plasma testosterone levels were measured using solid phase ELISA (MyBioSource) following manufacturer’s instructions and standards. In brief, individual 10 μl plasma samples were prepared with 100 μl of incubation buffer and 50 μl of enzyme conjugate and allowed to bind in testosterone antiserum coated wells of a microplate with shaking for 1 hour at ambient temperature. The plate was then washed 4 times (300 μl of wash buffer per wash) and incubated with substrate at ambient temperature in the dark. At 30 mins the reaction was stopped (with 50 μl of stop solution), and the absorbance measured in each well at 450 nm for both samples and standards. Standard curve was constructed using 4 Parameter Logistics (AAT Bioquest Inc. 2022), and individual sample concentrations were calculated.

Statistics

For quantitative comparisons, differences between two groups were compared with parametric pair-wise comparisons using the Student t-test. Comparisons for more than 2 groups were performed using ANOVA and post hoc Tukey’s test. All analyses were performed using Prism 5 (GraphPad; p<0.05 was considered significant).

Results

LHR-mediated steroidogenesis is muted in MA-10 cells

Compared to progesterone levels after stimulation with Bt2cAMP, MA-10 cells did not produce notable levels of progesterone in response to hCG (Figure 1A). The difference in progesterone production was >9-fold (p=0.0003) lower with hCG than that observed with Bt2cAMP. Although LHR protein was detectable in MA-10 cell Western blots (Figure 1B), there was only this muted response to hCG. The response could also be measured by the induction of STAR that was evident only with Bt2cAMP, but not detectable with hCG stimulation (Figure 1C). Nevertheless, compared to controls that did not produce any meaningful level of hormone, hCG stimulation did elicit some low levels of progesterone. Given prior knowledge regarding heterogeneity of the MA-10 parent cell line (Tu et al. 2015; Selvaraj & Stocco 2018), we evaluated whether this response might be indicative of the presence of sporadic rare clonal populations that responded to hCG.

Figure 1. MA-10 Leydig cells show minimal response to trophic stimulation.

Figure 1.

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(A) MA-10 cells only show a weak steroidogenic response with stimulation via LHR activation using hCG. Progesterone produced when stimulated with Bt2cAMP is >9-fold higher than this hCG response (n=5–6/group; groups not connected by same letter are significantly different, p<0.05). This is consistent with prior work that demonstrated loss of hCG sensitivity in this cell line (Hirakawa et al. 2002). (B) MA-10 Leydig cells show prominent baseline expression of LH receptor; representative Western blot from three independent cultures. (C) Robust STAR protein induction is observed post-stimulation only with Bt2cAMP, but not with hCG in MA-10 Leydig cells. STAR levels remain below detectable after attempts to stimulate with LHR activation using hCG. Representative Western blots for STAR and levels of β-actin (loading control) are shown.

Heterogeneity in parent MA-10 cells and LH-responsive clones

In separating 19 proliferative single cell clones from the parent MA-10 cell line and testing their progesterone production in response to Bt2cAMP and hCG stimulation, we confirmed the substantial heterogeneity in the cell population (Figure 2A). We found all extremes of progesterone production. Most clones responded to Bt2cAMP stimulation, but showed suboptimal responses to hCG stimulation. Some clones showed very low response to both Bt2cAMP and hCG stimulation indicating that there are populations within the MA-10 parent cell line that do not respond to any steroidogenic stimulation. We have previously noted how this can mislead core functional interpretations in studying clones after CRISPR/Cas9 targeting (Selvaraj & Stocco 2018). However, two clones, which we will refer to as MA-10Slip5 and MA-10Slip21, showed robust progesterone production in response to both hCG and Bt2cAMP stimulation. These two clones were selected for expansion and reevaluated for progesterone production. Their response to hCG remained unchanged after expansion (Figure 2B), and was sustained even after repeated passaging and freeze-thaws (>15 passages tested till date). The hCG-induced progesterone accumulation showed an increasing temporal pattern that extended across all the timepoints examined (30 min, 1-, 2-, 3-, and 6-hours) (Supplementary Figure S2). The ratio of progesterone measured with Bt2cAMP vs hCG stimulations were 1: 0.83 (MA-10Slip5), and 1:0.73 (MA-10Slip21). These ratios were significantly higher than that recorded for the parent MA-10 cell line, which was 1: 0.11 (Figure 1A). During this evaluation, it was also noted that MA-10Slip5 and MA-10Slip21 cells were morphologically similar to the parent MA-10 cell line (Figure 2C).

Figure 2. Screening single-cell MA-10 clones from parent line identified LH-responsive cells.

Figure 2.

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(A) Scatter plot showing results from single-cell screening for progesterone produced by MA-10 clones plotting Bt2cAMP against hCG-induction. Two clones MA-10Slip5 and MA-10Slip21 (red datapoints) make relatively high levels of progesterone in response to both hCG and Bt2cAMP. (B) Progesterone production by MA-10Slip5 and MA-10Slip21 cells in response to hCG was reproducible after expansion and propagation. Post-stimulation progesterone levels were not different between hCG and Bt2cAMP groups. However, MA-10Slip21 cells produced higher range of progesterone levels compared to MA-10slip5 cells. (C) Morphology of MA-10Slip5 and MA-10Slip21 clones were similar to parent MA-10 cells; representative phase contrast images are shown.

Serum lipoproteins are major contributors to acute progesterone biosynthesis

To test the ability of hCG to induce progesterone production in the presence of serum and the contribution of extracellular cholesterol sources to the acute regulation of steroidogenesis, we examined the effect of adding FBS on progesterone biosynthesis by MA-10Slip5 and MA-10Slip21 cells. Our results showed that hCG-induced progesterone production in medium containing serum (SER). The levels of progesterone measured with SER was also 2.37-fold and 2.71-fold higher compared to medium without serum (serum-free/SF) in MA-10Slip5 and MA-10Slip21 cells respectively (Figure 3A). As lipoproteins provide the major extracellular cholesterol source for uptake by cells, we tested progesterone production in lipoprotein depleted serum (LDS). Our results showed that LDS completely negated the increase in progesterone production observed in SER (Figure 3A). Although hormone levels were >2-fold and significantly different between SER vs SF and SER vs LDS conditions, induction of STAR after hCG stimulation in these cells were consistent showing similar levels of expression (Figure 3B).

Figure 3. Progesterone production by MA-10Slip5 and MA-10Slip21 clones is substantially increased in the presence of serum lipoproteins.

Figure 3.

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(A) In the presence of fetal bovine serum (SER), both MA-10Slip5 and MA-10Slip21 cells produced a significant 2.37-fold and 2.71-fold higher levels of progesterone, compared to serum free (SF) conditions when stimulated with hCG. Replacing SER with lipoprotein-depleted serum (LDS) did showed increase in progesterone production, levels measured were similar to SF conditions (n=6/group; groups not connected by same letter are significantly different, p<0.05). (B) STAR expression was consistently observed with hCG stimulation/LHR activation of MA-10Slip5 and MA-10Slip21 clones in contrast to the parent line. Representative Western blots for STAR and levels of β-actin (loading control) are shown for MA-10Slip5 and MA-10Slip21 clones.

Prominent potential for dynamic Rho signaling in the MA-10 cell proteome

Proteomics using whole cell lysates from MA-10 cells revealed a total of 1950 non-redundant proteins (Supplementary File 1, [Sheet I]). The protein landscape was first evaluated by categorizing protein classes (Supplementary File 1, [Sheet II]). This representation analysis performed using PANTHER revealed that the proteins were redundantly classified into multiple classes. The highest protein numbers were linked to enzymes that facilitate metabolite interconversions (262 proteins). Other prominent classes in the order of proteins identified were nucleic acid metabolism > translation > RNA metabolism > oxidoreductases and cytoskeletal proteins, that were all represented by more than 100 proteins. In enriching pathways represented in the MA-10 cell proteome using the Reactome database, 131 different pathways were identified as significant (FDR <0.05; Supplementary File 1, [Sheet III]). Examining the top 15 pathways (of highest significance) in this analysis, we identified Rho-GTPase signaling as being overrepresented in 8 of the 15 pathways (Figure 4). Examining the individual proteins identified, it was apparent that the MA-10 cell proteome uncovered the almost complete cohort of 35 Rho GTPase signaling components, indirect evidence for their possible relative abundance. The overlapping functions in the identified pathways are represented in the chord diagram (Figure 4).

Figure 4. Whole cell proteome of MA-10 cells uncovers the enrichment of Rho/Rock signaling components.

Figure 4.

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Proteomic profiling of MA-10 cells resulted in identification of 1950 non-redundant proteins. Enrichment analysis using the Reactome pathway database uncovered Rho signaling as predominant in MA-10 cells. Of the top 15 pathways identified, 8 were associated with Rho signaling (levels of significance as shown in the bar graph). The protein identity and overlap for these enriched Rho signaling pathways are represented as a chord diagram.

Disruption of Rho signaling regulation prevented the lipoprotein-induced increase in progesterone synthesis

Given the basis of Rho signaling in cellular responses influencing cytoskeletal and endocytic vesicular trafficking, we investigated its involvement in exogenous cholesterol acquisition for steroidogenesis. Progesterone production in MA-10Slip5 cells in the presence of a Rho activator II (Rho-a) showed no effect under SF medium after hCG stimulation (Figure 5A). However, under SER medium, presence of Rho-a resulted in a significant decrease in progesterone production at both 3- and 6-hours (p<0.05; Figure 5A), with mean levels almost indistinguishable to that observed in SF medium. Similarly, an inhibitor of Rho-associated coiled-coil containing kinase/ROCK (Rock-i) had no effect on progesterone production under SF medium with both hCG stimulation (Figure 5B). However, under SER medium, presence of Rock-i resulted in a significant decrease in progesterone production (p<0.05; Figure 5B), with mean levels that were almost indistinguishable to that observed in SF medium. The effects of Rho-a and Rock-i on MA-10Slip5 cells in SF and SER were also observed with Bt2cAMP stimulation (Supplementary Figure S3). These results indicated a direct and significant block to exogenous cholesterol acquisition by the disruption of Rho signaling in MA-10 cells.

Figure 5. Disrupting Rho signaling via overactivation or inhibition negated the lipoprotein-mediated increase in progesterone production.

Figure 5.

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(A) The significant increase in hCG-induced progesterone production in the presence of serum (SER) compared to serum-free (SF) conditions, could be significantly reversed (at 3 hours) or completely negated (at 6 hours) by Rho activation (Rho-a; treatment with Rho activator II). Rho-a had no effect on progesterone levels under SF conditions. For all conditions, n=6/group; groups not connected by same letter are significantly different, p<0.05. (B) The significant increase in hCG-induced progesterone production in SER compared to SF could also be significantly reversed (at 3 and 6 hours) by ROCK inhibition (Rock-i; treatment with Y-27632). Rock-i had no effect on progesterone levels under SF conditions. For all conditions, n=6/group; groups not connected by same letter are significantly different, p<0.05.

ROCK inhibition decreases in vivo hCG-induced testosterone production

In order to examine if the Leydig cell steroidogenic suppression by Rho signaling inhibition is reproducible in vivo, we evaluated hCG-induced testosterone production after treatment with Rock-i in adult male mice. We found that the levels of circulating testosterone concentration in Rock-i treated mice were significantly reduced (p<0.05) compared to control mice (Figure 6). Testosterone levels in Rock-i treated mice were 49.4% lower than that of control mice, proportionally similar to changes observed in MA-10Slip5 clones after Rho-a and Rock-i treatments (Figure 7). These parallels between in vitro and in vivo data not only signify the reproducible function of Rho-signaling in exogenous cholesterol acquisition by Leydig cells, but also the value of LH-responsive MA-10Slip clones in studying in vivo Leydig cell function.

Figure 6. Current model for LH-signaling and cholesterol sources contributing to steroidogenesis.

Figure 6.

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(A) Experimental timeline for the acute induction of steroidogenesis in adult male mice. Mice were treated with ROCK inhibitor (Rock-i/Y-27632, 30 mg/kg) or vehicle (control) for 1 hour and induced with hCG (7.5 IU) for another hour before collection of plasma for testosterone measurements. (B) Levels of hCG-induced testosterone measured in mice after Rock-i treatment were significantly reduced (p<0.05) compared to controls. Circulating testosterone levels were significantly decreased by 49.4% with Rock-i treatment.

Figure 7. Current model for LH-signaling and cholesterol sources contributing to steroidogenesis.

Figure 7.

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LH/hCG binding to the LHR (G-protein coupled receptor) activates adenylyl cyclase, prompting the synthesis of cyclic-AMP (cAMP). cAMP binds to regulatory subunits of protein kinase A (PKA), releasing its catalytic subunit. Signaling via PKA is considered primary for LH-induced steroidogenesis. In sourcing cholesterol for steroidogenesis: (1) PKA activates hormone sensitive lipase (HSL) that catalyzes release of free cholesterol from endogenous lipid droplet stores. This cholesterol is moved directly or indirectly to the endoplasmic reticulum for transfer to mitochondria. (2) There is also increase in de novo cholesterol synthesis in the endoplasmic reticulum, for transfer to mitochondria. (3) There is cholesterol acquisition from exogenous sources (LDL and HDL) via receptors (LDLR and SR-BI) followed by endosomal trafficking, or via direct movement from the plasma membrane to the endoplasmic reticulum (Gramd1b). Our results show that inhibiting Rho-GTPase signaling (Rho and ROCK), known modifiers of the membrane skeleton and vesicular trafficking, is critical for the acquisition of exogenous cholesterol. (4) Cholesterol in the endoplasmic reticulum is believed to gain access to the mitochondrial outer membrane at contact sites termed mitochondria-associated membranes (MAMs). STAR binds and transports cholesterol from the outer to the inner mitochondrial membrane. CYP11A1 enzyme that resides in the matrix side of the inner mitochondrial membrane converts cholesterol to pregnenolone (P5). Pregnenolone exits the mitochondria and is converted into progesterone (P4) by HSD3B in the endoplasmic reticulum and further processed/released. Results in this manuscript indicate that exogenous cholesterol sources contribute to a combined mean range of ~35–45% of Leydig cell steroidogenesis in vitro, and with mechanistic extrapolation, ~49% of Leydig cell steroidogenesis in vivo. For details on known pathways pictured see reviews: (Shen et al. 2016; Miller 2017; Selvaraj et al. 2018).

Discussion

Investigating inception of the practice of providing serum-free conditions, we uncovered three possible explanations: (a) Evidence was presented that stimulation of cultured primary bovine luteal cells only yielded LHR-mediated signaling and progesterone production under serum-free growth conditions (Orly et al. 1980; Pate & Condon 1982), suggesting that the presence of serum counters responsiveness to trophic hormones. (b) It was considered that avoiding serum eliminates the possibility of unknown factors including steroids and trophic hormones in culture medium that could introduce variability and artifacts (Hornsby & McAllister 1991). (c) It was postulated that serum could present pseudosubstrate effects due to high accumulating concentration of steroids (Hornsby 1980, 1982; Crivello et al. 1983); serum-free conditions would ensure a moderate rate of steroidogenesis. Although the first explanation could have been misconceived (Poff et al. 1988), it is also possible that specific responses supporting growth versus differentiation in luteal cells might not be extrapolatable to other steroidogenic cell types. Effect of serum on trophic stimulation of adrenocortical or Leydig cells has not been directly tested. For the second explanation, horse serum originally used in steroidogenic cell culture (Buonassisi et al. 1962; Ascoli 1981) is known to contain variable levels of steroids and circulating trophic factors such as follicle stimulating hormone (FSH) and LH (Evans and Irvine 1975). Use of fetal bovine serum (FBS) for culture of steroidogenic cells has been used only in more recent studies (Tu et al. 2015, 2016). For the third explanation, effects of excess steroid production could be prevented by selecting optimal early timepoints. Collectively, the approach of serum-free conditions in steroidogenesis experiments could misrepresent results in studies investigating upstream signaling mechanisms and cholesterol homeostasis.

Existence of cholesterol-enriched lipid stores as droplets is considered a core characteristic consistent across steroidogenic cells of the adrenals, ovaries and testes (Connell & Christensen 1975; Almahbobi et al. 1985, 1988; Silberzahn et al. 1985), albeit with exception of the hamster adrenal cortex (Alpert 1950; Lehoux & Lefebvre 1980; Koganti & Selvaraj 2020). The hormone sensitive lipase (HSL), a broad lipid hydrolase that also possesses cholesterol esterase activity (Yeaman 1990; Osuga et al. 2000; Kraemer et al. 2004), has been demonstrated to be critical for cholesterol mobilization from lipid droplets in steroidogenic cells (Kraemer et al. 2004; Manna et al. 2013). Although relevance of this core mechanism is undisputable, these studies have been performed by stimulating steroidogenic cells under serum-free conditions that forces cells to either synthesize cholesterol de novo or use stored forms (Freeman & Ascoli 1982). Moreover, demonstration that free cholesterol used for steroid hormone biosynthesis resides within the plasma membrane (Freeman 1989), the fact that this pool can be replenished by extracellular low-density lipoproteins (LDL) (Freeman 1987), and our results that lipoproteins provide cholesterol for nearly 50% of progesterone produced, are clear indications that use of medium containing lipoproteins could be crucial for the physiological equilibrium of cholesterol sourcing for steroidogenesis.

Similarly, circumventing LH-mediated signaling by using Bt2cAMP for stimulating steroidogenesis is not without limitations. Receptor-based signaling via LHR in cells is dynamic with aspects of both temporal and compartmental control (Cooke et al. 1992). Rapid internalization, recycling and degradation of LHR is known to occur early in the stimulation process (Rebois & Fishman 1984; Habberfield et al. 1986). Desensitization to gonadotropins, a known phenomenon that occurs post-stimulation goes beyond LHR loss indicating a cellular response decreasing steroidogenic capacity (Freeman & Ascoli 1981). Downstream signaling via LHR is primarily represented by cAMP/PKA signaling (McFarland et al. 1989); however, any cAMP formed is subject to rapid destruction (Sutherland & Rall 1958). Use of Bt2cAMP provides a persistent supraphysiological stimulation for steroidogenic cells. Moreover, Bt2cAMP is also a direct signal for lipolysis (Butcher et al. 1965; Carmen & Victor 2006), which might also present a rapid contribution to the available internal cholesterol pool. In support, we consistently observed higher levels of progesterone production with Bt2cAMP stimulation compared to hCG. This elevated reaction might not recapitulate the LH-induced precise and coordinated response that balances nutrient sensing, inter-organelle signaling, and transcriptional regulations. In corroboration, it has been demonstrated that LH could stimulate near-maximal steroidogenesis even before cAMP production is detectable (Catt & Dufau 1973; Moyle & Ramachandran 1973; Rommerts et al. 1973). Moreover, there is indeed accumulating evidence that cross-talk across multiple dependent and independent signaling mediators in steroidogenesis exist (Stocco et al. 2005).

Systems biology for predicting signaling that could regulate extracellular cholesterol acquisition pointed to the Rho-GTPase pathway. However, functional examination indicated that the process is controlled by an interplay of both Rho activation and ROCK inhibition events. In cells, Rho proteins are present in different membrane compartments (plasma membranes and endomembranes) (Michaelson et al. 2001). At the level of the plasma membrane they are known to induce membrane ruffling assembling focal complexes with actin (Ridley et al. 1992; Nobes & Hall 1995). Endocytosis driven by actin polymerization have obligate requirements for Rho-GTPases in both clathrin-dependent and clathrin-independent internalizations (Qualmann & Mellor 2003). Rho-GTPases have been previously implicated in the regulation of cellular LDL uptake, where its inhibition increased the cellular accumulation of LDL (Hrboticky et al. 2002). This is consistent with our finding that Rho activation negated the steroidogenic increase observed in the presence of external lipoprotein cholesterol. Aligned with physiological signaling, PKA is also known to inhibit RhoA activation (Qiao et al. 2003), indicating that LH-induced PKA activation might also support an endogenous mechanism to increase LDL uptake. In the endosomal compartments however, Rho-GTPase signaling is known to be complex and multifaceted (Phuyal & Farhan 2019). Albeit unexpected, our finding that inhibition of the Rho effector, ROCK could also negate the steroidogenic increase could be indicative a mechanism that interrupts delivery of vesicular cargo. ROCK inhibition is known to disrupt F-actin rearrangements necessary for trafficking components (Leung et al. 1996; Amano et al. 2010). Moreover, the recently discovered Gram1b protein that facilitates non-vesicular plasma membrane to endoplasmic reticulum cholesterol transport (Sandhu et al. 2018), is also dependent on the actin cytoskeleton that could be regulated by Rho-ROCK signaling. Corroboration provided by the in vivo suppression of hCG-induced testosterone production with inhibition of ROCK underscored the direct physiological reproducibility of this vital function in Leydig cell steroidogenesis. Together, these findings highlight a new multifaceted signaling mechanism associated with extracellular cholesterol acquisition for acute steroid biosynthesis.

Incorporating our findings into the current model (Figure 7), we highlight the previously unknown regulation of exogenous cholesterol supply by the RhoGTPase signaling modality linked to endocytic traffic in Leydig cells. On this mechanistic basis, we find that exogenous cholesterol contributes to substantial percentages of steroid hormone produced by Leydig cells both in vitro (~35–45%) and in vivo (~49%). This new understanding also signifies the need for including lipoproteins/serum when testing for steroidogenic capacity, and when studying any aspect signaling or transcriptional regulation relevant to steroid biosynthesis. Although MA-10 cells do not support the study of steroidogenic biotransformation beyond progesterone (Engeli et al. 2018), the LH-responsive MA-10Slip cells derived in this study provide a reformed tool to not only expand on existing knowledge of LH-mediated signaling, but also study steps and regulation leading to initial steroid biosynthesis with sufficient physiological biomimicry for the most relevant preclinical interpretations.

Supplementary Material

Supplementary File 1

File 1. MA-10 cell proteome profile, protein classification, and systems biology predicted pathways. [Sheet I] Full list of proteins identified in MA-10 Leydig cells. Data provided for three replicates used for proteomics. [Sheet II] PANTHER protein classification for the MA-10 cell proteome. [Sheet III] Pathways enriched in the MA-10 cell proteome by the Reactome algorithm.

Supplementary Figures

Acknowledgements

We sincerely thank Dr. Mario Ascoli, University of Iowa for sharing the MA-10 cell line for this work. The authors also thank Ms. Susanne Pelton for setup and assistance with the hormone assays.

Funding

Funding for this study was from the National Institutes of Health (Grant Number DK110059) to VS.

Footnotes

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary File 1

File 1. MA-10 cell proteome profile, protein classification, and systems biology predicted pathways. [Sheet I] Full list of proteins identified in MA-10 Leydig cells. Data provided for three replicates used for proteomics. [Sheet II] PANTHER protein classification for the MA-10 cell proteome. [Sheet III] Pathways enriched in the MA-10 cell proteome by the Reactome algorithm.

NIHMS1856174-supplement-Supplementary_File_1.xlsx (141.5KB, xlsx)
Supplementary Figures
NIHMS1856174-supplement-Supplementary_Figures.pdf (592.2KB, pdf)

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