*Article* **Comprehensive and Quantitative Analysis of the Changes in Proteomic and Phosphoproteomic Profiles during Stimulation and Repression of Steroidogenesis in MA-10 Leydig Cells †**

**Zoheir B. Demmouche <sup>1</sup> and Jacques J. Tremblay 1,2,\***


**Abstract:** Leydig cells produce testosterone, a hormone essential for male sex differentiation and spermatogenesis. The pituitary hormone, LH, stimulates testosterone production in Leydig cells by increasing the intracellular cAMP levels, which leads to the activation of various kinases and transcription factors, ultimately stimulating the expression of the genes involved in steroidogenesis. The second messenger, cAMP, is subsequently degraded to AMP, and the increase in the intracellular AMP levels activates AMP-dependent protein kinase (AMPK). Activated AMPK potently represses steroidogenesis. Despite the key roles played by the various stimulatory and inhibitory kinases, the proteins phosphorylated by these kinases during steroidogenesis remain poorly characterized. In the present study, we have used a quantitative LC-MS/MS approach, using total and phosphopeptideenriched proteins to identify the global changes that occur in the proteome and phosphoproteome of MA-10 Leydig cells during both the stimulatory phase (Fsk/cAMP treatment) and inhibitory phase (AICAR-mediated activation of AMPK) of steroidogenesis. The phosphorylation levels of several proteins, including some never before described in Leydig cells, were significantly altered during the stimulation and inhibition of steroidogenesis. Our data also provide new key insights into the finely tuned and dynamic processes that ensure adequate steroid hormone production.

**Keywords:** testis; Leydig cells; steroidogenesis; star; AMPK; phosphoproteomics; proteomics

#### **1. Introduction**

Leydig cells are located in the interstitial space between the seminiferous tubules of the mammalian testis [1,2]. These cells are the source of androgens, the main one being testosterone [3]. Steroidogenesis is the biological process of converting cholesterol into steroid hormones, which involves cholesterol transport into the mitochondria, where steroidogenesis is initiated. In males, androgens are essential for male sex differentiation during fetal life and for initiating and maintaining spermatogenesis from puberty onwards. Androgens are also needed to acquire male secondary sex characteristics during puberty. Inadequate androgen production is associated with some cases of differences/disorders of sex development (DSD) in males [4].

Since androgens have pleiotropic roles in male reproductive function and overall health, their synthesis is regulated tightly. Steroidogenesis in Leydig cells is stimulated mainly by the pituitary luteinizing hormone (LH) [5]. The binding of LH to its G proteincoupled receptor (LHCGR) on Leydig cells activates adenylate cyclase, which leads to an increase in intracellular cAMP levels [6]. LH/cAMP-induced steroidogenesis then triggers the activation of several kinases, including protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and calcium/calmodulin-dependent protein kinase I (CAMKI),

**Citation:** Demmouche, Z.B.; Tremblay, J.J. Comprehensive and Quantitative Analysis of the Changes in Proteomic and Phosphoproteomic Profiles during Stimulation and Repression of Steroidogenesis in MA-10 Leydig Cells. *Int. J. Mol. Sci.* **2022**, *23*, 12846. https://doi.org/ 10.3390/ijms232112846

Academic Editor: Antonio Lucacchini

Received: 29 September 2022 Accepted: 21 October 2022 Published: 25 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which in turn phosphorylates several proteins required for increased steroid hormone synthesis (reviewed in [7]). This includes several transcription factors, such as the cAMP response element-binding protein (CREB)/CRE modulator (CREM), GATA4, and SF1 (reviewed in [8]). Furthermore, the de novo synthesis of the NR4A1/NUR77 (nuclear receptor subfamily 4, group A, member 1) transcription factor is required for maximal hormoneinduced steroidogenesis [9]. The induction of *Nr4a1* expression and the stimulation of steroidogenesis in Leydig cells also requires the release of Ca2+ from internal stores through the ryanodine receptors, leading to the activation of CAMKI [10]. Once adequate testosterone levels are reached, stimulation of steroidogenesis is blunted by two mechanisms. The first is the classic negative feedback loop, where testosterone acts at the level of the hypothalamus and pituitary to inhibit LH production [11]. The second is at the level of the Leydig cell itself. In Leydig cells, cAMP is degraded into AMP by phosphodiesterase (PDE) 8A, PDE8B, and PDE4 [12]. The ensuing increase in the intracellular AMP levels activates AMP-activated protein kinase (AMPK), a ubiquitous serine/threonine kinase best known as an energy balance sensor. AMPK is a heterotrimeric complex containing one catalytic subunit (alpha) and two regulatory subunits (beta and gamma) [13]. Once activated, AMPK potently blunts the LH/cAMP-induced steroidogenesis in Leydig cells [14].

We previously compared the transcriptome of Leydig cells that were either untreated, stimulated with Forskolin (Fsk, an agonist of adenylate cyclase), or co-treated with Fsk+AICAR (an agonist of AMPK). This led to the identification and characterization of several genes that were upregulated by the LH/cAMP stimulatory pathway and subsequently downregulated by the AMPK repressive pathway [14]. In addition to the changes in the transcriptome, the protein levels are also affected by the stimulation/repression of steroidogenesis in Leydig cells. However, global changes in protein and phosphoprotein levels have never been reported in this context. In the present work, we have used a quantitative mass spectrometry approach to elucidate the global and dynamic changes in the phosphoproteome of Leydig cells in response to stimulatory (Fsk/cAMP) and inhibitory (AICAR/AMPK) treatments.

#### **2. Results**

#### *2.1. Validation of MA-10 Leydig Cell Responsiveness*

Before analyzing the samples by quantitative LC-MS/MS, we first validated the responsiveness of the MA-10 Leydig cells to the different treatments. MA-10 Leydig cells were treated for 1 h with the vehicle (DMSO), Forskolin alone (Fsk), or Fsk+AICAR. Fsk is an agonist of adenylate cyclase, leading to increased intracellular cAMP levels and stimulation of steroidogenesis. AICAR is an agonist of the AMPK kinase, which we have identified as a potent repressor of hormone-activated steroidogenesis [14]. Although 1 h is sufficient to detect changes in the protein phosphorylation levels, it is usually too short to detect changes in the total protein levels by Western blot. We, therefore, isolated total RNA, which was used in a qPCR to quantify the mRNA levels for the steroidogenic acute regulatory (*Star*) protein. The *Star* gene codes for the steroidogenic acute regulatory (STAR) protein, a protein essential for hormone-induced cholesterol transport into mitochondria and, consequently, steroidogenesis [15]. The *Star* gene is an excellent marker of the dynamic steroidogenic process in Leydig cells, as its expression is strongly induced by LH/Fsk/cAMP and repressed by AICAR/AMPK ([14] and reviewed in [8]). As shown in Figure 1, the *Star* mRNA levels from the three samples used in LC-MS/MS (described below) were increased sixto ten-fold in the presence of Fsk. As expected [14], this increase was potently repressed when the MA-10 Leydig cells were co-treated with AICAR in addition to Fsk (Figure 1). These results confirm the responsiveness of the MA-10 Leydig cells to Fsk and AICAR and validate that the protein samples isolated from these cells are suitable for LC-MS/MS analysis.

**Figure 1.** Validation of treatments on MA-10 Leydig cells by assessing *Star* mRNA levels. MA-10 Leydig cells were treated with either DMSO (control, grey bars), Forskolin alone (Fsk, 10 μM, blue bars) or Forskolin and AICAR (Fsk+AICAR, 10 μM, and 1 mM, respectively, red bars) for 1 h. Total RNA was extracted and reverse-transcribed, and qPCR was performed to quantify *Star* mRNA levels. *Rpl19* was used to normalize the data. The numbers on the x-axis refer to the three different samples used in the LC-MS/MS analysis. Results are displayed as the mean of three individual experiments, each performed in duplicate. For a given experiment, different letters indicate a statistically significant difference between groups (*p* < 0.05).

#### *2.2. Treatment of MA-10 Leydig Cells with Fsk or Fsk+AICAR Significantly Affects the Levels of 20 Proteins*

Total proteins from the MA-10 Leydig cells treated for 1 h with either the vehicle (DMSO), Fsk alone, or Fsk+AICAR were extracted, digested, and quantitively analyzed by LC-MS/MS. A total of 5887 proteins were identified, and 4819 were quantified (data not shown). The level of the majority of these proteins was not significantly changed by the treatments due to the short treatment time. Only 20 proteins (listed in Table 1) were significantly affected between treatments (DMSO vs. Fsk, Fsk vs. Fsk+AICAR, and DMSO vs. Fsk+AICAR). The protein levels either increased (shown in red in Table 1) or decreased (shown in blue in Table 1). In addition, the same protein was sometimes found in different comparison groups. For instance, the protein levels of the orphan nuclear receptor NR4A1 (NUR77), a known regulator of the hormone-induced steroidogenic gene expression in Leydig cells [10,16], was increased by 2.4-fold by the Fsk treatment (DMSO vs. Fsk group), and this increase was blunted after the AICAR treatment (down by 2.34-fold; Fsk vs. Fsk+AICAR group), as previously reported [14]. Since the levels of the NR4A1 protein are back down to the control levels after treatment with Fsk+AICAR, the NR4A1 protein levels were not significantly changed in the DMSO vs. Fsk+AICAR group. These changes in NR4A1 protein levels (increased by Fsk/cAMP and reduced upon activation of AMPK by AICAR) serve as a positive control and validate the quantitative LC-MS/MS approach used.


