Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro

Abstract Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post‐EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. It also decreased the levels of Leydig cell‐specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA‐Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell‐specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM‐mediated action was reversed by S3I‐201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.


Leydig cells and adult Leydig cells. Fetal Leydig cells develop during
the embryonic period and adult Leydig cells differentiate from stem Leydig cells during puberty. 3,4 The differentiation of adult Leydig cells includes four phases: stem, progenitor, immature and mature stage. 4 Adult Leydig cells, once formed, rarely divide and slowly maintain its population by spontaneous differentiation from stems Leydig cells. 4,5 However, when adult Leydig cells are depleted in the rat testis after a single intraperitoneal injection (75 mg/kg) of a drug, ethane dimethane sulfonate (EDS), adult Leydig cells can regenerate rapidly within 2 months. 6,7 Newly-formed progenitor Leydig cells arise from stem Leydig cells on the outer surface of the seminiferous tubule. 8 and 11β-hydroxysteroid dehydrogenase 1 (HSD11B1, encoded by Hsd11b1). [10][11][12] The regeneration of Leydig cells was similar to the pubertal developmental process of Leydig cells and it is a unique model for studying the effects of niche factors on Leydig cell development in the adult testis. 11 Recently, a seminiferous tubule culture system has been established to mimic the in vivo Leydig cell development after the culture of the seminiferous tubules in Leydig cell differentiation medium (LDM) and this system can be used for investigating effects of niche factors on stem Leydig cell differentiation and proliferation. 13 There are several niche factors that have been identified to regulate the stem Leydig cell development, including platelet-derived growth factor AA and BB, [13][14][15][16][17] desert hedgehog, 13,18,19 insulin-like growth factor 1 (encoded by Igf1), 20,21 kit ligand, 22 and cytokines. 23,24 After initial screening over 40 niche factors, oncostatin M (OSM), a 28-kDa protein that belongs to a member of the interleukin (IL)-6 cytokine family, is identified. The IL-6 family comprises IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, cardiotrophin-1 and neurotrophin-1/B-cell stimulatory factor-3. 25,26 Their effects are exerted via a common signal transducing receptor chain glycoprotein (gp) 130 (also called IL6ST, encoded by Il6st).
OSM binds to the type I OSM receptor, which forms a heterodimer with IL6ST to transduce the signal. OSM plays a biological role by activating Janus kinase (JAK)-signal transduction with transcriptional activator (STAT) signal transduction pathway and mitogen-activated protein kinase (MAPK) signaling pathway and may be involved in cell growth, differentiation, inflammatory response, hematopoietic processes and tissue remodeling. 27 OSM is produced mainly by activated T cells, neutrophils, monocytes and macrophages. It has also been found that OSM is highly expressed in the gonads of developing embryos, 28 in the late fetal and early neonatal rat testis as well as in the maturing and adult testis. 29 In the rat testis, OSM is mainly present in the Leydig cell lineage and is secreted by immature and adult Leydig cells. 30 It was found that OSM can regulate Sertoli cell and gonocyte proliferation. 31

| Serum testosterone assay in vivo
The objective of the assay is to investigate whether OSM affects the testosterone levels. The serum testosterone concentrations were measured by a chemiluminescence kit according to manufacturer's instruction (Siemens, Munich, Germany) as previously described. 35 The minimal detection concentration was 0.2 ng/mL.

| ELISA for serum LH and FSH levels in vivo
The objective of the assay is to quest the influence of serum LH and FSH levels after the treatment of OSM. Serum LH and FSH levels were detected with enzyme linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Chemicon, Temecula, CA, USA) as described previously. 36 Briefly, an aliquot of a sample and assay diluent were added to each well of the pre-coated 96-well plate. The plate was incubated for 2 hours at room temperature and

| Preparation of RNA-seq library in vivo
Total RNAs were extracted from testes using the Trizol Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction.
The concentration of total RNAs from each testis was measured using a NanoDrop ND-1000 (ThermoFisher Scientific, Shanghai, China). An aliquot of 1-2 μg total RNAs was used to prepare the sequencing library as follows: total RNAs were enriched by oligo-dT magnetic beads and RNA-seq library was prepared using KAPA

| Sequencing in vivo
We performed sequencing of testis and biological pathway analysis (the following section) to address the pathway of OSM-mediated actions in vivo. Image analysis and base calling were performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8, Illumina, Foster City, CA, USA). Sequence quality was examined using the FastQC software. 37 The trimmed reads (trimmed 5′, 3′-adaptor bases) were aligned to reference genome using Hisat2 software. 38 The transcript abundances for each sample was estimated with StringTie, 39 and the FPKM 40 value for gene and transcript levels were calculated with R package Ballgown. 41 The differentially expressed genes and transcripts were filtered using R package Ballgown. 41 The novel genes and transcripts were predicted from assembled results by comparing to the reference annotation using StringTie and Ballgown and the coding potential of those sequences was then assessed using CPAT. 40 Alternative splicing events and plots were detected using rMATS. 42 Principle Component Analysis and correlation analysis were based on gene expression level, Hierarchical Clustering, Gene Ontology, Pathway analysis, Gene Ontology, Pathway analysis, scatter plots and volcano plots were performed with the differentially expressed genes in R, Python or shell environment for statistical computing and graphics.

| Biological pathway analysis
Biological pathway analysis was performed as previously described. 24 The GenMAPP2.1 (San Francisco, CA, USA) was used to create a map of signal pathways for the potential pathways. We imported our statistical results into the program and illustrated biological pathways containing differentially expressed genes. The results of the differential gene expression profile were confirmed by qPCR.

| Quantitative real-time PCR (qPCR) in vivo and in vitro
We performed the qPCR of OSM-treated samples to verify the sequencing data of the testes and to investigate the effects of OSM on the gene expression of the seminiferous tubules. The first strand of cDNA was synthesized and used as the template for qPCR as previously described. 43 The mRNA levels of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, Hsd11b1, Dhh, Igf1, Pdgfa and Nr5a1 were analyzed using the SYBR Green qPCR Kit (Roche, Basel, Switzerland). The reaction mixture consisted of 7.5 μL SYBR Green Mix, 0.75 μL forward and 0.75 μL reverse primers, 0.02 μg diluted cDNAs, and 4 μL RNA-free water. The procedure of qPCR was set as the follows: 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, and 60°C for 30 seconds. The Bio-Rad CFX Manager Software was used to analyze the qPCR data.
The specificity of the fluorescence signal was determined by both melting curve analysis and gel electrophoresis. The mRNA levels were determined by a standard curve method. Ct values were collected for the standard curve and the target mRNA levels were calculated from the curve and were normalized to Rps16. The primers and gene names were listed in Table S1.

| Western blotting analysis in vivo
We performed Western blotting analysis of OSM-treated testes to investigate the effects of OSM on the protein expression of steroidogenesis-related genes. Western blotting was carried out as previously described. 44 Briefly, the testes were put into RIPA lysis buffer (Bocai Biotechnology, Shanghai, China) and homogenized. The protein concentrations of samples were measured using the BCA Protein Assay Kit (Takara, Japan) and bovine serum albumin as the protein standard. An aliquot (30 μg) of proteins was loaded and the proteins were electrophoresed on the 10% polyacrylamide gels containing sodium dodecyl sulfate and the proteins were transferred onto the nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBST buffer for 2 hours and incubated with primary antibodies against LHCGR, SCARB1, CYP11A1, HSD3B1, ACTB (the house-keeping protein) served as a control. The protein levels were quantified using ImageJ software and normalized to ACTB. All the antibody information was listed in Table S2.

| Immunohistochemical staining of the testis in vivo
We performed immunohistochemical staining of the testis and enu-

| Enumeration of Leydig cell number in the testis in vivo
In order to enumerate CYP11A1-positive or HSD11B1-positive Leydig cell numbers, sampling of the testis was performed according to a fractionator technique as previously described. 45 Each testis was cut in eight discs and two discs were randomly selected. Then discs were cut in four pieces and one piece was randomly selected from total eight pieces. These pieces of testis were embedded in paraffin in a tissue array as above. Paraffin blocks were sectioned in 6 μm-thick sections. About ten sections were randomly sampled from each testis per rat. Sections were used for immunofluorescent staining. Using the live image of a digital camera, under a 10× objective, and starting at a fixed point of the "upper" sections, total microscopic fields per section were counted. The total number of Leydig cells was calculated by multiplying the number of Leydig cells counted in a known fraction of the testis by the inverse of the sampling probability.

| Isolation and culture of seminiferous tubules in vitro
We performed culture of seminiferous tubules (including the following two sections) and treated them to investigate the effects of OSM on the proliferation and differentiation of stem Leydig cells.
One 70-day-old rat was injected intraperitoneally with a single dose of EDS (75 mg/kg body weight) for the in vitro experiment. The rat was killed by CO 2 euthanization 7 days after EDS treatment, when all Leydig cells were eliminated. 6,7 Testes were placed in MEM-199 medium and decapsulated. Seminiferous tubules were mechanically separated from the interstitium according to the described method. 15 The tubules were distributed randomly into a 12-well plate, with each well containing tubule fragments of equivalent total length (about 1 inch). Seminiferous tubules were cultured in Leydig cell differentiation medium (LDM), 13 which contains DMEM/F12 medium (pH 7.2) supplemented with 0.1% BSA, 15 mmol L −1 HEPES, 2.2 mg/ mL sodium bicarbonate and penicillin/streptomycin (100 U/mL and 100 μg/mL), insulin-transferrin-selenium (ITS), 5 ng/mL LH and 5 mmol/L lithium chloride, in a humidified atmosphere of 5% CO 2 at 37°C in a 12-well Costar culture plate with ultralow attachment surface (Corning, NY, USA) for up to 2 weeks. In LDM, different concentrations (0, 0.1, 1, 10, and 100 ng/mL) of OSM were added. We used S3I-201 (5 mmol L −1 ) and filgotinib (10 nmol L −1 ), the antagonists of OSM, to explore the underlying mechanism of OSM. S3I-201 is a STAT3 inhibitor, 46 while filgotinib is a JAK1 inhibitor. 47 After 2 weeks in LDM, the ability of the seminiferous tubules to produce testosterone was assayed by measuring testosterone concentration in the cultured medium as previously described. 48 2.14 | Stem Leydig cell proliferation assay in vitro

| Indirect SLC proliferative capacity assay in vitro
In order to perform the indirect proliferative assay of stem Leydig cells, the seminiferous tubules were cultured for 1 week to increase the number of stem Leydig cells and then the seminiferous tubules were switched into LDM for 2 weeks, where stem Leydig cells were induced to differentiate into adult Leydig cells to produce testosterone. 13 If stem Leydig cell number increases or decreases after treatment of a niche factor, the testosterone levels produced by Leydig cells in the medium will reflect the proliferative capacity. 13 Seminiferous tubules were treated with different concentration of OSM (0, 0.1, 1, 10, and 100 ng/mL) in M199 medium for a week, then the tubules were switched to LDM for 2 weeks and the media were collected and testosterone levels were assayed.

| Progenitor Leydig cell culture and proliferation assay in vitro
To investigate the effect of OSM on the proliferative capacity of progenitor Leydig cells, we isolated progenitor Leydig cells and cultured them in the 12-well plate with different concentrations of OSM (0, 1, 10, and 100 ng/mL) at the density of 5 × 10 5 cells/well for 24 hours. The isolation of progenitor Leydig cells was performed as described previously. 49 In brief, forty 21-day-old male Sprague

| Statistical analysis
Data were expressed as the mean ± SE, P < 0.05 was considered statistically significant. The differences of groups were evaluated by one-way ANOVA followed by ad hoc Dunnett's multiple comparisons test to compare with the control using SigmaStat software (Systat Software Inc., Richmond, CA, USA).

Leydig cell regeneration model in vivo
We used a Leydig cell regeneration model to explore the effects of OSM on rat Leydig cell regeneration in vivo. A single intraperitoneal  Figure 1A). After the treatment, OSM did not affect the body weights and testis weights when compared to the control (Table S3). OSM decreased serum testosterone levels at 10 and 100 ng/testis at post-EDS day 28 ( Figure 1B). However, it did not alter serum LH ( Figure 1C) and FSH ( Figure 1D) levels. These data suggest that OSM inhibits Leydig cell regeneration primarily via direct action on stem/progenitor Leydig cells.

| Sequencing analysis reveals OSM-mediated inhibition of Leydig cell regeneration in vivo
We examined the effects of OSM (10 ng/testis) on Leydig cell gene expression levels using RNA sequencing analysis. We sequenced the transcripts and 17,745 transcripts were detected in the testis of two groups. Of these transcripts, 997 transcripts were significantly up-regulated (P < 0.05) and 731 transcripts were significantly down-regulated (P < 0.05) in the 10 ng/testis OSM group when compared to the control (Figure 2A and B). In the Leydig cell steroidogenic pathway, gene expression of several Leydig cell genes (Star, Cyp11a1, Hsd3b1, and Cyp17a1) was down-regulated ( Figure 2C). In the hormone or growth factorreceptor complex, Lhcgr, Kit, and Smo were also significantly down-regulated. This indicates that the Leydig cell regeneration is blocked.

| OSM decreases Leydig cell protein levels during regeneration in vivo
We also detected the protein levels of Leydig cell gene products by Western blot and the result showed that LHCGR, CYP11A1, HSD3B1, CYP17A1 and HSD11B1 levels were lower in OSM-treated testis, which was similar to the respective mRNA levels (Figure 4).

| OSM does not affect the proliferation of Leydig cells in vivo
We used two biomarkers to label the Leydig cells: CYP11A1 (representing all the Leydig cells) and HSD11B1 (representing Leydig cells at the advanced stage). 11,12 We found that OSM did not affect both CYP11A1-positive and HSD11B1-positive cell numbers at ≥10 ng/ testis ( Figure S2). This indicates that OSM might not affect Leydig cell proliferation. Indeed, we further detected the PCNA-positive Leydig cells via PCNA-CYP11A1 double staining and found that OSM did not increase PCNA-positive Leydig cell number ( Figure S3).
This confirms that OSM does not affect Leydig cell proliferation. Since the Leydig cell regeneration during the 28-day period after EDS treatment covers the differentiation of stem into progenitor and immature Leydig cells, 11 we ask whether OSM blocks the differentiation of stem Leydig cells into the Leydig cell lineage. We previously developed an in vitro stem Leydig cell development model using the seminiferous tubule culture. 13 As shown in Figure 5A, OSM concentration-dependently inhibited steroidogenesis at doses of ≥0.1 ng/mL. In order to test the underlying mechanism, we treated stem Leydig cells with S3I-201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor) alone or in combination with 10 ng/mL OSM (Figure 5B). It showed that S3I-201 and filgotinib alone did not affect the medium testosterone levels. However, both S3I-201 and filgotinib reversed OSM-mediated suppression of steroidogenesis ( Figure 5B). These data indicate that OSM blocks stem/progenitor Leydig cell differentiation in vitro via JAK1-STAT3 pathway.

| OSM blocks stem Leydig differentiation by down-regulating Leydig cell gene expression in vitro
We measured the mRNA levels of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1 and Hsd11b1. We found that at ≥10 ng/mL OSM down-regulated Lhcgr, Scarb1 and Star levels and at 100 ng/mL it also down-regulated Cyp11a1, Hsd3b1 and Cyp17a1 levels ( Figure 6). This indicates that OSM inhibits stem Leydig cell differentiation by down-regulating Leydig cell specific gene expression.

| OSM does not alter stem Leydig cell proliferation in vitro
We used EdU incorporation into the proliferating cells to label the dividing stem Leydig cells on the surface of the seminiferous tubules.
As shown in Figure  In the in vitro seminiferous tubule culture system, Leydig cells cannot be formed in LH-free medium and supplement of LH to the medium significantly stimulates the differentiation of progenitor into adult Leydig cells, which are able to produce testosterone robustly. 22 This indicates that LHCGR is critical for the differentiation of Leydig cells once progenitor Leydig cells are committed. Kit encodes a receptor for Kit ligand, which was found to stimulate Leydig cell maturation. 22 Interestingly, in the rat testis, OSM has been reported to be present in the late Leydig cell lineage. 30 OSM itself stimulates stem and progenitor Leydig cell differentiation but blocks the LH-mediated stimulation. 30 In this regard, Leydig cells in the advanced stages secrete OSM, which negatively controls the maturation of stem/progenitor Leydig cells to counterbalance of population of Leydig cells in the testis once LHCGR is formed in the progenitor Leydig cells.
In summary, we demonstrate for the first time that OSM secreted by adult Leydig cells blocks stem or progenitor Leydig cell differentiation via IL6ST-JAK1-STAT3 signaling pathway, which leads to down-regulation of LHCGR, KIT and SMO that are critical factor for Leydig cell development (the schema is illustrated in Figure 7).