Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration

Abstract Objectives We aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)‐treated rat testis. Methods We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post‐EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro. Results FGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) on post‐EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA‐positive Leydig cells, and down‐regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down‐regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro. Conclusions These data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.


| INTRODUC TI ON
Leydig cells, existing in the interstitium surrounding the seminiferous tubules in the mammalian testis, produce testosterone. 1,2 Testosterone plays a critical role in stimulating spermatogenesis, maintaining secondary sexual characteristics and promoting muscle bulk and bone health. 3 Testosterone biosynthesis by Leydig cells relies on pituitary gland-secreted LH. 4 Leydig cells possess LH receptor (LHCGR), which is a G-protein on the cell plasma membrane and responds to LH stimulation. 5 After its stimulation, extracellular cholesterol is transported inside the Leydig cells via high-density lipoprotein receptor (SCARB1). Another protein, steroidogenic acute regulatory protein (STAR), is acutely motivated to transport free cholesterol into the inner mitochondrial membrane, where the first steroidogenic enzyme cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1) is present and catalyses the free cholesterol into pregnenolone. Pregnenolone is diffused into the smooth endoplasmic reticulum, where three steroidogenic enzymes, 3β-hydroxysteroid dehydrogenase (HSD3B1), cytochrome P450 17α-hydroxylase/C17-C20 lyase (CYP17A1) and 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) are present and they catalyse a series of androgen biosynthesis chain reaction of different steroid intermediates from pregnenolone to finally produce testosterone. 6,7 Besides the acute stimulation of steroidogenesis, LH is a hormone to promote the development of Leydig cells during puberty as the knockout of LHCGR in mice led to the Leydig cell hypoplasia with only the presence of progenitor Leydig cells. 8,9 However, LH is not required for the initial commitment of stem Leydig cells (the spindle-shaped cells), as they do not express LHCGR. 10 Progenitor Leydig cells (the spindle-shaped cells), which express Leydig cell biomarker LHCGR, CYP11A1, HSD3B1 and CYP17A1, 10 are still formed in the LHCGR knockout mice. 8,9 Other factors might be the candidates of the earlier regulation of the stem Leydig cell development.
Leydig cell regeneration can be achieved in adult rat testis after a single intraperitoneal injection of EDS. 11 During the earlier regeneration, stem Leydig cells highly proliferate and commit into the Leydig cell lineage starting on post-EDS day 14 by expressing Leydig cell biomarker CYP11A1. 12,13 On post-EDS day 28, ovoid immature Leydig cells are differentiated with an expression of a glucocorticoid metabolizing enzyme 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), a biomarker for Leydig cells at the advanced stages. 12 By searching the possible growth factors that might regulate Leydig cell development, we re-analysed the microarray data of transcriptome from EDS-treated rat testis during the course of Leydig cell regeneration. We found that all 21 fibroblast growth factors (fgf isoforms) are present in rat testis with the expression of Fgf16 being the highest among these factors. Fibroblast growth factors (FGFs) are secreted or anchored proteins that play critical roles in developmental cell processes, including proliferation and differentiation, and exert regulatory, morphological and endocrine and paracrine effects. 14 FGF16 is a paracrine factor that belongs to a subfamily of FGF9, which includes FGF9, FGF16 and FGF20. The FGF9 subfamily does not possess a classical N-terminal signal peptide but possesses an internal hydrophobic sequence that functions as a non-cleaved signal for transporting into the endoplasmic reticulum and secretion from cells. 15 Interestingly, knockout of FGF9 in mice creates a male-to-female sex reversal because of the Leydig cell hypoplasia, 16 indicating that FGF9 subfamily plays a critical role in Leydig cell development. However, knockout of FGF16 in mice does not have apparent dysfunction of reproduction but a decreased proliferation of heart cells. 17 Although the level of FGF16 in foetal rodent gonad is low, the abundant expression of FGF16 in adult rat testis indicates that it plays a role in Leydig cell function.
In the current study, we used an in vivo EDS-treated Leydig cell regeneration model and an in vitro stem Leydig cell culture to address the roles of FGF16 in Leydig cell development in the adult testis.

| Re-analysis of microarray data of cells in the Leydig cell lineage
Transcriptome dataset of rat testes during the course of Leydig cell regeneration after EDS treatment was previously published. 18 In the current study, we performed re-analysis of the dataset for the expression of Fgf members.

| Leydig cell regeneration model after EDS
Twenty-four 60-day-old male Sprague Dawley rats were used and acclimated to the new animal room for a week. To deplete Leydig cells from the adult testis, each rat was intraperitoneally injected EDS (75 mg/kg of body weight). EDS was dissolved in a mixture of dimethyl sulphoxide: H 2 O (1:3, v/v) and then an aliquot of 200 µL was injected.
Leydig cell-depleted rats were randomly divided into three groups with each group of eight rats. FGF16 was dissolved in normal saline and an aliquot of 20 µL for each testis was used for intratesticular injection. Each testis daily received an injection of 0 (normal saline), 10 or 100 ng/testis FGF16, respectively, from post-EDS day 14 for 14 days.
This time-course of administration regimen was adopted because progenitor Leydig cells begin to emerge from stem Leydig cells on post-EDS day 14. 19 Fourteen days after FGF16 treatment, rats were killed and drops of blood were collected. The serum samples were taken and stored at −20°C for the measurement of testosterone, LH and FSH levels. One testis per rat was frozen in −80°C for (quantitative real-time PCR) qPCR and Western blotting analysis. The contralateral testis was fixed in Bouin's solution for immunohistochemical staining.

| Measurement of serum and medium testosterone levels
Immulite2000 Total Testosterone kit was used to measure serum or medium testosterone concentrations as previously described. 20 The minimal detection limit of serum testosterone was 0.2 ng/mL.

| ELISA measurement of serum LH and FSH levels
Serum levels of LH and FSH were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Chemicon, Temecula, CA, USA) as previously described. 20 Briefly, serum sample and assay diluent were cultured in the 96-well plate at room temperature. Then, peroxidase-conjugated IgG anti-LH or anti-FSH agent was added and incubated, followed by washing steps and adding the substrate to initiate the reaction. A microplate reader was set at 550 nm with correction wavelength at 450 nm to read the data for LH or FSH.

| Immunohistochemical staining of the testis
Immunohistochemical staining kit (Vector, Burlingame, CA, USA) was used as previously described. 20 Eight testes per group were used and testis samples were prepared and embedded in paraffin in a tissue array block in TMA-MASTER (3Dhistech, Budapest, Hungary). Tissue-array samples were dehydrated in ethanol and xylene and then embedded in paraffin. Transverse sections (6 µm) were sliced and mounted on glass slides. Antigen retrieval was performed by heating for 10 minutes in

| Counting Leydig cells
To count CYP11A1-positive and HSD11B1-positive Leydig cells, the sampling of the testis was performed according to a fractionator technique as previously described. 20 In brief, each testis was sliced in eight blocks, of which two blocks were randomly selected. Then, blocks were further sliced in four pieces and one piece was randomly selected from a total eight pieces. These pieces of testis were embedded in paraffin in a tissue array as above. Paraffin blocks were sectioned as above. Ten sections were randomly sampled from each testis per rat. Sections were used for immunohistochemical staining as above. Pictures were taken and 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.

| Semi-quantitative measurement of CYP11A1 and HSD11B1 protein levels
CYP11A1 and HSD11B1 are the proteins in Leydig cells.
Immunohistochemical staining of CYP11A1 and HSD11B1 in the tissue array were performed as above. The measurement of CYP11A1 and HSD11B1 protein levels was performed as previously described. 20 Briefly, the densities of CYP11A1 or HSD11B1 and background area nearby were measured using the Image-Pro Plus6.0 (Media Cybernetics, Silver Spring, MD, USA). The net density of CYP11A1 or HSD11B1 was calculated after subtracting the density of the background area. More than 50 Leydig cells in each testis were counted and the density of each testis was averaged as one sample size.

| Isolation and culture of seminiferous tubules
In order to investigate whether FGF16 affects the stem Leydig cell development, an in vitro culture system of stem Leydig cells on the surface of the seminiferous tubules was used as previously described. 22 One 90-day-old male Sprague Dawley rat was treated with EDS (75 mg/kg body weight) as above. The rat was killed by CO 2 on post-EDS day 7 and Leydig cells were depleted. 22 Testes were placed in MEM-199 and decapsulated in the sterile condition. Seminiferous tubules were mechanically separated from the interstitial parts. The tubules were sliced into about one-inch long fragment and distributed randomly into a 12-well plate, with each well containing equivalent parts of tubules. Seminiferous tubules were cultured with Leydig cell differentiation medium (LDM), which contains 5 ng/mL LH, 5 mmol/L insulin-transferrinselenium, and 5 mmol/L lithium chloride in DMEM: F12 medium (1:1, v/v, pH 7.2) supplemented with 0.1% bovine serum albumin, 15 mmol/L HEPES, 2.2 mg/mL sodium bicarbonate, and penicillin/ streptomycin (100 U/mL and 100 mg/mL). The plate was placed in a humidified atmosphere of 5% CO 2 at 34°C for 2 weeks. At the end of 2 weeks, the stem Leydig cells differentiate into adult Leydig cells, which are capable of secreting testosterone to the medium, which can be detected. FGF16 was added to LDM together with LDM. Medium testosterone level was measured as above.

| EdU incorporation into stem Leydig cells
EdU incorporation into stem Leydig cells on the surface of the seminiferous tubules was measured using EdU Alexa Fluor Kit (Life Technologies, USA) as previously described. 23 In brief, the freshly isolated seminiferous tubules as above were cultured in M199 medium and treated with 0, 1, 10 ng/mL FGF16 for 7 days. Then, 1:1000

| Quantitative real-time PCR (qPCR)
Testis and seminiferous tubule samples were homogenized in Trizol agent (Invitrogen, Carlsbad, CA, USA) for total RNA purification as previously described. 20 After extraction, the concentration of total RNA was measured by a plate reader at OD260. The first strand of cDNA was biosynthesized and used as the template for qPCR am-

| Western blot analysis
Western blotting was carried out as previously described. 20  ACTB serves as a control. The protein levels were quantified using ImageJ software and normalized to ACTB.

| Statistical analysis
Data were expressed as the mean ± SEM P < 0.05 was considered statistically significant. The difference of two groups was evaluated by unpaired Student's t test when two groups were compared or by one-way ANOVA followed by ad hoc Dunnett's multiple comparisons to compare with the control when three or more groups were compared. GraphPad version 6 software (La Jolla, CA, USA) was used.

| The expression of Fgf members in the testis after EDS treatment
Our previous study demonstrated that EDS depleted all Leydig cells 7 days after treatment. 18 We re-analyed the transcriptome microarray data from the testes during the course of regeneration of Leydig cells to identify the differential expression of fibroblast growth factor members. 18 As shown in Figure S1, 21 Fgf members (Fgf1, Fgf2, Fgf3, Fgf4, Fgf5, Fgf6, Fgf7, Fgf8, Fgf9, Fgf10,   Fgf11, Fgf12, Fgf13, Fgf14, Fgf15, Fgf16, Fgf17, Fgf20, Fgf21, Fgf22 and Fgf23) of FGF family were identified and the Fgf16 level was the highest. The expression levels of all FGF members were significantly increased on post-EDS day 90 except Fgf21 that was the highest on post-EDS day 7. This suggests that Fgf16 is expressed in rat testis.

| FGF16 reduces testosterone levels in vivo
We used a Leydig cell regeneration model to study the effect of FGF16 on rat Leydig cell development in vivo ( Figure 1A). We intraperitoneally injected rats with EDS to deplete Leydig cells in the testis on post-EDS day 7 and intratesticularly injected FGF16 We selected this regimen to examine the effects of FGF16 on the earlier stage of regeneration. Intratesticular injection of FGF16 was performed in order to avoid its systemic action. After 14-day treatment, FGF16 did not affect rat body and testis weights when compared to the control (Table S3). FGF16 lowered serum testosterone level at a dose of 100 ng/testis ( Figure 1B). However, FGF16 did not alter serum LH and FSH levels ( Figure 1C and 1).
These data suggest that FGF16 delays Leydig cell regeneration via direct action on the testis.

| FGF16 does not alter Sertoli cell number in vivo
We counted Sertoli cells after staining these cells using SOX9 as the biomarker. As shown in Figure 2, FGF16 did not alter SOX9-positive Sertoli cell number. As the number of Sertoli cells is stable after 21 days postpartum, the data indicate that FGF16 does not affect Sertoli cell apoptosis.

| FGF16 increases Leydig cell mitosis in vivo
As progenitor and immature Leydig cells have the capacity of cell division, we asked whether the increased number of Leydig cells results from Leydig cell mitosis after FGF16 treatment. We labelled the mitotic cells using PCNA as the biomarker and Leydig cells using CYP11A1 as the biomarker and found that FGF16 significantly increased the percentage of PCNA-positive Leydig cells (Figure 3). This indicates that the increase of Leydig cell numbers comes from its mitosis.

| FGF16 affects the levels of Leydig and Sertoli cell proteins in vivo
Western blotting was conducted to detect the protein expression levels of LHCGR, SCARB1, STAR, CYP11A1, CYP17A1, HSD3B1, HSD11B1, HSD17B3, NR5A1, FSHR, DHH, SOX9 and ACTB in the testis after FGF16 treatment ( Figure 5A). Statistically, we found that FGF16 significantly lowered the levels of LHCGR, SCARB1, STAR, CYP11A1, CYP17A1, HSD3B1, HSD11B1, HSD17B3, NR5A1, FSHR, SOX9 and DHH when compared with control. We also determined the levels of CYP11A1 and HSD11B1 in Leydig cells and SOX9 in Sertoli cells per se using quantitative immunohistochemical measurement of their densities ( Figure 6). Again, the levels of CYP11A1 and HSD11B1 per Leydig cell or SOX9 per Sertoli cell were significantly lower in FGF16-treated testis. These data conform to their mRNA changes.

| FGF16 regulates AKT1, AKT2 and ERK1/2 pathways in vivo
Many studies have shown that AKT1, AKT2 and ERK1/2 pathways participate in Leydig cell development. 25,26 We explored the downstream signals after FGF16 treatment in the testis by investigating AKT1, AKT2, ERK1/2, and their phosphorylation status. FGF16 significantly increased pAKT1 and pAKT2 as well as pERK1/2 levels at doses of 10 and 100 ng/testis without affecting AKT1 and AKT2 as well as ERK1/2 levels although they trended lower (Figure 7). The results indicate that FGF16 acts primarily via AKT1/AKT2 and ERK1/2 phosphorylation pathways.

| FGF16 inhibits stem Leydig cell differentiation in vitro
As the Leydig cell regeneration during the 28-day period after EDS treatment covers the differentiation of stem into Leydig cells, 19 we asked whether FGF16 affects the differentiation of stem cells into the Leydig cell lineage. We used an in vitro stem Leydig cell differentiation model. 22 Stem Leydig cells can be induced to differentiate into Leydig cells in LDM, which secrete testosterone. 22 Indeed, FGF16 significantly lowered testosterone levels at 10 and 100 ng/ mL ( Figure 8B). Then, we performed qPCR to measure the mRNA levels. Again, FGF16 significantly down-regulated the mRNA levels of Insl3 at 1 ng/mL, Lhcgr, Cyp17a1 and Nr5a1 at 10 and 100 ng/mL, as well as Cyp11a1 at 100 ng/mL ( Figure 8C-G). We also measured the changes of LHCGR, CYP11A1, CYP17A1, INSL3 and NR5A1 levels and we found that they showed the similar trends to their mRNA levels ( Figure 9). This indicates that FGF16 blocks the differentiation of stem Leydig cells in vitro.

| FGF16 stimulates stem Leydig cell proliferation in vitro
We used EdU incorporation into proliferating cells to evaluate the proliferation of stem Leydig cells. We previously demon-  have an internal hydrophobic sequence that functions as a noncleaved signal for transporting into the endoplasmic reticulum and secretion from cells. 15 Herein, we reported that Fgf16 was expressed in rat adult testis as the highest level ( Figure S1). FGF16 has also been found to be present in the testis of the Nile tilapia, regulating and FGFR4, 30 suggesting that FGF16 can bind these FGFRs to regulate Leydig cell development. 30 In the EDS-treated Leydig cell regeneration model, there is a peak of proliferative wave of interstitial cells during the first several days because of the destruction of the existing Leydig cells. 31 Indeed, we treated EDS-treated testis with FGF16 and discovered that FGF16 significantly increased Leydig cell mitosis as shown by the increase of PCNA-labelling index (Figure 3) and Leydig cell number (Figure 2).
We also found that FGF16 increased EdU incorporation into stem Leydig cells (Figure 10 (Figures 4-6, 8, and 9). These results suggest that the steroidogenic capacity per Leydig cell is significantly reduced after FGF16 treatment. Therefore, the serum testosterone levels were significantly lowered after 100 ng/testis FGF16 treatment (Figure 1). Although the exact mechanism that FGF16 exerts is still not well understood, the critical transcription factor, NR5A1, was down-regulated by FGF16 (Figures 4, 5 We also asked what the down-stream signalling FGF16 exerts. FGF16/FGFR signalling is mediated by direct recruitment of signalling molecules that bind to tyrosine autophosphorylation sites on the activated receptor and/or indirect recruitment of docking molecules that have become tyrosine phosphorylated after FGFR activation. FGF1/FGFR signal transduction activates several intracellular signalling pathways, including the phosphatidylinositol 3kinase (PI3K)/AKT pathways. FGF16-FGFR signalling pathway may be mediated by down-stream AKT1 and AKT2 as well as ERK1/2 pathways. 37 AKT is a critical regulator of Leydig cell development.
So far, three AKT isoforms, AKT1-AKT3, were identified. AKT1 is the primary isoform in numerous mammalian tissues including testis. AKT2 is present in several insulin-responsive tissues, regulating glucose metabolism; and AKT3 is mainly expressed in the brain to regulate brain function. 38 Indeed, the AKT1 knockout in mice causes the testis abnormality. 39 However, AKT2-AKT3 double knockout in mice does not induce any abnormality of the testis, 40 indicating that AKT1 is a major down-stream signalling to regulate testis function.
Indeed, PI3K, when it is activated, can phosphorylate AKT1. 41 In the present study, we demonstrated that FGF16 significantly lowered the phosphorylation of AKT1 and AKT2.
The MEK-ERK1/2 pathway is a critical signalling pathway that mediates many signals from the surface receptors of many FGFs, including FGF16. 42 MEK phosphorylates ERK1/2, activating the down-stream cascades. It has been reported that a Leydig cell conditional double knockout of MEK1/2, the upstream kinases of ERK1/2, induces Leydig cell hypoplasia and the decreased androgen production as well as the down-regulation of steroidogenesis-related genes, including Cyp17a1, in mice. 43 Herein, we also observed a significant increase in phosphorylation of ERK1/2 after FGF16 treatment, indicating that this pathway may be involved in FGF16-mediated regulation of proliferation.
In conclusion, FGF16 increases the proliferation but inhibits the differentiation of Leydig cells during their regeneration. PI3K/AKT and MEK-ERK1/2 pathways are possibly involved in FGF16-mediated action (Figure 11).

ACK N OWLED G EM ENTS
This work was supported by NSFC (81730042 to R.S.G, 81771636, to C.L., and 81601266 to X.L.), Health and Family Planning Commission of Zhejiang Province (11-CX29 and 2013ZDA017 to R.S.G. and 2017KY483 to X.L.) and Zhejiang Provincial NSF F I G U R E 11 Illustration of the FGF16 signalling pathway to regulate Leydig cell development. FGF16 binds the FGFRs on the surface of stem/progenitor Leydig cells, triggering phosphorylation of PI3K, which phosphorylates AKT1 and AKT2. It also increases the ERK1/2 phosphorylation. As a result, the stem/progenitor Leydig cells undergo mitosis and exit from differentiation