Neurotrophin‐3 stimulates stem Leydig cell proliferation during regeneration in rats

Abstract Neurotrophin‐3 (NT‐3) acts as an important growth factor to stimulate and control tissue development. The NT‐3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT‐3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT‐3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT‐3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. NT‐3 increased CYP11A1‐positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT‐3 significantly down‐regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT‐3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT‐3 dose‐dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT‐3 antagonist Celitinib can antagonize NT‐3 action in vitro. In conclusion, the present study demonstrates that NT‐3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.


| INTRODUC TI ON
In mature male mammals, testicular adult Leydig cells (ALCs) are cells that primarily produce testosterone (T). 1 During the postnatal development, ALCs are differentiated from stem Leydig cells (SLCs). 2 Between ALCs and SLCs, there are two immediate stages, called progenitor Leydig cell (PLCs) and immature Leydig cells (ILC). 3 Previous studies have shown that ethane dimethyl sulphone (EDS) can accurately kill all ALCs in rat testis, and then start to regenerate ALCs. 4 lyase/17,20 desmolase (CYP17A1) and steroid 5α-reductase type I (SRD5A1). 6 At this stage, PLCs are unable to produce testosterone due to the lack of 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) enzyme catalysis, but they synthesize androsterone. 6 Twenty-eight days after EDS, the next advanced ILCs appear. 6 To judge that the PLC enters the ILC stage, two biomarkers, HSD17B3 and 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), a glucocorticoid metabolic enzyme, appear in ILCs. 6 Fifty-six days after EDS, ALCs are fully regenerated and the ability to produce T has also been completely restored. 6 Although several growth factors such as platelet-derived growth factors 7 and desert hedgehog 8  Some studies have shown that some nerve growth factors such as nerve growth factor (NGF) may also be critical for the development of ALCs. 11 Here, we described another neurotrophin, neurotrophin-3 (NT-3). NT-3 is the third member in the NGF series of neurotrophins. NT-3 has been shown to support the survival and differentiation of neurons. 12 Interestingly, NT-3 is also expressed in the mammalian testis. 13 NT-3 binds to the receptor TRKC, which is a member of the trk family of tyrosine protein kinase. 14 Before birth, NT-3 is involved in regulating the formation of seminiferous cords and germ cell differentiation, and in the determination of male gender. 14 NT-3 is secreted by Sertoli cells in the testis during the embryonic development. 14 NT-3 protomor contains the binding sites of Sertoli cell transcription factor, SOX9 and SOX9 stimulates the expression of NT-3. 15 In 13-14 embryonic days, large amounts of NT-3 are produced in the mouse testis and the knock out of NT-3 and TRKC can reduce testicular interstitium size. 16 This indicates that NT-3 regulates the development of Leydig cells. Previous studies have demonstrated that SLCs are mainly distributed on the surface of seminiferous tubules (STs) in rat testes. 17 SLCs can develop into ALCs under the induction of Leydig cell differentiation medium (LDM), which contains insulin-transferrin-selenium, luteinizing hormone (LH) and lithium ions. 17,18 In the current study, we used an in vivo EDS-treated ALC regeneration model and an in vitro SLC culture to study the role of NT-3 in SLC development.

| Chemicals and kits
Details of materials and methods are contained in Supporting information S1. Chemicals, test kits, equipment and software are included in Supporting information S2. The primers used for gene expression are included in Supporting information S3. Supporting information S4 contains antibodies for immunohistochemical staining and Western blotting.

| Animal study for EDS-treated SLC regeneration
Twenty four male Sprague Dawley rats were transported to Wenzhou Medical University and adapted for a week. ALCs were eliminated using 75 mg/kg EDS as previously described. 10 Male rats were then randomly divided into three groups, eight in each group. From the 14th to the 28th day after EDS administration, we injected 0 (normal saline), 10 or 100 ng/testis NT-3 into each testis. Intratesticular injection of NT-3 was chosen to avoid the systemic effects of NT-3 on the hypothalamic-pituitary-gonadal axis. On the 14th day after NT-3 treatment, rats were euthanized with carbon dioxide, and blood and testis were collected. The animal experiment protocol was approved by the Animal Protection and Use Committee of Wenzhou Medical University.

| Determination of serum and medium T
Both serum and medium T concentration was detected by Immulite2000 Total Testosterone kit as previously mentioned. 10 Normal male rat serum (2 ng/mL) was used as internal quality control. The minimum determine concentration of T is 0.2 ng/mL.

| ELISA for serum LH and FSH Levels
Luteinizing hormone and FSH levels were assayed using ELISA kits as described previously. 10 After sample reacts with peroxidase-conjugated IgG anti-LH or anti-FSH, substrate was added and plate was read.

| Preparation of testis tissue array for immunohistochemical staining and stereological counting of cells
A tissue array was prepared as described previously. 10 The tissuearray block was cut into 5 μm thick sections.

| Immunohistochemistry and immunofluorescence staining of testis
According to the previously reported method, 10 Immunohistochemical staining was performed. CYP11A1 (a biomarker for the Leydig cell lineage) or HSD11B1 (a biomarker of Leydig cells at the ILC and ALC stages) were used.

| Calculation of Leydig cell and Sertoli cell number per testis
In order to count CYP11A1-positive or HSD11B1-positive Leydig cells or SOX9-positive Sertoli cells, the fractionator technique was used for the above tissue-array section as described. 10

| ST culture for SLC developmental assay
To investigate whether NT-3 can affect the development of SLCs, an in vitro culture system of SLCs on the surface of STs was used as previously mentioned. 10 STs were cultured with various concentrations of NT-3 with or without antagonist Celitinib. Medium T concentrations were determined as above.

| Incorporation of EdU into SLCs
As mentioned earlier, 10 the incorporation of EdU into SLCs is measured by EdU Alaxa Fluor kit. EdU-positive cells were counted.

| Statistical analysis
The data are presented using the mean ± SEM P < .05 was considered statistically significant. One-way ANOVA then Sidak-adjusted Dunnett multiple comparison test or paired student t test (only for Western blotting analysis) was used to compare them with controls. Asterisks (**, ***) designate significant differences from the control (NT-3, 0 ng/ testis) at P < .01 and 0.001, respectively

| NT-3 inhibits testosterone secretion in vivo
In order to study the effect of NT-3 on the development of SLCs, we used the EDS model to eliminate ALCs. NT-3 (0, 10 or 100 ng/ testis/d) was administered to rats by intratesticular injection for 14 days ( Figure 1A). At the end of the treatment, NT-3 did not affect the weights of the body and testes and relative testes (divided by body weight) when compared with controls (Supporting information S5). NT-3 significantly inhibited serum T levels at doses of 10 and 100 ng/testis ( Figure 1B). However, NT-3 did not change serum LH ( Figure 1C) and FSH ( Figure 1D) levels. These data indicate that NT-3 exerts its effect in the testis.

| NT-3 affects the expression of Leydig cellspecific genes in vivo
The mRNA levels of ALC-specific genes were measured by qPCR.
The results showed that NT-3 significantly reduced Star and Hsd11b1 mRNA levels at 10 and 100 ng/testis and down-regulated Insl3 and Trkc expression at a dose of 100 ng/testis compared to the control group ( Figure 3). Since Leydig cell number is increased, we re-analysed the expression of all genes after adjustment to CYP11A1-positive  Figure S1).
This indicates that NT-3 reduces the level of T down-regulating the expression of Leydig cell genes. This also indicates that the down-regulation of Hsd11b1 is related to the delay of differentiation of SLCs.

| NT-3 affects Leydig cell-specific protein levels in vivo
The protein levels of ALCs were detected by Western blotting and it was demonstrated that the protein level change was according to their respective mRNA level (Figure 4). We further used semiquantitative immunohistochemical staining of HSD11B1 and SOX9 ( Figure S2). NT-3 lowered HSD11B1 density without affecting SOX9 density at a dose of 100 ng/testis, further confirming that the expression of HSD11B1 in the individual Leydig cell is down-regulated.

| NT-3 regulates AKT1 and mTOR pathways in vivo
Proteins in several signalling pathways were detected by Western blot (Figure 4) and it was found that after 100 ng/testis NT-3, pAKT1/AKT1 and pmTOR/mTOR ratios were increased and then the downstream p4EBP/4EBP ratio was decreased. ATP5O, a mitochondrial transcription-related gene, was analysed and it was found that its protein level was increased (Figure 4). It is speculated that NT-3 enhances mitochondrial function and promotes SLC proliferation and blocks the differentiation.

| NT-3 stimulates SLC proliferation in vitro
In vitro

| NT-3 blocks SLC differentiation in vitro
In vitro ST culture system was used to investigate the effect of NT-3 on the differentiation of SLCs. 17 STs were treated with NT-3 (1, 10 and  Figure 6A). Indeed, SLCs can be converted into Leydig cells, producing T ( Figure 6B). NT-3 dosedependently lowered medium T levels with significance at 10 and 100 ng/mL ( Figure 6B). LOX alone did not affect medium T levels while it reversed NT-3 (100 ng/mL) mediated T suppression ( Figure 6C). This suggests that NT-3 inhibits T production through TRKC receptor. We measured the expression of Leydig cell genes and proteins, and we found that NT-3 significantly down-regulated the expression of Lhcgr, Scarb1, Star, Insl3, Trkc and Nr5a1 at 10 and/or 100 ng/mL ( Figure 7A).
The proteins of LHCGR, SCARB1, TRKC, INSL3 were also lowered by NT-3 ( Figure 7B and C). These further confirm that NT-3 blocks SLC differentiation into the Leydig cell lineage.

| D ISCUSS I ON
NT-3 is a member of the NGF family. Previous studies have shown that NT-3 promoted the growth, proliferation and differentiation of neural stem cells. 20,21 In addition, NT-3 is secreted during the embryonic period, and NT-3 is involved in regulating the formation of seminiferous cords, the differentiation of germ cells, and the determination of male gender. 16 Our group has also shown that NGF induced SLCs to proliferate and differentiate during ALC regeneration. 11 Here, we show that NT-3 stimulates the prolifera- Medium T after 100 ng/mL NT-3 alone plus its antagonist LOXO-195 (LOX, 50 nmol/L). Mean ± SEM, n = 6. Identical letters indicate that there is no significant difference between two groups at P < .05 and reached the peak level 28 days after EDS but remained at a high level until post-EDS day 42. 29 Due to the decline of T, spermatogenesis was hindered, FSH level rose to the peak through negative feedback, and maintained the peak until T and spermatogenesis recovered, then serum FSH returned to normal level 49 days after EDS. 29 In this study, we found that serum FSH level was 6 ng/mL in the control, which was higher than the normal serum level (about 2-3 ng/mL). 29 Although serum T rose on post-EDS day 28 in the control but serum FSH levels were still higher, indicating that spermatogenesis is low in both control and NT-3 treated groups. Trkc expression was significantly reduced at a dose of 100 ng/testis NT-3 in vivo or 10-100 ng/mL NT-3 in vitro. This means that NT-3 may lead to a decrease in TRKC receptors, thereby affecting the normal development of SLCs.
In summary, we demonstrate that NT-3 binds to TRKC to stimulate the proliferation of SLCs, and block the differentiation of SLCs into the Leydig cell lineage. After binding to TRKC, NT-3 may activate the AKT1/ mTOR pathway.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article