The objective of the present study was to investigate the effects of piperine on the testis development in the pubertal rat. Immunohistochemical staining of HSD11B1 in testis sections of piperine (PIP) treated rats. Concentration-dependent effects of piperine (PIP) on basal androgen productions of Leydig cells. To examine whether PIP may have direct effects on Leydig cell steroidogenesis, we have treated immature Leydig cells isolated from 35-day-old rats with 50 μM PIP in the absence (basal) or presence of LH (10 ng/ml) or 8BR (10 mM) for 24 h. Western blot analysis of steroidogenic proteins with the total testis was performed as described (Wu et al., 2017). The expression levels of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Nr5a1) were measured using a SYBR Green qPCR Kit. Ten sections were randomly sampled from each testis per rat. These pieces of testis were embedded in paraffin in a tissue array as described above. Each testis was cut in eight disks, among which two were randomly selected. The numbers of CYP11A1-positive immature Leydig cells and HSD11B1-positive adult Leydig cells were evaluated according to a fractionator technique as previously described (Mendis-Handagama et al., 1989). CYP11A1 and HSD11B1 densities in the individual Leydig cells were calculated. The treatment of PIP promotes Leydig cell development and maturation but inhibits spermatogenesis. Further evidence that PIP may affect spermatogenesis with different mechanisms during puberty and at adulthood comes from the fact that FSH level was actually increased in adult rats after 10 mg/kg PIP treatment (Chinta et al., 2017). Blockade of kit ligand signaling also led to the reduced testosterone production in Leydig cells (Rothschild et al., 2003). Apparently, although the serum LH levels were not altered after PIP treatment, the significant increase of LHCGR could lead to the activation of ERK1/2 as shown in the present study (Figure 7). In the present study, we demonstrated that PIP significantly promoted Leydig cell development during puberty by increasing Leydig cell numbers, Leydig cell size, and the expression levels of steroidogenesis-related proteins. Herein, we investigated the downstream signals after PIP treatment in the testis. Many studies have demonstrated that ERK1/2 and AKT pathways participated in development of Leydig cells (Manna et al., 2006, 2007; Shiraishi and Ascoli, 2007). The androstanediol (DIOL) and testosterone production in rat immature Leydig cells after piperine treatment. The kinase and phosphorylated kinase protein levels of the testes of rats with or without piperine (PIP) treatment. Protein levels of the testes of rats with or without piperine (PIP) treatment. Gene expression levels in the testes of rats with or without piperine (PIP) treatment. Regimen of piperine (PIP) treatment and its effects on serum hormone levels. For in vitro studies, immature Leydig cells were isolated from 35-day-old male rats and treated with 50 μM piperine in the presence of different steroidogenic stimulators/substrates for 24 h. The doses of PIP used in the rats were 5 and 10 mg/kg which were adopted from another study for adult rats (Malini et al., 1999). Forced expression of NR5A1 can even convert stem cells or fibroblasts into steroidogenic cells by transcriptionally promoting the expression of LHCGR and other steroidogenic enzymes (CYP11A1, HSD11B1, CYP17A1, and HSD17B3) (Yang et al., 2017). NR5A1 is a ligand-free nuclear receptor and is a critical transcription factor for promoting Leydig cell development. PIP in vivo significantly increased NR5A1 mRNA and protein levels (Figures 4, 5). We found that testosterone (T)-stimulated DIOL level was not changed by PIP, indicating that PIP did not affect androgen-metabolizing enzyme activities. (B–D) Quantification of kinase and phosphorylated kinase protein levels. These results indicated that ERK1/2 and AKT pathways are involved in the PIP-mediated stimulation of Leydig cell development. PIP significantly increased the ratio of phospho-AKT1 (pAKT1)/AKT1, phosphos-AKT2 (pAKT2)/AKT2, and phospho-ERK1/2 (pERK1/2)/ERK1/2 in the PIP-treated testis (Figure 7). The purities of Leydig cell fractions were evaluated by histochemical staining for HSD3B1 (a biomarker of Leydig cells) with 0.4 mM etiocholanolone as the steroid substrate and NAD+ as a cofactor as described (Payne et al., 1980). The cells with density of 1.070–1.088 g/ml were collected and washed. After filtering with 100 μm nylon mesh, the cells were separated with Percoll gradient as previously described (Ge and Hardy, 1998). PIP treatment increased CYP11A1 density by 10 mg/kg and increased HSD11B1 density by 5 mg/kg and above. In general, the changes in these protein levels were in parallel with changes in their mRNA levels (Figure 5). However, the treatment did not affect Hsd17b3 by any doses (Figure 4). The morphological results of both testis and epididymis indicated clearly that PIP blocked spermatogenesis maturation. With 10 mg/kg PIP treatment, sperm count was reduced further, with sperms being aggregated together.
