RFamide-related peptide and messenger ribonucleic acid expression in mammalian testis: association with the spermatogenic cycle

Abstract

RFamide-related peptide (RFRP), the mammalian homolog of avian gonadotropin-inhibitory hormone, has a pronounced suppressive action on the reproductive axis across species. In mammals, RFRP acts directly on GnRH neurons, and likely at the level of the pituitary, to inhibit gonadotropin secretion. In the present study, we examined whether RFRP might act outside of mammalian brain on reproductive tissues directly. Using RT-PCR and in situ hybridization, we found that both RFRP and its receptors [G protein-coupled receptor (GPR) 147 and GPR74] are expressed in the testis of Syrian hamster. These results were confirmed and extended using double- and triple-label immunohistochemistry. RFRP expression was observed in spermatocytes and in round to early elongated spermatids. Significant expression of RFRP was not seen in Leydig cells. GPR147 protein was observed in myoid cells in all stages of spermatogenesis, pachytene spermatocytes, maturation division spermatocytes, and in round and late elongated spermatids. GPR74 proteins only appeared in late elongated spermatids. Additionally, we found that RFRP and its receptor mRNA are markedly altered by day length and reproductive condition. These findings highlight a possible novel autocrine and/or paracrine role for RFRP in Syrian hamster testis, potentially contributing to the differentiation of spermatids during spermiogenesis.

Figure 1

Expression of RFRP, GPR147, and GPR74 in Syrian hamster testis. All three genes, RFRP, GPR147, and GPR74, were found in both brain (Br) and testis (Te) of Syrian hamster by RT-PCR (A). Water (Ctr) was used as a negative control, and β-actin was used as a positive control. In situ hybridization revealed the presence of RFRP (B), GPR147 (C), and GPR74 (D) in hamster testis in a subset of seminiferous tubules. Hybridized probes were visualized by NBT/BCIP (arrows, bright field). Only background signal was observed using sense probes.

Figure 2

Expression of RFRP peptide and its receptor proteins in seminiferous tubules. Immunohistochemistry using specific antibodies against RFRP (A), GPR147 (B), and GPR74 (C) in hamster testis. RFRP ligand (A, green) was found around the nucleus of spermatocytes (Sc) and spermatids (Sd). GPR147 receptor (B, red) was found in myoid cells (Mi) along the edge of tubules, spermatocytes, and both round and elongated spermatids. GPR74 receptor (C, red) was only found in elongated spermatids. Nuclei were stained by DAPI (blue or white color). For each label, controls are shown as follows: no primary antibody (upper left), preadsorption with corresponding antigenic peptide (upper middle), and preadsorption with related peptides (upper right). Lower left, Primary antibody [RFRP (A), GPR147 (B), or GPR74 (C)]. Lower middle, High-power image of area outlined in lower left. Arrows indicate the positively stained tubules (upper right) or cells (lower middle). The highlighted DAPI channel for cell nuclear staining is shown in lower right.

Figure 3

RFRP peptide and its receptor proteins are expressed at specific stages during the seminiferous cycle. Triple-label immunohistochemistry staining for RFRP/GPR147/DAPI (A) and RFRP/GPR74/DAPI (B) in hamster testis reveals a temporal expression pattern during the seminiferous cycle. Spermatids at various steps of spermiogenesis are assigned numbers 1–17 as described in the text. RFRP ligand (A and B, green) is expressed in tubules from stages VIII to XIII surrounding the nucleus of both spermatocytes and spermatids during steps 8–13. GPR147 receptors (A, red) were found in myoid cells (Mi) of all tubules, P primary spermatocytes from stages VIII to XII, the MD spermatocytes in stage XIII, round spermatids a during steps 6–7 and elongated spermatids at during steps 16–17 from stages VI to VII. GPR74 receptors (B, red) were only found in elongated spermatids at late steps 16–17 from stages VI to VII. Cell nuclei were stained by DAPI (blue or white). The DAPI channel is shown in the right panel. An asterisk indicates the lumen side of the tubule.

Figure 4

RFRP mRNA and its receptors are altered by photoperiod and reproductive condition. Mean (±sem) relative paired testis, epididymis (Ep), EWAT, and seminal vesicles (SV) masses in animals held in either LD or SD. SD animals were classified as R or NR based on paired testis masses (A). Mean (±sem) RFRP (B), GPR147 (C), and GPR74 (D) mRNA levels in the testes of LD, SD-R, and SD-NR animals were quantified by real-time PCR. Data are presented relative to β-actin. Significant differences among animal groups were found for RFRP, GPR147, and GPR74 transcription but not the other genes (i.e. GnRH1, Kiss-1, and their receptors; supplemental Fig. S2) (*, P < 0.05; **, P < 0.01).

Figure 5

RFRP peptide and its receptor proteins are suppressed in SD-R animals. Triple-label immunohistochemistry staining for RFRP/GPR147/DAPI (A) and RFRP/GPR74/DAPI (B) in the testes of LD, SD-R, and SD-NR hamsters. For all genes, SD-NR animals showed a similar expression pattern to LD animals. Minimal RFRP expression (A and B, green) was seen in SD-R animals. GPR147 receptor (A, red) can be found in myoid cells (Mi) of all tubules in LD, SD-R, and SD-NR animals. In SD-R animals, P, primary spermatocytes, and MD spermatocytes exhibited GPR147 expression. GPR174 expression was not seen in round spermatids (Sd) in SD-R animals. GPR74 expression was found in not observed in SD-R animals. Spermatids at different steps of spermiogenesis were assigned values of 1–17. Cell nuclei were stained by DAPI (blue or white). The DAPI channel (white) is shown in the right panel. An asterisk indicates the lumen side of the tubule.

Figure 6

Diagram of the expression pattern of RFRP and its receptors pattern during the seminiferous epithelium cycle in Syrian hamsters. The map of the Syrian hamster seminiferous epithelium cycle was previous described by Clermont and Trott (27). The expression of RFRP, GPR147, and GPR74 are indicated by colored arrows. The density of the arrows represents the relative expression level of each gene in specific cell types across stages. Type A (A), intermediate (In), and Type B (B) spermatogonia; preleptotene primary spermatocytes (Pl), leptotene (L), zygotene (Z), P primary spermatocytes; MD spermatocytes; spermatids at various steps of spermiogenesis (1–17). The cell types that are absent in SD-R animals are indicated by a dashed line. Down-regulation of RFRP, GPR147, and GPR74 in SD-R animals is noted by X. Lightning bolts indicate times of down-regulation for RFRP and GPR147.