Seasonal variations of testis anatomy and of G-coupled oestrogen receptor 1 expression in Gerbillus gerbillus.

The gerbil, Gerbillus gerbillus, a nocturnal desert rodent of northern Africa, exhibits a seasonal reproductive cycle with marked anatomical and behavioural shifts between breeding season and resting season. The aim of this study is to investigate key elements involved in these seasonal changes, specifically in males: the histology of the testis as well as the expression of the G-protein-coupled oestrogen receptor 1 (GPER1) in the testis. During the breeding season, the seminiferous tubules were full of spermatozoa, and their epithelium contained germinal cells embedded in Sertoli cells. Amidst tubules, well-developed Leydig cells were observed around blood vessels, with peritubular myoid cells providing structural and dynamic support to the tubules. GPER1 was largely expressed throughout the testis. Notably, Leydig cells, spermatogonia and spermatocytes showed strong immunohistochemical signals. Sertoli cells, spermatozoa and peritubular myoid cells were moderately stained. During the resting season, spermatogenesis was blocked at the spermatocyte stage, spermatids and spermatozoa were absent and the interstitial space was reduced. The weight of the testis decreased significantly. At this stage, GPER1 was found in Leydig cells, spermatocytes and peritubular myoid cells. Sertoli cells and spermatogonia were not marked. Overall, the testis of the gerbil, Gerbillus gerbillus, has undergone noticeable histological, cellular and weight changes between seasons. In addition, the seasonal expression pattern of GPER1, with pronounced differences between resting season and breeding season, indicates that this receptor is involved in the regulation of the reproductive cycle.

Androgen and estrogen receptors immunolocalization in the sand rat (Psammomys Obesus) cauda epididymis.

Both androgens and estrogens play key, albeit incompletely described, roles in the functioning of the epididymis. Because this tightly-coiled tubular structure is compartmented, precise mapping of the distribution of sex steroid's receptors is important. Such receptors have been located in the first segments (caput, corpus), but the last part (cauda) remains poorly explored. We used immunochemistry to localize androgen (AR) and estrogen (ESR1 and ESR2) receptors in the cauda in the fat sand rat (Psammomys obesus). We compared results obtained during the breeding versus resting seasons. We also used individuals castrated, or castrated then treated with testosterone, or subjected to the ligation of their efferent ducts. During the breeding season, in principal cells, we found strong staining both for AR and ESR1 in the apical cytoplasm, and strong staining for ESR2 in the nucleus. During the resting season, principal cells were positive for AR and ESR1, but negative for ESR2. In castrated animals, staining was null for ESR2 and AR, and weak for ESR1. In castrated then treated animals, immuno-expression was restored but only for AR and ESR1. Following efferent duct ligation, AR reactivity decreased while ESR1 and ESR2 provided strong staining. Broadly similar, but not fully identical patterns were observed in basal cells. They were positive for ESR2 and AR during the breeding season, but not for ESR1. During the resting season, staining was modest for ESR1 and AR and negative for ESR2. In all experimentally treated animals, we observed weak staining for AR and ESR1, and a lack of signal for ESR2. Overall, this study provides strong evidence that androgens and estrogens are involved in the seasonal regulation of the whole epididymis in the fat sand rat, with marked differences between caput and cauda (the corpus is highly reduced in rodent).

GPER1 in sand rat epididymis: Effects of seasonal variations, castration and efferent ducts ligation.

Estrogen plays a crucial role in regulating epididymal function and development. Estrogen signaling is mediated via two main receptors essentially involved in the genomic regulating pathway: ERα and ERß. Recent studies revealed the contribution of a novel estrogen receptor involved in the non-genomic pathway: GPER1. This receptor belongs to the family of seven-transmembrane G-protein-coupled receptors and it triggers rapid cellular responses. Immuno-histochemical studies and Western Blot analyses were performed to investigate the GPER1 expression in the caput and cauda epididymis of free-ranging fat sand rats (Psammomys obesus) captured during the breeding and resting seasons. We also investigated the effect of castration (C), castration followed by testosterone treatment (C+T), and ligation of the efferent ducts (L). During the breeding season, a marked positive GPER1 immunoreactivity was detected in the cytoplasm of principal cells and basal cells; this signal persisted during the resting season, attenuated however, meanwhile the clear cells were not immuno-reactive. In C animals, the immuno-histochemical staining underwent nuclear translocation. In C+T animals, this response became nuclear and cytoplasmic. In the L group, the expression of the GPER1 was mainly located in the cytoplasm of principal cells and in the nuclei of basal cells; the sperm was also immune-positive in the cauda epididymis. Western blot analysis showed that GPER1 has a molecular weight of 55kDa in the caput and cauda epididymis during the breeding season, and it persisted during the resting season in the caput epididymis with a decrease in the cauda epididymis. These results suggest that GPER1 mediate a specific cellular estrogen signaling with marked differences between the breeding and resting seasons. Experimental groups suggest that testosterone is involved in the regulation of the expression of GPER1, in addition to other estrogen signalization pathways.

Seasonal variations of aromatase and estrogen receptors expression in the testis of free-ranging sand rats.

An increasing number of studies revealed the importance of estrogen in male reproduction. However, most research was conducted in laboratory rodents subjected to standardized environmental conditions. Therefore, seasonal regulations of estrogen pathways remain poorly understood under natural conditions. Using immunohistochemistry, the expression of several molecules involved in the functioning of testis (i.e. 17-ß estradiol [E2], P450 aromatase, estrogen receptors ESR1, ESR2, and GPER1 [also known as GPR30]) were investigated in free-ranging fat sand rats, Psammomys obesus, during the breeding and resting seasons. Leydig cells showed a strong immunoreactivity for aromatase in the testis sampled during the breeding season only; however, E2, ESR1, ESR2 and GPER1 were present during both seasons. Sertoli cells showed a positive signal for E2 and ESR2 during the breeding season; though, all molecules, except GPER1, were present during the resting season. Spermatogonia were reactive for E2, ESR2 and GPER1 during the breeding season and for ESR1 and GPER1 during the resting season. During both seasons, spermatocytes-I presented a moderate reactivity for E2, ESR1, ESR2 and a strong reactivity for GPER1; aromatase was detected during the resting season only. Spermatids and spermatozoa were present exclusively during breeding season and were reactive for all molecules; except round spermatids that were negative for aromatase. The functioning of the testis depends on finely tuned stimulation and inhibition systems. Our results suggest that differential expression of aromatase, ESR1, ESR2, and GPER1 across cells types is involved in the seasonal activation/inactivation cycle of spermatogenesis in a free-ranging species.

Immunolocalization of estrogen and androgen receptors in the caput epididymidis of the fat sand rat (Psammomys obesus): Effects of seasonal variations, castration and efferent duct ligation.

The fat sand rat (Psammomys obesus) is a model to study seasonal reproductive cycle changes and several metabolic disorders. In order to show a possible involvement of estrogens in the male reproductive functions, the expression of estrogen receptors (ESR1 and ESR2) and androgen receptor (AR) were investigated in the caput epididymidis of fat sand rats during the breeding season, resting season, after castration, after castration followed by testosterone treatment, and after ligation of efferent ducts. In the breeding season, principal cells presented a strong immunostaining of AR in both nuclei and cytoplasm, a strong staining of ESR1, mainly in the apical zone, and a strong immunoexpression of ESR2, mainly in nuclei. In the resting season, a moderate immunostaining of AR in both cytoplasm and nuclei was observed. ESR1 staining showed a strong immunoreactivity in the nuclei. In contrast, the nuclei were negative for ESR2. After castration, a low and selective signal distribution was observed: the nuclei were moderately positive for AR and ESR2, and negative for ESR1. After castration and testosterone treatment, an androgen-dependence for AR and the restoration of ESR1 but not ESR2 immunoexpression were observed. After ligation of the efferent ducts, a considerable reduction of AR immunoreactivity was observed in contrast to ESR1 and ESR2, which gave a strong immunostaining signal. These results illustrate the complexity of the regulation of the androgen and estrogen receptor expression in the epididymis and argue for the coexistence of both androgenic and estrogenic pathways.