Apoptosis in epididymis of sand rat Psammomys obesus, Cretzschmar, 1828: Effects of seasonal variations, castration and efferent duct ligation.

The aim of this study was to visualize apoptosis throughout the reproductive cycle and after castration, castration then treatment with testosterone, and ligation of efferent ducts. The sand rat, Psammomysobesus, Cretzschmar 1828, is a diurnal rodent belonging to the family Gerbillidae. Its breeding cycle is seasonal with reproduction in autumn, winter and early spring and a short resting period from late spring to early summer. Five groups of males were studied: (1) animals captured during the breeding season; (2) animals captured during the resting season; (3) animals castrated and kept 30 days; (4) animals castrated, kept 30 days, and then treated with testosterone for 15 days; (5) animals subjected to the ligation of efferent ducts and kept 30 days. Epididymis were removed and the presence of apoptotic cells was explored using the "Apostain" immunohistochemical method. Histological results showed cell and tissue remodeling. During the breeding season, a positive apoptotic signal was observed mainly in smooth muscle cells of caput and cauda epididymis. This signal persisted throughout the resting season. The orchiectomy induced apoptosis in almost of epithelial and connective cells. However, this intense cell death was not reversed by treatment with testosterone. In animals that experienced efferent duct ligation, principal cells and smooth muscle cells showed a positive signal for apoptosis. Our results converge to qualify the sand rat epididymis as an excellent model for the study of apoptosis and argue for continued cell death, at least independent of circulating testosterone levels.

G protein-coupled oestrogen receptor 1, oestrogen receptors and androgen receptor in the sand rat (Psammomys obesus) efferent ducts.

BACKGROUND: The efferent ducts are mainly involved in the reabsorption of the seminiferous tubular fluid. Testosterone and oestrogens regulate efferent ducts functions via their receptors. MATERIALS AND METHODS: This paper presents an experimental investigation on the location of the P450 aromatase, the 17-b oestradiol (E2), the androgen receptor (AR), the oestrogen receptor 1 (ESR1), the oestrogen receptor 2 (ESR2) and the G protein-coupled oestrogen receptor 1 (GPER1) in the efferent ducts using Psammomys obesus as an animal model to highlight the effect of the season on the histology and the distribution of these receptors. RESULTS: We observed a proliferation of the connective tissue, decreasing in the height of the epithelium during the resting season compared to the breeding season. Ciliated cells expressed P450 aromatase, AR, E2, ESR1, ESR2 and GPER1 during both seasons. Basal cells showed a positive staining for the ESR1 and the GPER1 during both season, the AR and E2 during the breeding season and ESR2 during the resting season. CONCLUSIONS: Our result shows that the expression of androgen receptor and oestrogen receptors in the efferent ducts vary by season witch suggest that they are largely involved in the regulation of the efferent ducts functions.

Ovarian function of the Algerian wild Libyan jird, Meriones libycus during seasonal reproductive cycle: histological and immunohistochemical expression.

Meriones libycus (Libyan jird), a nocturnal Saharan rodent, is characterized by a seasonal reproductive cycle with a short active phase (spring and early summer) and a long resting period (late summer, autumn, winter). Histological and immunohistochemical techniques were performed in order to study the seasonal variations in mature ovaries. During the breeding season, the ovary showed a continuous cyclical activity, the various stages of folliculogenesis from primordial to preovulatory follicles were observed; broken follicles and corpora lutea were also observed. During sexual quiescence, the ovarian cycle was interrupted; anovulation was observed without any corpus luteum. Non mature antral follicles entered the atretic process. Steroid and steroidogenic enzyme activities were studied using indirect immunohistochemistry. 17ß-estradiol, progesterone, testosterone hormones and P450 aromatase (P450 arom) were detected in the different components of the ovary and in various stages of healthy and atretic follicles during the seasonal reproductive cycle. Our results indicate that during ovarian folliculogenesis in breeding season steroids hormone and P450 arom present important activities. In comparison with the resting period, steroidogenesis and steroidogenic enzyme activity became less pronounced in the healthy preantral follicle; it seemed that steroid biosynthesis was reduced and could be involved in the stimulation and maintenance of the ovarian structural integrity in early follicle development. In conclusion, the histological and immunohistochemical seasonal variations of ovaries in Meriones libycus support the hypothesis that seasonal fluctuations are indirectly involved in regulating reproduction, inducing significant changes in both ovarian morphology and its hormonal function.

Immunoexpression of gelatinase (MMP-2 and MMP-9) in the seminal vesicles and ventral prostate of Libyan jird (Meriones libycus) during the seasonal cycle of reproduction.

An immunohistochemical study of matrix metalloproteinases (MMP-2 and MMP-9) or gelatinase (gelatinase A and gelatinase B) was performed on the seminal vesicles and ventral prostate of the Libyan jird (Meriones libycus) collected in the Beni-Abbes area during breeding period (spring and early summer), during resting phase (late summer, autumn, winter) and from castrated animals in the spring. The work was done using the indirect immunohistochemistry protocol by amplification with streptavidin-biotin-peroxidase and AEC as chromogen. In the seminal vesicles, during the breeding period, an important immunohistochemical signal of MMP-2 and MMP-9 was observed in epithelial cells and smooth muscle cells (SMC) without any immunoexpression in the extracellular matrix (ECM) and secretion. During resting phase and in thirty days castrated Meriones libycus, the MMP-2 and MMP-9 immunoexpression was weak in the epithelial cells and persisted with the same intensity in the SMC. The ECM, with no immunostaining in active season, showed a pronounced immunoresponse of both the two gelatinase. Three days after castration, the MMP-9 immunohistochemical reaction in epithelial cells and SMC was as intense as during active season. A prolonged castration of 50 and 90 days resulted in the maintenance of the MMP-9 immunostaining in epithelial cells and SMC and its disappearance from the ECM, suggesting a slow process of regression. During the breeding period, in the ventral prostate, MMP-2 immunostaining was more important in the SMC than in epithelial cells. The MMP-9 immunoexpression pattern was the opposite, the epithelial cells showed a higher immunoreaction than SMC. ECM and secretion lacked MMP-2 and MMP-9 immunostaining. The ventral prostate lumen contained a granular secretion without any gelatinase immunolabelling and was hollowed by empty circular forms reflecting the disappearance of the product in these areas. Part of the secretion showed a positive MMP-2 and MMP-9 immunoreaction. The latter was subsequently filled and seemed involved in the progression of the secretion in the tubules, preventing their filling. In resting phase and in animals castrated since thirty days, the immunoreactivity of both the two gelatinases was maintained in the epithelial cells and in the SMC, and was absent in the ECM. The gelatinases are involved in the seasonal reproductive cycle of Meriones libycus.

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles.

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.

Ecophysiological responses of the seminal vesicle of Libyan jird (Meriones libycus) to the Saharan conditions: histological, morphometric and immunohistochemical analysis.

The Libyan jird (Meriones libycus) is a nocturnal Saharan Rodent submitted to a seasonal cycle of reproduction characterized by a short active period during spring and beginning of summer, and a long phase of sexual quiescence from the end of summer until the end of winter. During this cycle, the male reproductive organs, and more particularly seminal vesicles, experience some important weight and histological variations. During the breeding period, the wall of each seminal vesicle describes several folds radiating inside a broad lumen filled with a very abundant secretion. The wall is limited with high columnar epithelial cells surrounded with extracellular matrix restricted to some connnective fibres located in the narrow axis of the folds and in the chorion. The fibro-muscular wall is narrow. During sexual quiescence, the seminal vesicles regress. No secretion has been observed inside the lumen. The wall of lumen is now surrounded with a single cubic epithelium. The persistent epithelial folds possess a wide axis. The hypertrophied extracellular matrix is constituted with a very tight and abundant connective tissue. The fibro-muscular wall is thick. A quantitative morphometric study was performed with automatic image analysis that allowed to quantify the seasonal variations of the histological components. The numerical values obtained agree with the histological images observed, the epithelial surface area (microm2) is high in spring and significantly weak during sexual quiescence. The stroma and the fibro-muscular wall occupy an important surface area on sections during the resting period compared with the value collected during the active phase. The study of the apoptosis by TUNEL method revealed the presence of a considerable number of apoptotic nuclei in the epithelial fraction during the resting phase. The indirect immunohistochemical method allowed us to visualize the presence of types I and III collagen in the extracellular matrix, weak during the period of breeding, intense and diffuse during the resting season like in castrated Meriones libycus.