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).

Immunodistribution of RFamide-related peptide-3 (RFRP-3) during the seminiferous epithelium cycle in a desert rodent Psammomys obesus.

The Sand rat, Psammomys obesus, living northwest of the Algerian Sahara, presents a seasonal reproductive cycle. The purposes of this study were firstly to determine the stages of seminiferous epithelium cycle (SEC) by histological and morphometric analysis and secondly to investigate, for the first time, the testicular expression of RFamide-related peptide-3 (RFRP-3) during the SEC by immunohistochemistry. The results showed that the SEC consists of 14 stages according to the tubular morphology method. RFRP-3 was observed in both testicular compartments: the tubular and the interstitial. Leydig cells exhibited the highest RFRP-3 signal (30.73 % ± 4.80) compared to Sertoli cells (13-15 %). In the germline, RFRP-3 was detected during the late prophase I of meiosis in late pachytene, diplotene and metaphasic spermatocytes I. In addition, only round and triangular spermatids were positive during spermiogenesis. Referring to the SEC, it was found that the increased staining of RFRP-3 in spermatocytes I coincided with late pachytene of XI and XII stages (16.90 % ± 0.69 and 16.61 % ± 0.28, respectively). In spermatids, the labeling decreased in the triangular ones at stage IX (8.04 % ± 0.42). These results suggest the involvement of RFRP-3 in the control of SEC in P. obesus.

Kidney functional morphology variations between spring and winter in the Saharan male lizard Uromastyx acanthinura (Sauria, Agamidae), with special reference to body water economy.

To better understand the adaptive mechanisms in Uromastyx acanthinura to the seasonal variations in the arid environment, the present study aimed to explore the kidney functional morphology involved in body water economy. These investigations were carried out by the histological, histochemical and immuno-histochemical methods using conventional light microscopy. The glomeruli number is estimated at 2000 per kidney. The glomeruli size is rather small and decreases significantly in winter. Interestingly, the proximal convoluted tubule (PCT) is long and divided into two different segments which is one of the particularities of this species. Both of the distal convoluted tubule (DCT), connecting tubule (CnT) and collecting duct (CD) epithelium contains mucous cells. The nature and intensity of these mucous secretions vary according to seasons. The evident hypertrophy of the secondary collecting duct (SCD) and tertiary collecting duct (TCD) epithelium is related to the high secretory activity during spring, corresponding to the sexual segment of kidney (SSK). Labeling with anti α-smooth muscle actin-1 showed a thick layer of mucularis surrounded the entire CD. Also, the mesangium of glomeruli contains myofibroblasts. All these renal structural characteristics involved in body water economy may be considered as an adaptive mechanisms of U. acanthinura to resist to dehydration and cope with seasonal variations in the arid environment.

Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus.

OBJECTIVE: Numerous studies have shown that a methionine-rich diet induces hyperhomocysteinemia (Hhcy), a risk factor for cardiovascular diseases. The objective of the present study was to determine the involvement of Hhcy in cardiac remodeling in the sand rat Psammomys obesus. MATERIALS AND METHODS: An experimental Hhcy was induced, in the sand rat Psammomys obesus, by intraperitoneal injection of 300 mg/kg of body weight/day of methionine for 1 month. The impact of Hhcy on the cellular and matricial structures of the myocardium was analyzed with histological techniques (Masson trichrome and Sirius red staining). Immunohistochemistry allowed us to analyze several factors involved in myocardial remodeling, such as fibrillar collagen I and III, metalloproteases (MMP-2 and -9) and their inhibitors (TIMP-1 and -2), TGF-ß1 and activated caspase 3. RESULTS: Our results show that Hhcy induced by an excess of methionine causes, in the myocardium of Psammomys obesus, a significant accumulation of fibrillar collagens I and III at the interstitial and perivascular scales, indicating the appearance of fibrosis, which is associated with an immuno-expression increase of TGF-ß1, MMP-9 and TIMP-2 and an immuno-expression decrease of MMP-2 and TIMP-1. Also, Hhcy induces apoptosis of some cardiomyocytes and cardiac fibroblasts by increasing of activated caspase 3 expression. These results highlight a remodeling of cardiac tissue in hyperhomocysteinemic Psammomys obesus.

Immunohistochemical Localization of Calpains in the Amphibian Xenopus laevis.

Though histochemical techniques have been used for decades, they are still very important in basic research. They make it possible to work on fixed tissues and provide a large amount of information in a relatively short time and at a low cost. Here we describe methods for indirect immunohistochemistry and immunofluorescence on sections of tadpoles and tissues of adult amphibians belonging to the species Xenopus laevis. The objective is to localize calpains within tissues in order to understand their involvement in cellular processes.

Immunolocalization of RFamide-related peptide 3 in a desert rodent Gerbillus tarabuli during seminiferous epithelium cycle.

Gerbillus tarabuli is a nocturnal seasonal breeder desert rodent with a main breeding season in spring and summer, and sexual quiescence in winter. This species is an interesting model for studying testis function in rodents. Therefore, the present study was performed firstly to investigate the stages of seminiferous epithelium cycle of Gerbillus tarabuli with a histological, morphometric and statistical study. And secondly to investigate the expression and possible variations in cellular distribution of RFamide-related peptide-3 (RFRP-3) - the mammalian ortholog of avian gonadotropin-inhibitory hormone (GnIH) - during seminiferous epithelium cycle using immunohistochimestry. Our results showed for the first time that the seminiferous epithelium cycle in Gerbillus tarabuli comprises 14 well-defined stages according to the tubular morphology method. The seminiferous epithelium thickness showed a significant difference during the epithelium cycle, thus it was the only morphometric classification criterion of seminiferous epithelium cycle in Gerbillus tarabuli. The immunohistochemical study reveals, for the first time, the presence of RFRP-3 in Gerbillus tarabuli testes, in both testicular compartments: the tubular and the interstitial. RFRP-3 is expressed differently according to the seminiferous epithelium cycle, RFRP-3 seemed to be more expressed at the stages V-VII and XIII. RFRP-3 was detected in Sertoli cells (≈12%), spermatocytes I (≈19%), round and elongated spermatids (≈13%), and with a more important signal in Leydig cells (26.87%±0.07). These results indicated the importance of RFRP-3 in testicular function in Gerbillus tarabuli; its expression at the interstitial and germinal levels argues in favor of an involvement in androgens synthesis and in spermatogenesis specifically in meiosis and spermiogenesis. This action seems primordial from stages V-VII and XIII. Also, the study of the seminiferous epithelium cycle will enrich the histological identity of the species.

Study of the hydromineral regulation of Typhlonectes compressicauda according to the seasonal variation.

INTRODUCTION: Typhlonectes compressicauda is a viviparous gymnophionan amphibian living in tropical areas of South America. This lengthened amphibian is submitted to seasonal variations characterized by the rainy season (from January to June) and the dry season (from July to December). The mineral homeostasis in amphibians is partly ensured by the neurohormones arginine-vasotocin (AVT), and mesotocin (MST). These two hormones were localized in the hypothalamus, and their receptors, mesotocin receptors (MTR) and vasotocin receptors (VTR2) in the kidney. The aim of the study was to better understand the physiology of the hydromineral regulation of the studied species. MATERIAL AND METHODS: The specimens of T. compressicauda male and female adult were divided into 6 groups: males in the rainy season, males in the dry season, females pregnant in the rainy season, females pregnant in the dry season, females not pregnant in the rainy season, females not pregnant in the dry season. We studied the expression of hormones (AVT, MST) and their receptors (MTR, VTR2) in the hypothalamus and the kidney, respectively, by immunohistochemical and histological techniques. We also studied the expression of aquaporin-2 (AQP2), a water-channel protein in the kidney. RESULTS: We found that the MST (diuretic hormone) and its receptor were more intensively expressed during the rainy season, whereas the period of maximal AVT (anti-diuretic hormone) and VTR2 expression was the dry season. A quantitative analysis showed significant differences in the number of labeled cells in the hypothalamus depending on the seasonal variation. The expression of AQP2 was observed in renal tubules during both seasons with an increased intensity during the dry season. CONCLUSION: The expression of the MST/AVT in brain, their receptors MTRs/VTR2, and AQP2 in kidney changed in T. compressicauda according to the seasonal variations. A direct relationship between the seasonal cycle and reproduction cycle was demonstrated in this species.

Seasonal changes in plasma testosterone and cortisol suggest an androgen mediated regulation of the pituitary adrenal axis in the Tarabul's gerbil Gerbillus tarabuli (Thomas, 1902).

In the desert gerbil Gerbillus tarabuli (Thomas, 1902), cortisol is the main glucocorticosteroid produced by the adrenal glands. Plasma cortisol concentrations show highest values when testosterone is reduced and lowest values during the breeding season which occurs from early winter to late spring. In order to specify the implication of testicular androgens in these corticosteroid seasonal variations we investigated the effects induced by gonadectomy performed during the breeding season on the pituitary adrenal axis. The animals collected in winter were assessed into three groups: sham-operated (Controls; n=13), gonadectomised (GDX; n=13) and testosterone replaced gonadectomised (GDX+T; n=13). Physiological replacement of testosterone enanthate (75µg/100gb.w./twice daily) was applied during one week, while GDX group received the vehicle (40µL sesame oil) alone. The right adrenal glands removed from euthanized animals were fixed for histomorphometry and androgen receptors (ARs) immunohistochemistry and the left ones were frozen with plasma samples until hormonal assays. Gonadectomy induces the enlargement of the adrenal cortex essentially due to that of zonae fasciculata (ZF) and reticularis (ZR) and perimedullary connective tissue which is abundant in the gerbil adrenals. The ARs immunostaining present at both cytoplasmic and nucleus level, is enhanced intensely in the ZR and moderately in the ZF and zona glomerulosa (ZG) cells. GDX group shows reduced plasma ACTH concentration (p=0.0126) by 61% despite the increase in cortisol concentration occurring both in plasma (+216%; p=0.0436) and adrenal tissue (+117%; p=0.0348). Plasma aldosterone is also enhanced significantly (p=0.0147) by 189% but androstenedione synthesis increased in adrenal tissue (p=0.0459) by 65% instead a decrease at circulatory level (p=0.0355) by 58% due to lack of testicular origin. So, testosterone deprivation activates corticosteroidogenesis also evidenced by the adrenal structure changes and the gonadectomy-induced increase in the plasma cholesterol. All of the gonadectomy-induced responses are reversible after physiological testosterone replacement. We conclude that the assessment of circulating adrenocorticotropic hormone (ACTH) concentrations together with cortisol levels essentially, reflecting the hypothalamic-pituitary adrenal (HPA) axis feedback loop control during the annual endogenous changes of testosterone secretion, represents a well-adapted response of this desert species living in an extreme environment.

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.

The role of homocysteine in seminal vesicles remodeling in rat.

INTRODUCTION: Elevated plasma homocysteine (Hcy) levels have been associated with several tissue injuries including heart and liver fibrosis. In these diseases, hyperhomocysteinemia (Hhcy) plays a major role in modulating the alteration of the balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMPs), leading to the pathological accumulation of extracellular matrix (ECM) proteins. Since the effect of Hhcy on ECM of seminal vesicle was not studied, the aim of our research was to check if Hcy can induce a remodeling within seminal vesicles ECM. MATERIAL AND METHODS: The study was conducted in 22 adult male Wistar rats. The rats were divided into two groups: a control group, which received standard diet and tap water; the treated group received the same diet and water supplemented with solution of L-methionine (200 mg/kg b.w./day) for 6 months. Plasma homocysteine concentration was measured. Histological changes were observed with light microscope. The presence of collagen I and III and metalloproteinases (2, 3, 7 and 9) in the seminal vesicles was examined using immunohistochemistry and Western blotting. RESULTS: Plasma Hcy levels increased significantly after methionine treatment and interfered significantly with body weight in treated rats. The content of fibrillar collagens (I and III) in the wall of seminal vesicles was elevated in hyperhomocysteinemic rats. Moreover, we found that hyperhomocysteinemia increased the expression of MMP-2, -3, -7 and -9 in seminal vesicles of experimental rats. CONCLUSIONS: Increased plasma concentration of Hcy accompanied by the accumulation of collagen and upregulation of MMPs in rat seminal vesicles might contribute to the remodeling of seminal vesicles.

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.

Seasonal morphophysiological variations in the prostatic complex of the Tarabul's gerbil (Gerbillus tarabuli).

Gerbillus tarabuli is a nocturnal Saharan rodent which has an annual reproductive cycle characterized by the reproductive activity in spring and a long phase of sexual quiescence in other seasons. We describe the morphology and hormonal regulation of the prostatic complex of this rodent in the two periods, based on anatomical, histological, morphometric, and immunohistochemical analyses. The organisation of this prostatic complex is similar to that reported for Meriones unguiculatus, but different from the prostate of Psammomys obesus, the rat, and the mouse. In addition to the anterior lobes, ventral lobes, and dorsal lobes, the prostatic complex of Gerbillus tarabuli, also includes dorsolateral lobe. Each lobe is composed of a fibro-muscular stroma surrounding a glandular epithelium. Dorsolateral lobes are easily distinguishable by their big volume. The prostate grows and regresses cyclically throughout the year. During the resting season, ventral lobes and anterior lobes showed atrophy, with a significant decrease in both epithelial height and supranuclear area size, and a strong thickening of the fibro-muscular compartment. In dorsal lobes, the epithelial and stromal compartments atrophied and regenerated simultaneously, whereas in dorsolateral lobe the thickness of the epithelium, the supranuclear zone and the stroma increased during resting period. Furthermore, seasonal variations were observed in the distribution and expression of both androgen receptors, and estrogens receptors. Expression patterns of all receptors were lobe-specific. In conclusion, both androgens and estrogens are involved in the homeostasis and regulation of the prostate in Gerbillus tarabuli. Dorsolateral lobe seems to be controlled by a different mechanism than other lobes.

17ß-estradiol effect on testicular ß-endorphin expression in Psammomys obesus.

INTRODUCTION: Testicular function in the sand rodent Psammomys obesus is subjected to seasonal alternations with a trigger of spermatogenesis in winter and a total quiescence which extends from late spring to summer. The aim of this study was to investigate the distribution of b-endorphin in the testis at the period of winter sexual activity and at its summer regression, and assess the effect of 17 ß-estradiol treatment on testicular morphology and b-endorphin expression. MATERIAL AND METHODS: The adult males were grouped into 4 groups (rest group, sexually active group, rest treated with 17ß-estradiol group and controls at sexual rest injected with olive oil, n = 5 in each group). Using anti-serum against b-endorphin, we studied its testicular expression by Western blot and cellular location by immunohistochemical (IHC) method, respectively. RESULTS: We detected by Western blot a peptide of 3.5 kDa molecular weight corresponding to b-endorphin only in sexually resting and control males. The 17ß-estradiol treatment induced a clear reduction in the b-endorphin band expression compared with the latter. These results were confirmed by the IHC analysis since b-endorphin was only observed in the testis at sexual rest and in controls, in majority of seminiferous tubules at the level of germ cells. The intensity of IHC labeling was significantly different between spermatogonia and spermatocytes I or round spermatids which revealed the strongest labeling. The intense immunoreactivity was also located in Leydig cells and highly significantly varied compared to the germ cells. The 17 ß-estradiol treatment decreased significantly the ß-endorphin signal in germ cells but not in Leydig cells. CONCLUSION: The 17ß-estradiol treatment induces a repressive effect on seasonal testicular endorphinergic system in P. obesus and this action targets exclusively the germ cells.

Protective effects of probiotic Lactobacillus plantarum BJ0021 on liver and kidney oxidative stress and apoptosis induced by endosulfan in pregnant rats.

Endosulfan (EDS) is one of the most widely organochlorine insecticide used in many parts of the world, although it is currently banned or severely restricted in use in some countries. EDS causes a variety of negative effects in non-target species including humans. Therefore, the aim of the present study was to investigate the possible protective effects of Lactobacillus plantarum BJ0021 on toxicity, oxidative stress, and apoptosis induced by EDS intoxication on liver and kidneys of pregnant rats. This pesticide induced a significant increase in total cholesterol, alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea and creatinine in serum, while urinary urea and creatinine were lower than those of the control group. In the liver and kidney, lipid peroxidation increased significantly, the antioxidant levels, such as superoxide dismutase (SOD) and catalase (CAT) were markedly depressed and TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) revealed more apoptotic cells. In contrast, co-administration of L. plantarum BJ0021 to EDS-treated animals ameliorated most of these biochemical parameters, but the activity of antioxidant enzymes (SOD, CAT) did not modify and the number of apoptotic nuclei remained significantly raised in kidney compared to control. In conclusion, the administration of L. plantarum BJ0021 decreased apoptosis and might play a protective role in reducing toxicity of EDS in pregnant rats.

Protective effects of probiotic Lactobacillus plantarum BJ0021 on liver and kidney oxidative stress and apoptosis induced by endosulfan in pregnant rats.

Endosulfan (EDS) is one of the most widely organochlorine insecticide used in many parts of the world, although it is currently banned or severely restricted in use in some countries. EDS causes a variety of negative effects in non-target species including humans. Therefore, the aim of the present study was to investigate the possible protective effects of Lactobacillus plantarum BJ0021 on toxicity, oxidative stress, and apoptosis induced by EDS intoxication on liver and kidneys of pregnant rats. This pesticide induced a significant increase in total cholesterol, alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea and creatinine in serum, while urinary urea and creatinine were lower than those of the control group. In the liver and kidney, lipid peroxidation increased significantly, the antioxidant levels, such as superoxide dismutase (SOD) and catalase (CAT) were markedly depressed and TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) revealed more apoptotic cells. In contrast, co-administration of L. plantarum BJ0021 to EDS-treated animals ameliorated most of these biochemical parameters, but the activity of antioxidant enzymes (SOD, CAT) did not modify and the number of apoptotic nuclei remained significantly raised in kidney compared to control. In conclusion, the administration of L. plantarum BJ0021 decreased apoptosis and might play a protective role in reducing toxicity of EDS in pregnant rats.

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.

Relationships between calpains and glutamate- or kainate-induced apoptosis in Xenopus laevis tadpoles.

Cell glutamate-damage induced by overstimulation of ionotropic receptors is initiated by modification of the intracellular Ca2+ homeostasis and the concomitant activation of Ca2+-dependent cysteine proteases, the calpain and caspase families. The resultant cleavage of target molecules mediates a critical function in the execution of the cell death. In this work, we investigated relationships between the activity of calpain and glutamate-orkainate-induced apoptosis in several organs of Xenopus laevis tadpole. Animals (stage 48) were incubated for 3 hours with glutamate (30-120 mM) or kainate (0.015-0.75 mM) and the rise of both apoptosis and calpain was observed in several organs. Our results indicated that glutamate (120 mM) or kainate (0.15 mM) exposure induced cell death with apoptotic features. The toxic effects of drugs into the organs were variable. Apoptosis was probably not the only form of cell death and option of necrosis or apoptosis was depending on the stimulation degree of the receptor, i.e. the receptor type, intensity and time course of molecule exposure. The increase of ubiquitous calpain was not correlated with the peak of apoptosis, suggesting the role of calpain in cell death was complex: calpain and caspase pathways were tightly interrelated in the glutamate- or kainate-induced cell death and the contribution of calpain to another type of death than apoptosis was perhaps preferred.

Postnatal changes in testicular development and androgen receptors immunolocalization in D'Man ram lambs.

The purpose of this study was to characterize testicular development in D'Man ram lambs, focusing primarily on androgen receptors (ARs) immunolocalization in the adenohypophysis and testis that is not still known in the D'Man ram lamb. Lambs (n = 12) were divided into four groups (three lambs per group). Adenohypophysis and testis were fixed and paraffin embedded; cross-section (3 µm) were stained and evaluated with immunohistochemistry. Testis weight increased at a greater rate between two and five months after birth, which was associated with remarkable changes in testicular histology, including significant increases in the diameter of seminiferous tubules. Spermatogenesis started between three and five months after birth; lumen and elongated spermatids were observed for the first time in three and four months-old animals respectively. ARs detected with immunohistochemistry were located in the nuclei and cytoplasm of adenohypophysis cells, and only in nuclei of testis cells (Leydig, Sertoli, peritubular myoid and germ cells).

Ecotoxicological effects of an aged TiO2 nanocomposite measured as apoptosis in the anecic earthworm Lumbricus terrestris after exposure through water, food and soil.

Titanium dioxide nanoparticles seem to have a low toxicity to terrestrial organisms, though few studies are published in this area. TiO(2) used in sunscreens are nanocomposites where TiO(2) has been coated with magnesium, silica or alumina, as well as amphiphilic organics like polydimethyl siloxane (PDMS), and these coatings are modified by ageing. We assessed the ecotoxicity and propensity for bioaccumulation of an aged TiO(2) nanocomposite used in sunscreen cosmetics, and its potential effect on the frequency of apoptosis in different earthworm tissues. The earthworm Lumbricus terrestris was exposed to the TiO(2) nanocomposite for 7 days in water or 2-8 weeks in soil with the nanocomposite mixed either into food or soil at concentrations ranging from 0 to 100 mg kg(-1). Apoptosis was then measured by immunohistochemistry and Ti localized by XRF microscopy. Results showed no mortality, but an enhanced apoptotic frequency which was higher in the cuticule, intestinal epithelium and chloragogenous tissue than in the longitudinal and circular musculature. TiO(2) nanoparticles did not seem to cross the intestinal epithelium/chloragogenous matrix barrier to enter the coelomic liquid, or the cuticule barrier to reach the muscular layers. No bioaccumulation of TiO(2) nanocomposites could thus be observed.