Distribution of the Novel RFRP-3/receptors system in the epididymis of the seasonal desert rodent, Gerbillus tarabuli, during sexual activity.

RFamide-related peptide (RFRP-3), the Mammalian ortholog of the Avian gonadotropin-inhibitory hormone (GnIH), is a novel neuropeptide known for its inhibitory regulatory effect on reproduction in various mammalian species. However, a stimulatory action has been reported. This paper aims to: i) study the histology of the epididymis (caput) of Gerbillus tarabuli during the breeding period; and ii) to determine the distribution of the "RFRP-3/receptors system" in the epididymis (caput) of this desert rodent during the active season, and thus, to inspect its potential local interfering in sperm maturation. For that, immunohistochemistry was performed to detect the epididymal immunolocalizations of the three molecules, RFRP-3, GPR147, and GPR74. This is the first report of the epididymis histology in Gerbillus tarabuli, as it is the first evidence of the existence of the RFRP-3/Receptor system in the same organ of the same species. During the breeding season, moderate immunostaining of the RFRP-3/receptors system was present in the caput epididymis' epithelial parts (basal and principal cells) and spermatozoa. In contrast, these three molecules were absent in the peritubular and muscle coat's myoid cells and of the interstitial part of the caput epididymis. The results suggest that the epididymis is a potential source of RFRP-3 in the desert Rodent, Gerbillus tarabuli, which may function as a paracrine and/or autocrine factor affecting the main epididymis' function: sperm maturation.

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.

Calpains expression during Xenopus laevis development.

Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.

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.

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.

Immunocytochemical localization and ultrastructural study of gonadotroph cells in the female desert lizard Uromastyx acanthinura.

The pars distalis from the pituitary gland of adult female desert lizards (Uromastyx acanthinura), captured during vitellogenesis (late may) and hivernal period, was studied with immunocytochemical methods using specific antisera against human FSH (hFSH) and LH (hLH). The immunostaining with anti-hLH and anti-hFSH allowed the identification of only FSH-like containing cells. The FSH-like immunoreactive cells were affected differently by a physiological stage and showed some heterogenous cytological characteristics. During vitellogenesis, four aspects of rostral FSH-like immunoreactive cells could be recognized. The expression of FSH-like in mainly immunoreactive cells was parallel to an intense synthetic activity and to the presence of ultrastructural features indicating an intense release of the hormone. This release was considerably altered in winter, the immunoreactive cells stored an important amount of secretion granules which increased in size and undergo a crinophagic process.

Factorial designs: an efficient approach to choosing the main factors influencing growth and hydrolase production by Tetrahymena thermophila.

A fractional factorial design with eight trials was applied to select and model the effects of major factors, individually and in combination, on improving Tetrahymena thermophila growth and enzyme production. Regulated pH at 6.8 and olive oil at 0.5% (v/v) showed positive effects on fermentation, and increased cell growth parameters including generation time and maximal population formation. Lipase and protease production were also improved by these factors and were favoured by cultivation of Tetrahymena in darkness. This statistical experiment offers a beneficial and rapid screening procedure to select the most effective combination of factors influencing fermentation processes.

Synaptic and non-synaptic AMPA receptors permeable to calcium.

For a long time, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors permeable to calcium have been considered to be either non-existent or as "atypical". There is now ample evidence that these receptors exist in numerous regions of the nervous system and in many neuronal as well as non-neuronal cell populations. This evidence has been accumulated by several methods, including electrophysiological recording, calcium imaging and cobalt-loading. Functional AMPA receptors permeable to calcium are already expressed at very early stages of embryonic development, well before the onset of synaptogenesis. They are probably involved in the paracrine signaling necessary for construction of the nervous system before becoming involved in synaptic transmission. In immature cells, cyclothiazide strongly increases the steady-state level of responses not only to AMPA, but also to kainate. Ingestion, during pregnancy, of food or drug substances that can cross the placental barrier and act upon the embryonic receptors may constitute a risk for normal development. In the adult nervous system, synaptic as well as non-synaptic (paracrine) AMPA receptors permeable to calcium are probably widely expressed in both glial and neuronal cells. They may also participate in controlling some aspects related to adult neurogenesis, in particular the migration of newly formed neurons.

Expression of inducible nitric oxide synthase in tumors in relation with their regression induced by lipid A in rats.

It is well documented that nitric oxide (NO) is an effector molecule of macrophage-mediated tumor cell toxicity in vitro; however, little is known about the role of NO in the antitumor immune response in vivo. We have developed a treatment protocol using lipid A. We have investigated the effects of lipid A on inducible NO synthase (NOS II) expression and evolution inside tumors during the course of treatment. Lipid A (OM-174) treatment induced tumor regression in rats bearing established colon tumors. Furthermore, NO was synthesized and secreted inside the tumors of lipid A-treated rats, as demonstrated by the increase of NOS II mRNA and NOS II content in the tumors, as well as of NOS II activity and NO production. During treatment, NOS II was localized in tumor cells only. Lipid A had no direct effect on tumor cells in vitro, while the combination of interferon gamma (IFN-gamma) plus interleukin-1 beta (IL-1beta) induced production of NO by tumor cells which was cytostatic. The content of IFN-gamma and IL-1beta in tumors was enhanced during lipid A treatment; this is in agreement with an indirect effect of lipid A in vivo via the IFN-gamma and IL-1beta pathways.

AMPA/kainate receptors permeable to divalent cations in amphibian central nervous system.

Glutamate receptors have been studied extensively in mammals but less explored in lower vertebrates. These receptors are present in amphibians. Using a recent method based upon agonist-induced cobalt uptake, we were able to detect the presence of functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors permeable to divalent cations in tadpoles and in adults. The uptake specificity was checked by co-application of an antagonist. We studied the distribution of receptor-bearing cells in the principal brain regions. The distribution was similar in the two species studied: Rana esculenta (green frog) and Bufo bufo (common toad). The high number of cobalt-positive cells suggests that the AMPA/kainate receptors permeable to divalent cations play an important role in the anuran nervous system.

Visualization of gene expression of prolactin receptors (PRL-R) by in situ hybridization, in Typhlonectes compressicaudus, a gymnophionan amphibian.

The expression of the short and long forms of prolactin receptors (PRL-R) mRNA was studied in various types of tissue from Typhlonectes compressicaudus, an amphibian, by quantitative in situ hybridization. Both forms were expressed in all the types of tissues studied. In the liver, small intestine and hypophysis, the mRNA coding for the short form of PRL-R was more strongly expressed than the mRNA coding for the long form and vice-versa for the stomach, spleen and kidneys. In the female liver, quantification showed a higher value of mRNA expression mid-way through pregnancy than during the sexual inactivity period. This result was found to be correlated with the reserve function of the liver. In the kidney and small intestine, the presence of PRL-R was correlated with the hydromineral function. A comparison with certain mammals was also established. These results confirm the ubiquity of PRL effects on metabolic regulation, and suggest a phylogenic conservation of its receptors.

Effect of iturin A, a lipopeptide from Bacillus subtilis on morphology and ultrastructure of human erythrocytes.

Iturin A, a lipopeptide from Bacillus subtilis, induced morphological modifications of human erythrocytes and a concomitant release of exovesicles. The modifications depended on the lipopeptide concentration and on the incubation time. Crenation of cells occurred while invaginations appeared when the incubation time was increased. This suggested that iturin A was gradually translocated from the outer layer into the inner half of the bilayer. For one lipopeptide concentration, heterogeneity in the form of the erythrocytes was observed which could result from heterogeneity in the self-association of iturin A. The lipid composition of exovesicles was markedly different from that of the native erythrocytes; they were enriched in cholesterol, sphingomyelin and phosphatidylcholine. Freeze-fracture showed that the ultrastructure of the protoplasmic face of the membrane was not modified by iturin A but, when erythrocytes were prefixed with glutaraldehyde prior to iturin A treatment, irregular smooth protuberances appeared with a decrease of intramembranous particles density. These protuberances which covered the complete area of the cell may have been complex associations of iturin A, phospholipids and cholesterol.