Defining the boundaries of the testis angiotensin I-converting enzyme ectodomain

Numerous cytokines, receptors, and ectoenzymes, including angiotensin I-converting enzyme (ACE), are shed from the cell surface by limited proteolysis at the juxtamembrane stalk region. The membrane-proximal C domain of ACE has been implicated in sheddase-substrate recognition. We mapped the functional boundaries of the testis ACE ectodomain (identical to the C domain of somatic ACE) by progressive deletions from the N- and C-termini and analysing the effects on catalytic activity, stability, and shedding in transfected cells. We found that deletions extending beyond Leu37 at the N-terminus and Trp616 at the C-terminus abolished catalytic activity and shedding, either by disturbing the ectodomain conformation or by inhibiting maturation and surface expression. Based on these data and on sequence alignments, we propose that the boundaries of the ACE ectodomain are Asp40 at the N-terminus and Gly615 at the C-terminus.     

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.     

Molecular and genetic regulation of testis descent and external genitalia development

Testicular descent as a prerequisite for the production of mature spermatozoa and normal external genitalia morphogenesis, and therefore facilitating copulation and internal fertilization, are essential developmental steps in reproduction of vertebrate species. Cryptorchidism, the failure of testis descent, and feminization of external genitalia in the male, usually in the form of hypospadias, in which the opening of the urethra occurs along the ventral aspect of the penis, are the most frequent pediatric complications. Thus, elucidating the molecular mechanisms involved in the regulation of testis descent and the formation of external genitalia merits a special focus. Natural and transgenic rodent models have demonstrated both morphogenic processes to be under the control of a plethora of genetic factors with complex time-, space-, and dose-restricted expression pattern. The review elucidates the molecular mechanisms involved in the regulation of testis descent and the formation of external genitalia and, wherever possible, assesses the differences between these rodent animal models and other mammalian species, including human.     

Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice

We investigated the modifications in endogenous antioxidant capacity and oxidative damage in the brain, liver, kidney and testis in mice exposed to bisphenol A (BPA), an environmental endocrine disrupter. Mice were exposed to BPA throughout embryonic/fetal life and during lactation by feeding their pregnant/lactating mothers BPA at 5 or 10 microg per milliliter of drinking water. At the age of four weeks, male mice were sacrificed. Exposure to BPA increased the activity of catalase and glutathione peroxidase in the liver and kidney, respectively. It also increased thiobarbituric acid-reactive substances in the brain, kidney and testis, and decreased the wet weight of the brain, kidney and testis. Our results suggest that exposure to BPA throughout embryonic/fetal life and during infancy induces tissue oxidative stress and peroxidation, ultimately leading to underdevelopment of the brain, kidney and testis.     

Effects of gold on testicular steroidogenic and gametogenic functions in immature male albino rats

The present study was undertaken to evaluate the effects of gold chloride, a metallic earth salt, on steroidogenic and gametogenic functions of testis in immature rats. Immature rats of Wistar strain, were injected (s.c.) with gold chloride at the dose of 0.3 mg and 0.5 mg/kg body weight/day for 26 days. All the treated animals along with the vehicle-treated controls were sacrificed 24 hours after last injections. Testicular steroidogenic activity was evaluated by measuring the activities of two steroidogenic key enzymes, Delta5-3beta-hydroxysteroid dehydrogenase (Delta5-3beta-HSD) and 17-beta hydroxysteroid dehydrogenase (17-beta HSD). Gametogenic capacity was determined by counting the number of germ cells at stage VII of seminiferous cycle. Plasma levels of testosterone (T) was measured by radioimmunoassay (RIA). Administration of gold chloride at a dose of 0.3 mg/ kg body weight for 26 days led to insignificant changes of testicular Delta5-3beta-HSD,17beta-HSD activities and gametogenesis along with plasma T. In contrast 0.5 mg gold chloride treatment for 26 days caused a significant increase in plasma T (p < 0.001) along with stimulation of testicular Delta5-3beta-HSD activity (p < 0.001) and 17beta-HSD activity (p < 0.001). Gametogenic activity exhibited a significant increase in the number of step 7 spermatids (7Sd) (p < 0.001) at stage VII of seminiferous cycle when compared to control. The results of our experiment suggest that gold chloride treatment might be associated with significant stimulatory effects on testicular activities. Furthermore, since hormonal changes, altered steroidogenic enzymes and gametogenic activities were evident to a specific dose of gold chloride treatment, our data may have some clinical implication on the stimulation of fertility.     

Involvement of K+ channels and calcium-dependent pathways in the action of T3 on amino acid accumulation and membrane potential in Sertoli cells of immature rat testis

The purpose of this study was to investigate the involvement of calcium in K+ currents and its effects on amino acid accumulation and on the membrane potential regulated by tri-iodo-L-thyronine (T3) in Sertoli cells. Immature rat testes were pre-incubated for 30 min in Krebs-Ringer bicarbonate buffer and incubated for 60 min in the presence of [14C]methylaminoisobutyric acid with and without T3 or T4 (dose-response curve). Specific channel blockers or chelating agents were added at different concentrations during pre-incubation and incubation periods to study the basal amino acid accumulation and a selected concentration of each drug was chosen to analyze the influence on the stimulatory hormone action. All amino acid accumulation experiments were carried out in a Dubnoff metabolic incubator at 32 degrees C, pH 7.4 and gassed with O2:CO2 (95:5; v/v). Seminiferous tubules from immature Sertoli cell-enriched testes were used for the electrophysiology experiments. Intracellular recording of the Sertoli cells was carried out in a chamber perfused with KRb with/without T3, T4 or blockers and the membrane potential was monitored. We found that T3 and T4 stimulated alpha-[1-14C] methylaminoisobutyric acid accumulation in immature rat testes and induced a membrane hyperpolarization in Sertoli cells. The action of T3 on amino acid accumulation and on the hyperpolarizing effect was inhibited by the K(+)-ATP channel blocker tolbutamide as well as the voltage-dependent Ca2+ channel blocker verapamil. These results clearly demonstrate for the first time the existence of an ionic mechanism related to Ca2+ and K+ fluxes in the rapid, nongenomic action of T3.     

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin <Emphasis Type="Italic">Anthocidaris crassispina</Emphasis>

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles (zymogen granules) and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in empty vacuoles inside the remnant body. The empty vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of cheese-hole-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (no. 7000989). This work also was supported by a grant from Minpromnayka NS 1219.2003.4     

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.This work was supported by Research Initiate Grant funded by Faculty of Science, Mahidol University to W.W.