Cold-inducible RNA-binding protein (CIRP) regulates target mRNA stabilization in the mouse testis

Cold-inducible RNA-binding protein (CIRP) is an RNA-binding protein that is expressed in normal testis and down-regulated after heat stress. Recent studies suggest that CIRP contributes to male fertility problems but the mechanisms are unclear. The purpose of this study was to identify the likely mechanism of CIRP in reproduction. Based on the RNA-Binding Protein Immunoprecipitation-Microarray (Chip) Profiling (RIP-Chip) and biotin pull-down assays, we found that the mRNAs binding with CIRP in testis were mostly associated with translation regulator activity, antioxidant activity, envelope and reproduction, including important mRNAs related to male infertility. We also discovered that (Un)(n ? 2) was the possible core recognition sequence, and the binding mRNAs increased their stabilization. Our results improve our understanding of the mechanism by which heat stress causes male infertility.     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

Immunohistochemical analysis of cAMP response element-binding protein in mouse testis during postnatal development and spermatogenesis

Basal activity and cellular localization of cAMP response element-binding protein (CREB) was examined in mouse testis during postnatal development and spermatogenesis. Testes of ICR mice sampled on postnatal day (PND) 3, 7, 14, 21, 28, 35, 42, and 49 were analyzed using Western blotting. Basal CREB activity was significantly higher in early phase (PND 3–7) developing testes than in intermediate- and late-phase developing (PND 14–42) and adult testes (PND 49). Furthermore, immunohistochemical analysis demonstrated the change of CREB phosphorylation in various testicular cell types during postnatal development. In particular, CREB phosphorylation in seminiferous tubules of the adult testis varied according to the spermatogenic cycle, while phosphorylation was evident in spermatogonia during all stages. Phosphorylation was moderate in pachytene spermatocytes of stages I–III and intense in round and elongate spermatids of spermiogenesis in stages XII–IX. These results suggest that CREB plays an important role in cell proliferation and differentiation in the early phase of postnatal development and spermatogenesis of mouse testis.     

Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development

Up to the midblastula stage, cell division in Xenopus is directed by maternal mRNAs and proteins that are inherited by the fertilized egg. We have isolated cDNA clones for mRNAs that undergo either adenylation or deadenylation during this developmental period. Coincidental with this poly(A) metabolism are changes in polysomal localization. The relationship between polyadenylation and translation is discussed.     

Galactosyl receptor in human testis and sperm is antigenically related to the minor C-type (Ca2+-dependent) lectin variant of human and rat liver

Galactosyl receptor, a cell surface Ca²⁺-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca²⁺-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization. © 1995 Wiley-Liss, Inc.     

Neonatal exposure to endocrine disruptors suppresses juvenile testis weight and steroidogenesis but spermatogenesis is considerably restored during puberty

Neonatal exposure to endocrine disruptors induces developmental abnormalities in the male reproductive system. As to investigate whether neonatal exposure affects spermatogenesis in juvenile and pubertal testes, Sprague-Dawley rat pups were given various endocrine disruptors by a single injection on the day of birth at concentrations ranging between 4 microM and 40 mM and sacrificed on day 21 (juvenile) or 50 (puberty). The testes were weighed and examined histologically at each stage. Further, the metabolites of steroidogenesis were analyzed using normal-phase high performance liquid chromatography. Neonatal exposure significantly reduced testis weights and steroid biosynthesis of juveniles, but they were highly restored at puberty.     

A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis

The urokinase receptor, uPAR, which binds to the urinary-type plasminogen activator, controls matrix degradation in the processes of tissue remodeling, cell migration, and invasion. In the present study, we found a new urokinase receptor gene that encodes a 249-amino acid putative protein. Northern blot analysis showed specific expression in the testis of this gene, which we named the spermatogenesis-related gene (SGRG). In situ hybridization revealed a strong expression signal for SGRG in spermatogonia, but not in spermatocytes. Therefore, we conjecture that SGRG may regulate spermatocyte migration through breakdown of extracellular matrix protein barriers in spermatogenesis. Since SGRG is specifically expressed in spermatogonia, it provides an attractive candidate for development of a contraceptive vaccine.     

Temporal germ cell development strategy during spermatogenesis within the testis of the Ground Skink, Scincella lateralis (Sauria: Scincidae)

Ground Skink (Scincella lateralis) testes were examined histologically to determine the testicular organization and germ cell development strategy employed during spermatogenesis. Testicular tissues were collected from 19 ground skinks from Aiken County, South Carolina during the months of March-June, August, and October. The testes consisted of seminiferous tubules lined with germinal epithelia in which germ cells matured in close association with Sertoli cells. As germ cells matured, they migrated away from the basal lamina of the epithelia towards the lumina of the seminiferous tubules. The testes were spermatogenically active during the months of March, April, May, June, and October (largest seminiferous tubule diameters and epithelial heights), but entered a quiescent period in August (smallest seminiferous tubule diameter and epithelial height) where only spermatogonia type A and B and early spermatocytes were present in low numbers within the seminiferous epithelium. Although the testicular organization was similar to other amniotes, a temporal germ cell development strategy was employed during spermatogenesis within Ground Skinks, similar to that of anamniotes. Thus, this skink's germ cell development strategy, which also has been recently reported in all other major reptilian clades, may represent an evolutionary intermediate in terms of testicular organization between anamniotes and birds and mammals.     

Site-specific phosphorylation of Tau protein is associated with deacetylation of microtubules in mouse spermatogenic cells during meiosis

Tau is one of the microtubule-associated proteins and a major component of paired helical filaments, a hallmark of Alzheimer’s disease. Its expression has also been indicated in the testis. However, its function and modification in the testis have not been established. Here, we analyzed the dynamics of phosphorylation patterns during spermatogenesis. The expression of Tau protein and its phosphorylation were shown in the mouse testis. Immunohistochemistry revealed that the phosphorylation was strongly detected during meiosis. Correspondingly, the expression of acetylated tubulin was inversely weakened during meiosis. These results suggest that phosphorylation of Tau protein contributes to spermatogenesis, especially in meiosis.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

2‐D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild‐caught and hormone‐treated F1 fish

In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild‐caught males. To gain insights into the molecular mechanisms that may be altered in the F1 testis, we used 2‐D DIGE to compare the protein profiling of the testis of wild‐caught males at the spermiation stage with that of F1 males showing different stages of germ cell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins by MS/MS revealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germ cell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males.     

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa

Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry.Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.     

Belle is a Drosophila DEAD-box protein required for viability and in the germ line

DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development.     

Postnatal differentiation of the gametogenic and endocrine functions of the testis in the tree-shrew (Tupaia belangeri)

Summary Testicular development was studied in Tupaia belangeri (tree-shrew) from birth to sexual maturity. At birth the seminiferous cords contained peripheral supporting cells and centrally located gonocytes. Large foetal Leydig cells were prominent in the interstitium. The mitotic index of the gonocytes was low at birth and rose to peak levels at Day 20, following the regression of the foetal generation of Leydig cells, and during the nadir in circulating testosterone concentrations. Mitotic activity returned to low levels at Day 30 in association with the reappearance of differentiated Leydig cells and the first signs of increased androgenesis. The negative temporal relationship between mitogenesis and androgenic function suggests that the proliferation of the gonocytes does not require, and may be inhibited by, high titres of androgens. Post-mitotic development of the gonocytes occurred during a period of rising testosterone levels, and the first appearance of spermatogonia coincided with peak testosterone levels. This indicates that androgens may be specifically involved in the initiation of spermatogenesis. Spermatogenesis progressed to completion during a phase of declining testosterone levels. The precise temporal correlations established during post-natal development suggest that the tree-shrew is a suitable animal model for studies on the endocrine control of the initiation of spermatogenesis in primates.     

Stages and duration of the cycle of the seminiferous epithelium in oncilla (Leopardus tigrinus, Schreber, 1775)

Six adult Leopardus tigrinus (oncilla) were studied to characterize stages of the seminiferous epithelium cycle and its relative frequency and duration, as well as morphometric parameters of the testes. Testicular fragments were obtained (incisional biopsy), embedded (glycol methacrylate), and histologic sections examined with light microscopy. The cycle of the seminiferous epithelium was categorized into eight stages (based on the tubular morphology method). The duration of one seminiferous epithelium cycle was 9.19 d, and approximately 41.37 d were required for development of sperm from spermatogonia. On average, diameter of the seminiferous tubules was 228.29 μm, epithelium height was 78.86 μm, and there were 16.99 m of testicular tubules per gram of testis. Body weight averaged 2.589 kg, of which 0.06 and 0.04% were attributed to the testis and seminiferous tubules, respectively. In conclusion, there were eight distinct stages in the seminiferous epithelium, the length of the seminiferous epithelium cycle was close to that in domestic cats and cougars, and testicular and somatic indexes were similar to those of other carnivores of similar size.