Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

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.     

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

Background

Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues.

Results

In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis.

Conclusions

Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.     

Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats

Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5?mg/kg of DXR and were sacrificed at seven, 14, 21 and 28?days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14?days. At 21 and 28?days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.     

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.     

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.     

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma. Olufunmilade A. Omisanjo is a scholarship holder of the German Academic Exchange Service (DAAD). Sonja Hartmann is a member of the German Research Foundation (DFG) Research Training Group 533 Cell–cell-Interaction in Reproduction.     

Testosterone and FSH have independent,synergistic and stage-dependent effects upon spermatogenesis in the rat testis

Summary Adult rats were hypophysectomized and treated with ethane dimethanesulphonate (EDS) selectively to eliminate the Leydig cells in the testis. By removing the source of endogenous gonadotrophins and androgens, the subsequent effects on the seminiferous epithelium were studied after 20 days of treatment with vehicle, or FSH (2x50 g/day) or a low dose of testosterone (0.6 mg testosterone esters every 3rd day) alone or in combination. Compared to vehicle-treated hypophysectomized rats with Leydig cells, testis weight in saline-treated hypophysectomized rats treated with EDS declined by 50%, spermatogenesis was disrupted severely and only 18% of the tubules contained spermatids, these being confined to stages I–VI of the spermatogenic cycle. Treatment with either FSH or testosterone esters alone significantly (P<0.01) increased testis weight compared to vehicle-treated hypophysectomized rats treated with EDS and 40% of tubules contained spermatids either at stages I–VI after FSH, or at all stages I–XIV after testosterone treatment. Treatment with FSH and testosterone esters together maintained testis weights approximately 20% above vehicle-treated hypophysectomized controls; over 70% of the seminiferous tubules contained spermatids and there was a marked stimulation of spermatogenesis at all stages of the spermatogenic cycle. The results suggest, that in the absence of the pituitary gland and the Leydig cells, FSH alone partially supports spermatogenesis up to the development of round spermatids whereas testosterone is capable of maintaining spermatid development at all 14 stages of the cycle. When FSH and testosterone were administered in combination, the effects upon spermatogenesis were far greater than the response expected if their individual effects were simply additive. It is therefore concluded that FSH may play a role in normal spermatogenesis and that this role is essentially that of augmenting the response of the testis to testosterone. The biochemical mechanisms via which this might occur are discussed and hypophysectomized rats treated with EDS used in the present studies should provide a useful approach for their identification.     

Sertoli cell Dicer is essential for spermatogenesis in mice

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.     

The effect of donor age on progression of spermatogenesis in canine testicular tissue after xenografting into immunodeficient mice

The objective of this study was to examine the effect of donor age on progression of spermatogenesis in dog (Canis lupus familiaris) testis tissue after xenografting. In Experiment 1, canine testes were obtained by surgical castration. Based on developmental pattern of spermatogenesis at the time of grafting, donors were categorized as immature, young, and adult (<4, 4 to 6, and >6 mo old, respectively). Fragments of testis tissue were implanted subcutaneously on the back of immunodeficient mice; xenografts were retrieved and analyzed 4, 6, or 8 mo later. At 4 mo postgrafting, immature and young groups had higher graft recovery rates, graft weights, vesicular gland indices, seminiferous tubule numbers, and larger seminiferous tubular diameters compared with those of adult donor xenografts. At 8 mo postgrafting, immature donor xenografts had maintained growth and development as exhibited by greater graft weights, vesicular gland indices, seminiferous tubule numbers, and tubular diameters compared with those of adult donor xenografts. At this time point, growth and development of xenografts did not differ between immature and young donors, whereas those from young donors had greater seminiferous tubule numbers and diameters compared with those of adult donor xenografts. Elongated spermatids were the most advanced germ cell type present at 4 and 8 mo postgrafting in xenografts of immature age groups. In Experiment 2, the longer-term efficiency of spermatogenesis and the potential sperm production in xenografts from immature donor dogs were determined. Testis tissue from 2-mo-old donor dogs were grafted into recipient mice, and xenografts were retrieved after 13 mo. Complete spermatogenesis was present in 5 of 29 recovered xenografts, with isolation of fully formed sperm (up to 36.3 × 10⁶ per gram tissue). In conclusion, immature and young donors (<6 mo of age) were the most promising donors for dog testis tissue xenografting. This strategy may offer an alternative for male germ-line preservation for canids that die prematurely or must be castrated before maturation.     

Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis

Summary The structure of guppy (Poecilia reticulata) spermatogonia and spermatocytes has been studied using electron microscopy. The spermatogonia, situated at the apex of the seminiferous tubule, are almost all surrounded by a network of Sertoli cells; they have very diffuse chromatin and one or two large nucleoli. The cytoplasm contains relatively few organelles, although annulate lamellae are found. The mitochondria have few cristae and are concentrated at one pole of the cell; they are sometimes found with intermitochondrial cement. These spermatogonia are separated from each other, having no intercellular bridges or inclusion in Sertoli cells, and are relatively undifferentiated; they correspond to stem cells. The spermatogonia beneath the apex are organized into cysts. First-generation spermatogonia are more dense and heterogeneous, their nuclei becoming smaller and their chromatin becoming denser during successive generations. In spermatocytes, the synaptinemal complex exists as a modified form until metaphase. The concentration of organelles in the cytoplasm increases and the organelles become more diversified as spermatogenesis progresses. Many cytoplasmic bridges are observed (several per cell), indicating that the cells remain in contact after several divisions. These changes in germ cell structure have been related to some of the characteristic features of spermatogenesis in guppy, e.g. the large number of spermatogonial generations and the complexity of spermiogenesis.     

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.