Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)     

细鳞鱼精巢超微结构和精子发生

细鳞鱼Brachymystax lenok(Pallas)精巢为叶型;支持细胞(Sertoli cell)和Leydig细胞具有典型内分泌细胞的一些结构特征;A型精原细胞的细胞器是区域性分布,精原细胞线粒体的发育与拟染色质 (chromatoid bodies)有关;生精细胞(spermatogenic cell)核膜孔由均匀排布最终演变为区域性聚集。重复注射绒毛膜促性腺激素(hCG)可促进雄鱼性成熟;精子质量受到多种因素的影响。     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

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.     

Genes expressed in Blue Fin Tuna (Thunnus thynnus) liver and gonads

Blue Fin Tuna (BFT), Thunnus thynnus, has been seriously endangered by global massive overfishing and by the pollution of marine environment. Feeding and fattening of caught tuna in marine cages is a recent resource, but the development of a self-sustained aquaculture activity, being independent from the supply of wild fish, is required from both industrial and conservation perspectives. At this scope, several technical problems have to be solved and the control of reproduction is the cardinal one. Beside the technological developments of farming facilities and protocols, a molecular approach seems promising for the studies of appropriate nutritional strategies, reproduction physiology and animal welfare, as well as lifestyle and response to endocrine disruptor pollutants. In this context, we have started an EST project on this species sequencing 2743, 2907, and 3014 clones from expression libraries of ovary, testis and liver, respectively, and 1499 clones from an ovary normalized library. Thanks to this project, we have identified several sequences with known function in other organisms, but not previously described in this species. Among the new genes, 712 were found only in the expression library of the ovary, 613 in that of the testis and 318 in that of the liver, while 324 additional genes were shared by two or more expression libraries; other 127 genes not found in the expression libraries were obtained from the ovary normalized library. This represents a contribution to the knowledge of the molecular basis of BFT and a necessary step for facilitating further molecular studies on this species. Accession numbers: EC 091633 to EC 093160; EG 629962 to EG 631176; EC 917676 to EC 919417; EG 999340 to EG 999999; EH 000001 to EH 000505; EH 667253 to EH 668984; EL 610526 to EL 611807; EC 42144 to EC 422414; and EH 379568 to EH 380065.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Completion of meiosis in male zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) despite lack of DNA mismatch repair gene <Emphasis Type="Italic">mlh1</Emphasis>

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization. Marcelo C. Leal and Harma Feitsma contributed equally to this work. This work was supported by the Brazilian Foundation CAPES, the Cancer Genomics Center (Nationaal Regie Orgaan Genomics), the European Union-funded FP6 Integrated Project ZF-MODELS, and Utrecht University.     

Species Comparison of Adenosine Receptor Subtypes in Brain and Testis

We aimed at comparing the binding characteristics of adenosine A₁ and A_2A receptors (A₁Rs and A_2ARs) in high-expressing cerebral areas, the cortex and striatum respectively, of human, bovine and rat brain. Adenosine A₃ receptor (A₃R) binding was studied in rat and bovine testis. Results confirmed species differences in AR saturation-displacement binding parameters. To investigate A₃Rs in CNS, we carried out immunoblot in human brain, resolving two signals, a 52 KDa band with the highest density in hippocampus and a 48 KDa one, slightly more expressed in cortex. Subsequently, A₃R binding was performed by [¹²⁵I]-4-aminobenzyl-5′-N-methylcarboxamidoadenosine ([¹²⁵I]-AB-MECA) in human hippocampus, revealing an high affinity population of sites and another non saturable component. [¹²⁵I]-AB-MECA first site displacement by N⁶ (3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and 1,3-dipropyl-8-cyclopenthyl-xanthine (DPCPX) distinguished two affinity sites, being only in part identified as A₃Rs. Therefore, A₃Rs result clearly expressed by Western blot in human brain, but their full CNS characterization needs further investigation.