Spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs

Starting from the period of testis differentiation, the Sertoli cell plays a pivotal role in the development of a functional testis. FSH is the major mitotic factor for Sertoli cells. Because the supporting capacity of Sertoli cells is relatively fixed for each species, their total number per testis, established just before puberty (approximately 4 months in pigs), dictates the potential for sperm production. In contrast to Sertoli cells that are still undifferentiated, mature Leydig cells are already present at birth in pigs. Spermatogenesis lasts from 30 to 75 days in mammals, and this time period is under the control of the germ cell genotype. In boars, each spermatogenic cycle and the entire spermatogenic process lasts 8.6-9.0 and approximately 40 days, respectively. The sperm transit through the epididymis takes approximately 10 days in pigs and this is within the range cited for most mammals. Germ cell loss occurs normally during spermatogenesis, mainly during the spermatogonial and meiotic phases. In pigs, significant germ cell loss also takes place during spermiogenesis. In mammals in general, including pigs, only 2-3 out of a possible 10 spermatozoa are produced from each differentiated type A1 spermatogonium. The high supporting capacity of Sertoli cells and the short duration of the spermatogenic cycle are the main factors responsible for the comparatively high spermatogenic efficiency of pigs.     

Identification of GABA(B) receptor in rat testis and sperm

gamma-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABA(A)R, in mammalian sperm. In the present study a second subtype, GABA(B)R, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABA(B)R1 (GABA(B)R1a and GABA(B)R1c) and GABA(B)R2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABA(B)R1a and GABA(B)R1c/lb, respectively, determined by Western blot using polyclonal anti-GABA(B)R1 antibody. By an indirect immunofluorescence technique, GABA(B)R1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABA(B)R.     

LDH-C：睾丸重要的特异表达基因

乳酸脱氢酶C(LDHC)是目前已知的最早在男性生精细胞中发现的睾丸特异同功酶。LDHC最早通过凝胶电泳技术在人精子及睾丸生精细胞中被发现。免疫组化结果显示LDHC最早出现在早期的粗线期初级精母细胞中,其数量随着减数分裂逐渐增加。成熟精子中LDHC主要定位在精子尾部的主段区域。研究显示,乳酸脱氢酶家族的同功酶在哺乳动物细胞中无所不在,他们受到发育的调控,具有组织细胞的特异性,且功能多样。本文就LDHC的发展史及他们在帮助精子完成受精过程中的作用作一综述。     

Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.     

Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle- and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G(s) (cAMP) and G(q) (inositol phosphates) signal transduction pathways during spermatogenesis.     

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.     

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3

The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.     

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.     

LDH—C：睾丸重要的特异表达基因

乳酸脱氢酶C（u）Hc）是目前已知的最早在男性生精细胞中发现的睾丸特异同功酶。LDHC最早通过凝胶电泳技术在人精子及睾丸生精细胞中被发现。免疫组化结果显示LDHC最早出现在早期的粗线期初级精母细胞中,其数量随着减数分裂逐渐增加。成熟精子中LDHC主要定位在精子尾部的主段区域。研究显示,乳酸脱氢酶家族的同功酶在哺乳动物细胞中无所不在,他们受到发育的调控,具有组织细胞的特异性,且功能多样。本文就LDHC的发展史及他们在帮助精子完成受精过程中的作用作一综述。     

Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia.

To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.