Murine Asb-17 expression during mouse testis development and spermatogenesis

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.     

Gene expression profiles of mouse spermatogenesis during recovery from irradiation

Background  

Irradiation or chemotherapy that suspend normal spermatogenesis is commonly used to treat various cancers. Fortunately, spermatogenesis in many cases can be restored after such treatments but knowledge is limited about the re-initiation process. Earlier studies have described the cellular changes that happen during recovery from irradiation by means of histology. We have earlier generated gene expression profiles during induction of spermatogenesis in mouse postnatal developing testes and found a correlation between profiles and the expressing cell types. The aim of the present work was to utilize the link between expression profile and cell types to follow the cellular changes that occur during post-irradiation recovery of spermatogenesis in order to describe recovery by means of gene expression.     

Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

Cell- and stage-specific high-level expression of TBP-related factor 2 (TRF2) during mouse spermatogenesis

Mice lacking the TBP-related factor 2 (TRF2) gene, which is highly expressed in the testis, have a severe defect in spermiogenesis. Here we show that the expression of TRF2 is both cell type- and stage-specific. TRF2 expression was first detected in the late pachytene spermatocytes at stage VIII and increased throughout the subsequent stages. After meiotic divisions, the TRF2 expression declined continuously in round spermatids during progression from stage I to stage V. This observation is consistent with an essential regulatory role of TRF2 in male germ cell differentiation during spermatogenesis.     

Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis

The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII-VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX-I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.     

Analysis of cell-type-specific gene expression during mouse spermatogenesis

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of specific cells, or changes in the cell-type composition. This reflects the cellularity of the tissue. Here we have combined techniques that assess the expression profiles of genes at the whole-tissue level, differential display and DNA array, and, at the level of cellularity, in situ hybridization. Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII-XI). This pachytene cluster was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination of these cell-type-specific expression patterns.     

Ontogeny of tyrosine phosphorylation-signaling pathways during spermatogenesis and epididymal maturation in the mouse

The objectives of this study were to map the ontogeny of tyrosine phosphorylation signal transduction pathways during germ cell development and to determine their association with the differentiation of a functional gamete. Until testicular germ cells differentiate into spermatozoa, cAMP-induced tyrosine phosphorylation is not detectable. Entry of these cells into the epididymis is accompanied by sudden activation of the tyrosine phosphorylation pathway, initially in the principal piece of the cell and subsequently in the midpiece. In the caput and corpus epididymides, the potential to express this pathway is inhibited by the presence of calcium in the extracellular medium. However, calcium has no effect on the expression of this pathway in caudal epididymal sperm. The competence of these cells to phosphorylate the entire sperm tail, from the neck to the tail-end piece, is accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation, involving the acrosomal domain of the sperm head, is invoked as spermatozoa enter the caput epididymis, and phosphorylation remains high until these cells enter the distal corpus and cauda. The proportion of cells exhibiting this form of tyrosine phosphorylation is not affected by extracellular calcium or cAMP but is negatively correlated (R2 = 0.99) with their ability to acrosome-react. However, this relationship is not causative. Our findings indicate that the development of functional spermatozoa is accompanied by carefully orchestrated changes in tyrosine phosphorylation, controlled by independent regulatory mechanisms in distinct subcellular compartments of these highly specialized cells.     

Demonstration of a stage-specific expression of the ZFY protein in fetal mouse testis using anti-peptide antibodies.

The zinc finger Y (Zfy) gene is located on the Y chromosome of all placental mammals. Although it is phylogenetically conserved and is expressed in mouse fetal testis, it is not the sex determining Y (Tdy) gene. To address the possible function of the Zfy gene in mice, the distribution of Zfy protein in fetal mice was investigated by immunocytochemical staining using several specific antisera against synthetic peptides of the mouse Zfy protein. Analysis of various fetal tissues at different embryonic stages demonstrated a specific staining only in fetal testis. In particular, reactive protein was initially observed in male fetal gonads at day 11.5 postcoitum (p.c.). The immuno-staining intensified in fetal testes at day 12 and 12.5 p.c., decreased drastically in those at day 13 and 14 p.c. and became undetectable in those at day 15 p.c. and beyond. The reactive molecules were distributed mostly within the seminiferous tubules of the embryonic testis. The present observations confirm the previous findings with RT-PCR analysis and indicate that Zfy or Zfy-like protein is expressed in stage-specific manner during early testis differentiation. Its location in the seminiferous tubules suggests a possible role in early germ cell development.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

Analyses of stage-specific multiple forms of lactate dehydrogenase and of cytochrome c during spermatogenesis in the mouse.

Spermatogenesis is a programmed developmental process characterized by the inactivation of certain genes and the activation of other, testis-specific genes. Synthesis of unique gene products such as LDH-C4 and cytochrome ct occurs only at precise stage of germ cell formation. The developmental sequences of gene activation for these proteins was observed by immunohistochemical procedures. LDH-C4 is first detectable during mid-pachytene of the primary spermatocyte. The C subunits appear to be uniformly distributed throughout the cytoplasm of the spermatocyte. The mid-pachytene stage also marks the first appearance of cytochrome ct. The association of this electron transport protein with spermatocyte mitochondria is reflected in a granular fluorescence of specific antibody-treated sections of testis. Neither the C subunit of LDH, nor cytochrome ct appear in leptotene or early pachytene primary spermatocytes. These analyses indicate that there is stage-specific protein synthesis in the primary spermatocyte which is characterized by differential activation of the LDH-C locus and of the gene coding for cytochrome ct.     

Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification

The developmental expression of 2-keto-3-deoxy-D-glycero-D-galacto-nononicacid-containing glycosphingolipids (KDN-gangliosides) in rainbowtrout testis during spermatogenesis was studied using a monoclonalantibody, mAb.kdn3G, which recognizes the KDN     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.