Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.     

Nuclear envelope remodeling during mouse spermiogenesis: postmeiotic expression and redistribution of germline lamin B3

Lamins are members of a multigene family of structural nuclear envelope (NE) proteins. Differentiated mammalian somatic cells express lamins A, C, B1, and B2. The composition and organization of the nuclear lamina of mammalian spermatogenic cells differ significantly from that of somatic cells as they express lamin B1 as well as two short germ line-specific isoforms, namely lamins B3 and C2. Here we describe in detail the expression pattern and localization of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, we show that lamin B3 is selectively expressed during spermiogenesis (i.e., postmeiotic stages of spermatogenesis). In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. Our results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely the ubiquitous lamin B1 and the germline-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis.     

The differential expression of the actins and tubulins during spermatogenesis in the mouse

Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.     

DNA polymerase-beta and poly(ADP)ribose polymerase mRNAs are differentially expressed during the development of male germinal cells.

We have examined the steady-state mRNA levels in spermatogenic cells of two nuclear enzymes that appear to be involved in DNA repair, DNA polymerase-beta (pol-beta) and poly(ADP)ribose polymerase (PADPRP). Two pol-beta mRNAs of 1.3 kb and 1.4 kb were detected in extracts from mouse testes. In leptotene/zygotene spermatocytes a low level of the 1.4-kb mRNA was observed. Both pol-beta mRNAs were found in meiotic pachytene spermatocytes, with the 1.3-kb form being more abundant. In contrast, the 1.4-kb form was more abundant in haploid round spermatids. Polysome gradient analyses indicated that the two pol-beta mRNAs were predominantly present in the nonpolysomal fractions of spermatocytes. In round spermatids, a larger fraction of the 1.4-kb pol-beta mRNA was associated with polysomes, correlating well with the higher levels of pol-beta enzyme detected during spermiogenesis. The pattern of PADPRP mRNA expression differed from the expression of pol-beta mRNA. The two PADPRP mRNAs of 3.7 and 3.8 kb were present in type A and type B spermatogonia, reached their highest levels in pachytene spermatocytes, and were greatly reduced in haploid round and elongating spermatids. Most of the pachytene spermatocyte PADPRP and mRNAs were present in polysomes, whereas a greater percentage of PADPRP mRNAs in round spermatids were detected in the nonpolysomal fractions. This finding correlates with the immunocytochemical nuclear localization of this enzyme in pachytene spermatocytes. These data demonstrate that different developmental patterns of mRNA expression and translational regulation exist for the pol-beta and PADPRP mRNAs during differentiation of male germinal cells.     

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455–464; Hecht et al. (1986) Exp. Cell Res. 164, 183–190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.     

The role of histones in chromatin remodelling during mammalian spermiogenesis.

One of the most dramatic chromatin remodelling processes takes place during mammalian spermatogenesis. Indeed, during the postmeiotic maturation of male haploid germ cells, or spermiogenesis, histones are replaced by small basic proteins, which in mammals are transition proteins and protamines. However, nothing is known of the mechanisms controlling the process of histone replacement. Two hints from the literature could help to shed light on the underlying molecular events: one is the massive synthesis of histone variants, including testis-specific members, and the second is a stage specific post-translational modification of histones. A new testis-specific 'histone code' can therefore be generated combining both histone variants and histone post-translational modifications. This review will detail these two phenomena and discuss possible functional significance of the global chromatin alterations occurring prior to histone replacement during spermiogenesis.     

The Enzymatic Activity of Cu/Zn Superoxide Dismutase Does Not Fluctuate in Mouse Spermatogenic Cells Despite mRNA Changes

In the mammalian testis, multiple mRNAs encoding the copper zinc superoxide dismutase (SOD-1) are expressed in postmeiotic male germ cells. Here we relate SOD-1 mRNA levels to SOD-1 protein and enzyme activity levels in mouse spermatogenic cells. Although the sizes and relative amounts of the multiple SOD-1 mRNAs vary as male germ cells enter meiosis and proceed into the postmeiotic stages of spermatogenesis, the amount of SOD-1 protein and enzyme activity does not fluctuate significantly, suggesting a precise control of SOD-1 activity in male germ cells.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.     

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.     

In postmeiotic male germ cells poly (A) shortening accompanies translation of mRNA encoding γ enteric actin but not cytoplasmic β and γ actin mRNAs

In the mammalian testis the cytoplasmic β and γ actins are expressed in all stages of germ-cell differentiation, whereas γ enteric actin is expressed in germ cells solely in postmeiotic stages. Northern blot analysis of mouse testicular RNAs reveals actin mRNAs of about 2.1, 1.5, and 1.4 kB. The 2.1-kB mRNAs encode the cytoplasmic β and γ actins, whereas the two faster-migrating actin mRNAs encode γ enteric actin. When post-mitochondrial mouse testis extracts are fractionated by sucrose gradient centrifugation, the 1.5-kB γ enteric actin mRNA is primarily found in the nonpolysomal fraction, whereas the 1.4-kB γ enteric actin is polysomal. When the poly (A) tails are removed, the nonpolysomal and polysomal γ enteric actin mRNAs both migrate at 1.3 kB, indicating that the difference in electrophoretic mobilities of the two γ enteric actin mRNAs is caused by poly (A) length differences. The nonpolysomal and polysomal forms of the cytoplasmic β and γ actins show similar electrophoretic mobilities before and after deadenylation. Sequence comparison of the 3′ untranslated region of the mouse γ enteric actin to the 3′ untranslated regions of other testicular mRNAs that undergo partial deadenylation reveals three highly-conserved sequence elements. These data demonstrate that the poly (A) shortening of polysomal mRNAs previously seen only with testis-specific mRNAs that are stored as mRNPs also occurs with mRNAs of widely-expressed genes that are expressed in postmeiotic male germ cells. The mRNAs all contain specific conserved sequence elements in their 3′ untranslated regions. © 1996 Wiley-Liss, Inc.     

Synthesis of mouse t complex proteins during haploid stages of spermatogenesis

We have analyzed the expression, through spermiogenesis, of a series of testicular cell polypeptides encoded by genes within the mouse t complex. Two of these polypeptides, TCP-3 and TCP-7, are synthesized in a testes-specific manner with highest levels of expression during haploid stages of spermatogenesis. A third, TCP-1, is also expressed at highest levels in haploid cells, and expression of this polypeptide continues until the last residual body stage of spermiogenesis. The genes that encode these polypeptides have been correlated with the t phenotype of transmission ratio distortion.