Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.     

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.     

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12–15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4′,6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis. (J Histochem Cytochem 57:951–962, 2009)     

Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids

The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.     

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.     

Transformation of ram spermatid chromatin

In order to investigate the sequence of changes in nuclear basic proteins throughout ram spermiogenesis, we have used different techniques to obtain populations of spermatid nuclei in specific stages of maturation. Sedimentation of testis cells at 1 gravity followed by treatment with Triton X-100 resulted in one population of round spermatid nuclei (steps 1–a), one of non-round spermatid nuclei (steps 8b-15), and one of elongated spermatid nuclei (steps 12–15). Populations of non-round spermatid nuclei were obtained by treatment with EDTA (steps 9–15), by sonication (steps 12–15) and digestion by DNase (steps 13–15). Nuclear proteins, extracted either directly with dilute acid or following a reducing treatment with 2-mercaptoethanol were characterized by polyacrylamide gel electrophoresis.The most striking alterations in protein composition occur during the elongation phase (steps 8–12). The five histones are displaced from chromatin at the same rate. When they are freed of histones (step 12), the nuclei start to accumulate the sperm-specific protein (BNSP) which is then partly extractable by dilute acid without a thiol that is required for its extraction from more mature nuclei. This stepwise replacement process is accompanied by a reduction of the basic protein amount bound to DNA. As soon as histones begin to disappear, eight spermatidal protein fractions are present in the nuclei until the BNSP synthesis reaches its maximum rate. Except for one, they all contain cysteine and are partially intermolecularly cross-linked in the chromatin. After in vivo and in vitro labelling experiments, they are synthesized in elongating spermatids (steps 8–11). None are degradation products of histones.Correlations of the times of onset of EDTA, sonication and DNase resistances with changes in the basic nuclear proteins point out that stabilization and condensation of spermatid chromatin is promoted through a progressive increase in disulfide bridges.     

Spermatogenesis in the mouse. I. Autoradiographic studies of nuclear incorporation and loss of 3H-amino acids

Autoradiographic and electron microscope methods were used to correlate changes in nucleoproteins with nuclear fine structure during spermatogenesis in the mouse. Testes were fixed at daily intervals after intratesticular injectionwith labeled amino acid. [3H]Arginine, lysine, valine, and proline were rapidly incorporated into primary spermatocyte nuclei, retained through subsequent spermatocyte divisions and through spermatid differentiation to step 12 of spermiogenesis, but were lost with spermatid differentiation beyond step 12. Arginine and lysine (not valine or proline) also were rapidly incorporated into certain elongated spermatid nuclei but differed strikingly in their distribution and fate. Nuclei of late step-12 through step-15 spermatids were initially labeled with arginine. This label was retained through subsequent spermatid differentiation and sperm maturation in the epididymis. By contrast, lysine was initially incorporated only into late step-12 and step-13 spermatid nuclei, and was retained only to early step 14 of spermiogenesis. Spermatid incorporation of lysine coincided with the initiation of chromatin condensation in late step-12 nuclei, and loss of lysine coincided with the completion of condensation in step-14 nuclei.     

SYNTHETIC ACTIVITIES DURING SPERMATOGENESIS IN THE LOCUST

Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.     

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.     

Stage-specific expression of dynein light chain-1 and its interacting kinase, p21-activated kinase-1, in rodent testes: implications in spermiogenesis.

Mammalian spermatogenesis is a complex process involving regulatory interactions of many gene products. In this study, we found that dynein light chain-1 (DLC1), a component of the dynein motor complex, is highly expressed in mouse and rat testes. Immunohistochemically detectable levels of DLC1 are observed specifically in spermatids in steps 9-16 in distinct subcellular compartments: in steps 9-11, DLC1 is predominantly localized in the nucleus; in steps 12 and 13, it is found in both nucleus and cytoplasm; and in step 14-16, it is present exclusively in the cytoplasm. In addition, we found p21-activated kinase 1 (Pak1), a protein kinase that activates DLC1 by phosphorylating DLC1 at Serine 88, was also expressed during these stages of spermatogenesis. Pak1 was also expressed in Leydig cells, in preleptotene primary spermatocytes, and in round spermatids. The spermiogenic stage-specific expression of DLC1 suggests a role for DLC1 in chromatin condensation, spermatid shaping, and the final release of sperm from the spermatogenic epithelium. Further, Pak1 may also play a role in spermiogenesis by regulating DLC1 phosphorylation and, consequently, its function.     

Structural organization and distribution of the symbiotic bacteria Wolbachia during spermatogenesis of Drosophila simulans

Electron microscopy and morphometric analysis have shown that the symbiotic bacteria Wolbachia occur the testis cells during spermatogenesis and are absent in mature spermatids. Bacteria did not affect the structural organization of testis cells, which have a typical morphology during morphogenesis. Bacteria were distributed along the meiotic spindle microtubules near the mitochondria. They increased in number in spermatids at the stage of elongation. Endosymbionts aggregated at the spermatid distal end and contained many vacuoles but were absent at the spermatid proximal end near the nuclei. It was shown for the first time that the diameter of spermatids in a strongly infected line was two of three times that in a noninfected line. We hypothesize that the increase in the number of endosymbionts during spermatid elongation can affect the chromatin condensation in the spermatozoon.     

Stage-specific expression of rat transition protein 2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe.

The present study has used methoxyacetic acid (MAA)-induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7-12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACT

During spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.