Characterization of Sptrx, a novel member of the thioredoxin family specifically expressed in human spermatozoa.

Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis.

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking.

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

Protein composition of the ventral processes on the sperm head of Australian hydromyine rodents.

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.

On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.

Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme.

Outer dense fibers are structures unique to the sperm tail. No definite function for these fibers has been found, but they may play a role in motility and provide elastic recoil. Their composition had been described before, but only two of the fiber proteins, Odf1 and Odf2, are cloned. We cloned Odf2 by virtue of its functional and specific interaction with Odf1, which, we show, is mediated by a leucine zipper. Further work demonstrated that the 84-kDa Odf2 protein localizes to both the cortex and the medulla of the fibers, whereas the 27-kDa Odf1 protein is present only in the medulla. Here we report the cloning and characterization of a new Odf1-interacting protein, Spag4. Spag4 mRNA is spermatid specific, and the 49-kDa Spag4 protein complexes specifically with Odf1, but not Odf2, mediated by a leucine zipper. It also self-associates. In contrast to Odf1 and Odf2, Spag4 protein localizes to two microtubule-containing spermatid structures. Spag4 is detectable in the transient manchette and it is associated with the axoneme in elongating spermatids and epididymal sperm. Our data suggest a role for Spag4 in protein localization to two major sperm tail structures.

Molecular cloning and developmental expression of the major fibrous sheath protein (FS 75) of rat sperm.

The fibrous sheath (FS) is a cytoskeletal structure that encases the axoneme in the principal piece of the spermatozoon tail. In the rat, it is composed of several proteins, of which a 75-kDa polypeptide (FS 75), as estimated by PAGE, is the most prominent. The objectives of this study were to clone and sequence this protein and to characterize its transcriptional and translational origins during spermatogenesis. Initially, we isolated two overlapping cDNA segments that encoded a large part of the FS 75 protein but lacked the initiation codon for translation. Both clones were obtained by screening a rat testicular phagemid cDNA library with an anti-FS 75 polyclonal antibody. An upstream portion of the FS 75 mRNA containing the initiation codon was obtained by polymerase chain reaction with a pair of specific primers. Accounting for the overlap in all segments, 2786 nt of an approximately 3-kb FS 75 mRNA was obtained. The amino acid sequence of the longest possible open reading frame of the rat FS 75 was found to be similar to two previously cloned variants of the major FS polypeptide of mouse spermatozoa. Sequence analysis of the rat FS cDNA revealed anchoring kinase A protein domains and several kinase phosphorylation sites, supporting the idea that this protein plays a crucial role in the motility of spermatozoa. The presence of a potential N-myristoylation site suggests that this protein may covalently bind to the inner leaflet of the plasma membrane (PM), which in turn may explain the close association of the FS and PM from early development. Developmental northern blot analysis and in situ hybridization revealed that the FS 75 mRNA is haploid expressed, with an abundant level of mRNA in late round spermatids. Maximum levels of the FS 75 polypeptide, as determined by immunocytochemistry, correlated with a rapid decline in corresponding mRNA levels in step 14-16 spermatids. Since transcription termination occurs several steps earlier, the bulk of FS 75 mRNA appears to be translationally regulated.

Androgen binding protein secretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development.

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

Occurrence and formation of cytoskeletal proteins in mammalian spermatozoa.

Mammalian spermatozoa are composed of specialized cytoskeletal elements, which appear to have no structural or protein counterparts in somatic cells. Most evident are the outer dense fibres (ODF) and fibrous sheath (FS) of the sperm tail and the perinuclear theca (PT) of the sperm head. The purpose of this study is to review our results on the occurrence and assembly of proteins making up these three elements during spermatogenesis. Our approach was to raise antibodies against the prominent proteins of these elements and to immunolocalize them on testicular sections prepared for histological and ultrastructural analyses. We found that all of the cytoskeletal proteins considered were expressed exclusively during the haploid phase of development and that the proteins of each element had similar if not identical patterns of expression. The PT proteins were synthesized in the first half of spermiogenesis and were associated with acrosome formation, while the ODF and FS proteins were synthesized in the second half of spermiogenesis. The ODF proteins assembled in a proximal-distal direction along the length of the axoneme, while the FS proteins assembled in the opposite direction; both assemblies eventually meeting and overlapping within the periaxonemal cytoplasmic compartment. During assembly the ODF proteins appeared to be temporarily stored in granulated bodies of the cytoplasmic lobe, while the FS proteins were randomly distributed throughout the cytoplasm. In the case of the PT, there appeared to be an interdependence between PT assembly and acrosome formation. The developmental protein distribution patterns observed for each of the elements suggest unique cellular targeting mechanisms adapted by the spermatid to regulate the assemblies of the respective cytoskeletal proteins.

Developmental expression of the 84-kDa ODF sperm protein: localization to both the cortex and medulla of outer dense fibers and to the connecting piece.

Outer dense fibers (ODF) are specialized cytoskeletal elements of the mammalian sperm tail which are composed of several prominent proteins. We previously reported the isolation of a cDNA (111-450) encoding a putative 84-kDa ODF protein. Here we demonstrate by independent cDNA isolations and by translational/immunoprecipitation of testicular mRNAs using anti-ODF 84 antibodies that 111-450 cDNA encodes the 84-kDa protein. We then analyzed the testicular expression of the ODF 84 mRNA and protein. Riboprobes generated from the clones recognized four testicular-specific transcripts of 1.6, 2.2, 2.4, and 2.8 kb in both rat and bull of which the immunoprecipitable product of the 2.4-kb mRNA comigrates with ODF 84 protein. Developmental Northerns indicated that the 2.2- and 2.4-kb mRNAs are first transcribed during meiotic prophase while the other two species are first expressed in round spermatids. The levels of all the transcripts steadily increased up to elongated spermatids. Immunocytochemistry revealed that the anti-84 reactive ODF proteins were synthesized and assembled in the cytoplasm of elongated spermatids (steps 9-18) with peak activity occurring in step 16 of spermiogenesis. Immunogold labeling was selective to the assembling ODF and connecting piece of the tail and to granulated bodies of the cytoplasmic lobe. Both the striated collar and capitulum of the connecting piece were immunolabeled as well as the basal plate of the implantation fossa. A combination of pre- and postembedding immunogold labeling provided evidence that the 84-kDa ODF protein is localized to both the cortex and medulla of the ODF in contrast to the sole medullary localization of the major 27-kDa ODF protein. Thus the 84-kDa ODF protein, encoded by the 2.4 transcript, is translationally regulated, packaged after synthesis into granulated bodies, assembled in a proximal to distal direction along the axoneme and may interact by means of leucine zippers specifically with the 27-kDa ODF protein during assembly. Its localization to both the cortex and medulla of the ODF, as opposed to exclusive medullary localization of the 27-kDa ODF protein, and the presence of two leucine zippers, only one of which interacts with the 27-kDa ODF, suggests that it could act as a link between proteins of the two regions of the ODF.

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to impair reproductive function of males in animal models, possibly due to a reduction in serum androgen levels. Thus, TCDD may alter the testosterone biosynthetic pathway in the testis or the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) in androgen target tissues. Pregnant Sprague Dawley rats were gavaged with TCDD (0, 0.2 or 1.0 microg/kg) on day 15 of gestation only. TCDD caused a reduction in the body weight gain of the dams in both dose groups and a significant reduction in litter size in the higher dose group. Litters delivered normally and TCDD exposed male offspring grew at the same rate as controls. Males were sacrificed at 15, 30, 45, 60, 90 and 120 d of age. Steroidogenic enzyme activities were determined in testicular microsomes and androgen target tissue nuclear fractions. Serum androgens were measured by radioimmunoassay (RIA). At 30 d of age, rats exposed to 1.0 microg/kg TCDD exhibited lower 17-hydroxylase activity (P < 0.05) and lower caput-corpus epididymal weights (P < 0.05). At 45 d of age, the same treatment resulted in testicular 3beta-HSD, 17beta-HSD and 5alpha-reductase activities that were significantly greater (P < 0.05) but, conversely, serum androgens were one quarter the values evident in controls (P < 0.05). At the other ages, no differences were observed in serum androgens and, with the exception of lower 17beta-HSD activity at 90 d of age (P < 0.05), no other differences in testicular steroidogenic enzyme activities were found. 5Alpha-reductase activities in the androgen target tissues were also unchanged. Histological examination of testes showed that the spermatogenic profile was identical to controls at all ages.

Molecular cloning and structural analysis of the gene encoding PERF 15 protein present in the perinuclear theca of the rat spermatozoa.

We have cloned the PERF 15 gene, which encodes the most abundant protein of the perinuclear theca of rat spermatozoa. PERF 15 is related to the superfamily of lipophilic transport proteins. It has a molecular weight of 15,060 and is present exclusively in the subacrosomal region of the sperm head. Northern blot analysis demonstrated that this gene transcribed PERF 15 mRNA in meiotic and postmeiotic cells. The PERF 15 gene contains four exons and three introns. Exon 1 codes for amino acids 1-24, exon 2 for amino acids 25-82, exon 3 for amino acids 83-116, and exon 4 for amino acids 117-132. The three introns are composed of 2241, 547, and 164 base pairs (bp), respectively. The exon/intron boundaries are identical to those found in the mouse myelin P2 gene, but there is no resemblance in size and sequence between the corresponding introns of the PERF 15 and myelin P2 genes. Localization of the initiation transcription site by primer extension showed that the 5'-untranslated region of this gene is 67 bp upstream of the translation initiation site. Primer extension analysis also suggests that there is one transcription start site for this gene. Inverse polymerase chain reaction generated a 204-bp fragment, located upstream of the translation initiation codon, that has some homology with regions of other mammalian genes.

The removal of the sperm perinuclear theca and its association with the bovine oocyte surface during fertilization.

The perinuclear theca (PT) is a unique cytoskeletal structure whose anterior part is intercalated between the inner acrosomal membrane and the nuclear envelope of the mammalian sperm head and is important for spermiogenesis and stabilization of sperm structures (Oko and Maravei, Biol. Reprod. 50, 1000-1014, 1994; Oko and Maravei, Microsc. Res. Tech. 32, 520-532, 1995). Using immunofluorescence labeling of inseminated bovine oocytes and serial sectioning-ultrastructural analysis, we demonstrate that the PT is removed from the sperm nucleus following the loss of the sperm plasma membrane and the interaction of oocyte cortex with the PT. These events precede the development of the male pronucleus. The removal of the PT involves the elongated oocyte microvilli, rich in actin microfilaments, since it can be blocked by the microfilament-disrupting drug cytochalasin B. Reduction of disulfide bonds, which is a major factor supporting the disassembly of the sperm nucleus and accessory structures during mammalian fertilization, seems to exert little effect on the PT in vitro, as evidenced by the treatment of isolated bull sperm with the disulfide bond-reducing agent dithiothreitol. In vivo, intact bull sperm microinjected into mature oocytes do not undergo disassembly of the PT. Consequently, the decondensation of the sperm nucleus does not occur. These data suggest that the binding of the PT to the oocyte microvillar region and its removal from the sperm nucleus constitute an early step in mammalian fertilization, which is required for the conversion of the sperm nucleus into a male pronucleus.

Molecular cloning and developmental expression of a small ribonuclear protein in the mouse testis.

U1 RNP C polypeptide is a ubiquitous and highly conserved protein that is found associated to the U1 small nuclear ribonuclear particle (U1 snRNP). The U1 snRNP is involved in pre-mRNA splicing by defining introns and exons and by binding to consensus sequences within the pre-mRNA. In the present study we immunoscreened a mouse testicular phagemid cDNA library with an anti-Sm serum from patients with systemic lupus erythematosus. Sequence analysis of a positive clone containing a 0.75 kb cDNA insert revealed that it encodes the entire amino acid sequence of the U1 RNP C polypeptide. Northern blots of total RNA isolated from testes and various adult mouse tissues demonstrated that the 0.75 kb transcript is highly expressed in the testes and that it begins developmentally at day 18 postpartum, corresponding to the appearance of preleptotene spermatocytes. In situ hybridization confirmed the meiotic and post-meiotic expression of this transcript. LM immunoperoxidase staining with the anti-Sm serum localized spliceosome snRNPs predominantly in the nuclei of somatic and germinal testicular cells but not in elongated spermatids. EM immunogold labeling confirmed the LM observations but additionally showed that snRNP content peaked in the nuclei of pachytene spermatocytes and that 2 cytoplasmic components found exclusively in meiotic and post-meiotic germ cells were intensively reactive. Immunoblots of testicular homogenates probed with the anti-Sm serum revealed several reactive proteins, of which one, a 21 kDa polypeptide, could be the U1 RNP C based on its predicted molecular weight. In summary we report an isoform of U1 RNP C which is testis specific and which may play a role in mRNA splicing exclusively in meiotic and post-meiotic germ cells during spermatogenesis.

Interactional cloning of the 84-kDa major outer dense fiber protein Odf84. Leucine zippers mediate associations of Odf84 and Odf27.

The study of mammalian sperm tail outer dense fibers (ODF), a structure of unknown function, is hampered by the insoluble nature of ODF proteins and the availability of only one cloned component, Odf27. We report here the first use of the Odf27 leucine zipper as bait in a yeast two-hybrid screen to isolate a novel testis-specific protein whose interaction with Odf27 depends critically on the Odf27 leucine zipper. We find that the novel gene, 111-450, encodes a product that localizes to ODF as determined by fluorescence microscopy and immunoelectron microscopy and that the gene 111-450 product is identical to the major ODF protein, Odf84. Interestingly, Odf84 contains two C-terminal leucine zippers, and we demonstrate that all leucine residues in the upstream leucine zipper are required for interaction with Odf27, demonstrating the strategic validity of our approach. The use of the yeast screening approach to isolate leucine zipper containing proteins should be useful in other systems, and our findings have implications for ODF structural models.

Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent.

The perinuclear theca (PT) is a cytoskeletal structure that covers the nucleus of mammalian spermatozoa and is believed to have a membrane-binding role. The objectives of this study were to analyze the protein composition of the mouse PT, to identify and sequence its major protein component, and to characterize this protein's transcriptional and translational origins during spermatogenesis. The PT was extracted from demembranated and acrosome-depleted mouse sperm heads by alkaline treatment. The protein profile of the PT extract was composed of several polypeptides, of which a 15-kDa subacrosomal protein predominated and was found to be immunocross-reactive with a previously cloned 15-kDa PT protein of the rat (PERF 15) that belongs to a family of lipid-binding proteins. A primer pair designed from rat PERF 15 cDNA was then used to screen a mouse testicular cDNA library by polymerase chain reaction (PCR). The deduced amino acid sequence obtained from the PCR product was almost identical to the testicular-specific rat PERF 15. Developmental Northern blots and in situ hybridization studies performed with riboprobes encoding the mouse PERF 15 cDNA revealed that mRNA levels were highest in round and early elongating mouse spermatids. Immunohistochemistry indicated that PERF 15 began to be expressed in the cytoplasm of mid-pachytene spermatocytes and appeared to reach maximum expression in the distal cytoplasm of late elongating mouse spermatids, long after transcriptional arrest. During the development of round and early elongated spermatids, the immunolabel became progressively concentrated over the anterior half of the spermatid nucleus, suggesting a subacrosomal deposition of PERF 15 during this phase of mouse spermiogenesis.

Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.