Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.     

Changes in chromatin structure of boar late spermatids to mature spermatozoa by using modification with dansyl chloride

Changes in the chromatin structure of boar late spermatids maturing to spermatozoa were studied by chemical modification of their nuclei with dansyl (Dns) chloride. Protamine was isolated from the dansylated boar spermatid and sperm nuclei, and its dansylated sites and degrees of dansylation were determined by sequence analysis. The N-terminal Ala-1, Tyr-3 and Tyr-42 of the protamine molecule in cauda epididymal sperm nuclei were dansylated 27%, 22% and 40%, respectively, whereas the respective residues in late spermatid nuclei were about 1.5-times as reactive as those in cauda epididymal sperm nuclei. However, the dansyl ratio of Tyr-3 to Tyr-42 remained unchanged from the late spermatid to mature sperm nuclei. SDS treatment did not affect the reactivity of cauda epididymal protamine and that of Ala-1 of caput epididymal protamine, but raised that of Tyr-3 and Tyr-42 of caput epididymal protamine by a factor of about 1.5. As a result of the SDS treatment, caput epididymal protamine came to have almost the same reactivity as late spermatid protamine. These facts suggest that the fundamental structure, in terms of DNA-protamine interaction, of sperm chromatin was already formed at the stage of the late spermatid, and then during epididymal transit the sperm chromatin was more tightly condensed, with increasing disulfide cross-links, thereby acquiring insensitivity towards the SDS-treatment.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Meiotic DNA synthesis during mouse spermatogenesis

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

Identification and validation of mouse sperm proteins correlated with epididymal maturation

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Developmental expression of G protein alpha subunits in mouse spermatogenic cells: evidence that G alpha i is associated with the developing acrosome.

Guanine nucleotide-binding proteins (G proteins) are important signal transducing molecules found in all cells. G proteins are associated with the plasma membrane/outer acrosomal membrane region of acrosome-intact sperm and at least one G protein is involved in the zona pellucida-induced acrosome reaction. With the goal of elucidating the functions of these proteins during spermatogenesis, we investigated the types of G proteins present in spermatogenic cells and when they first become associated with the developing acrosome. Using bacterial toxin-catalyzed [32P]ADP-ribosylation in conjunction with immunoprecipitation and immunofluorescence utilizing antibodies directed against specific regions of various G protein isotypes, the alpha subunits of Gi1, Gi2, Gi3, and G(o) were detected in mouse spermatocytes and spermatids. An antiserum recognizing a conserved sequence of G alpha i subtypes localized to the proacrosomal granules of spermatocytes and the developing acrosome of spermatids. Levels of G alpha o diminished as spermatocytes developed into spermatids such that G alpha o was not detected in cauda epididymal sperm. Immunoreactivity using G alpha o-specific antisera did not display a distinct regionalization within any of the spermatogenic cell types. G alpha s was not detected in the developing spermatogenic cells or sperm. The association of G alpha i with the developing acrosome suggests a role for G proteins may have a role in acrosome biogenesis as well as being part of a complex required later for signal transduction leading to acrosomal exocytosis.     

Characterization of fucosyltransferase activity during mouse spermatogenesis: evidence for a cell surface fucosyltransferase

Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltransferase activity of mixed germ cells displayed an apparent Vmax of 17 pmol (mg of protein)-1 min-1 and an apparent Km of approximately 13 microM for GDP-L-[14C]fucose in the presence of saturating amounts of asialofetuin at 33 degrees C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization.     

Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice

Previous studies have demonstrated the importance of transition nuclear proteins, TP1 and TP2, in spermatogenesis and male fertility. However, importance of the overall level of transition proteins and their level of redundancy in the production of normal sperm is not clear. Epididymal sperm from the nine possible Tnp1 and Tnp2 null genotypes demonstrated a general decrease in normal morphology, motility, chromatin condensation, and degree of protamine 2 processing with decreasing levels of transition proteins in mutant sperm. Nuclei of some mutant epididymal sperm stained poorly with hematoxylin and DNA fluorochromes, suggesting that the DNA of these sperm underwent degradation during epididymal transport. When epididymal sperm were injected directly into oocytes, fertilization and embryonic development were reduced only in the two most severely affected genotypes. These phenotypes indicated some functional redundancy of transition proteins; however, redundancy of transition protein function was not complete, as, for example, sperm from double heterozygous males had fewer abnormalities than sperm from males homozygous for a single Tnp null mutation. Our study suggests that each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect. Similarities between sperm defects found in Tnp mutants and infertile patients make the Tnp mutants a valuable tool with which to study outcomes following fertilization using sperm with compromised DNA integrity.     

Surface changes in chimpanzee sperm during epididymal transit

Intact chimpanzee caput and cauda epididymal sperm, sperm cell lysates, and caput and cauda epididymal fluid were radiolabeled by enzymatic iodination with lactoperoxidase and Na125 I and were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Caput epididymal sperm showed nine labeled macromolecular components of 90, 64, 56, 48, 38, 31, 20, 18 and 16 Kd and cauda epididymal sperm showed eleven macromolecular components of 90, 64, 55, 47, 42, 33, 27, 18, 17, 15 and 11 Kd. Six of the components labeled on caput sperm (90, 64, 56, 48, 18 and 16 Kd) were detected in equal amounts of cauda sperm and two (38 and 20 Kd) were detected at greatly reduced labeling intensities. In the cauda epididymidis, four new components (33, 27, 17 and 11 Kd) became prominent features of the sperm surface. Analysis of labeled caput and cauda sperm cell lysates resolved components distinct from those detected on sperm surfaces. Electrophoresis of caput epididymal fluid showed five labeled components of 66, 56, 47, 41 and 37 Kd, while electrophoresis of cauda epididymal fluid showed eight labeled components of 92, 66, 56, 48, 31, 27, 24 and 11 Kd. Three components (66, 56 and 47 Kd) were present in both caput and cauda fluid, two (41 and 37 Kd) in caput fluid only, and five (92, 31, 27, 24 and 11 Kd) in cauda fluid only. Components of 37 Kd were labeled in caput fluid and on caput sperm but not on cauda sperm, whereas components of 27 Kd and 11 Kd were labeled in cauda fluid and on cauda sperm but not on caput sperm. These data show that chimpanzee sperm undergo extensive surface modifications during epididymal maturation and that some of these modifications may be related to exogenous proteins/glycoproteins in epididymal fluids.     

Role of follitropin receptor signaling in nuclear protein transitions and chromatin condensation during spermatogenesis

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.     

The secretion of two sperm maturation-related glycoproteins in BALB/c mouse epididymis

Summary A well-developed Golgi apparatus and rough and smooth endoplasmic reticulum in the principal cells of the mouse epididymis indicate active protein synthesis. Studies have shown that epididymal secretions are essential for sperm maturation. In a previous study, two wheat-germ agglutinin (WGA)-binding glycoproteins, GP-49 and GP-83, were identified on the surface of mature mouse sperm. In this study, synthesis and secretion of these two glycoproteins were investigated. Apparent WGA-binding was found on the stereocilia and in the apical region of principal cells in the corpus and cauda of epididymis. Post-fixation and pre-embedding cytochemical localization revealed that WGA-binding sites were situated in the Golgi apparatus, multivesicular bodies and stereocilia of principal cells. GP-49 and GP-83 were identified in the Nonidet P-40 homogenates of corpus and cauda epididymidis. In the epididymides of which ductuli efferentes had been ligated for more than 4 weeks, no sperm were found in the lumina of epididymal tubules. WGA-binding sites were present in the corpus and cauda; GP-49 and GP-83 were identified in tissue homogenates of the corpus and cauda as well. These findings suggest that GP-49 and GP-83 of mature sperm may be secreted by the principal cells of the corpus and cauda. These two molecules apparently conjugate to sperm whilst sperm transit through the epididymis.