Difference in Ca2+ oscillation-inducing activity and nuclear translocation ability of PLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human, and medaka fish

Mouse phospholipase C, zeta 1 (PLCZ1), a strong candidate of egg-activating sperm factor, induces Ca(2+) oscillations and accumulates into formed pronucleus (PN) when expressed by cRNA injection. These activities were compared among mouse and human PLCZ1, newly cloned rat Plcz1, and medaka fish plcz1. The PLCZ1 proteins of the four species have an approximately homologous sequence of nuclear localization signal. However, the nuclear translocation ability was defective in rat, human, and medaka PLCZ1 expressed in mouse eggs. Rat PLCZ1 could not enter rat PN, whereas mouse PLCZ1 could. Mouse and human PLCZ1 translocated into the nucleus of COS-7 cells transfected with cDNA. There was little medaka PLCZ1 accumulated in the nucleus, and rat PLCZ1 was never located in the nucleus. All PLCZ1 proteins including fish could induce Ca(2+) oscillations in mouse eggs, but the activity was variable in the order of human > mouse > medaka > rat, estimated from minimal RNA concentration to induce Ca(2+) spikes. Ca(2+) oscillations by human PLCZ1 continued far beyond the time of PN formation (T(PN)), whereas those by mouse PLCZ1 ceased slightly before T(PN). High-frequency Ca(2+) spikes by overexpressed rat PLCZ1 stopped far before T(PN), possibly by feedback inhibition. Ca(2+) oscillations by fertilization of rat eggs stopped at T(PN), despite defective nuclear translocation of rat PLCZ1. Thus, PLCZ1 sequestration into PN participates in termination of Ca(2+) oscillations at the interphase of mouse embryos but does not always operate in other mammals, notably in rat embryos.     

Small nuclear ribonucleoprotein polypeptides in rat sperm.

1. Immunoblot analysis of rat sperm head proteins revealed the presence of polypeptides recognized by anti-Sm serum obtained from patients with systemic lupus erythematosus. 2. Two of these polypeptides have molecular weights of 26,000 and 15,000 and they were identified as small nuclear ribonucleoprotein components present in other rat tissues. 3. When the autoimmune serum was used in the immuno-gold procedure for electron microscopy, gold particles were found only on the sperm nucleus. 4. The results indicate that some polypeptides of the small nuclear ribonucleoprotein complex are components of the rat sperm chromatin.     

The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

A possible role for sperm RNA in early embryo development

Recently it has been demonstrated that, along with sperm, some of its RNA can be introduced into the oocyte during fertilization, which stays stable until the activation of the embryonic genome. Originally it was thought that RNA present in semen relates to contamination from somatic cells and/or immature sperm both containing substantially higher amounts of RNA than the fertilizing sperm. However, RNA is still found after stringent washing through density gradients resulting in a sperm fraction that is translational silenced and devoid of cytosolic rRNA and thus of potential RNA contamination-which is not transferable to the oocyte. Sperm only delivers a relatively small amount of paternal RNA (5-10 fg) into the fertilized oocyte when compared to the amount of maternal RNA (approximately 1 ng). Pooled human sperm contains about 5000 different mRNA sequences of which half are common between ejaculates. Besides mRNA sperm also contains small sperm RNA molecules that might interfere in gene expression (iRNA). In human sperm already more than 68 putative iRNAs have been identified and 15 of them may specifically inhibit genes that are only active during early embryonic development. The composition and quantity of sperm RNA is considered to be a valuable diagnostic tool for male fertility. However, only a subpopulation of the purified mature sperm fraction (with a yet unknown composition and quantity of RNA) will appropriately respond to capacitation media to become competent to fertilize the oocyte. In this review the origin and function of sperm borne RNA transferred into the oocyte is discussed along with their putative role in early embryogenesis, which still needs to be experimentally proven.     

Changes in the rat sperm head during epididymal transit

Morphological changes in sperm are one aspect of a maturation process during epididymal transit in marnrnals. The literature mentions only, for different strains of rats, a remodeling and decrease in size of the acrosome. In the present work, the sperm were obtained from caput, corpus, and cauda epididymis of the albino rat. Samples were processed for scanning electron microscopy, with routine techniques, and for light microscopy and video microscopy. It appeared, with these techniques, that the acrosomal curvature and the whole head surface area of the rat sperm decrease during the epididymal transit. To measure these changes, a geometrical method was designed, and surface measurements were made using a computer program. It was found that the caput sperm head has the greatest surface area and a sharper acrosome bend than the cauda sperm. In an attempt to explain the above-mentioned changes, the suggestion is offered that some compactation of the nucleus and acrosomal material could be related to the decrease of the surface area.     

Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.     

Sperm head structure of a murid rodent from Southern Africa: The red veld rat Aethomys chrysophilus

The morphology of spermatozoa from the red veld rat, Aethomys chrysophilus, of Southern Africa is described; two very different types were found, which came from animals from two separate, as-yet-undescribed, species. In individuals from South Africa the sperm head had a somewhat disc-shaped nucleus and a large acrosome with a huge apical segment that, during epididymal transit, changed in form from initially projecting anteriorly to a highly complex structure that was flexed caudad and lay alongside part of the rest of the sperm head. In addition, the chromatin generally appeared to be not fully condensed. Spermatozoa from animals collected in Malawi were very different in morphology and had a head with a typical apical hook, a perforatorium, fully condensed chromatin, and a 4-μm-long ventral spur. Its sperm tail was also significantly longer. The time of divergence of these two groups of animals from a common ancestor is not known, but the present results show that a considerable morphological change in the sperm nucleus, acrosome, and subacrosomal space can evolve even between two, presumably closely related, species.     

Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3

The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.     

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.     

Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

Phospholipase C-zeta (PLCzeta), a strong candidate of the egg-activating sperm factor, causes intracellular Ca2+ oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLCzeta. Changes in the localization of expressed PLCzeta were investigated by tagging with a fluorescent protein. PLCzeta began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLCzeta in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLCzeta was recognized in every embryo up to blastocyst. Thus, PLCzeta exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca2+ oscillations in early embryogenesis.     

Antifertility effects in rats actively immunized with 80 kDa human semen glycoprotein.

A 80 kDa human sperm antigen has been identified using the serum of an infertile woman having circulating antisperm antibodies. The antigen was then purified to homogeneity by gel permeation chromatography using HPLC (protein PAK-125 column) system and on FPLC (superose-12 column) system. The antigen was found to be a glycoprotein. The antigen was mainly localized in the postacrosomal region of the human sperm, while it was localized in the head region of the rat sperm as demonstrated by immunofluorescent staining. The presence of this antigen was also demonstrated in the human prostate and endometrium and in the rat testis; epididymis and the prostate by immunocytochemical staining. The purified protein upon active immunization in female rats caused infertility in 100 percent animals. While in male rats it caused infertility in 90 percent animals. On morphometric analysis of testicular tissue it was observed that there was no significant change in spermatogonia and spermatocytes, but significant decrease in spermatids and sperm number as well as daily sperm production in the immunized male rats. The epididymal spermatozoa were markedly reduced in number and were largely found to be agglutinated. The results suggest that 80 kDa human sperm antigen appears to be a suitable candidate for immunocontraception both in male and female.     

The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

Studies on Nucleic Acid and Protein Synthesis and D evelopment of Male Gametnphyte of Clivia nobilis Cultured in Vitro

A simple method for culture male gametophyte (MG) of Clivia nobilis in vitro was established and the process of their development was observed. After research on dynamic of nucleic acid and protein synthesis of MG in various developmental stages by using of inhibitors and autoradiography authors found that DNA synthesis fro mrelease of tetrad to sperm only takes place in nucleus of interphase. There is no 3H-tymidine incorporation into vegetative nucleus (Vn), generative nucleus (Gn) or nucleus of sperm in 96 hours before dehiscence of anthers (BDA). The dynamic of protein synthesis is similar to the same of RNA’s. Both of them have three peaks and two intermissions. The 1st peak is in 12–9 days BDA. The 2nd is in 7–5 days BDA and intermitted from 48 hours BDA. The 3rd begins from the 1st hour after culture (AC) decleases at 6th hour AC and stops before 20th hour AC. The kind of inhibitor, the time and quanlity of treatment are affected the morphogenesis, showing the relationship to synthesis among DNA, RNA and protein and to the same between biomacromolecular and morphological development of MG.     

Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons

The reactivation of the chick erythrocyte nucleus was studied after erythrocytes were induced to fuse with rat epithelial cells in the presence of Sendai virus. The chick nucleus swells, shows an increase in dry mass and protein content and resumes RNA synthesis. Nucleoplasmic antigens characteristic of the rat cell are found to migrate into the erythrocyte nucleus. The rate of uptake of these molecules, which are believed to be proteins, appears to be directly related to increases in nuclear size, ³H-uridine incorporation and RNA polymerase activity. The polymerase activity which increases during the first days after cell fusion is sensitive to α-amanitin but relatively resistant to actinomycin D. At later time points there is an increase in α-amanitin resistant polymerase activity which probably reflects the appearance of ribosomal RNA synthesis.When heterokaryons containing different proportions of rat: chick nuclei are compared, reactivation is found to proceed most rapidly in those containing a high rat: chick nuclear ratio. As the number of erythrocyte nuclei in heterokaryons increases, the rate of reactivation in the individual nuclei is progressively reduced suggesting that the erythrocyte nuclei compete with each other for macromolecules of specific importance for the activation process.     

Widespread occurrence of calicin, a basic cytoskeletal protein of sperm cells, in diverse mammalian species

A novel cytoskeletal element consisting of dense webs of thin (3-14 nm) filaments surrounding the nucleus of the sperm head has recently been isolated and shown to be associated with certain major basic proteins. Using antibodies specific for calicin, a prominent Mr-60,000 cytoskeletal protein of the posterior calyx of bull sperm heads detected in immunoblotting on gel electrophoretically separated polypeptides as well as in immunofluorescence and immunoelectron microscopy, we show that the same--or an immunologically related--polypeptide occurs in sperm heads of other species with greatly different morphology, including human, boar, guinea pig, hamster, rat and mouse. The calicin localization in the various species is described and discussed in relation to the specific sperm morphology.