Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry.

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.

Nucleotide sequence and exon-intron organization of the human proacrosin gene.

Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb--4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three activesite residues His, Asp and Ser are encoded by three different exons. According to the exon-intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsin and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active-site residue together with the proacrosin-specific proline-rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline-rich domain was generated during evolution by intron-exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at -74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis-specific genes, typical TATA and CAAT box sequences in convential distances from the 5' end of the transcription start site could not be evaluated in the proacrosin gene.

Differences in lipoprotein composition between heads and tails of human sperm: an infrared spectroscopy study.

Human spermatozoa and their fractions (heads and tails) have been studied by infrared spectroscopy. Protein conformation in isolated human spermatozoa heads, although predominantly of the alpha helix or random coil type, has a significant proportion of antiparallel B structure. Spectra of isolated spermatozoa tails show that proteins exist in this fraction preponderantly in pleated-sheet conformation (parallel and antiparallel). The quantity and type of lipids seem to be drastically different between heads and tails of spermatozoa. Head lipids are scarce and difficult to extract, and they are apparently tightly bound to proteins, highly unsaturated, and rich in free hydroxyl and carboxyl groups. Tail lipids are more abundant and more easily extractable. Head phospholipids are probably phosphatidylcholine, cephalins, and inositols, and tail phospholipids are preponderantly plasmalogen-type lecithins and sphingomyelins. The presence of specific infrared bands points to the existence in tails of important amounts of sulfur compounds, probably sulfolipids or sulfoglycolipids.

Stimulation of phospholipid turnover in isolated sea urchin sperm heads by the fucose-sulfate glycoconjugate that induces an acrosome reaction.

The fucose-sulfate glycoconjugate (FSG) component of sea urchin egg jelly that induces an acrosome reaction in spermatozoa-stimulated multiple Ca2+-dependent phospholipid changes in the sperm cell head and flagellum. When cells were radiolabeled with myo-[3H]inositol, FSG treatment decreased radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate within 30 s. In addition, FSG treatment elevated concentrations of phosphatidic acid in spermatozoa. The Ca2+-channel antagonist, verapamil, inhibited the effects of FSG on [3H]polyphosphoinositides and phosphatidic acid. To investigate the possible compartmentalization of phospholipid turnover, isolated heads and flagella were prepared. Treatment of sperm heads with FSG or the monovalent cation ionophore, gramicidin S, caused increased [3H]inositol phosphate and phosphatidic acid accumulation and induction of an acrosome reaction. Effects of FSG and gramicidin S on phosphatidic acid elevations in sperm heads and intact cells were inhibited by verapamil. FSG failed to cause detectable changes in [3H]inositol phosphate or phosphatidic acid concentrations in isolated flagellar preparations. However, when cells were treated with FSG and the flagella were isolated subsequently, phosphatidic acid concentrations in the flagellar preparations were increased.

Isolation, structure and protein composition of the perforatorium of rat spermatozoa.

Heads of spermatozoa were sonically separated from tails and treated in 1 N NaOH until the perforatoria were partially detached from the nucleus. Their complete detachment was then assured by repeatedly passing the suspension through a 22-gauge needle. The perforatoria were then separated from nuclei on sucrose gradients and the purity of the fraction was verified by electron microscopy. The isolated perforatoria were denatured and used to raise antibodies or run on polycrylamide gels. Such gels revealed many polypeptide bands, six of which were most prominent (Mr approximately 13,000, 13,400, 16,000, 33,000, 35,000, and 43,000). Of these, the 16,000 Mr polypeptide was major. Anti-perforatorium serum reacted with the perforatoria of fixed spermatozoa, with a substance found between the plasmalemma and the outer acrosomal membrane of the acrosomal head cap and with the inner component of the ventral spur, but not with the postacrosomal dense lamina. This observation indicated that the perforatorium and dense lamina, although structurally continuous to form the perinuclear theca, are biochemically distinct. On Western blots, the anti-perforatorium serum reacted with the prominent polypeptides of the perforatorium and cross-reacted with some less prominent polypeptides of the fibrous sheath (FS) and outer dense fibers (ODF), most notably with a 16,000 Mr polypeptide found in both. Likewise anti-FS or anti-ODF serum cross-reacted with some major and minor polypeptides of the perforatorium, again most notably with a major 16,000 Mr polypeptide. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. These results demonstrate that the perforatorium is composed of several polypeptides, is immunologically distinct from the postacrosomal dense lamina, may be immunologically similar to a substance found between the plasmalemma and outer acrosomal membrane and to a substance found on the inner aspect of the ventral spur, has antigenic determinants in common with the FS and ODF, and may share a 16,000 Mr polypeptide with these two cytoskeletal structures of the flagellum.

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.

Zinc in sperm chromatin and chromatin stability in fertile men and men in barren unions.

The stability and the content of zinc of the chromatin were studied in spermatozoa from ten men with unexplained infertility, and in spermatozoa from five fertile donors. A positive relation was found between zinc in sperm nuclei (X-ray microanalysis) and the resistance of the chromatin to decondense in sodium dodecylsulfate (SDS). The infertile men had lower degree of sperm chromatin stability and lower sperm zinc content than the fertile donors. A subgroup of the infertile men, which all had minor clinical signs of prostatic inflammatory reaction, had the lowest content of zinc in the chromatin and the lowest degree of chromatin stability. A low content of nuclear zinc would impair the structural stability of the chromatin and thereby increase the vulnerability of the male genome. This mechanism may be one explanation for the reduced fertility of the men with minor inflammation of the prostate.

Localization and distribution of actin in mammalian sperm heads.

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed.

Nuclear zinc in human epididymal and ejaculated spermatozoa.

Sperm nuclear zinc content (expressed as zinc/phosphorus and zinc/sulphur ratios) was determined with X-ray microanalysis in individual, air-dried, epididymal spermatozoa from elderly men, and in ejaculated spermatozoa from healthy donors. Ejaculated sperm nuclei contained (also after treatment with sodium dodecyl sulphate) more zinc than epididymal spermatozoa. The results indicate that the human spermatozoon accumulates zinc from the prostatic fluid upon ejaculation.

Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component.

A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.

Characterization of fibronectin on human spermatozoa.

Ejaculated human spermatozoa were shown to have fibronectin polypeptides on their surface. Immunofluorescence studies revealed fibronectin as a belt-like fluorescent band on the post-acrosomal area of sperm heads, whereas none was found in sperm tails. The location of the fluorescent band corresponded to the equatorial segment of the spermatozoon. Fibronectin polypeptides were heterogeneous with Mr ranging from 35000 to 210000, as revealed by immunoblotting and by immunoprecipitation of detergent extracts from surface-radioiodinated spermatozoa.

Calmodulin, a calmodulin acceptor protein, and calcimedins: unique antibody localizations in hamster sperm.

A calmodulin acceptor protein has been identified in isolated hamster caudal sperm by immunofluorescence and Western transfer techniques. The protein shows a localization in sperm heads identical to calmodulin. Fluorescence of both calmodulin and the acceptor protein are lost by treatment with MgCl2, conditions which release the acrosome. These results are consistent with the proposed function of calmodulin in a sperm function.

Localization of epididymal secretory proteins on rat spermatozoa.

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.

Biochemical factors affecting the fertility of cryopreserved human semen.

The effects of dilution and cryopreservation on biochemical, fine structural and conventional semen parameters were studied and correlated with an index of fertility. Dilution of semen had major effects on the parameters studied. Freezing and thawing further reduced motility and the proportion of viable spermatozoa, and increased the proportion of spermatozoa with damaged membranes. The fertility index of donors was positively correlated with sperm concentration, seminal plasma protein concentration, damage to sperm head membranes and the proportion of spermatozoa with intact acrosomes prior to dilution or after freezing and thawing. Seminal plasma [Na+], [Ca2+], Ca2+/Mg2+-ratios and the change in [K+] were negatively correlated with fertility index. Multiple regression analysis revealed that [Na+] in seminal plasma, and sperm concentration in semen prior to dilution and freezing, contribute to 68% of the variation in fertility of cryopreserved semen.

Proteins of de-membraned protamine-depleted mouse sperm. Homology with proteins of somatic cell nuclear envelope/matrix.

Following treatment with Triton X/1 M NaCl/2-mercaptoethanol, mouse sperm heads are divested of protamines and other basic proteins; the residual structure is one in which the general morphological organization of the decondensed chromatin and the nuclear boundaries are conserved [1]. In this study, the protein complement of that residual structure has been characterized and subdivided into two sets: 1. Those that are sperm-unique, including constituents of the sperm head that may be intrinsically nuclear (or extra-nuclear, but exceedingly adherent to the nuclear envelope). 2. Those that display corresponding electrophoretic properties and immunologic cross-reactivity with proteins of similarly treated mouse somatic cell nuclei. Among the latter are proteins of molecular weight 52, 63 and 69 kD, two of which (63 and 69 kD) appear to be homologous to polypeptides of somatic nuclear envelope/pore complex lamina. Absence from sperm nuclei of the third of the characteristic predominant triplet of somatic nuclear lamina polypeptides of mammalian cells, here designated 67 kD, indicates cell-type variation in these structures. On the other hand, the identification of homologous polypeptides in the sperm and somatic complements suggests that those are specific instances of conservation and may represent the paternal contribution to the pool of polypeptides for assembly of the envelopes of the pronuclei of the one-cell embryo.

Localization of actin in the sperm head of the plains mouse, Pseudomys australis.

Actin was localized in the sperm head of the plains mouse, Pseudomys australis, using NBD-phallacidin, a fluorescent-labeled phallotoxin. It was found to be present in the two ventral hooks in sperm from the testis, epididymis, and vas deferens. Faint fluorescence was observed in other regions of the sperm head, whereas autofluorescence occurred in the midpiece of the tail in some preparations. The material in the ventral hooks was also found to be birefringent, which is consistent with it showing preferential orientation.

Investigation of fading and recovery of fluorescence intensity at 73.5 K.

Fading and recovery of Af-feulgen stained sperm heads are investigated at 73.5 K. The fast fluorescence signals are measured and stored by two coupled transient recorders. 100% recovery was reached after a dark time of 3 s. This shows that the photodecomposition is mainly caused by change of the probability density of energy level and not by destruction of the chromophore groups. The recovery effect allows measurement of the fluorescence intensity of the same sample more than 50 times. Therefore the unaffected spectrum can be measured directly, provided that between the short-term measurements at constant wavelength an appropriate dark phase has been put into operation.