Filipin-sterol complexes in golden hamster sperm membranes with special reference to epididymal maturation

Summary The distribution of membrane filipin-sterol complexes (FSCs) was qualitatively surveyed on freeze-fracture replicas of spermatozoa from the male reproductive tract and ejaculates of golden hamster. In the head, the acrosomal plasma membrane showed the strongest filipin labeling on the principal segment, but it was absent in the quilt-like pattern areas. These latter were observed in both caput and corpus epididymal spermatozoa, but were absent in mature spermatozoa. The postacrosomal plasma membrane had few FSCs and both the outer and inner acrosomal membranes were always negative to filipin. The nuclear membrane of the principal segment was constantly filipinpositive. The nuclear membrane of the postacrosomal region had more FSCs than that of the principal segment, particularly in mature spermatozoa. Many linear, rod-like FSCs were observed on the postacrosomal nuclear membrane of mature spermatozoa, especially in the uterine spermatozoan samples. In the neck, the plasma membrane had only a few FSCs. The redundant nuclear membrane was slightly filipin-positive, while the membrane scroll of mature spermatozoa was heavily labeled. In the tail, the plasma membrane of both the middle and principal piece was moderately labeled.     

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.     

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.     

Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies

The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.     

Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X-100-insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK2 cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.     

Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: Its potential for tyrosine as well as serine phosphorylation towards microtubule-associated protein 2 and histone H 2B

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 μM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149–154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M_r 42,000, is stimulated by Mg²⁺ > Mn²⁺ but not by Ca²⁺, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa. © 1994 Wiley-Liss, Inc.     

Fate of spermatozoa that do not participate in fertilization in the domestic fowl

Summary The fate of spermatozoa that do not participate in fertilization was investigated by electron microscopy. After artificial insemination, we observed several spermatozoa between the fibers of the outer layer of the vitelline membrane of the ovum. One or more spermatozoa were also found in a phagocytic vesicle of macrophages located in the intercellular space of the mucosal epithelium of the infundibulum or in the outer layer of the vitelline membrane.From these observations, we assume that the superfluous spermatozoa in the lumen of the anterior part of the oviduct might be removed by inclusion into the outer layer of the vitelline membrane and by phagocytosis by macrophages.The authors are greatly indebted to Assoc. Prof. Osamu Koga for his invaluable advice. The authors also wish to thank Mr. Takayuki Mri for his helpful suggestions and technical advice. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Scanning electron microscopic study of spermatozoa from gossypol-treated rats

Summary Spermatozoa from the cauda epididymidis of gossypol-treated rats exhibit distinctive departures from the morphology of spermatozoa from control rats: wrinkled and disorganized cell membrane in the head and tail regions, cell membrane missing from segments of the tail midpiece and principal piece regions, malformed heads, decapitate spermatozoa, retention of a cytoplasmic droplet at variable loci along tail midpieces, and looped tails. The observations suggest that gossypol exerts its contraceptive effect during spermatocytogenesis and spermiogenesis, including the posttesticular development and maturation of spermatozoa in the epididymis.     

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.     

Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization.

An easy assay for quantification of Y chromosome-bearing sperm (Y-sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male-specific DNA fragment was tested for identification of Y-sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y-sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.