Basic protein changes during the final stages of sperm maturation in the house cricket.
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Animals↗
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Cytofluorometric study of nuclear sulphydryl and disulphide groups during sperm maturation in the mouse.
The amount of nuclear sulphydryl and disulphide groups was determined by cytofluorometry on single mouse spermatozoa from the caput, corpus and cauda epididymidis, and the vas deferens. N-(7-Dimethylamino-4-methylcoumarinyl) maleimide was used as a specific and quantitative fluorescent reagent for thiols. The sperm nuclear content of free sulphydryl groups decreased sharply from the caput epididymidis to the vas deferens. The amounts of cysteine residues present as the thiol form in the spermatozoa from the caput, corpus and cauda epididymidis and vas deferens were 50, 15, 5 and 3% respectively.
Animals↗
Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation.
In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation.
Animals↗
Plasma membrane structure and remodelling during sperm maturation in the epididymis.
All mammalian spermatozoa are surrounded by a limiting plasma membrane that mediates many of the early events during fertilization. The membrane is derived originally from spermatogonia/spermatocytes in the testis but is then modified considerably during spermiogenesis, epididymal maturation and capacitation. It is characterized by an unusually high proportion of polyunsaturated phospholipids that give it special physical characteristics and compartmentalization of many of its component proteins and lipids into discrete domains on the head and tail. During passage of spermatozoa through the epididymis remodelling of the plasma membrane is commensurate with acquisition of motility and fertilizing capacity. Remodelling processes include uptake of secreted epididymal glycoproteins, removal or utilization of specific phospholipids from the inner leaflet of the bilayer, processing of existing or acquired glycoproteins by endoproteolysis and re-positioning of both protein and lipid components to different membrane domains. These modifications are carefully coordinated at different zones of the epididymis and indirectly affect intracellular membranes, organelles and even nucleoprotein. Maturation changes are described and appraised within the context of current concepts of membrane structure and fertilization.
Animals↗
Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation.
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Animals↗
Effects of egg and sperm maturation and spawning on the distribution and elimination of a polychlorinated biphenyl in rainbow trout (Salmo gairdneri).
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Adipose Tissue↗
Function of human epididymal proteins in sperm maturation.
Human post-testicular proteins were cloned by subtractive screening of epididymal cDNA libraries, employing testis as the primary negative control. This method identified six human epididymal cDNAs, named HE1-HE6, which are derived from abundant epididymal mRNAs. With the exception of HE5, which turned out to be identical to the lymphocyte surface antigen CD52, they represented completely novel human gene products. To date, there is little information on their function and the mechanism of their deposition on the sperm surface. Unlike the sperm coating antigens, CD52 binds firmly to the sperm membrane via its GPI anchor during epididymal passage. Its synthesis is carefully regulated by the epididymal epithelium. From the results of both in vivo and in vitro studies it was concluded that androgen and temperature are principal factors synergistically modulating epididymal CD52 expression. The human counterparts of two well-known major rodent epididymal proteins, secretory epididymal glutathione peroxidase (sGPX) and acidic epididymal glycoprotein (AEG = Protein DE), were not cloned by the subtractive screening approach, but by RT-PCR amplification.
Antigens, CD↗
Sperm maturation in human semen: role of transglutaminase-mediated reactions.
A Ca2+-dependent, transglutaminase-like activity has been detected both free in the human seminal plasma and bound on the spermatozoon surface. A marked variability of the two enzymatic activities in the semen of different normal subjects was observed; but limited changes occurred in various ejaculates of the same individual. Moreover, we report evidence of the ability of several seminal plasma proteins to act as acyl donor substrates for endogenous transglutaminase, whereas human ejaculated spermatozoa have been shown to possess polyamine-binding sites specifically involved in transglutaminase-catalyzed reactions. It is postulated that semen transglutaminase may play a role in suppressing sperm antigenicity and in the male gamete's acquiring biological features of a fully differentiated and fertile cell.
Adult↗
The maturation of sperm motility in the epididymis and vas deferens of the vervet monkey, Cercopithecus aethiops.
Among the diverse facets of sperm maturation, changes in motility are conspicuous and hence studies of sperm kinematics might provide good indices for sperm maturation. Accordingly, the maturation of sperm motility in the epididymis and vas deferens of the vervet monkey, Cercopithecus aethiops, was assessed using a computer-aided sperm motility analysis system. The results revealed clear trends in the development of both sperm motility per se and in the movement characteristics of motile spermatozoa from different regions of the epididymis, the vas deferens and the ejaculate, reflecting maturational changes associated with the attainment of functional motility and fertility. Motion of spermatozoa from the caput epididymis was sluggish and irregular. As the spermatozoa moved through the corpus epididymis, motility increased sharply, and continued to improve through the cauda epididymis and vas deferens. Despite the high proportion of motile cells, full maturation of motion capabilities was not completed in spermatozoa from the corpus epididymis. Only once spermatozoa had reached the cauda epididymis and vas deferens did they attain their full vigour, and swam rapidly (greater VCL, VSL and VAP) with straightline trajectories (greater LIN, WOB and STR; lower ALH, MAD and CURV). After acquiring their maximal percentage motility and progressive velocity in the cauda epididymis and vas deferens, a slight decline in motility and vigour occurred in ejaculated spermatozoa, and was possibly associated with the ageing of stored spermatozoa. The results from this investigation have revealed clear trends in the maturation of the motility of vervet monkey spermatozoa during their transit through the epididymis and vas deferens and final emergence in the ejaculate, and have provided crucial baseline information on the reproductive physiology of this potentially valuable biomedical model to serve as a reference for future studies in reproductive toxicology.
Animals↗
Two genes, three messengers: hybrid transcript between a gene expressed at specific stages of T-cell and sperm maturation and an unrelated adjacent gene.
To identify new genes that are turned on following the expression of a pre-T-cell antigen receptor in early thymocytes, we developed a PCR-based subtractive approach that selects for mRNAs that are differentially expressed between thymocytes at the CD4(-)CD8(-) and CD4(+)CD8(+) stages. This approach led to the characterization of a cDNA, Tssn1, which is predominantly expressed in CD4(+)CD8(+) cells and at specific stages of spermatogenesis. We analyzed the structure of the Tssn1 gene in mouse and human and found that it is highly conserved. Of interest, Tssn1 is adjacent to an unrelated gene named 2310047L21Rik and gives rise to hybrid 2310047L21Rik--Tssn1transcripts. Therefore, this analysis offers insights into the critical steps that may precede the birth of new genes endowed with either compound functions or novel tissue distribution.
Amino Acid Sequence↗
Results of microsurgical vasoepididymostomy: role of epididymis in sperm maturation.
One-hundred-and-ninety patients with obstructive azoospermia caused by bilateral epididymal blockage have been followed for greater than or equal to 4 years after undergoing 'specific tubule' vasoepididymostomy. When anastomosis was required in the corpus epididymidis, the 'patency' rate was 78% and the overall pregnancy rate was 56%. The pregnancy rate for 'patent' cases was 72%, indicating that a high fertility rate can be obtained with spermatozoa that have not passed through the full length of corpus epididymidis. By contrast, with vasoepididymostomy to the caput epididymidis there was a 73% 'patency' rate, but the overall pregnancy rate was only 31%. The pregnancy rate for 'patent' cases was 43%. Spermatozoa from the corpus epididymidis have a higher rate of fertility than spermatozoa from the caput epididymidis, but spermatozoa from proximal areas of the corpus have no less fertility than spermatozoa from the distal corpus epididymidis. The most remarkable observation is that in almost half the cases, spermatozoa which have never journeyed beyond the caput epididymidis seem to be capable of causing pregnancy.
Epididymis↗
Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation.
This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotin-streptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.
Animals↗
Identification of post-translational modifications that occur during sperm maturation using difference in two-dimensional gel electrophoresis.
Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three samples in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa.
Adenosine Triphosphatases↗
Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors.
OBJECTIVES: Hitherto, patients with testicular dysfunction and azoospermia had to resort to adoption, donor sperm insemination, or child-free living. The realization that a proportion of such men harbor spermatozoa in their testicular parenchyma, combined with the ability of intracytoplasmic sperm injection (ICSI) to effect pregnancy with single sperm, has prompted male infertility clinicians to explore testicular sperm extraction (TESE) in this patient population. We sought to investigate the likelihood of finding spermatozoa during TESE from men presenting with nonobstructive azoospermia and to define if any factors existed that were predictive of eventual sperm presence or absence. METHODS: Thirty patients with nonobstructive azoospermia underwent TESE and simultaneous formal testis biopsy, cytologic analysis, and wet preparation analysis. Tissue obtained from TESE was analyzed according to a rigorous protocol, followed by exhaustive searching by trained embryologists. RESULTS: Twenty-one patients (70%) had spermatozoa found on testicular tissue analysis. Neither patient age nor follicle-stimulating hormone (FSH) level was predictive of the ability to find sperm. With regard to histologic pattern, 50% of men with Sertoli cell-only, 75% of patients with maturation arrest, and 100% of patients with spermatids seen on histologic analysis had sperm retrieved from their testicular tissue during TESE. Absence of sperm on cytologic smear and wet preparation analysis failed to predict the presence of sperm on formal testicular tissue analysis in 40% of patients. CONCLUSIONS: Men with nonobstructive azoospermia may have mature spermatozoa present within their testicular parenchyma. Relying on these data, patients should not be excluded from TESE based on serum FSH level, age, prior histopathologic pattern, or cytology/wet preparation results. These figures will allow clinicians to counsel patients with nonobstructive azoospermia informatively regarding TESE and their chances of having testicular sperm retrieved.
Adult↗
Maturation of sperm motility in the epididymis of the common marmoset (Callithrix jacchus) and the cynomolgus monkey (Macaca fascicularis).
The acquisition by spermatozoa of the capacity for movement upon liberation from the epididymal duct into buffer is one of the obvious changes associated with sperm maturation and, hence, studies of sperm kinematics might provide good indices for sperm maturation. Profiles of development in the movement pattern of spermatozoa taken from six regions of the epididymis were established in marmoset and cynomolgus monkeys using computer-assisted sperm analysis. Motion of spermatozoa from initial regions was sluggish and irregular, then developed in the more distal region into erratic movement effected by an increase in the vigour of flagellation as reflected by curvilinear velocity. The tendency of immature spermatozoa to display circular motion was more common in the marmoset than in the cynomolgus monkey. In the marmoset the most drastic changes in percentage motility and kinematic parameters of spermatozoa occurred between the distal caput and the proximal corpus; maximal values for these parameters were found around the distal corpus epididymis. For the cynomolgus spermatozoa the sharpest gain in percentage motility was also achieved between the caput and proximal corpus epididymis, but with full kinematic development in the proximal cauda epididymis. These maturational changes were not synchronous among spermatozoa from the same site as they moved along the epididymis, as reflected in the heterogeneity of each sample in the kinematic properties of individual spermatozoa, especially those from the proximal corpus epididymis. The maturational profiles in the monkeys are comparable to the situation in the human, for which they may provide a useful model.
Animals↗
Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.
It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
Animals↗
Changes of the DNA packaging mode during boar sperm maturation.
Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.
Animals↗
Immunohistochemical localization of endothelial nitric oxide synthase in human testis, epididymis, and vas deferens suggests a possible role for nitric oxide in spermatogenesis, sperm maturation, and programmed cell death.
Recent work has implicated nitric oxide (NO) in several aspects of male genital physiology including erectile function and androgen secretion, as well as in vitro effects on sperm motility and capacitation. The objectives of this study were to characterize the distribution of endothelial nitric oxide synthase (eNOS) in "normal" human testis, epididymis, and vas deferens and in testis pathology. Nitric oxide synthase protein was localized immunohistochemically using an eNOS monoclonal antibody. Endothelial NOS protein co-localized to areas that showed positive NADPH diaphorase activity. Within the testis, eNOS protein was localized to the cytoplasm of Leydig cells and Sertoli cells at all stages of spermatogenesis. Within the epididymis and vas deferens, eNOS was localized to the epithelium. Endothelial NOS was also localized to endothelial cells in all tissues; it was not detectable in normal germ cells. Endothelial NOS and diaphorase activity were, however, detected in degenerating or apoptotic intraepithelial germ cells. In addition, prematurely shed spermatocytes and spermatids had intense eNOS expression. Previous studies have suggested a role for NOS in the contractile, hemodynamic, and hormonal aspects of testicular function as well as in epididymal secretion. The studies reported herein suggest a role for eNOS in spermatogenesis and germ cell degeneration.
Antibody Specificity↗