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The role of the human epididymis in sperm maturation and sperm storage as reflected in the consequences of epididymovasostomy.

Epididymovasostomy has been used for examination of the role of particular regions of the epididymis in sperm maturation and storage, as reflected in fertility and in the motility, structural, and surface character, and also the number of the spermatozoa ejaculated. Human spermatozoa need be exposed only to the environment in the caput before passing into the vas deferens, in order to complete their maturation. However, the chance of pregnancy appears greater where the anastomosis is lower. Among the sperm characteristics examined, only motility was affected by the level of anastomosis, and there was a trend to higher numbers where the anastomosis was established beyond the upper caput. The storage function of the cauda is discussed in light of the finding that epididymovasostomy patients sometimes ejaculate sperm numbers seen in normal men.

Ejaculation

Changes in porcine sperm lactate dehydrogenase isoenzymes during sperm maturation.

The changes in sperm lactate dehydrogenase (LDH-lactate: NAD oxidoreductase, EC 1.1.1.27) activity and the relative occurrence of LDH isoenzymes during sperm maturation were studied using pubertal German improved Landrace boars. The LDH of spermatozoa liberated from the testis, caput epididymis, proximal and distal corpus epididymidis and cauda epididymidis was spectrophotometrically quantified while the LDH isoenzymes were separated on fine cellulose acetate membrane strips with the Sartorius Sartophor system. The LDH content of sperm dropped drastically as they moved from the testis to the caput epididymidis. Thereafter, only little and insignificant changes were observed. Testicular sperm was composed more of the fastest anodically-migrating isoenzyme (LDH1) while with sperm maturation, the least or slowest migrating isoenzymes (LDH4 and 5) became progressively more dominant. This loss in LDH content in sperm and the shifts in the LDH isoenzyme patterns indicate that the development of sperm during maturation is dependent on a delicate balance between lactate and pyruvate, such that the cathodic isoenzymes involved in the anaerobic energy-supplying metabolic processes are sufficiently available for sperm activity and survival.

Animals

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.

Agglutination

Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation.

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.

Acrosome

The timing of the restitution of chromosome breaks induced by X-rays in the mature sperm of Drosophila melanogaster.

Drosophila melanogaster males with marked X and Y chromosomes were irradiated, and mature sperm sampled by mating the males to females carrying attached-X chromosomes. Induced loss and partial loss of the paternal sex chromosomes was studied. F1 females were scored according to their phenotype, and transmitted fragments were analyzed genetically. Half of the exceptional F1 females could be scored as "partial losses". Of the apparent total loss exceptions, which were tested, half were carrying detectable fragments. 21% of the transmissible fragments is an under-estimate because only 6 of the 10 chromosome tips were marked in such a way that duplications could be detected. In addition, the markers used were located near, but not at, the chromosome ends. These data are interpreted as indicating that a high proportion of the chromosome loss and partial loss, induced by irradiation of mature sperm, is a consequence of chromatid rearrangements arising from chromosome breaks which stay open until replication. It is suggested that, during the transition from sperm head to mature pronucleus, repair of breaks and chromosome replication are two processes that occur in overlapping time intervals. It is therefore possible for chromosome breaks induced in mature sperm to give rise to chromosome and chromatid rearrangements.

Animals

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.

Animals

Dose-response relationships and R.B.E. values of dominant lethals induced by X-rays and 1.5 MeV neutrons in prophase-1 oocytes and in mature sperm of the two-spotted spider mite Tetranychus urticae Koch (acari, tetranychidae).

Mature sperm and prophase-1 oocytes of Tetranychus urticae Koch were irradiated with 250-kVp X-rays or 1.5 MeV fast neutrons. The X-ray doses ranged from 0.5 to 24.0 krad, and those of the fast neutrons from 0.1 to 16.0 krad. The genetic endpoint measured was lethality, expressed in the stages from egg to adulthood in the F1 progeny. The frequency of recessive lethals in female germ cells was estimated by comparing survival of fertilized versus unfertilized F1 eggs, after irradiation with the same dosage. X-Rays induce dominant lethals in prophase-1 oocytes by the action of both single hits on single targets and multiple hits on multiple targets. 1.5-MeV neutrons induce these effects predominantly by the action of multiple tracks on multiple targets. Dominant lethals were induced in mature sperm by X-rays and by fast neutrons by the action of both single hits on single targets and multiple hits on multiple targets. Both for prophase-1 oocytes and for mature sperm the low R.B.E. value corresponded with the relatively large multiple-target component of induction of dominant lethals by fast neutrons. The nature of dominant lethality in relation to the kinetochore organization of the chromosome is discussed. A non-linear trend in the dose--effect relationship was observed for both X-rays and fast neutrons for the estimated frequency of recessive lethals induced in prophase-1 oocytes. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad.

Animals

How is the flagellar length of mature sperm determined? II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro.

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 mum, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 mum, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.

Acrosome

Mecobalamin promotes mouse sperm maturation.

The effect of Mecobalamin (alpha-(5,6-dimethyl benzimidazolyl)-Co-methyl-cobamide: Me-B 12) on sperm production in the oligozoospermic mice experimentally induced by the treatment with adriamycin (0.3 mg/kg, three times a week for 5 weeks) was evaluated quantitatively by means of equilibrium sedimentation in Percoll. After centrifugation, the distribution profile of the sperm showed two peaks, i.e. the first peak near the bottom consisting of mature sperm with good motility and the second peak containing immature and/or immotile sperm. By oral administration of Me B 12 (1.0 mg/kg/day) to the oligozoospermic mice for 10 weeks, the sperm count, sperm motility, motile sperm count, diameter of seminiferous tubules and the percentage of good motile sperm with higher apparent density were increased as compared with those of the control. These results suggest that Me-B 12 enhanced the testicular function, resulting in an increased output of mature sperm.

Animals

Regulation of epididymal function and sperm maturation--endocrine approach to fertility control in male.

The structural and functional integrity of the epididymis, the acquisition of fertilizing ability by spermatozoa and their viability within the epididymis are androgen dependent phenomena. Although the precise mechanism by which sperm maturation and viability in the epididymis are brought about by androgen are not clearly understood, it is generally held that specific epididymal secretions produced under the influence of androgen affect these events. Though the spermatozoa appear to remain viable in a low androgen environment, sperm maturation requires a relatively high androgen environment. Against this background the potentiality of antiandrogens as extragonadal antifertility agents has been discussed. Studies with steroidal and nonsteroidal antiandrogens have revealed that in adult animals the secretory activity of the epididymis, as evidenced by the level of glycerylphosphorylcholine, either remains unaffected or is stimulated under their influence. These studies have further indicated that the extragonadal antifertility action of antiandrogens will depend upon their ability to (1) lower the testicular androgen synthesis and/or androgen binding protein, which possibly serves as a carrier of androgen from the testis to epididymis; (2) to lower local androgen synthesis as a result of reduced levels of circulating androgen, and (3) to inhibit 5 alpha-reduction of testosterone to dihydrotestosterone and/or to inhibit androgen binding to receptors. Success in the rational development of new antifertility agents for male which will act by controlling epididymal function will depend upon a clear understanding of the factors that regulate epididymal secretion and the role of epididymal secretions in sperm maturation and survival.

Androgen Antagonists

Spermiation and sperm maturation in the marmoset.

The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hook-shaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.

Animals

Characterization of fibronectin as a marker for human epididymal sperm maturation.

Fertilization involves adhesive interactions between gametes similar to those mediated by fibronectin (FN) in other cellular systems. Fibronectin has been found on the equatorial segment of ejaculated human serum. As sperm capacity to interact with the oocyte is acquired during epididymal transit, the possible participation of FN in human sperm maturation was studied. The presence of FN in both epididymal sperm and fluid was demonstrated by the detection of a major component of 220 kD in immunoblot studies using anti-FN antisera. The concentration of FN in soluble tissue extracts of epididymis was determined by enzyme-linked immunosorbent assay (ELISA). A gradual increase along the length of the organ, averaging 12-fold from proximal caput to distal corpus, was detected. Immunocytochemistry assays indicated that the number of spermatozoa with immunoreactive FN over the equatorial segment increased from 18% in caput to 64% in distal corpus epididymis. Immunoprecipitation of medium from epididymal explants culture with anti-FN antiserum demonstrated the de novo synthesis of FN in vitro. The greater number of FN-positive sperm coincident with FN accumulation in distal regions of the epididymis supports the role of FN in sperm maturation.

Biomarkers

Membrane remodelling during sperm maturation in the epididymis.

Identification of molecules or factors on spermatozoa that are involved in the recognition and binding to the zona pellucida of the egg is one of the central problems in current research on mammalian fertilization. This information is important, not just from the standpoint of scientific interest, but also for treatment of male infertility and design of future contraceptive technology. One of the most informative approaches to solving this puzzle has been to study the maturation of spermatozoa in the epididymis. The gradual development of motility and zona-binding capacity by spermatozoa after they leave the testis affords an opportunity to investigate what distinguishes a fertile from an infertile spermatozoon and what mechanisms are involved in the transformation from one state to the other. This chapter has reviewed some of the current information on sperm maturation in the epididymis and attempted to correlate it with concepts emerging from in vitro fertilization experiments. Emphasis has been placed on mechanistic aspects of maturation changes to the sperm plasma membrane, not only because in many species it is the surface membrane that encounters the zona and it is therefore likely to be the site of potential recognition molecules, but also because the plasma membrane influences many intracellular events and has a bearing on the maturation of the nucleus and axoneme as well. An example of the latter is shown by the induction of motility in demembranated testicular sperm by the addition of ATP and Mg2+ (Mohri and Yanagimachi 1980; White and Voglmayr 1986). This suggests that the motor apparatus for motility is essentially present in immature spermatozoa and that one of the requirements for it to become fully operative are subtle alterations to the permeability properties of the plasma membrane. A similar mechanism may also be true of the maturing sperm nucleus to which only passing reference has been made here. Very little is known about this aspect of sperm maturation beyond the increasing stabilization of the nucleoprotein by formation of disulphide bonds (Bedford 1975). The fact that isolated heads from immature spermatozoa fail to form pronuclei if injected directly into the egg (Uehara and Yanagimachi 1977) may explain, in part, the high embryonic mortality observed when not fully mature spermatozoa are induced to fertilize eggs (Orgebin-Crist et al. 1975). Thus maturation events within the nucleus are just as important as those on the surface membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals

An evolutionary view of the male reproductive tract and sperm maturation in a monotreme mammal--the echidna, Tachyglossus aculeatus.

In exploring the evolution and adaptive significance of epididymal function, we have studied the male excurrent duct and spermatozoa of a monotreme mammal--the echidna. Sperm maturation in the echidna excurrent duct appears simpler than that in most therians examined. Furthermore, neither the duct nor the spermatozoa of the echidna display specific therian characteristics; they bear a much closer resemblance to those of non-passerine birds. The echidna spermatozoon is filiform, the sperm tail has no distinctive features, and the anterior seventh of the undulating nucleus is covered by a modest acrosome. Immediately behind this a restricted apposition between plasma membrane and nuclear envelope constitutes a post-acrosomal ring. This is evident also in some reptiles and marsupials, whereas in Eutheria such a membrane association appears as the posterior ring at the base of the sperm nucleus. Maturation of spermatozoa in the Wolffian duct of the echidna appears to be expressed only in a changing capacity for motility and in loss of the cytoplasmic droplet. Neither surface, structural nor acrosomal changes that characterize sperm maturation in therian mammals have been detected in maturing echidna spermatozoa. The echidna duct displays little of the regional complexity of the epithelium that typifies this duct in the Theria. Of five regions distinguishable on the basis of epithelial morphology, the first two appear to be counterparts of efferent ducts by virtue of a low columnar, partially ciliated epithelium. The tall pseudo-stratified Golgi-rich epithelium of the major portion of the duct broadly resembles that of the therian epididymis, but it displays only two structurally distinguishable regions, the more distal being the site of a dense luminal secretion. The foamy epithelial cells of the fifth and terminal region, characterized by a mass of supra-nuclear vesicles and rough ER, suggest a secretory function that may in some way contribute significantly to the ejaculate, for accessory glands are poorly developed in monotremes. The possibility is considered that the relative complexity of epididymal function and sperm structure in therian mammals could have been determined by evolutionary change in the milieu of the female tract, and/or in the character of the egg vestments that the fertilizing spermatozoon must penetrate.

Animals

Integrating transcriptomics and metabolomics reveals the molecular landscape of sperm maturation driven by regional differentiation in the epididymis of Guizhou-Guiqian semi-fine wool sheep.

Epididymal regionalized differentiation is crucial for sperm maturation. However, little is known about the synergistic remodeling mechanisms of different epididymal segments at the transcriptional and metabolic levels during sexual maturation in ruminants (especially sheep). We investigated the caput, corpus, and cauda epididymidis of pre-pubertal (2-month-old) and post-pubertal (7-month-old) Guizhou-Guiqian semi-fine wool sheep using histology, RNA sequencing, and metabolomics. Post-pubertal tissues exhibited increased luminal diameters, cilia lengths, and abundant cauda spermatozoa. Transcriptomic analysis revealed increasing differentially expressed genes (DEGs) along the caput-corpus-cauda axis (4642, 6103, and 7698 DEGs, respectively). Metabolomics detected 786 unique differentially accumulated metabolites (DAMs). Region-specific analysis showed that in the caput, up-regulated pathways (fructose/mannose metabolism; HK2, ALDOA, HKDC1) provide energy and substrates for initial sperm motility. In the corpus, down-regulated genes associated with extracellular matrix and tight junctions suggested epithelial barrier remodeling to establish an immune-tolerant microenvironment. The cauda specifically up-regulated the pentose phosphate pathway (FBP1, GPI) and glutathione metabolism, maintaining redox homeostasis for long-term sperm storage. Additionally, glycerophospholipid metabolism was enriched across all segments, where PEMT, AGPAT5, and LCAT likely regulate sperm plasma membrane fluidity. In conclusion, during sexual maturation, the caput drives energy metabolism and glycosylation, the corpus establishes immune tolerance, and the cauda maintains antioxidant homeostasis. The glycerophospholipid network throughout the across all epididymal segments synergistically remodels sperm membrane. This study reveals the underlying multi-omics regulatory mechanisms of epididymal functional differentiation, providing a theoretical basis for elucidating the molecular mechanisms of sperm maturation in this breed and for the molecular breeding of early reproductive performance in rams.

Animals

Sperm maturation and storage in the male rat after acute treatment with aflatoxin B1.

The effects of acute aflatoxicosis on sperm maturation in the rat were evaluated using sperm GOT, GPT and LDH activities as indices. The levels of these enzymes in sperm undergoing maturation were stable like the sperm populations in the testis and epididymis. Acute aflatoxicosis enhanced corpus epididymal sperm transaminase but not sperm transit.

Aflatoxin B1

Fine structure of spermiogenesis in the coccid insect Aspidiotus perniciosus: late stages of differentiation and structure of the mature sperm bundles.

Spermatids of A. perniciousus are organized into bundles each containing 32 cells. During the process of differentiation, nuclear elongation and chromatin condensation take place. The mature sperm bundles are finally surrounded by the secondary sheath which is composed of a trilaminar membrane. The mature sperm is not differentiated into head, mid piece or tail but has two tapering ends and an electron dense central core. Centrioles, the nuclear membrane, mitochondria and a flagellum appear to be lacking. Microtubules are arranged in a spiral pattern forming the enveloping microtubular sheath.

Animals

The non-mature sperm cells: evaluation of surface markers and interaction in in vitro cultures.

The ejaculated human seminal plasma cells may be classified into subpopulations on the basis of spermatogenesis or biological features. In this paper we divided these cells into two main groups: one of them is obviously predestinated to fertilization as mature sperm cells, viable and motile ejaculated cells. Second group of unknown biological role is very heterogeneous; simplifying its variety we call them non-mature sperm cells. Morphological studies by electron microscopy and evaluation of surface markers of these cells by monoclonal antibodies did not reveal any known biological features or markers for cells of leukocyte origin. They did not express any antigenic determinants of HLA system and they did not react with autologous or allogeneic lymphocytes.

Antibodies, Monoclonal