Is lithium essential for epididymal sperm maturation?

A wider biological role of ultratrace element lithium in the mammalian reproduction has been reported, however, presence of lithium in the epididymal luminal fluid (ELF) and its influence on sperm during maturation events in the epididymal regions are still unknown. A pilot study was carried out in Jamunapari buck which revealed that levels of lithium in the ELF diminished gradually and significantly (P < 0.01) from caput to cauda epididymis, concomitantly, a distinct increase (P < 0.01) in the spermatozoan motility, viability and hypo-osmotic reactive sperm were observed, except spermatozoan motility that was found absent in the caput epididymis. Therefore, we hypothesize that levels of lithium in the epididymal regions is one of the motility initiation and/or regulatory factor for epididymal sperm maturation essential for acquiring fertilizing competence of sperm cells, hence, lithium could also be considered as one of the biomarker of sperm maturation in any species.     

Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI

BACKGROUND: Recent studies showed that ICSI with cauda epididymal or ejaculated sperm of infertile mice or men, respectively, was less effective in fertilization and normal embryo development than ICSI using sperm from the testes. These studies suggested that sperm nuclear quality declined after release from the testis, but the site where this loss of fertility occurs has not been localized. METHODS: We performed ICSI with testicular, caput, and cauda epididymal sperm from infertile Tnp1-/-Tnp2+/- mutant mice, which have a minimal level of transition nuclear proteins and are sterile by natural mating. RESULTS: When the heads of motile sperm from the testis or caput epididymis of Tnp1-/-Tnp2+/- males were injected into enucleated mouse oocytes, sperm chromosomes showed no difference from those of wild-type mice, but the chromosomes from sperm taken from the cauda epididymis of mutant males showed increased abnormalities. Injection of testicular or caput epididymal sperm from Tnp1-/-Tnp2+/- males into intact oocytes resulted in normal embryonic and fetal development and yields of liveborn equivalent to wild-type, but cauda sperm from Tnp1-/-Tnp2-/- mice produced lower implantation rates and yields of liveborn than did those from wild-type mice. CONCLUSIONS: These results demonstrate that in mice with sperm chromatin abnormalities, the decline in fertility of sperm with ICSI occurs after the caput epididymis. The advantage of using caput epididymal sperm for ICSI in certain situations may be considered as an approach to be tested in human assisted reproduction.     

Androgen regulation of glycosidase secretion in epithelial cell cultures from human epididymis.

The human epididymis and its secretions actively promote sperm fertilizing capacity and provide protection for spermatozoa against harmful influences. Among epididymal secretions, glycosidases have been recently studied and associated with molecular changes on the sperm surface. In the present work, we studied the influence of different concentrations of testosterone, dihydrotestosterone and cyproterone acetate on the secretion of alpha-glucosidase, N-acetyl-glucosaminidase, beta-glucuronidase and alpha-mannosidase by isolated and cultured epithelial cells from human caput, corpus and cauda epididymides. Cell cultures were obtained from aggregates of isolated tubule fragments plated on extracellular matrix-covered multi-well plates. Activities of the glycosidases were measured in conditioned culture media and were higher in the distal regions of the epididymis. Testosterone and dihydrotestosterone significantly increase the enzyme secretion in a concentration-dependent manner. This increase was higher in corpus and/or cauda than in caput epididymis. Cyproterone acetate caused a dose-dependent decrease in glycosidase secretion in cultures from all epididymal regions. It is concluded that the secretion of epididymal glycosidases is regulated by androgen, being stimulated by dihydrotestosterone and testosterone and inhibited by the androgen antagonist cyproterone acetate.     

Andrology: Evaluation of motility, fertilizing ability and embryonic development of murine epididymal sperm after coculture with epididymal epithelium

Murine sperm from the caput, corpus and cauda epididymis werecocultured with epididymal epithelial cells of their own regionor more distal regions, in the presence and absence of androgens(testosterone and dihydrotestosterone). Epitheial cell cultureswere used 3 or 10 days after preparation in a complex tissueculture medium (Chang's) as plated tubules. The coculture studiesinvolving spermatozoa and oocytes with epithelial cells werecarried out in T6 medium. Motility of caput spermatozoa wasmaintained for 24 h in the presence of day 3 corpus and caudaepithelial cells and hormones but not under other conditions.Likewise, the motility of corpus spermatozoa was maintainedfor 24 h in the presence of day 3 cauda epithelial cells andhormones but not other conditions. Fertilization of zonaintactoocytes by epididymal spermatozoa was not affected by theircoculture for 24 h with epithelial cells but fertilization ratesfor zona-free oocytes were increased for caput spermatozoa coculturedwith more distal epithelial cells. Fertilization rates for bothzona-intact and zona-free oocytes were increased for corpusspermatozoa cocultured with more distal cauda epithelial cells.The developmental capacity of embryos derived from caput spermatozoawas not significantly increased by coculture with epithelialcells but those derived from corpus spermatozoa cocultured withcauda epithelial cells were signilicantly increased. We concludethat the presence of more distal epithelial cells of the mouseepididymis maintains motility in culture, increases the abilityof caput and corpus spermatozoa to fertilize zona-free oocytesand increases the developmental capacity of embryos formed fromcorpus spermatozoa. These observations demonstrate the functionof epididymal regions in the maturation of murine spermatozoafor fertilization and embryo development.     

Morphometric analysis of intact sperm heads and of sperm nuclei in the mouse

A morphometric analysis of mouse sperm and of their nuclei was undertaken to investigate their respective post-testicular maturation. Sperm were collected from the testis, caput and cauda epididymidis, and their corresponding nuclei were isolated. Results indicate that the post-testicular maturation of sperm is distinct from that of nuclei. The size of intact sperm heads increases in the caput followed by a subsequent decrease in the cauda. In contrast, sperm nuclei decrease progressively in size. In general, a greater magnitude and number of alterations in intact heads and nuclei occur while in transit from the testis to the caput than during passage to the cauda epididymis. These results suggest that the period immediately following their release from the testis is crucial to the complete morphological maturation of sperm heads and nuclei. © 1993 Wiley-Liss, Inc.     

Autometallographic demonstration of zinc ions in rat sperm cells

An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.      

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.     

Mouse Sperm Rosette: Assembling During Epididymal Transit,in vitro Disassemble,and Oligosaccharide Participation in the Linkage Material

In many mammals, sperm associations had been observed, but not in the mouse. In this work, mouse sperm rosettes are morphologically described inside the epididymis and during its dissolution in a culture medium. Also characterized are the saccharides present in the linking material. Sperm association and other epididymal actions are supported by sperm during epididymal transit and are verified at the caudal region, suggesting a relation between epididymal transit and sperm maturation. In drops of epididymal content obtained from distal (cauda), but not from proximal (caput and corpus) regions; dissolved in culture medium, rosettes appear to be 10 to 15 motile sperm joined by their heads. After 3 min, sperm progressively detach, disassembling the rosette. These structures are studied by several techniques, including optic, electronic (scanning electron microscopy and transmission electron microscopy), and video microscopy. At the ultrastructural level, a dense network of electron‐dense material was observed between sperm heads, joining them. Based on previous works in rat, several lectins were used to characterize the type of saccharides present in this linking material. To avoid the contact between sperm and epididymal fluid from distal region—that probably exerts an influence on sperm association—a ligature was placed between caput and corpus. This epididymal content isolated from caput did not display any rosettes after 28 days. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acro somes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Ontogeny of mouse caudal proteins identified by a monoclonal antibody

A monoclonal antibody (mAb) D2G4 directed to human spermatozoa recognized antigens on the acrosomal region of both human and mouse spermatozoa and reacted with two proteins of molecular weights 45 kd and 26 kd. Immunohistochemical staining with this antibody indicated that only the epithelial cells of the cauda epididymis were stained and not the sections of testis, caput or corpus epididymis. These observations suggest that antigens recognized by D2G4 were acquired by the spermatozoa during their passage through the cauda epididymis and appear to have a role in the maturation of spermatozoa. The ontogeny of these antigens in mice was studied during sexual maturation. These antigens could be detected in cauda epididymis from day 50 onwards by immunohistochemistry. The highest concentration of these antigens was observed in the cauda epididymis of 80-day-old mice. SDS-PAGE analysis indicated that the 26 kd protein recognized by D2G4 was visible from day 50 onwards, confirming the immunohistochemical observations. The plasma testosterone levels showed a significant increase from days 40 to 60 followed by a decrease. The fact that these epididymal proteins appear during sexual maturation and at the time of the testosterone surge indicates that they are androgen dependent.     

Sperm maturation in the male reproductive tract: Development of motility

Rabbit spermatozoa were removed from various levels of the male reproductive tract. They were examined in Hanks' solution at room temperature with a phase contrast microscope and their motility characteristics were recorded cinematographically. Spermatozoa from the seminiferous tubules and ductuli efferentes show weak, vibratory movements with no forward progress. Little change in motility occurs until the sperm reach the flexure of the caput epididymidis where some are capable of moving more vigorously in a circular fashion. Samples from the distal caput epididymidis show a sudden increase in sperm activity and a consistent pattern of tight, circular movement. As the sperm traverse the corpus epididymidis, increasing numbers show progressive, forward movement with longitudinal rotation. The proportion of such sperm becomes significant only in samples from the upper cauda epididymidis and more distal regions. Sperm from the ductus deferens rarely retain the circular movement. It is concluded that rabbit spermatozoa undergo a distinct sequence of changes in their swimming movements as they mature in the epididymis. A similar change was noted in epididymal spermatozoa from the rat and guinea pig suggesting that this process is fundamental to sperm maturation in several species.     

Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation

Previously it was demonstrated that immature and immotile human spermatozoa from the caput epididymides developed a good progressive motility after in-vitro stimulation with phosphatidylcholine (PC). In order to define the role of PC and membrane anisotropy in epididymal maturation and to determine the exact lipid composition of human spermatozoa during epididymal maturation, spermatozoa from seven epididymides from patients who underwent orchiectomy because of prostatic cancer were investigated. Lipids were determined by high- performance thin-layer chromatography and gas chromatography. Membrane anisotropy was measured by fluorescence polarization. The ratio between PC and phosphatidylserine (PS) plus phosphatidyl ethanolamine (PE) plus sphingomyelin (SM) was significantly higher in spermatozoa from the cauda compared to those from the caput and corpus. This was due to an increase of PC and a decrease of the concentration of PS plus PE plus SM. With regard to fatty acids, those with saturated chains predominated in caput spermatozoa while the highest concentration of unsaturated long-chain fatty acids was in cauda spermatozoa. A lower membrane anisotropy of cauda spermatozoa compared with caput or corpus spermatozoa was found. In conclusion, during epididymal maturation human spermatozoa integrate lipids, particularly PC, which is strongly associated with the induction of progressive motility. A change in the pattern of fatty acids and a decrease in the cholesterol/phospholipid molar ratio cause a decrease in membrane anisotropy in cauda spermatozoa.      

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus.

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.     

Relationship of interstitial edema with L-cysteine-induced sperm granulomas in the pubertal rat epididymis.

Although the pathogenesis of sperm granulomas is complicated, the leakage of spermatozoa into extraluminal tissues is regarded as a crucial event. It has been previously shown that pubertal rats injected with L-cysteine develop interstitial edema followed by sperm granulomas in the epididymis. In this study we investigated the relationships between these two lesions in 6-week old rats given daily intraperitoneal injections of L-cysteine (1,000 mg/kg body weight) for 4 weeks. Rats were examined during weeks 0, 1, 2, 3 and 4 after the first injection. Interstitial edema (moderate or severe) and sperm granulomas were seen in the corpus and cauda epididymis of L-cysteine-treated rats in study weeks 2, 3, and 4. There was no marked alteration of basement membrane of the epididymal ducts in the edematous tissues as shown by immunohistochemistry with an antilaminin antibody. However, the extravasation of Evans blue dye given I hour before necropsy suggested that the severe interstitial edema was due to increased vascular permeability. In addition, a small number of neutrophils were seen in the edematous tissues, suggesting that they might play a role in the increased vascular permeability and leakage of epididymal fluid. Interestingly, slight interstitial edema was observed in the caput epididymis in both control and L-cysteine-treated rats in early study weeks 0, 1, and 2. It is speculated that this change was related to the leakage of epididymal fluid due to increased intraluminal pressure depending on rat epididymal maturation. Taken together, these findings suggest that the severe interstitial edema results from increased vascular permeability. This, along with increased intraluminal pressure, might be the trigger for duct rupture, the prerequisite for sperm granuloma formation associated with excessive doses of L-cysteine.     

Maturation antigen of the mouse sperm flagellum: II. Origin from holocrine cells of the distal caput epididymis

During epididymal transit, the mouse sperm flagellum acquires a surface glycoprotein (SMA4) from epididymal fluid that functions as a sperm antiagglutinin. To determine the origin of this molecule, testes and epididymides of male mice were sectioned for light microscopy and stained with wheat germ agglutinin (WGA)-peroxidase, a probe that has been used previously to examine the biology of SMA4. WGA reactivity was localized to the cytoplasm in a small population of cells in the distal caput epididymis. Testis cells and principle cells of the caput were nonreactive with WGA, while stereocilia were stained on principle cells in the corpus and cauda. The WGA-positive cells in the distal caput were identified as holocrine cells on the basis of morphology, distribution, and PAS + reaction. At high magnification, intense WGA reactivity was due to the presence of numerous apical granules in the cytoplasm. The location of the cells in distal caput coincided exactly with the region of tubule in which sperm first acquired SMA4 on their flagellae. These data suggest that holocrine cells near the junction of caput and corpus epididymis are the source of the sperm antiagglutinin SMA4.     

Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 ± 44 FSC/μm² (mean ± standard deviation, S. D. ), 322 ± 41 FSC/μm² and 355 ± 31 FSC/μm² in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 ± 39 FSC/μm²) and uterine sperm (243 ± 50 FSC/μm²). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.     

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat.

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosomal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1-5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35-50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64-71 K) was present in all cells of the epididymis, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the length of the epididymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.     

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosmal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1–5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35–50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64–71 K) was present in all cells of the epidiyms, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the lenght of the epidiymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.     

Short-term effects of androgen withdrawal on the structure of different epithelial cells in the rat epididymis

Rat spermatozoa are highly dependent on the milieu of the normal epididymis for their maturation and survival, and die within a few days after androgenic support of the epididymal epithelium is withdrawn. The immediate changes in the ultrastructural organization of the epithelial cells of the rat epididymis, 2, 4, 6 and 14 days following castration have been monitored by morphometric analysis of localized regions of the caput and cauda epididymidis. While castration results in greater endocytosis by principal cells (Moore and Bedford, '79), many of their early structural changes following androgen withdrawal (disappearance of vesicles from the cell apex, reduction in rough endoplasmic reticulum, a drop in the volume of the Golgi cisternae and increase in lysosome content) seem indicative of inhibition of a secretory function. By contrast with the regressive response of the principal cell, the ultrastructure of clear cells in the cauda and of apical cells in the caput region appeared unchanged up to 14 days after castration. The implications of this evidence for specialized functions, and the suggestion of a differential androgen dependence among major cell types of the epididymal epithelium, are discussed briefly.