Sperm maturation in the domestic cat

The epididymis is essential for sperm development and maturation, and, subsequently, the ability of spermatozoa to penetrate and fertilize the female gamete. Functional differences in segments of the long tubule are reflected by histological differences among epididymal regions. The feline epididymis can be divided into six different regions according to their histological differences. A marked increase in sperm concentration occurs between regions 2 and 3, indicating resorption of fluid in region 2, a concept supported by the histological characteristics of the epithelium. At the transition between regions 4 and 5, located between the caput and corpus epididymides, histological characteristics change from being that of a maturation function to being typical of a storage function. Migration of the cytoplasmic droplet and induction of motility occur in this same region. Proteins are secreted from epithelial cells in the feline epididymis by merocrine and apocrine secretion, although the functions of different feline epididymal proteins have not been determined. Hypotaurine, taurine and, probably, alkaline phosphatase are produced by the feline epididymis. During epididymal transit the percentage of immature, unviable and morphologically abnormal spermatozoa decreases, indicating the existence of a mechanism that removes abnormal spermatozoa. In contrast, the percentage of spermatozoa with abnormal tails increases slightly during epididymal transit. Most of the distal droplets present on spermatozoa in the cauda epididymis are lost at or after ejaculation. Additional knowledge of the feline epididymis should be beneficial for developing sperm preservation protocols and advance the prospects for effective male contraceptive methods.     

小鼠附睾头精子获得与卵子质膜融合能力的物质基础研究

随着精子在附睾中的转运,它们与卵子质膜的融合能力逐渐增加。怩证明２附睾体和附睾尾的精子均具有相当高的膜融合能力,而附睾头中的精了奶少能与卵子质膜融合,这是还说明附睾头中的精子不具备与云透明带卵子融合的物质条件呢？利用附睾结扎留并延长体外获能时间,可使附睾头远端精子的融合能力明显地提高;在精子培养液中加入ＡＴＰ,并延长精卵共培养时间,也可使一少部分附睾头近端的精子获得与卵子质膜融合的能力。这表明附睾     

Le transit epididymo-deferentiel

The epididymis and vas deferens constitute not only a simple conduit for sperm transport but also play an important physiological role in the development of sperm fertilizing ability. The epithelial compartment plays a major functional role in determining the biochemical composition of the luminal fluid in which the spermatozoa undergo a series of structural, biochemical and metabolic changes. During epididymal transit spermatozoa acquire their capacity for motility and also their ability to attach and bind to the zona pellucida and fertilize the oocyte. In man, sperm maturation may occur in the extreme proximal region of the epididymis. The regulation of epididymal and vasa deferential function, as well as sperm maturation, are under androgenic control     

Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa

The testis produces male gametes in the germinal epithelium through the development of spermatogonia and spermatocytes into spermatids and immature spermatozoa with the support of Sertoli cells. The flow of spermatozoa into the epididymis is aided by testicular secretions. In the epididymal lumen, spermatozoa and testicular secretions combine with epididymal secretions that promote sperm maturation and storage. We refer to the combined secretions in the epididymis as the sperm-milieu. With two-dimensional-PAGE matrix-assisted laser desorption ionization time-of-flight MS analysis of healthy testes from fertile accident victims, 725 unique proteins were identified from 1920 two-dimensional-gel spots, and a corresponding antibody library was established. This revealed the presence of 240 proteins in the sperm-milieu by Western blotting and the localization of 167 proteins in mature spermatozoa by ICC. These proteins, and those from the epididymal proteome (Li et al. 2010), form the proteomes of the sperm-milieu and the spermatozoa, comprising 525 and 319 proteins, respectively. Individual mapping of the 319 sperm-located proteins to various testicular cell types by immunohistochemistry suggested that 47% were intrinsic sperm proteins (from their presence in spermatids) and 23% were extrinsic sperm proteins, originating from the epididymis and acquired during maturation (from their absence from the germinal epithelium and presence in the epididymal tissue and sperm-milieu). Whereas 408 of 525 proteins in the sperm-milieu proteome were previously identified as abundant epididymal proteins, the remaining 22%, detected by the use of new testicular antibodies, were more likely to be minor proteins common to the testicular proteome, rather than proteins of testicular origin added to spermatozoa during maturation in the epididymis. The characterization of the sperm-milieu proteome and testicular mapping of the sperm-located proteins presented here provide the molecular basis for further studies on the production and maturation of spermatozoa. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or targets for male contraception.     

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.     

Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.     

Effects of castration on thiol status in rat spermatozoa and epididymal fluid

Mammalian spermatozoa gain their fertilizing ability as they mature in the epididymis, a process which is accompanied by oxidation of sperm protein thiols. Since sperm maturation is dependent upon normal androgenic support to the epididymis, the present work was designed to study the effects of castration on thiol status. Spermatozoa and epididymal fluid were isolated from the epididymides of male rats 5 days after castration or after 11 daily injections of the antiandrogen, cyproterone acetate. Spermatozoa and epididymal fluid were labeled with the fluorescent thiol labeling agent monobromobimane. Intact spermatozoa were evaluated by fluorescence microscopy, protein thiols were analyzed by electrophoresis, and fertilizing ability was examined after insemination of sperm suspension into the uterine horns of immature superovulated female rats. We found that both treatments resulted in an increase in cauda sperm thiols as shown by increased fluorescence in the intact spermatozoa. Protamines and nonbasic proteins were found to have increased levels of reactive thiols. The protein profiles of epididymal fluid from castrated rats were different from those of the controls, and the fluorescence patterns corresponded to the protein profiles. Our results indicate that testosterone withdrawal leads to inhibition of sperm thiol oxidation. Mol. Reprod. Dev. 47:295–301, 1997. © 1997 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 acrosomes 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.     

Characteristics of donkey spermatozoa along the length of the epididymis

In mammals, the epididymis has numerous interrelated functions including absorptive and secretory activity that affect luminal environment and cell membrane, and the maturation and storage of sperm. Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymis and the motility of epididymal spermatozoa should be a balance between the maturation of flagellum and the inhibition of the flagellar machinery. In this study maturational change in sperm characteristics were evaluated in the epididymis of donkey. Spermatozoa collected from four portions of the epididymis (head, cranial corpus, caudal corpus, tail) were compared before and after ejaculation for viability, mitochondrial activity, kinetic parameters, and morphology. A significant increase in the mitochondrial activity along the epididymis was reported, suggesting a possible involvement in the motion mechanism. This should be corroborated by the significant correlation between mitochondrial activity and the total and progressive motility and the increase in velocities of spermatozoa recorded by computer-assisted sperm analysis. The percentage of most of the abnormal spermatozoa were similar in all tracts, with a great variability between jackasses. Only the bent midpiece percentage decreased significantly along epididymis. A significant increase in the percentage of distal cytoplasmic droplets (DCD), and a simultaneous decrease in the proximal cytoplasmic droplets (PCD), was found. The DCD fell down after ejaculation suggesting the late loss of the cytoplasmic residual (DCD) in the donkey, as hypothesized in the stallion. Because the prevalence of PCD were similar in both tail epididymal and ejaculated spermatozoa, a defect of the maturative process in the PCD sperm should be speculated.     

Physiology of the epididymis and spermatozoa

The epididymis is an ideal extragonadal target site to inhibit fertility in the male. Synthesis and secretion of constituents like sialic acids, protein and glycerylphosphoryl choline by the epididymal epithelium under androgen control provide an ideal fluid environment for sperm maturation. An optimal level of sialic acid secretion by the epididymal epithelium is needed to maintain functional integrity of sperm. The existence of specific androgen receptors in the epididymis and spermatozoa are related to their ability to metabolise androgens.     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice

We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.     

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

Developing spermatozoa require a series of posttesticular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation-dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5 kDa wheat germ agglutinin (WGA)-binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA-stained bands, the presence of a 17.5 kDa WGA-binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5 kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5 kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide.     

Régulation des fonctions de l’épithélium épididymaire des mammifères: état des lieux

Male gametes formed in the testis, the site of spermatogenesis, continue their maturation outside of the gonad in the genital tract. The epididymis is at the centre of this process of post-testicular maturation of spermatozoa, responsible for acquisition of their mobility and their ability to recognize and penetrate an oocyte, essential events for fertilization. During their transit in the epididymal ducts, gametes evolve in a perpetually changing luminal environment due to complex interactions of secretion and reabsorption activities of the epididymal epithelium. These numerous secretion and reabsorption activities are closely regulated in space and time, which makes this epididymal epithelium a very complex tissue subject to an elaborate network of cross-regulations. Apart from classical endocrine and paracrine regulations, the proximal part of the epididymis is also subject to lumicrine regulation by testicular factors which increase the complexity of the regulation networks involved. The present study tries to present an exhaustive review of the factors demonstrated in the literature to exert a modulation of epididymal activity.     

The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.     

Epididymis as a target for contraception

Advantage of using a vaccine based on sperm antigens is that it can be used both in males and females as individuals who have antisperm antibodies are usually infertile but otherwise healthy. Several sperm specific antigens identified as prospective candidates for immunocontraception are of testicular origin. For the purpose of immunocontraception it may be desirable not to disrupt spermatogenesis and testicular function. Concept of post testicular maturation of spermatozoa has been very well established. During post testicular voyage spermatozoa undergo a series of complex and sequential events which transforms the immature immotile spermatozoa into mature sperm. Acquisition of functional maturity is necessary for progressive motility, zona pellucida recognition culminating in sperm egg binding. Importance of epididymal maturation is highlighted by the fact that high percentage of male infertility in human originates from the malfunction of the epididymis. The epididymis has also shown to be involved in sperm storage and provides an adequate environment for final maturation of the sperm. It provides a conducive microenvironment by virtue of which the spermatozoa are protected during the storage. In view of this it is imperative that more attention needs to be focused on epididymal antigens. The information obtained will enable us to identify epididymal antigens relevant to fertility and also help in infertility diagnosis.     

Identification of a major secretory glycoprotein from rat epididymis: interaction with spermatozoa

A polypeptide with molecular mass of 17 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-beta-N-acetylglucosaminidase H without prior denaturation or protease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 x 10(9) M-1 and 17,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GP17. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.     

Effects of epididymal maturation, zinc (II) and copper (II) on the reactive sulfhydryl content of structural elements in rat spermatozoa

The sulfhydryl content of rat spermatozoa at different stages of their maturation in the epididymis was determined by alkylation with ¹⁴C-iodoacetamide. Inhibition of this reaction by reagents having an affinity for thiols verified its specificity. The results support previous conclusions that epididymal maturation in eutherian mammals involves oxidation of -SH groups to -S-S- crosslinks in sperm heads and tails, imparting unusual stability to these structures. The heads and tails of immature rat spermatozoa displayed more than 20 and 5 times as many reactive -SH groups respectively as did those of mature spermatozoa. Fractionation of sonicated spermatozoa revealed that most of the reactive thiols are in the tails. Zn²⁺, Cu²⁺ and Cd²⁺ inhibited the alkylation of -SH groups by iodoacetamide. Although the Zn²⁺ inhibition could be reversed by EDTA, the effect of Cu²⁺, believed to involve oxidation, was not reversible and could be largely prevented by a sufficient excess of Zn²⁺. Thus, Zn²⁺ may retard the oxidation of sperm -SH groups in vivo.     

Effect of daily spermatozoan production but not age on transit time of spermatozoa through the human epididymis

Daily spermatozoan production, numbers of epididymal spermatozoa, and transit times of spermatozoa through different regions of the epididymis were determined in 38 men, aged 20-49 or 50-79 yr. Specimens were obtained at autopsy within 24 h of death due to traumatic injury or heart failure. Subjects were in apparent good health prior to death, and death was not preceded by an extended period of hospitalization. Daily spermatozoan production per testis (DSP/T) and numbers of epididymal spermatozoa were determined from counts of maturation-phase spermatids or epididymal spermatozoa in tissue homogenized in a Waring blender. Epididymal transit time was calculated as the number of spermatozoa in a given region of the epididymis or in the entire epididymis divided by DSP/T of the connected testis. Parenchymal weight, spermatozoan production rate, numbers of epididymal spermatozoa, and epididymal transit time were similar (p greater than 0.05) between paired testes or epididymides. Men were divided into four groups on the basis of age and DSP/T. Since there was no (p greater than 0.05) effect of age on epididymal transit time, men in different age groups were combined within their respective group on the basis of DSP/T. In the group with high DSP/T, DSP/g parenchyma was much higher and epididymal transit time was much faster. However, parenchymal weights and numbers of epididymal spermatozoa were similar (p greater than 0.05) between DSP/T groups. The similarity in number of spermatozoa in epididymides of men whose DSP/T differed by threefold is consistent with the inability of the human epididymis to store many spermatozoa when no blockage is present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.