An epididymis-specific beta-defensin is important for the initiation of sperm maturation

Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific beta-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca(2+), a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, beta-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.     

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

Background  

As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

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.     

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on rat epididymal sperm maturation and fertility

Sperm entering the epididymis gain progressive motility and fertilizing ability in a process termed maturation. The functional dependence of the epididymis on dihydrotestosterone (DHT) is well established, yet few studies have examined the consequences on the epididymis of inhibiting DHT formation. We have shown that inhibition of both isoforms of 5alpha-reductase (types 1 and 2), the enzyme that converts testosterone to DHT, has pronounced effects on epididymal gene expression. In the present study, we investigate whether inhibiting 5alpha-reductase has consequences on epididymal sperm maturation. Rats were treated with vehicle or 10 mg/kg/day PNU157706, a dual-type inhibitor, for 28 days. Fertility and several key facets of sperm maturation were analyzed. Changes in sperm motility were assessed by computer-assisted sperm analysis (CASA). Changes in sperm morphology were assessed by CASA and electron microscopy. The motility of spermatozoa from the cauda epididymidis of treated animals showed a significant decrease in both the percentage of motile and progressively motile sperm as well as altered motion parameters. The morphology of cauda epididymal spermatozoa was also adversely affected by the treatment; the most prominent effect was a markedly elevated proportion of sperm that retained their cytoplasmic droplet. Matings with treated males resulted in fewer successful pregnancies and a higher rate of preimplantation loss. Progeny outcome was unaffected. The compromised sperm motility and morphology likely contribute to the subfertility of inhibitor-treated rats. Our results indicate a role for dual 5alpha-reductase inhibitors in further studies of epididymal physiology and as a potential component of a male contraceptive.     

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process of Corynorhinus mexicanus bat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.     

附睾局部组织肾素血管紧张素系统(英文)

附睾内液体微环境对精子的成熟和贮藏是相当重要的。附睾液体的形成取决于附睾上皮的吸收与分泌功能,而先前的实验已经证明：这些吸收与分泌的活动是受到除神经激素以外旁分泌与自分泌的调控。虽然肾素血管紧张素系统（ＲＡＳ）在很多组织上旁分泌与自分泌的作用已多有报导,但其在附睾的存在及作用仍鲜为人知。本综述总结了本实验室在这方面的研究结果,通过使用不同的实验方法,例如,免疫组织化学,放射免疫分析,分子生物学及短路电流电生理学方法,我们得到的结果显示了ＲＡＳ主要成员在附睾的分布（Ｆｉｇ．１）和表达,并阐明了血管紧张素ＩＩ对附睾阴离子分泌的调控作用（Ｆｉｇｓ．２＆３）。本综述还就血管紧张素ＩＩ对附睾旁分泌与自分泌的作用及其机制（Ｆｉｇ．４）,以及对精子功能的作用进行了讨论。     

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.     

Disrupted sperm function and fertilin beta processing in mice deficient in the inositol polyphosphate 5-phosphatase Inpp5b.

Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b(-/-) males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin beta, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b(-/-) animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b(-/-) mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.     

Mouse cysteine-rich secretory protein 4 (CRISP4): a member of the Crisp family exclusively expressed in the epididymis in an androgen-dependent manner

The final maturation of spermatozoa produced in the testis takes place during their passage through the epididymis. In this process, the proteins secreted into the epididymal lumen along with changes in the pH and salt composition of the epididymal fluid cause several biochemical changes and remodeling of the sperm plasma membrane. The Crisp family is a group of cysteine-rich secretory proteins that previously consisted of three members, one of which-CRISP1-is an epididymal protein shown to attach to the sperm surface in the epididymal lumen and to inhibit gamete membrane fusion. In the present paper, we introduce a new member of the Crisp protein family, CRISP4. The new gene was discovered through in silico analysis of the epididymal expressed sequence tag library deposited in the UniGene database. The peptide sequence of CRISP4 has a signal sequence suggesting that it is secreted into the epididymal lumen and might thus interact with sperm. Unlike the other members of the family, Crisp4 is located on chromosome 1 in a cluster of genes encoding for cysteine-rich proteins. Crisp4 is expressed in the mouse exclusively in epithelial cells of the epididymis in an androgen-dependent manner, and the expression of the gene starts at puberty along with the onset of sperm maturation. The identified murine CRISP4 peptide has high homology with human CRISP1, and the homology is higher than that between murine and human CRISP1, suggesting that CRISP4 represents the mouse counterpart of human CRISP1 and could have similar effects on sperm membrane as mouse and human CRISP1.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Epididymal functions and their hormonal regulation

The epididymis is a complex organ which maintains a specific intraluminal environment thought to be important for effecting sperm maturation in proximal regions and sperm storage in distal regions of the duct. The composition of the internal milieu is achieved both by transport between blood and lumen (and vice versa) and by synthesis and secretion into the lumen. Several low-molecular weight organic molecules achieve high concentration in the epididymal lumen, but their functions in the events of sperm maturation and storage still remain unclear. Metabolic processes occurring within epididymal tissue and the absorptive and secretory activity of the epididymal epithelium are regulated by androgens. The synthesis of some, but not all, secretory proteins is also androgen-dependent. In addition to androgens, other hormones and local testicular factors may influence epididymal function. There is now increasing evidence that epididymal-specific and androgen-dependent secretory proteins play a fundamental role in modifying the surface characteristics of sperm in preparation for the events of fertilization.     

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.