Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Comparison between epididymosomes collected in the intraluminal compartment of the bovine caput and cauda epididymidis

During their transit along the epididymidis, mammalian spermatozoa acquire new proteins involved in the acquisition of male gamete fertilizing ability. We previously described membranous vesicles called epididymosomes, which are secreted in an apocrine manner by the epididymal epithelium. Some selected proteins associated with epididymosomes are transferred to spermatozoa during epididymal transit. The present study compared epididymosomes collected from caput epididymal fluid with vesicles from the cauda epididymidis in the bull. Two-dimensional gel electrophoresis revealed major differences in protein composition of epididymosomes isolated from the caput and cauda epididymidis. LC-QToF analysis of major protein spots as well as Western blot analysis confirmed the differences in proteins associated with these two populations of epididymosomes. Biotinylated proteins associated with caput and cauda epididymosomes also revealed differences. When incubated with caput epididymal spermatozoa, epididymosomes prepared from these two segments transferred different protein patterns. By contrast, cauda epididymosomes transferred the same pattern of proteins to spermatozoa from the caput and cauda epididymidis. Transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa decreased in a dose-dependent manner when biotinylated epididymosomes were diluted with unbiotinylated vesicles. Caput epididymosomes added in excess were unable to inhibit transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa. Following transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa, addition of unbiotinylated cauda epididymosomes was unable to displace already transferred biotinylated proteins. These results established that epididymosomes from caput and cauda epididymidis have different protein composition and interact differently with maturing spermatozoa.     

CD9-Positive Microvesicles Mediate the Transfer of Molecules to Bovine Spermatozoa during Epididymal Maturation

Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30–120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn²⁺ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.     

Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.     

Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida

Glioma pathogenesis‐related 1‐like protein1 (GliPr1L1) was identified by liquid chromatography‐tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine‐rich secretory proteins, Antigen 5 and pathogenesis‐related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N‐glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N‐glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti‐GliPr1L1 showed that this protein is involved in sperm–zona pellucida interaction. J. Cell. Physiol. 227: 3876–3886, 2012. © 2012 Wiley Periodicals, Inc.     

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     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.     

The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm

The epididymis is a long, tightly coiled tube within the lumen of which sperm matures. Sperm maturation involves morphological and biochemical changes in the sperm plasma membrane in response to epididymal secretions and their various proteins. Some of these proteins become outer membrane components while others become integral membrane proteins; transfer of some proteins to the sperm plasma membrane may be mediated by epididymosomes. Nevertheless, the molecular pathways by which spermatozoa acquire fertilizing capacity during their transit through the epididymis remain ambiguous. In a recent study of stallion epididymal sperm, we found that sperm harvested from different parts of the epididymis (caput, corpus and cauda) had a varying, but generally poor, ability to undergo the acrosome reaction in vitro. At ejaculation, however, sperm mix with seminal plasma which contains various components, including the small membranous vesicles known as prostasomes, that may enable the sperm to undergo physiological activation. Seminal plasma components may have a 'washing' effect and help to remove 'de-capacitation' factors that coat the sperm during storage in the cauda epididymis; alternatively seminal plasma and prostasomes may contain factors that more directly promote sperm activation. This article reviews current information on the roles of epididymal and accessory gland fluids on the acquisition of fertilizing capacity by stallion sperm.     

A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases

When the plasma membranes of caput and cauda epididymal spermatozoa of hamster were evaluated for their ability to undergo phosphorylation, a differential phosphorylation of the membrane proteins was observed. In the plasma membranes of the caput epididymal spermatozoa (immature), twelve proteins were phosphorylated (100, 76, 67, 65, 55, 52, 47, 42, 38, 32, 30, and 20 kD), whereas in the plasma membranes of cauda epididymal spermatozoa (mature), a differential phosphorylation pattern was observed with respect to the 94, 67, 52, and 47 kD proteins. The 94 kD protein was found to be phosphorylated and the 67 kD protein was found to be not phosphorylated in cauda spermatozoal plasma membrane (Cd SPM) in contrast to this protein in caput spermatozoal plasma membrane (Cpt SPM). The 52 and 47 kD proteins were also more intensely phosphorylated in Cd SPM than Cpt SPM. The 100 kilodalton protein, although present in both Cpt and Cd sperm plasma membranes, was found to be phosphorylated at the tyrosine residues only in the Cd SPM, as indicated by the Western blot using antiphosphotyrosine antibody. Further, a differential phosphorylation of the substrate proteins present in the Cpt and Cd SPM was seen when Mg²⁺ in the assay buffer was replaced by other divalent cations. For instance, Zn²⁺ stimulated the phosphorylation of an 85 kD protein in cauda SPM and not in the caput SPM and Ca²⁺ stimulated the phosphorylation of a 76 kD protein only in the cauda SPM. The phosphoproteins in both the plasma membranes were found to be phosphorylated predominantly at the tyrosine residue. The differential phosphorylation of a 100 kD protein at tyrosine in the Cd SPM (Western blot), which is absent in the immature Cpt SPM, also indicated that certain proteins in the hamster spermatozoa are phosphorylated in a maturation-specific manner. Mol. Reprod. Dev. 47:341–350, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

Background  

Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.     

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.     

Effect of diabetes mellitus on epididymal enzymes of adult rats.

Diabetes mellitus caused significant reduction in serum testosterone and accessory sex glands weight. The sperm content of epididymal regions also decreased. Among the epididymal regions, the cauda epididymidal tissue alone showed significant reduction in Na(+)-K+ ATPase activity. However, Mg2+ ATPase activity was lowered in caput epididymidis only. Specific activity of Ca2+ ATPase significantly decreased in caput and cauda epididymides. All three ATPases decreased significantly in caput epididymidal spermatozoa leaving cauda epididymidal spermatozoa unaffected. Specific activity of alkaline phosphatase was suppressed in caput epididymidis and in the spermatozoa collected from caput and cauda epididymides, while the acid phosphatase was unaffected. In general, the results are suggestive of definite influence of diabetes on epididymal phosphatases which is region specific. Diabetes induced decrease in phosphatases may have an impact on secretory and absorptive functions of epididymis and thus on sperm maturation.     

Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (⁴⁵Ca²⁺) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ～ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.     

OBSERVATION ON REGIONAL DIFFERENCES OF SOME DEHYDROGENASES AND HYDROLASES IN RAT EPIDIDYMIS

The epididymal epithelia, by secretion, fluid reabsorption and transition, provide a favorable environment for sperm maturation. We observed, with histochemical method, the regional differences of four hydrolases and five dehydrogenases in caput, corpus and cauda of rat epididymis to     

Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.     

Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)*

Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca²⁺ efflux pump, plasma membrane Ca²⁺ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4–64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.     

Responses of monkey epididymal sperm of different maturational status to second messengers mediating protein tyrosine phosphorylation,acrosome reaction,and motility

The maturation of various aspects of sperm function have been demonstrated in monkey and human epididymal sperm, including the ability to undergo the acrosome reaction. The present study aimed to investigate the maturational changes in non‐human primate sperm in the signal transduction mechanisms leading to the acrosome reaction involving cyclic AMP, Ca²⁺ influx, protein kinase C, and protein tyrosine phosphorylation. Sperm from the caput, corpus, and cauda epididymidis of cynomolgus monkeys were incubated in a complete medium for 2.5 hr, followed by 30 min stimulation with 1 mM dibutyryl cAMP and 1 mM caffeine, 50 μM 1,2‐dioctanoyl‐sn‐glycerol (DOG), and 50 μM Ca²⁺‐ionophore A23187. Quantitative Western blotting revealed little difference in tyrosine phosphorylated proteins among the caput, corpus, and cauda sperm without stimulation. Incubation with cAMP increased the amount of tyrosine phosphorylated proteins up to 10‐fold in the corpus and cauda sperm, but to a lower extent in the caput sperm. Ca²⁺‐ionophore attenuated the cAMP stimulation but had no effect on its own. Such responses in tyrosine phosphorylated proteins were in great contrast to the responses in the acrosome reaction, where A23187 was the strongest stimulant, resulting in induction of the reaction in 50 ± 5%, 11 ± 5%, and 8 ± 4% cauda, corpus and caput sperm, respectively (mean ± sem, n = 6). DOG and cAMP in combination induced acrosome reactions in about 10% of viable cells in the cauda and corpus but not caput sperm. Caput sperm responded to cAMP with increases in percentage motility without forward progression whereas cauda sperm displayed marked kinematic changes expected of hyperactivation. Comparisons of responses suggest that the major tyrosine phosphorylated proteins detected are unlikely to be involved immediately in the precipitation of the acrosome reaction, but more related to flagellar motion. Development of signal transduction pathways is part of the epididymal maturational process. Mol. Reprod. Dev. 54:194–202, 1999. © 1999 Wiley‐Liss, Inc.