Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Chromatin condensation in cat spermatozoa during epididymal transit as studied by aniline blue and acridine orange staining

Summary.  Chromatin stability and DNA-resistance to acidic denaturation was evaluated by acidic aniline blue and acridine orange staining of cat sperm from different regions of the epididymis. The results were related to conventional sperm parameters. The percentage of aniline blue-stained spermatozoa (persisting histones) decreased significantly from the caput to the cauda region (31.8% and 7.8%, respectively; P <0.0001). The percentage of stained heads of cauda sperm was much lower in populations of morphologically normal forms than in those with abnormal forms (4.1% and 13.8%, respectively, P <0.0001). Among spermatozoa with abnormalities, the percentage of stained heads was significantly higher in cells with head abnormalities than in sperm with only tail abnormalities (87.1% and 10.3%, respectively; P >0.0001). With acridine orange fluorescence staining, 86.5% of cauda epididymal sperm were found with well-condensed chromatin, stabile against acid-induced denaturation. Chromatin stability increased significantly from the caput epididymal region (51.1%) to the cauda epididymal region (86.5%). The percentage of cauda epididymal sperm with normal condensed chromatin was neither linked to testicular sperm count, motility nor to age of the cats. The parameter of chromatin condensation and stability can be a valuable index of sperm quality, reflecting the possible disorders of spermatogenesis and epididymal sperm maturation, frequently observed in feline species.     

Alterations in protein profile of rat spermatozoa during maturation

Alterations in the polypeptide pattern of rat spermatozoa during epididymal transit were studied by SDS-PAGE and compared with that of epididymal cytosol and luminal fluid. The total number of cytosol and luminal fluid polypeptides increase from caput to cauda epididymidis but sperm associated polypeptides decrease during epididymal transit. Changes in polypeptide pattern of spermatozoa are due to their acquisition, loss or modification. Spermatozoa acquire seven polypeptides, of which six are acquired in corpus (MW 16.5, 38, 41, 72, 75 and 100 Kdal) and one (MW 28.5 Kdal) in cauda epididymidis. Spermatozoa lose one polypeptide of MW 72.5 Kdal in caput and two polypeptides of MW 70 and 115 Kdal in cauda epididymidis. Four polypeptides of MW 18.5, 19.5, 64 and 67.5 Kdal disappear from cauda spermatozoa without appearing in the luminal fluid. Polypeptide of MW 62.5 Kdal is observed only in spermatozoa and luminal fluid from cauda epididymidis.     

Functional role of hepatocyte growth factor receptor during sperm maturation

Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during the first hour of culture, whereas it is maintained for at least 3 hours when HGF is present in the culture medium. We also report that HGF does not influence spermatozoa viability as indicated by the cytometrical analysis of propidium iodide-labeled sperm; an equal number of dead cells appeared in control and in HGF-treated preparations. In conclusion, our data strongly support the hypothesis that HGF positively influences sperm motility maintenance during sperm transit through the epididymis, indicating that c-met receptor and its ligand, HGF, have a role in male fertility.     

Localization of antigenic determinants recognized by a monoclonal antibody in rat epididymal spermatozoa, with particular reference to sperm maturation

Summary.  This study localized antigenic determinants recognized by a mouse anti-human sperm monoclonal antibody TüS10 immunocytochemically and immunoelectron microscopically in the rat sperm recovered from the caput and cauda epididymidis. Immunocytochemistry showed that the antibody bound specifically to the plasma membrane overlying the principal piece of membrane-intact sperm from the caput and cauda epididymidis. Demembranation by Triton X-100 significantly decreased the affinity of the monoclonal antibody TüS10 to the caput sperm but did not obviously change that to the cauda sperm. Immunoelectron microscopy with biotinstreptavidin peroxidase complex pre-embedding method confirmed the localization of the antigenic determinants over the cell surface of the principal piece of the membrane-intact spermatozoa from the caput and cauda epididymidis. The demembranated sperm from the caput epididymidis showed no intracellular labelling, while those from the cauda displayed labelling on their external surface of the fibrous sheath. Using monoclonal antibody TüS10 as a probe, we detected different distribution patterns of the antigenic determinants between the spermatozoa in the caput and cauda epididymidis. These results suggest that spermatozoa mature with immunologically detectable changes in the fibrous sheath during their epididymal transit.     

Immunolocalization of heat shock protein 70 in bovine spermatozoa

Heat shock protein 70 (HSP70) is part of a superfamily of molecular chaperones, which protect cells from chemical and heat shock. The objectives of this study were to determine the presence of HSP70 in bovine spermatozoa and its subcellular localization during different stages of spermatogenesis. Analysis of sperm proteins by Western blotting using a monoclonal antibody to the inducible form of HSP70 revealed a single immunoreactive band with an estimated molecular weight of 70 kDa in samples from 18 of 18 bulls. Using immunofluorescence microscopy and the same antibody, HSP70 was localized to the cytoplasm of prophase spermatocytes and elongating spermatids, to cytoplasmic droplets of caput epididymal spermatozoa, and to cytoplasmic droplets, acrosome, post-acrosomal region and middle piece of corpus and cauda epididymal spermatozoa. The pattern of distribution changed in freshly ejaculated spermatozoa as HSP70 was detected on the acrosome only. During capacitation and acrosome reaction, HSP70 was once again redistributed, and was localized to the equatorial segment, post-acrosomal region and middle piece. Thus, HSP70 is present in the spermatozoa of mature bulls and redistribution of the protein occurs during capacitation and the acrosome reaction.     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa

Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1–3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer‐assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Organ culture of rat caput epididymal tubules in a perifusion chamber

We have been able to culture caput epididymal tubules from rats by modifying an organ culture system (Orgebin-Crist et al, 1980) which was used to culture rabbit corpus epididymal tubule segments. The morphology of the epithelium was consistently good throughout seven days in culture, although sloughed epithelium was commonly seen during the first 24 hours. Evidence of this sloughing was much less frequent thereafter. Throughout seven days of culture, autoradiography of SDS-PAGE of luminal fluid obtained from tubules cultured in medium containing 14C-L-leucine showed incorporation into bands identical to those stained by Coomassie Blue. Rat epididymal alpha-lactalbumin was consistently localized on the luminal surface of the epithelium and the middle piece of the spermatozoa. Spermatozoa appeared to have normal morphology throughout the first three days in culture; thereafter, decapitated spermatozoa were frequently seen. Caput spermatozoa only quiver in place prior to culture, but after three days in culture, 53% of the spermatozoa from distal caput tubules are progressively motile upon dilution in a balanced salt solution. Since the transit time for spermatozoa in the caput epididymidis of the rat is approximately three days, it should be possible with this culture system to study maturational events involving interactions between spermatozoa and the epididymal epithelium.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Mobility shift assay of calcium-binding proteins of mouse epididymal spermatozoa

The calcium-binding proteins (CBPs) of mouse epididymal spermatozoa were analysed by mobility changes in the presence of added Ca2+ in two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The expression patterns of relatively high molecular weight CBPs (Mr > 20 kDa) were different between caput and cauda epididymal spermatozoa. There was a constitutive expression of low molecular weight CBPs (Mr < 20 kDa) regardless of the epididymal region. Most of the CBPs disappeared after the acrosome reaction (AR) induced by Ca2+ ionophore A23187, suggesting that they originated from the acrosome and/or the plasma membrane overlaying the acrosome. Taken together, it can be suggested that changes in CBPs of spermatozoa are important features of sperm maturation during epididymal transit, and that they may be related to the fertilizing ability of mouse epididymal spermatozoa.     

Maturation of sperm volume regulation in the rat epididymis

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality （measured to be 343 ± 13 and 365 ± 19 mmol kg^-1, respectively） to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measure- ments of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg l both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume.     

Changes in surface protein structure of bull spermatozoa during epididymal maturation

The surface proteins of bull spermatozoa from caput and cauda epididymidis were labelled by lactoperoxidase-catalyzed radioiodination and solubilized and analysed by SDS-PAG-electrophoresis. The surface protein patterns of caput and cauda epididymal spermatozoa resembled each other but some distinct differences could be found. Caput epididymal spermatozoa revealed a protein peak with molecular weight of 15 000 - 18 000 daltons but this peak was not found on cauda epididymal spermatozoa. On caput epididymal epermatozoa the most intensely labelled protein peak was located between 90 000 and 100 000 daltons but on cauda epididymal spermatozoa the corresponding peak was only weakly labelled and had a molecular weight of 80 000–90 000 daltons. Surface protein with molecular weight of 42 000–47 000 daltons was dominating on cauda epididymal spermatozoa. The surface protein structure of cytoplasmic droplets did not drastically differ from that of epididymal spermatozoa.     

Development of the oocyte-penetrating capacity of spermatozoa in the human epididymis

The development of the penetrating capacity of human epididymal spermatozoa has been assessed in vitro using zona-free hamster oocytes. Spermatozoa were recovered from epididymal tissue of men undergoing vasectomy or epididymovasostomy. The results suggest that human spermatozoa first develop the potential to penetrate oocytes in the proximal corpus region. Spermatozoa recovered from more proximal sites of the excurrent duct failed to bind or fuse with zona-free hamster eggs although a small proportion (3%) in the caput region were capable of progressive motility. The data are discussed in relation to biochemical changes to human spermatozoa during maturation.     

In vitro initiated sperm forward motility in caput spermatozoa: weak and transient

Testicular spermatozoa during journey through epididymis acquire forward motility, which is essential for fertility. To understand the biochemistry of sperm motility initiation, various initiation media have been developed that permitted high level of motility induction (55-60%) in the immature caput-spermatozoa in presence of activating principles: theophylline, bicarbonate and epididymal plasma (EP) when analysed microscopically. Here, we show for the first time using caprine model that stability and quality of in vitro-induced motility in the caput spermatozoa is insignificant in contrast to naturally induced motility in mature cauda spermatozoa. In vitro-induced motility of the immature spermatozoa was lost completely upon the removal of these activators by centrifugation. Selective withdrawal of either EP or HCO(3) by dilution retains 50-60% of the in vitro-induced motility. Spectrophotometric analysis revealed that in vitro-induced vertical motility in immature spermatozoa is too little when compared to mature spermatozoa. In in vitro-initiated caput spermatozoa, cyclic adenosine monophosphate level becomes doubled but lesser than cauda spermatozoa. This revelation concludes that scientific knowledge generated over the years on the basis of in vitro initiation method is insignificant and needs improvisation to delineate biochemical regulation of sperm motility which in turn has remarkable potential in wide biological fields, especially in infertility treatment.     

Purification and Localization of Acidic Epididymal Glycoprotein (AEG): A Sperm Coating Protein Secreted by the Rat Epididymis

A protein designated acidic epididymal glycoprotein (AEG) was purified from rat epididymis using ion exchange chromatography on DEAE-Sephadex, gel filtration in Sephadex G-75 and affinity chromatography on Concanavalin-A Sepharose. AEG is a major secretory product of the epididymis making up 2–3 per cent of total soluble protein. Antibody to AEG was raised in rabbits and purified by affinity chromatography on AEG-Sepharose. Quantitation of AEG in cytosol using "rocket" immunoelectrophoresis showed AEG to increase in epididymal segments from caput to cauda. Ligation of the midcorpus decreased AEG in the cauda. Localization of AEG using an immunoperoxidase method revealed that it is secreted largely by the epithelium of caput and corpus beginning with the region distal to the initial segment. It appears to be secreted by a specific cell type, probably the so-called "principal" cell. Specific staining of AEG was also noted in "clear" cells in the cauda. Spermatozoa become coated with AEG as they leave the initial segment and remain so during passage through the cauda.     

大鼠精子在附睾成熟过程中肉毒碱量变化的研究

肉毒碱被认为是与精子在附睾中成熟有关的成熟因子。本文报告测定大鼠附睾头部,体部及尾部精子中的肉毒碱。结果表明精子在循附睾头、体、尾运行过程中,精子中肉毒碱含量逐步增加,在附睾体一头部,附睾尾一头部的精子内的肉毒碱量呈现十分显著性差异。结果提示附睾体部或体一头部交界部位可能是精子在附睾中成熟的关键部位。     

葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有

哺乳动物睾丸中的精子经过“附睾成熟”期,由静止状态转变成为运动状态。该过程中精子从附睾头部向附睾尾部移动,同时精子发生了一系列的形态、生理和生化改变,如蛋白组成和蛋白修饰的改变可能会影响精子获能的潜能。本实验使用基质辅助激光解吸／电离串联质谱（MALDI-MS／MS）法分析仓鼠睾丸头部和尾部精子的蛋白组学,成功发现了113个蛋白质点。对113个蛋白质点进一步对照比较发现30个蛋白质点（对应20个蛋白）的密度发生了显著改变,其中附睾尾部精子5个蛋白的密度增加,11个蛋白密度减少;此外,葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有,而纤维蛋白原样蛋白1为附睾尾部精子所特有。几个蛋白密度增加可能与附睾成熟过程中精子代谢和ATP产生相关。一些蛋白如ERp57,GRP78,GP96,Hsp60,Hsp70和二氢硫辛酰胺S-乙酰转移酶的密度改变通过免疫印迹法得到验证。本研究首次报道了仓鼠精子的蛋白质组学研究,全面展示了仓鼠精子附睾成熟过程中的蛋白结构改变。     

Biochemical parameters of initiation and regulation of sperm motility

Studies of in vitro models demonstrate that a forward motility protein (FMP) is required for the initiation of forward motility in the immature epididymal spermatozoa. FMP is a heat-stable glycoprotein derived from epididymal plasma. During the epididymal maturation of spermatozoa in vivo, there is a marked increase of intrasperm pH and level of cyclic adenosine monophosphate (cAMP). Several studies suggest that exogenous FMP in concert with elevated intrasperm pH and level of cAMP initiates flagellar motility during the epididymal transit of sperm. cAMP activates sperm cytosolic cAMP-dependent protein kinases, which in turn phosphorylate multiple intrasperm phosphoproteins that may regulate flagellar motility. Exogenous calcium ion activates intact sperm motility, although it inhibits motility of demembranated cells on reactivation. Occurrence of cAMP-dependent type I and II protein kinases, a novel cAMP-independent protein kinase, and a phosphoprotein phosphatase has been demonstrated on the external surface of spermatozoa. The sperm surface has a coupled-enzyme system: ecto-cAMP-independent protein kinase and phosphoprotein phosphatase that regulate the phosphorylation and dephosphorylation of endogenous sperm ectophosphoproteins. The specific activities of these ecto-enzymes increase markedly during forward progression, suggesting that they may have a role in regulating flagellar motility.