Sperm morphological abnormalities appearing in the male rabbit reproductive tract

The role of the excurrent duct system in producing and/or eliminating morphologically abnormal spermatozoa may modify the semen parameters and interfere with sperm fertilizing capacity. To study this process, changes in the morphology of spermatozoa during their transit through the reproductive tract in sexually mature rabbits were investigated. The incidence of head, midpiece and tail abnormalities as well as of multiple defects in a single spermatozoon, and the position of the cytoplasmic droplet along the sperm midpiece were evaluated in samples from the testis, 6 regions of the epididymis and the vas deferens. Spermatozoa were characterized by rapid migration of the cytoplasmic droplet when passing from the proximal to the distal caput of the epididymis, and spermatozoa with no droplet predominated in the distal epididymis and vas deferens. In passing from the testis to the proximal caput of the epididymis, the incidence of spermatozoa with an abnormal midpiece and those with multiple defects decreased significantly. The proportion of spermatozoa with abnormal heads was also lower in the testis, but no statistically significant differences were found, whereas there was no change in the proportion of those with abnormal tails. These results indicate that there must be a mechanism for the disposal of defective spermatozoa. No evidence of spermiophagy by luminal macrophages was observed in the extracts, although a few spermatozoa exhibited signs of degeneration, suggesting, that although intraepithelial phagocytosis has not been clearly demonstrated in the nonexperimental rabbit, sperm cells may undergo a form of autolysis within the lumen of the duct.     

Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility

Protease activities with specificity toward synthetic substrates, Suc-Gly-Pro-Leu-Gly-Pro-MCA for prolyl endopeptidase or collagenase-like peptidase, and Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin were identified in the detergent-soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm-activating protein (HSAP). Among them a prolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity from herring testis. The molecular mass of the enzyme was 79 kDa and the properties of the enzyme were quite similar to prolyl endopeptidase from other tissues or cells. Both the enzyme activation and the sperm motility activation by HSAP were inhibited by benzyloxycarbonyl-L-thioproline-thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility activation by HSAP was inhibited by substrates of the prolyl endopeptidase. Western blotting with mouse anti-prolyl endopeptidase serum revealed the presence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm. These results suggest that prolyl endopeptidase exists on the surface of the sperm tail and interacts with the HSAP.     

Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

The antibody from an infertile woman that induces sperm agglutination interacts with rat testicular cells and sperm

The serum obtained from an infertile woman induced a specific head-to-head agglutination of human and rat sperm. The immunoglobulin G (IgG) fraction of the serum was obtained and found to interact with the proteins of rat sperm in testis and epididymis. Using an indirect immunofluorescent method with rat sperm from vas deferens, we determined that the antibody recognized the protein on the convex and concave regions of the acrosome and over the entire tail. However, with testicular spermatozoa, the antibody recognized only the distal end of the tails. In paraffin sections of adult rat testis, sperm tails located at the luminal region of the seminiferous tubules stained intensely. Weak but significant staining also occurred on late spermatids. In the epididymal sections, staining was restricted to spermatozoa in the lumen. On the other hand, sections of testes from 25-day-old rats containing spermatogonia and early spermatocytes had a completely negative reaction. Testicular somatic cells, including Sertoli cells, peritubular myoid cells and interstitial cells, did not stain. To identify the testicular protein interacting with the antibody, adult rat testis proteins were prepared and analyzed by a sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblot technique. The antibody interacted with a protein with an estimated molecular weight of 82,000 in the testicular homogenate and particulate fraction, whereas the reaction was considerably weaker with the testicular cytosol fraction.     

磁处理水对家兔生殖影响的研究

目的:探讨磁处理水对家免睾丸、附睾重量以及精子活力、沃纱、密度的影响。方法:新西兰雄性大白免36只,随机分为实验组和对照组。对照组饮用自来水,实验组饮用磁处理水,磁处理水每天更换,实验进行90天。光镜下检测每只家免附睾尾精子密度,精子活力,精子活率,最后将睾丸、附睾称重。结果:实验组与对照组比较,其睾丸附睾重量明显增加,精子密度增加14 % ,精子活力增加8% ,精子活率增加11% ,两组差异有显著性(P <0 .0 5 )。结论:实验结果提示饮用磁处理水能显著提高家免的生殖能力。     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

Effects of breeding season and mating on total number and distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.     

Cauda epididymal sperm motility: a comparison among five species

Rat spermatozoa are immotile in the cauda epididymidis and are kept quiescent by a protein which increases viscoelasticity of cauda luminal fluid. How species-specific this phenomenon is, is unknown. In the present study, the motility of cauda epididymal spermatozoa of rats, hamsters, guinea pigs, rabbits and humans have been investigated. Sperm motility was observed in undiluted cauda sperm samples and in samples diluted with physiological diluents with or without Ca++, among others. Hamster sperm were studied in further detail to determine if the motility inhibiting factor in hamster cauda lumen fluid had characteristics similar to those previously described in the rat. Cauda fluid protein concentrations and apparent viscoelasticity were also determined and related to cauda sperm motility in all species. The results demonstrated that all species studied except rabbits have immotile sperm in their native cauda fluid and that additional Ca++ is not a factor in the initiation of motility. Cauda sperm immotility is not always related to fluid viscosity, however, so other as yet unknown mechanisms must be called upon in some species. The vigorous motility of rabbit spermatozoa in their native fluid implies that a fundamental difference exists in the relationship between epididymis and spermatozoa in rabbits from that observed in other species.     

Changes in distribution of phosphomannosyl receptors during maturation of rat spermatozoa

The aim of the present work was to study the distribution of the cation-independent (CI) and cation-dependent (CD) mannose-6-phosphate receptors (MPRs) in spermatozoa obtained from either rete testis or three regions of rat epididymis. We observed that both receptors underwent changes in distribution as spermatozoa passed from rete testis to cauda epididymis. CI-MPR was concentrated in the dorsal region of the head in rete testis sperm and that this labeling extended to the equatorial segment of epididymal spermatozoa. CD-MPR, however, changed from a dorsal distribution in rete testis, caput, and corpus to a double labeling on the dorsal and ventral regions in cauda spermatozoa. The percentages of spermatozoa that showed staining for either CI-MPR or CD-MPR increased from rete testis to epididymis. The observed changes were probably the result of a redistribution during transit rather than an unmasking of receptors. The fluorescence corresponding to CD-MPR and CI-MPR on the dorsal region disappeared when caudal spermatozoa underwent the acrosomal reaction. Receptors were localized on the plasmalemma of spermatozoa, as observed by immunoelectron microscopy. Changes in distribution may be related to a maturation process, which suggests new roles for the phosphomannosyl receptors.     

DNA stability and thiol-disulphide status of rat sperm nuclei during epididymal maturation and penetration of oocytes.

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 +/- 0.16) and testicular spermatozoa (4.03 +/- 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 +/- 0.03). However, the ratio was not different among corpus (0.69 +/- 0.01), cauda epididymis (0.68 +/- 0.11) and ejaculated spermatozoa (0.63 +/- 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 +/- 0.16) through epididymis (caput, 2.72 +/- 0.08; corpus, 1.07 +/- 0.03; cauda, -0.05 +/- 0.05; ejaculated, 0.08 +/- 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 +/- 0.09; perivitelline sperm, 0.64 +/- 0.16) and sperm decondensation (decondensed sperm, 0.69 +/- 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.     

The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa

CLK3, a member of the LAMMER family of dual-specificity protein kinases, is abundantly expressed in the reproductive system of male mice. Specifically, high levels of CLK3 protein expression are found in mature spermatozoa in the testis and epididymis. The majority of the CLK3 protein in the testis is a full-length kinase-containing form, and only a small amount of a catalytically inactive N-terminally truncated splice variant protein product is observed. Within the mature spermatozoa CLK3 is localized to the acrosome and tail. CLK3 is expelled from the sperm following the acrosome reaction and inactivated, likely by degradation by the proteases released by the sperm during the acrosome reaction. The CLK family of kinases has previously been implicated in mRNA splicing; however, the bulk of the CLK3 protein in these cells is located in the cytoplasm, suggesting that CLK3 may have additional roles in the cell.     

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.     

ABCA17 mediates sterol efflux from mouse spermatozoa plasma membranes

Mammalian spermatozoa lose plasma membrane cholesterol during maturation in the epididymis and during capacitation in the female reproductive tract. While cholesterol acceptors such as high-density lipoproteins (HDL) and apolipoproteins A-I (apoA-I) and J (Apo J) have been found in male and female reproductive tracts, transporters that mediate cholesterol efflux from plasma membranes of spermatozoa to acceptors are not well defined. Candidates include members of the ATP-binding cassette (ABC) transporter superfamily including ABCA1, ABCA7, ABCA17, and ABCG1. In this study, we utilize immunocytochemistry on sections of adult mouse testis and epididymis and RT-PCR on isolated germ cells. The data reveal that ABCA17 is expressed by steps 12-16 elongated spermatids in the mouse in testis and by spermatozoa in the lumen of the epididymis where ABCA17 localizes to the sperm head and tail midpiece. It also localizes on these areas of mouse sperm isolated from the epididymis. Moreover, ABCA17 antibody interferes with cholesterol efflux from spermatozoa to lipid acceptors apoA-I. Taken together, these results suggest that ABCA17 plays an important role in the process of sterol efflux which renders spermatozoa capable of fertilizing an oocyte.     

Isolation and characterization of rat sperm tail outer dense fibres and comparison with rabbit and human spermatozoa using a polyclonal antiserum

Rat outer dense fibres were isolated from cauda epididymal spermatozoa using mechanical and chemical dissection methods. Sperm tail isolation procedures were monitored by phase-contrast microscopy and the purity of the outer dense fibres was verified by electron microscopy. SDS-PAGE of isolated outer dense fibres revealed at least nine Coomassie brilliant blue stained bands, and 12 silver staining bands. The most abundant proteins were a large band between 26.5 and 32.5 kDa, and 84 kDa, 21.5 kDa and 15.5 kDa bands. The amino acid composition of the total rat outer dense fibres and seven isolated proteins showed similar compositions, being abundant in aspartic and glutamic acid, serine, glycine and leucine. However, the content of cysteine and proline was highly variable among the isolated proteins. Immunofluorescence microscopy demonstrated that a polyclonal antiserum to isolated rat outer dense fibres showed positive staining localized to the mid-piece of rat and rabbit spermatozoa. However, there was crossreactivity in the principal piece as well as the mid-piece of the human spermatozoa. The antiserum also showed crossreactivity in the perforatorium of rat sperm heads and the acrosome and equatorial segment of rabbit sperm heads. These data indicate that it is technically possible to isolate proteins from the outer dense fibres that will enable further studies of the amino acid sequences of sperm tail proteins.     

Spermiogenic,epididymal, and spermatozoal damage induced by a peritesticular injection of ethylene dibromide to rams

The effects of a peritesticular injection of ethylene dibromide (EDB) on the germ cells, epididymis, and spermatozoa of rams was examined by removing each injected testis and epididymis at different times after treatment and by monitoring the seminal characteristics of ejaculates.A high incidence of abnormal elongating and elongated spermatids was observed in the testes of treated rams 48 h after injection. At this time the epithelium of the corpus and cauda epididymidis was damaged in the majority of observed rams, and the spermatozoa in these segments were abnormal, mainly with acrosomal defects, denuded forms and denuded tailless forms. Apparent phagocytosis of spermatozoa was also observed in these portions of the epididymal duct. Ejaculates collected as early as 48 h after injection had a very low sperm density and large percentages of spermatozoa with acrosomal and tail abnormalities. A dose-reponse effect was obtained, and the reversibility of the effect of a low dose of EDB on sperm morphology was demonstrated in the rams by semen examination.     

White tea intake prevents prediabetes-induced metabolic dysfunctions in testis and epididymis preserving sperm quality

Prediabetes has been associated with alterations in male reproductive tract, especially in testis and epididymis. Moreover, in vitro studies described a promising action of tea (Camellia sinensis L.) against metabolic dysfunctions. Herein, we hypothesized that white tea (WTEA) ingestion by prediabetic animals could ameliorate the metabolic alterations induced by the disease in testicular and epididymal tissues, preserving sperm quality. WTEA infusion was prepared and its phytochemical profile was evaluated by ¹H-NMR. A streptozotocin-induced prediabetic rat model was developed and three experimental groups were defined: control, prediabetic (PreDM) and prediabetic drinking WTEA (PreDM+WTEA). Metabolic profiles of testis and epididymis were evaluated by determining the metabolites content (¹H-NMR), protein levels (western blot) and enzymatic activities of key metabolic intervenient. The quality of spermatozoa from cauda epididymis was also assessed. Prediabetes increased glucose transporter 3 protein levels and decreased lactate dehydrogenase activity in testis, resulting in a lower lactate content. WTEA ingestion led to a metabolic adaptation to restore testicular lactate content. Concerning epididymis, prediabetes decreased the protein levels of several metabolic intervenient, resulting in decreased lactate and alanine content. WTEA consumption restored most of the evidenced alterations, however, not lactate content. WTEA also improved epididymal sperm motility and restored sperm viability. Prediabetes strongly affected testicular and epididymal metabolic status and most of these alterations were restored by WTEA consumption, resulting in the improvement of sperm quality. Our results suggest that WTEA consumption can be a cost-effective strategy to improve prediabetes-induced reproductive dysfunction.