Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X-100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.     

Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

Features of a seminal proteinase inhibitor- zona pellucida-binding component on murine spermatozoa

Murine cauda epididymal sperm contain sites on the plasma membrane over the apical portion of the acrosome that recognize proteinase inhibitors and the homologous zona pellucida. Ten times more of the component can be extracted from cauda and ductus sperm than from equal numbers of caput and corpus sperm. Likewise, few sperm from the upper epididymal regions are able to bind seminal inhibitor, while the majority of sperm from the cauda and ductus do bind. Cauda epididymal and ductus sperm lose little of their ability to bind inhibitor after a 4-hour in vitro incubation in either a capacitating or a noncapacitating medium. The percentage of naturally inseminated sperm with the seminal inhibitor bound to their surface decreases to about 10 after 4 hours in utero. Approximately 80% of these sperm show positive fluorescence when given the opportunity to rebind the inhibitor, and these sperm do have an intact plasma membrane over the apical portion of the acrosome. Furthermore, after 4 hours in utero, the inhibitor bound in the same region of the sperm head as it did on freshly ejaculated sperm. The seminal inhibitor inhibits the binding of sperm to the zona if added during the first 15 minutes of incubation but has no effect on attachment. The data indicate that sperm gain the ability to bind the seminal inhibitor during the epididymal sojourn. Furthermore, this binding capacity is not lost during in vitro or in utero incubation. The site is not involved in sperm-zona attachment but does participate in the binding of sperm to the zona.     

Lipid phase transition in the plasma membrane of the goat epididymal maturing spermatozoa

Microviscosity of the highly purified plasma membranes isolated from the maturing goat caput, corpus and cauda epididymal sperm, was measured using l,6-diphenyl-l,3,5-hexatriene as the lipophilic probe at varying temperatures (12–42°C). As shown by the Arrhenius plot of the data each of the maturing sperm membranes had two distinct lipid phase transitions in the temperature zones 19–25°C and 34–37°C. The low-temperature transitions for the immature caput- and mature cauda-sperm membranes were noted at 19–20°C, and 24–25°C, respectively, whereas both these membranes showed high temperature transition at 36–37°C. The maturing corpus-sperm membrane had phase transitions at 21–22°C and 35–36°C that were significantly different from those of the immature/mature sperm membranes. The data implicate significant alteration of the sperm membrane structure during epididymal maturation. The phase transition of the mature male gametes at 36–37°C may have a great impact on the subsequent events of the sperm life cycle since the mature spermatozoa that are stored in the epididymis a few degrees below the body temperature, experience higher temperature when ejaculated into the female reproductive tract.     

Interaction of the human epididymal protein CD52 (HE5) with epididymal spermatozoa from men and cynomolgus monkeys

A monoclonal antibody (CAMPATH-1G) against the human lymphocyte surface protein CD52, which is similar to the epididymal secretion HE5, was used to ascertain the presence of this protein on maturing primate spermatozoa by flow cytometry. The percentage of human viable spermatozoa stained specifically with this antibody increased from sperm in spermatocoeles (0.5%), to the efferent ducts (3.8%), corpus (47.2%), and cauda (85.7%) epididymidis. Positive cells revealed staining mainly over the whole tail and postacrosomal region of the sperm head. Spermatozoa (∼10%) from both the efferent ducts and corpus epididymidis took up additional antigen when incubated with human distal cauda epididymidal plasma as a source of CD52, and 12–22% of human testicular sperm (from spermatocoeles) took up CD52 from human seminal plasma. In the cynomolgus monkey, nonspecific binding of control IgG was greater than that in human males and net CD52 staining was measurable only on ∼30% of corpus sperm where it was mainly on the principal piece. Neither caput nor cauda sperm took up human CD52 upon incubation with human seminal plasma, but an additional 27% of corpus sperm expressed CD52. Such uptake of CD52 was drastically reduced, or did not occur, when seminal plasma had been fractionated by filtration through 0.1 μm filters (filtrate II) or 300,000 Da cutoff filters (filtrate III), respectively. Western blots revealed that CD52 contents were much reduced in filtrate II and nondetectable in filtrate III of seminal plasma. Similar reduction of CD52 in the filtrate of cauda epididymidal plasma indicates the association of this epididymal secretion with large molecular factors and suggests their involvement as carriers in the in vivo transfer of the secretion onto the epididymal sperm surface. The in vitro uptake of CD52 by some but not all immature sperm and the detection by Western blotting of much less CD52 in the corpus than the cauda luminal plasma suggest that the acquisition of this epididymal secretion by spermatozoa depends on their maturation status as well as the availability of the protein in the epididymal lumen. Mol. Reprod. Dev. 48:267–275, 1997. © 1997 Wiley-Liss, Inc.     

Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro

Normal epididymal function, such as protein expression and secretion, is primarily regulated by testicular androgens and temperature. However, the role of spermatozoa in this critical process has never been studied. In order to determine whether sperm itself could regulate epididymal function, we have developed a cell culture system of bovine epididymal cells to study the interactions between spermatozoa and the epididymal epithelium. Primary cells from caput, corpus, and cauda epididymal tissues were cultured in the presence of androgens at 32 degrees C (scrotal) and 37 degrees C (abdominal). Newly synthesized proteins were metabolically labeled with (35)S-methionine after sperm co-incubation and the pattern of secreted proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proliferation rate, protein secretion rate and electrophoretic patterns of secreted proteins were evaluated 48 hr post-co-incubation. Incubation at 32 degrees C indicated that spermatozoa stimulation increases the level of protein secretion of cultured cells from all epididymal sections while it slightly decreases proliferation of corpus cells. At 37 degrees C, spermatozoa co-incubation significantly decreases the protein secretion rate of cultured cells from all epididymal sections. Independently of cell incubation temperature, spermatozoa stimulation induces both an increase in the intensity of radiolabeled proteins and the appearance of new secreted proteins of caput cells without affecting the protein pattern of corpus or cauda cells. Incubation at 37 degrees C, however, greatly modifies the pattern of proteins expressed at 32 degrees C by cauda cells. Taken together, these results support the hypothesis that spermatozoa themselves affect epididymal cell function, most importantly for caput epididymides.     

Characterization of anti-sticking factor-II from goat epididymal plasma

A previous study has characterized the major 47 kDa anti-sticking factor (ASF-I) from goat cauda-epididymal plasma (Roy, N., and Majumder, G.C., Biochim. Biophys. Acta, 991:114-122, 1989). This study reports the purification and characterization of ASF-II, another anti-sticking factor from the goat epididymal plasma. ASF-II was purified to apparent homogeneity by using concanavalin A-agarose affinity chromatography, DEAE-cellulose chromatography, alumina gel adsorption, and isoelectric focussing techniques. It showed a single protein band by both non-denaturing and SDS-polyacrylamide gel electrophoresis. ASF-II showed a molecular weight of 36,000 and a sedimentation constant of 2.4S. ASF-II is largely stable to heat treatment and it is a specific glycoprotein having high affinity and specificity for its anti-sticking action. At saturating concentration (1 nM) it inhibited adhesion of nearly 50% of spermatozoa to the glass surface of the haemocytometer counting chamber. Both the protein and sugar parts of the factor are essential for the anti-sticking activity since it lost its activity completely when treated with trypsin, L-fucosidase, or mannosidase. ASF-II does not coat the glass surface and it binds to spermatozoa. Data are consistent with the view that ASF-II has not been derived from the larger ASF-I molecule due to its enzymic modifications. Both ASF-I and -II had no effect on sperm forward motility as evidenced by spectrophotometric motility assays, indicating thereby the suitability of the factors to improve the existing sperm motility assays by eliminating the possibility of cell-sticking artifacts.     

Ascorbic acid induces spectrin reorganization in bull epididymal spermatozoa

The acrosome, a complex organelle, plays a key regulatory role in the sperm–egg interaction. We have previously shown that ascorbic acid affects both motility and spectrin protein patterns in sperm. In this study, we further characterized the changes in spectrin in sperm challenged with ascorbic acid, using SDS-PAGE, western blots, and immunofluorescence. Ascorbic acid shifts spectrin to a higher-molecular-weight species based on western blot studies. This shift in the spectrin band correlates with a striking series of changes in spectrin immunofluorescence patterns. Upon ascorbic acid challenge, spectrin localization changes, eventually resulting in the formation of vesicles. These vesicles can reach sizes up to five times the original volume of the sperm cell and sometimes show multiple spikes. These findings indicate that a novel process is taking place in the acrosome upon ascorbic acid challenge and suggest that the cytoskeleton may be a useful target for studying and hopefully controlling the sperm–egg interaction. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.     

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85–90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re‐evaluated to determine the true taxonomic and phylogenetic position of this species.     

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

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

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

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing–thawing.     

Ecto-cyclic AMP-receptor in goat epididymal intact spermatozoa and its change in activity during forward motility

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.     

Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

Changes in the rat sperm head during epididymal transit

Morphological changes in sperm are one aspect of a maturation process during epididymal transit in marnrnals. The literature mentions only, for different strains of rats, a remodeling and decrease in size of the acrosome. In the present work, the sperm were obtained from caput, corpus, and cauda epididymis of the albino rat. Samples were processed for scanning electron microscopy, with routine techniques, and for light microscopy and video microscopy. It appeared, with these techniques, that the acrosomal curvature and the whole head surface area of the rat sperm decrease during the epididymal transit. To measure these changes, a geometrical method was designed, and surface measurements were made using a computer program. It was found that the caput sperm head has the greatest surface area and a sharper acrosome bend than the cauda sperm. In an attempt to explain the above-mentioned changes, the suggestion is offered that some compactation of the nucleus and acrosomal material could be related to the decrease of the surface area.     

Changes of murine sperm phospholipid composition during epididymal maturation determined by MALDI-TOF mass spectrometry

After leaving the testis, spermatozoa undergo several important steps of biochemical maturation during the passage through the epididymis, increasing their motility and fertilizing ability. These changes comprise (among others) the modification of the phospholipid composition of the sperm membrane. This process is thought to be important for the achievement of motility and fertilizing capacity. The lipids of the sperm membrane are characterized by a significant content of unsaturated fatty acyl residues, resulting in a high sensitivity against oxidative stress. This is evidenced by the appearance of lysolipids, for example, lysophosphatidylcholine, which acts like a detergent and is normally present in only very small amounts in biological membranes. The epididymis represents a tubular system comprising three main parts (caput, corpus, and cauda), through which the spermatozoa are consecutively transported undergoing distinct maturation stages. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we established three striking differences in the lipid composition of murine spermatozoa from the different epididymal regions: in comparison to the caput sperm, sperm from the cauda are characterized by (1) a higher degree of unsaturation (PC 18:0/22:5 and 18:0/22:6 vs. 18:0/20:4 and 18:0/18:1), (2) an enhanced plasmalogen content, and (3) an enhanced content of lysolipids. These changes are likely to be of physiological relevance and potentially useful as diagnostic markers of sperm maturation and acquisition of motility.     

Birth of a chimpanzee (Pan troglodytes) after artificial insemination with cryopreserved epididymal spermatozoa collected postmortem

A male chimpanzee (Pan troglodytes) suddenly died of acute hemorrhagic enteritis at the age of 18 years. Within 24 hours after its death, a large quantity of spermatozoa of excellent quality was recovered from the distal cauda epididymides and was subsequently cryopreserved. After storage for 67 days, the frozen spermatozoa were thawed and inseminated in an adult, normal cycling, nulliparous female. The optimal day for insemination was estimated by monitoring the swelling of the female’s sex skin and urinary luteinizing hormone concentrations. Pregnancy was confirmed by a urinary chorionic gonadotropin test, and a normal female infant was born after 214 days of gestation. This birth demonstrates that distal cauda epididymal spermatozoa recovered from a dead male can be cryopreserved and successfully inseminated in a female chimpanzee. Zoo Biol 20:135–143, 2001. © 2001 Wiley‐Liss, Inc.