Rabbit Epididymal Spermatozoa Research Articles

Identification and characterisation of mitochondrial proteins isolated from rabbit epididymal spermatozoa - a preliminary study.

This is the first study to identify 23 protein spots corresponding to 13 proteins in mitochondria isolated from rabbit epididymal spermatozoa. In the group of protein spots identified in stress-induced samples, the abundance of 20 protein spots increased, whereas the abundance of three protein spots (GSTM3, CUNH9orf172, ODF1) decreased relative to the control. The results of this study provide valuable inputs for future research into the molecular mechanisms implicated in pathological processes during oxidative stress (OS).

Cytochrome Oxidase and Lactate Dehydrogenase Activities in Peroxidized Rabbit Epididymal Spermatozoa/Cytochromoxydase- und Lactatdehydrogenase-Aktivität bei peroxydierten Kaninchen Spermatozoen aus dem Nebenhoden

We report a quantitative cytochemical study on cytochrome oxidase and lactate dehydrogenase activities on rabbit epididymal spermatozoa during spontaneous lipid peroxidation. Our data show that during aerobic incubation both in NTP and KTP media the sperm cytochrome oxidase activity undergoes a significant decrease. The lactate dehydrogenase activity shows different cytochemical patterns in comparison between the two media considered. Such activity significantly increases in rabbit spermatozoa suspended in NTP medium from the first until the sixteenth hour of incubation time. At the following times the lactate dehydrogenase activity significantly declines showing yet until the later times of incubation integrated optical density values fairly high. During the whole period of the aerobic incubation, the spermatozoa suspended in medium KTP show lactate dehydrogenase integrated optical density values which not significantly differ from those of the control in spite of an initial enhancement from the first until the thirteenth hour of the experimental treatment.

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Distribution of DNA, nuclear micro-heterogeneities and compaction of the chromatin in rabbit epididymal spermatozoa. Ultrastructural evaluation of the Feulgen-like technique using osmium ammine.

An adaptation of the Feulgen procedure to visualise DNA at the ultrastructural level, using osmium ammine instead of Schiff reagent, was applied to ultrathin sections of rabbit epididymal spermatozoa, known to display increasing chromatin compaction as they progress through the epididymis. In contrast with the somatic cell nuclei of the epididymal epithelium, which display classical staining, the chromatin of spermatozoa is partly or fully destroyed during the hydrolysis step of the technique. The sperm chromatin resistance towards destruction is a function of the initial sperm nuclear compaction and of the duration of hydrolysis prior to Feulgen-like staining. For a given nucleus, the maximum staining intensity is only obtained after an optimal duration of hydrolysis. However, because of this duration and local differences in chromatin compaction, the total DNA of a section is never completely visualized. The most compact parts of the nuclei are not yet stained, when the less compact parts have already been destroyed. This gives rise to a sperm-specific pattern which corresponds to the enhancement of microheterogeneities present in all sperm nuclei and to the local depolymerisation of the nuclear material during hydrolysis. The depolymerised parts of nuclei sit over the sections, and they also bind uranyl acetate, ethidium bromide and anti-protamine antibodies. Therefore, in sperm nuclei, the Feulgen-like staining at the ultrastructural level does not reveal the true DNA distribution. However, the amount of staining can be quantified to evaluate sperm chromatin compaction.

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine.

The question of whether diplasmalogens [1,2-di(O-1'-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29 +/- 2 micrograms/10(8) cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1'-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104 +/- 5 mu/10(8) cells, of which 50% was monoplasmalogen with the 1'-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

The phospholipid-binding protein of the reproductive tract of the bull — Effect on the removal of spermatozoal cytoplasm droplets in other species and influence of antibodies on its reactivity

The phospholipid-binding protein (PBP) isolated from bull seminal vesicle fluid removed cytoplasm droplets not only from bull, but also from ram, boar and rabbit epididymal spermatozoa. However, the presence of a protein cross-reacting with anti-PBP antisera was demonstrated by immunofluorescent staining in ram seminal vesicles and ampullae. In contrast to PBP from bull, the ram PBP-like protein did not lyse bull or ram erythrocytes. Rabbit antiserum against PBP only negligibly reduced the ability of PBP to remove cytoplasm droplets from bull epididymal spermatozoa, but it inhibited the haemolytic effect of the protein.

Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility.

Loss of forward motility of rabbit epididymal spermatozoa in high K+ phosphate buffer is inhibited by taurine, hypotaurine, epinephrine and bovine serum albumin. Pyruvate and lactate also show this effect. The rate of lipid peroxidation in these spermatozoa, as measured by rate of formation of malondialdehyde, is also inhibited by these agents. A close linear correlation between percent inert spermatozoa and malondialdehyde was found, which was independent of the rate of peroxidation. Complete cessation of motility was observed at 0.5 nmol malondialdehyde/10(8) cells in the absence or presence of these agents, which is the same value found in other suspending media in a previous study [Alvarez and Storey (1982) Biol. Reprod. 27:1102-1108]. Albumin was the most effective agent in preventing loss of motility and inhibiting lipid peroxidation. Hypotaurine was the next most effective, followed by taurine, epinephrine, pyruvate and lactate. Hypotaurine reduces the amount of rate of superoxide production, as measured by the rate of reduction of acetylated ferricytochrome c by O(2), from rabbit sperm under these conditions and concomitantly reduces inactivation of the superoxide dismutase in these cells. Since superoxide seems to be the major inducer of lipid peroxidation in rabbit sperm, the protective effect of hypotaurine, which should be readily permeant to the plasma membrane, may be ascribed to scavenging of intracellular superoxide. The mechanism of the protective action of albumin is not known. Rabbit epididymal spermatozoa lose motility over time if Ca2+ or Mg2+ are omitted from the suspending medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of superoxide dismutase in protecting rabbit spermatozoa from O2 toxicity due to lipid peroxidation.

Cyanide-sensitive superoxide dismutase (SOD) activity in rabbit epididymal spermatozoa decreases with time of aerobic incubation of a washed cell suspension. The inactivation of SOD activity correlates linearly with the increasing percent immotile sperm observed under these con- ditions. We had previously shown a linear correlation between percent immotile sperm and spon- taneous lipid peroxidation in both high Naand high i��' media, the peroxidation rate being eightfold higher in the high Kmedium as compared to the high Ni' medium (Alvarez and Storey, 1982). These two linear correlations connect SOD inactivation and lipid peroxidation. In this study, the reaction of 03 with the sperm cells was characterized by a second order rate constant k5. The value of k5 for fresh cells is 1.2 X io� (cells/ml�'min'' in high Na4' medium and 12.5 X 10� (cel!s/m!F' minin high K' medium. This difference is reflected in the lipid peroxidation rate in the two media. As SOD is inactivated during aerobic incubation in the high Na+ medium, the value of k5 increases to 18.3 X 10-8 (cells/mli'mincorresponding to 100% inactivation of SOD. This value is close to that of 19.9 X 10 cells/mIT' mirf' obtained in the high Na4' medium and 22.9 X 10' (cells/mW' min' obtained in the high K' medium with fresh cells in the pres- ence of CN to inactivate the SOD. We suggest that attack of 02�' to induce lipid peroxidation in fresh cells occurs at the plasma membrane and is sensitive to the ionic composition of the medium, but that as SOD becomes inactivated during aerobic incubation, more intracellular lipid peroxidation occurs, which is not affected by the ionic composition of the medium. These results imply that SOD activity is the primary enzymic defense against oxidative degradation available to rabbit spermatozoa.

Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa.

Rabbit spermatozoa from the cauda epididymis produced 0.7-0.8nmol of H(2)O(2)/min per 10(8) cells at cell concentrations below 10(7) cells/ml with linear dependence on cell concentration. Above 2 x 10(7) cells/ml, the rate again became linear with cell concentration but decreased to 0.1-0.2nmol/min per 10(8) cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H(2)O(2) production rate on cell concentration; below 10(7) cells/ml the rate was 0.3-0.4nmol/min per 10(8) cells; above 2 x 10(7) cells/ml, the rate was 0.1-0.2nmol/min per 10(8) cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta305, 557-565] produced 0.1-0.2nmol/min per 10(8) cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 10(7) to 2.2 x 10(8) cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H(2)O(2) production to 0.3nmol/min per 10(8) cells. The decreased rate of H(2)O(2) production observed with intact sperm at high cell concentrations is attributed to reaction of H(2)O(2) with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H(2)O(2) generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H(2)O(2) production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell.

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.

Detection of Autoimmune Response to Rabbit Epididymal and Seminal Spermatozoa

Conventional and anaphylactic autoantibodies to rabbit epididymal and seminal spermatozoa were detected in sera of rabbits autoimmunized by several procedures. Antibodies against epididymal spermatozoa were found in 85% of rabbits showing lesions of varying degrees in testes. When the comparison was made with antibodies to antigens of spermatozoa from semen, the correlation was markedly lower. The data prove the importance of a proper selection of the antigenic material when looking for antibodies to spermatozoa in studies of autoimmunity and fertility.

The maturation of rabbit epididymal spermatozoa in organ culture: stimulation by epididymal cytoplasmic extracts.

Infertile spermatozoa from the proximal corpus epididymidis do not become fertile when this epididymal segment is cultured 24 h in vitro. Addition of a cytoplasmic extract from the distal corpus significantly increases their fertilizing ability. Extracts prepared from cauda epididymides or from liver were effective; however, liver extract was less effective than extract from cauda epididymides in increasing fertilizing ability. The factor(s) responsible for this effect is not a steroid or another charcoal extractable substance. These observations stress the importance of the epididymal contribution to the maturation of spermatozoa.

The maturation of rabbit epididymal spermatozoa in organ culture: inhibition by antiandrogens and inhibitors of ribonucleic acid and protein synthesis.

Infertile spermatozoa from the proximal corpus epididymidis will become fertile when this epididymal segment is cultured 24 h in vitro with 5alpha-dihydrotestosterone (5alpha-DHT). The effect of antiandrogens and inhibitors of RNA and protein synthesis upon this 5alpha-DHT-induced sperm maturation was investigated. The response to 5alpha-DHT was abolished by cyproterone acetate, SKF 7690, actinomycin D, cycloheximide, puromycin, and the aminonucleoside analog of puromycin. Addition of cyproterone acetate or actinomycin D to a suspension of distal corpus spermatozoa did not change their fertilizing ability. Culture of segments of the distal corpus for 24 h with the same antiandrogens and RNA and protein synthesis inhibitors did not change the fertilizing ability of spermatozoa. These results indicate that the development of the sperm-fertilizing ability observed in the proximal corpus epididymidis in vitro in response to 5alpha-DHT is dependent upon binding of 5alpha-DHT and synthesis of new RNA and protein molecules by the target cell. They also indicate that antiandrogen and inhibitors of RNA and protein synthesis do not have a nonspecific toxic effect on spermatozoa during the time period studied. It is likely that the target cells are the epididymal epithelial cells rather than the spermatozoa themselves.

Simultaneous measurement of glucose and O2 consumption in cell suspensions with minielectrodes.

Separation procedures which yield cell suspensions with homogeneous cell populations greatly facilitate the study of cell metabolism, but yield a limited amount of material. Multiple recording of metabolic parameters from minimum volumes is therefore useful. We have developed a method for the simultaneous recording of glucose and O2 concentrations with Polarographic electrodes in 0.26 ml suspensions of rabbit epididymal spermatozoa (RES). These cells are found “pre-separated” as a homogeneous population in the cauda epididymidis. They consume glucose and O2 and produce lactate (Murdoch and White, 1967) and so provide a useful system to test the method as well as a system in which to study the effect of perturbations on aerobic glycolysis.

The effects of testosterone, 5alpha-dihydrotestosterone, 3alpha-androstanediol, and 3beta-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture.

The fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3-4 days when testosterone (0.5 mug/ml) or 5alpha-dihydrotestosterone (0.5 mug/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5alpha-dihydrotesterone, 3alpha-androstanediol, or 3beta-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 mug/ml. Only with 5alpha-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5alpha-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.

Energy Metabolism of Spermatozoa: III. Energy-Linked Uptake of Calcium Ion by the Mitochondria of Rabbit Epididymal Spermatozoa

A study of energy-linked calcium (Ca++) uptake by rabbit sperm mitochondria is reported. The respiration rate of hypotonically treated rabbit epididymal spermatozoa with succinate in the presence of oligomycin and rotenone was increased by 50% upon addition of Ca++ and an additional 30% by the introduction of KPi. Compared with mitochondria from other tissues, the uptake with sperm mitochondria is quite inefficient, evidenced by a Ca++ ratio of .3 compared with values of 1.7 to 2.0 with liver mitochondria. This is probably due to an ineffeciency in the Ca++ carrier, during maturation of the spermatozoa, to utilize high energy intermediates generated by substrate oxidation. The uptake of Ca++ was not inhibited by La+++ nor by concentrations of ruthenium red which prevent Ca++ uptake in other mitochondria. The Ca++ uptake reaction is known to be genetically determined in all mammalian mitochondria such that modifications in the Ca++ carrier provide a specific biochemical correlate for the study of alterations in rabbit spermatozoa mitochondria.

The kinetics of inhibition of rabbit sperm acrosomal proteinase by 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK).

The interaction of acrosomal proteinase, a trypsin-like enzyme extracted from rabbit epididymal spermatozoa, BAEE (N-benzoyl-L-arginine ethyl ester), a synthetic substrate, and TLCK (L-1-chloro-3-tosylamido-7-amino-2-heptanone), a synthetic trypsin inhibitor, was analyzed by kinetic methods. TLCK is a competitive inhibitor, forming a reversible complex at the active site of the proteinase. The Michaelis (Km) and dissociation (Ki) constants are similar to those for trypsin. With time, the proteinase-TLCK complex becomes irreversible. TLCK thus labels the enzyme active site, a mechanism previously established for its inhibition of trypsin.

Energy conservation capacity and morphological integrity of mitochondria in hypotonically treated rabbit epididymal spermatozoa

Hypotonic treatment of rabbit epididymal spermatozoa in 10 mM phosphate buffer disrupts the plasma membrane and removes the cytoplasmic droplet from those cells to which it is still attached. There is, however, no effect on the mitochondria, which retain their helical configuration around the axial filament complex, have intact inner and outer membranes, and show the same cristal morphology as do the mitochondria in untreated cells. Hypotonically treated spermatozoa respire with added malate-pyruvate, succinate, and ascorbate plus N, N′-tetramethyl- p-phenylenediamine, but not with added fructose or NADH. Respiration is inhibited by oligomycin and stimulated by uncoupler, showing that the mitochondria have retained their capacity for energy conservation. The uncoupled respiration rate is not further stimulated by added cytochrome c. Reduced- minus-oxidized difference spectra obtained at −196 °C of the hypotonically treated cells show a full complement of cytochromes, including cytochrome c. This result implies that the cytochrome c lost from mammalian spermatozoa on storage or chilling [Mann, T. (1951) Biochem. J. 48, 386–388] is cytoplasmic cytochrome c not yet incorporated into the mitochondria. The mitochondrial cytochrome c remains firmly held inside the intact outer membrane.

IDENTIFICATION AND SUBCELLULAR LOCALIZATION OF THE ENZYMES EFFECTING PENETRATION OF THE ZONA PELLUCIDA BY RABBIT SPERMATOZOA

Summary. A sucrose density gradient centrifugation procedure for separating the subcellular components of washed and sonicated rabbit epididymal spermatozoa has been developed and applied to the subcellular localization of several sperm enzymes, including the acrosomal enzymesinvolved in penetration of the zona pellucida by the spermatozoon. These zona penetration enzymes were identified as hyaluronidase and a trypsin-like enzyme. They were extracted as a single complex, which sedimented in a sucrose gradient in the vicinity of proteins with molecular weights of 59,000. The combined action of these two enzymes in the acrosomal extract produces a rapid and complete dissolution of the zona pellucida, which can be completely inhibited by soybean or lima bean trypsin inhibitors. Thus, penetration of the zona pellucida must be an enzymatic process involving mainly the trypsin-like enzyme of the acrosome. The subcellular localization of catalase, carbonic anhydrase, and the two kinetic forms of lactic dehydrogenase are also described to demonstrate the contrast in subcellular localization.