Human sperm testicular angiotensin-converting enzyme helps determine human embryo quality / 亚洲男科学杂志(英文版)

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.

Período de permanência de espermatozoides em glândulas hospedeiras de espermatozoides e glândulas infundibulares em codorna de corte / Stay period of sperm in sperm-storage glands and in infundibular glands in quails

Para determinar o tempo de permanência de espermatozoides nas glândulas hospedeiras de espermatozoides (GHEs) e nas glândulas infundibulares (GIs) de codorna de corte (Coturnix coturnix coturnix), foram utilizados 12 machos e 66 fêmeas, totalizando 78 codornas em fase reprodutiva. As fêmeas foram distribuídas em 11 grupos e acasaladas por 24 horas em gaiolas individuais. Os machos, utilizados de modo intercalado, foram separados do contato com as fêmeas e colocados em descanso. As aves do grupo-controle (G0 - seis fêmeas) foram abatidas no início do experimento, enquanto as 60 fêmeas acasaladas foram distribuídas em 10 grupos (G1 a G10, com seis fêmeas cada) e abatidas a cada período de 24 horas, de forma sequencial. Fragmentos foram obtidos da região uterovaginal e do infundíbulo e submetidos às análises histológica, histoquímica e histométrica com técnicas de rotina. Os resultados morfométricos mostraram que 46% das GHEs continham espermatozoides em seu lume no primeiro dia após o acasalamento, diminuindo gradativamente nos dias posteriores chegando a 3% no quinto dia. Nesse período, os espermatozoides ascendem em direção às GIs, onde permanecem viáveis e férteis por, pelo menos, 96 horas após deixarem as GHEs, possibilitando a postura de ovos férteis por 10 dias, em média, após o acasalamento.

Sperm-Storage Tubules (SSPs) and Infundibular Tubules (ITs) are the structures responsible for sperm storage in the oviduct of birds, snakes, alligators and turtles after mating. Aiming to determine length of stay of sperm-storage tubules (SSPs) and infundibular tubules (ITs) cutting quail, Coturnix coturnix coturnix, we used 12 males and 66 females, totaling 79 quails in the reproductive phase. The females were allocated into 11 groups and mated for 24 hours in individual cages. The males used were merged and separated from contact with females and placed at rest. The poultry of the control-group (G0 six females) was slaughtered at the beginning of the experiment, the 60 previously mated females were allocated into 10 groups (G1 to G10, with six females each) and were slaughtered sequentially. On the 10th day, the last group (G10) was shot. The fragments obtained from the utero-vaginal region and the infundibulum of each female underwent histological techniques, immunohistochemistry and morphometry routine. The morphometric results showed that GHEs had 46% of the sperm in his heat on day 1 after mating, decreasing gradually in the after days reaching 3% on day 5. At this time they increase toward the infundibular tubules, where they remain viable and fertile for at least another 96 hours (4 days) after leaving the SSPs, allowing these birds to lay fertile eggs for 10 days on average after mating.

Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm

We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation.

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.

[Comparison of anti-oxidant enzymes activity and levels of zinc and selenium in sperm and seminal plasma between fertile and idiopathic infertile men]

Reactive oxygen species [ROS]-induced lipidperoxidation can lead to dysfunction of sperm and thereby, infertility may be occurred. So, always there is a balance between amount of ROS and anti-oxidant molecules in semen. Anti-oxidant enzymes of sperm; superoxide dismutase [SOD], glutathione peroxidase [GPX], catalse and zinc and selenium can protect it from destructive effects of ROS. Hence, the present study was designed to compare the activities of these enzymes and trace elements between fertile and idiopathic infertile men. Semen specimens were collected from 30 infertile men with proven infertility by an urologist, and 30 fertile men as control donors, with age range between 20-40 years old. Semen analysis was conducted by CASA method. Atomic absorption method was used for measuring of zinc and selenium concentration. Activity assays of SOD and GPX were performed by Randox Kits. Aebi method also was applied for evaluation of catalase activity. There was no difference between the activities of enzymes in fertile men and infertile ones. Also, it wasn't seen any difference in the selenium and zinc levels of seminal plasma. There was no relationship between evaluated items with sperm parameters. Only, in asthenoteratospermic individuals negative correlations were found between GPX and sperm motility, selenium and sperm morphology. Also, in these individuals, there was a positive correlation between SOD and catalse activity. Measuring activities of SOD, GPx, and catalase and the contents of zinc and selenium of seminal plasma do not appear to be suitable tools for determining the fertility potential of sperm

Role of CK2 alpha subunit in idiopathic male infertility

Protein kinase CK2 which was formerly known as casein kinase is a ubiquitously expressed protein kinase. Its expression and its activity as a protein kinase is closely connected with proliferation i.e. rapidly proliferating cells have high amounts of the enzyme and its activity in general higher than in normal cells. Protein kinase CK2 is composed of two regulatory beta subunits and two Catalytic alpha, alpha subunits, but these subunits are also found in its free form. Although CK2 is expressed in every tissue, there are some differences in the expression level in various tissues. CK2alpha is most highly expressed in brain and testes whereas only low amounts of CK2alpha are found in other tissues. The regulatory beta subunit and the catalytic alpha subunit are both absolutely necessary for the survival of cells, because the Knockouts are lethal during embryogenesis. Mice with a CK2alpha Knockout are viable however the male mice are infertile. Materials and Methods: 83 men were involved in this study, they were 17 normal men and 66 men with idiopathic infertility problems. Required sperm samples were obtained from an in vitro fertilization unit. The sperms were extracted and their protein content is determined. Equal amounts of protein will be loaded on SDS-polyacrylamide gel. After a Western Blotting, the CK2alpha, CK2alpha and CK2beta subunits were detected by specific antibodies. Results: the presence percentage of CK2alpha was 12.1% in infertile men group, and it was significantly lower compared to control group, which was 100 %. Conclusion: the absence of CK2alpha from the sperm would be used as a marker for the identification of idiopathic men infertility

[ effect of oxamate on LDH-C4 activity of sperm in rats]

LDH-C4 is one of the lactate dehydrogenase isoenzymes found in mature testis and spermatozoa of many species. The Physiological function of these isoenzymes indicates its role in creating energy for sperm motility and survival. In this research, the effects of oxamate as specific inhibitors of the LDH-C4 of rat were studied in vivo. A total of 20 adult rats were divided into four equal groups. One group as the control group received saline only, and different amounts of oxamate were injected into other three groups [600, 300, 150mg/kg] daily for 45 days intraperitoneally. The rats were then killed with chloroform and the caudal part of epididym was separated. By making several cuts in caudal part of the epididymis, the sperms were isolated and put in T6 medium+5mg/ml[-1] BSA. Later, the sperms were incubated under 37§C and 5% CO2 for one hour. LDH-C4 enzyme was extracted using the Erwin Goldberg and the protein amounts were measured by Lowry's method. Relative purification was done in two stages including ammonium sulfate precipitation and column chromatography by DEAE-Sephadex-A50. All stages of extraction, the amount of total protein, LDH enzyme activity in the oxamate-exposed groups, and specified recipient, were then compared with the control group. In this study, the total enzyme LDH-C4 activity in the control group was 11.8 +/- 0.3 and the oxamate recipient groups [150, 300 and 600mg/kg] were 8.3 +/- 0.3, 6.9 +/- 0.2 and 3.2 +/- 0.1 IU, respectively. The concentration of 600mg/kg oxamate was inhibited about 63% enzyme activity compared to control group. This study showed that the oxamate may reduce in vivo enzyme activity through LDH-C4 with increasing concentration and this effect is proportional. Therefore, with the effect of the competitive inhibitors of oxamate on LDH-C4, this substrate can be used as a contraceptive for males

Estrogens and male reproduction: a new concept

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (alpha and ß) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30 percent lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.

Effect of exogenous selenium on the testicular toxicity induced by ethanol in rats.

The effects of supplementation of selenium at a dose of 10 microg/ kg body weight were investigated on ethanol induced testicular toxicity in rats. In the present study, four groups of male albino rats were maintained for 60 days, as follows: (1) Control group (normal diet) (2) Ethanol group (4g/kg body weight) (3) Selenium (10 microg/kg body weight) (4) Ethanol + Selenium (4g/kg body weight + 10 microg/kg body weight). Results revealed that ethanol intake caused drastic changes in the sperm count, sperm motility and sperm morphology. It also reduced the levels of testosterone and fructose. The activities of 3betaHSD, 17betaHSD in the testis and SDH in the seminal plasma were also reduced. Lipid peroxidation was also enhanced as the lipid peroxidation products were increased and the activities of the scavenging enzymes were reduced. But on coadministration of selenium along with alcohol all the biochemical parameters were altered to near normal levels indicating a protective effect of selenium. These results were reinforced by the histopathological studies.

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome recated cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96 per cent of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.

Seminal LDH-C4 isoenzyme and sperm mitochondrial activity: a study in male partners of infertile couples.

Lactate dehydrogenase isoenzyme patterns were obtained in the seman of 93 male partners of infertile couples and 28 proven fertile subjects as a control group. Sperm mitochondrial activity index (SMAI) alongwith the conventional seminal parameters was studied for all the subjects excepting the azoospermic and vasectomised males. Only LDH-C4, a germ cell specific isoenzyme activity varied with the variation in sperm density. LDH-C4 activity varied significantly (p<0.001) within and between different groups. Lactate dehydrogenase-C4 activity was absent in 17 azoospermic samples, confirming its germinal epithelial origin, as well as in 8 samples of vasectomised males. In one azoospermic sample, there were many immature germ cells along with surprisingly high LDH-C4 activity suggesting more activity of germinal epithelium associated with high LDH-C4 activity. LDH-C4 activity was reduced significantly in oligozoospermic samples in proportion to sperm density, thus confirming strong correlation (p<0.001) between LDH-C4 and sperm density. There was statistically significant correlation between LDH-C4 and percentage sperm motility as well as between LDH-C4 and Sperm Mitochondrial Activity Index (SMAI) (probability varying from p<0.05 to p<0.01 in different groups), but no such correlation was found between LDH-C4 and sperm morphology. The data confirms LDH-C4 as a germinal epithelial marker. Its relationship with percentage sperm motility is suggestive of definite role of LDH-C4 in evaluation of the spermatozoal quality, similarly its relationship with Sperm Mitochondrial Activity Index (SMAI score) suggest the role of LDH-C4 in metabolism of the spermatocytes and sperms, though further studies are required for clear and detailed understanding of its metabolic role in semen.

Lipid peroxidation and antioxidant enzymes in male infertility.

BACKGROUND AND AIM: Mammalian spermatozoa are rich in polyunsaturated fatty acids and are very susceptible to attack by reactive oxygen species (ROS) and membrane lipid peroxide ion. Normally a balance is maintained between the amount of ROS produced and that scavenged. Cellular damage arises when this equilibrium is disturbed. A shift in the levels of ROS towards pro-oxidants in semen and vaginal secretions can induce an oxidative stress on spermatozoa. The aim was to study lipid peroxidation and antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase (SOD) and to correlate the same, with the 'water test', in male infertility. SETTINGS: Experimental study. SUBJECTS AND METHODS: Ejaculates from a total of 83 infertile and fertile healthy individuals were obtained. Lipid peroxidation and antioxidant enzyme levels were studied and correlated with water test. RESULTS: The results indicate that (i) the antioxidant enzyme catalase showed no significant changes in the various pathological samples, (ii) antioxidant enzymes SOD and glutathione peroxidase correlate positively with asthenozoospermic samples and (iii) the degree of lipid peroxidation also correlates positively with the poorly swollen sperm tails. The increase in SOD and glutathione peroxidase values, in the pathological cases represents an attempt made to overcome the reactive oxygen species. CONCLUSION: Water test could be used as a preliminary marker test for sperm tail damage by reactive oxygen species, since it correlates very well with lipid peroxidation and antioxidant enzymes.

Human spermatozoal motility and lipid peroxidation.

Correlation between human spermatozoal motility and lipid peroxidation is worked out following their suspension in native seminal plasma and in Biggers, Whitten and Whittengham (BWW) medium. Spermatozoa suspended in BWW showed higher motility and lesser degree of lipid peroxidation than those suspended in seminal plasma. Further, higher activities of antioxidant enzymes are recorded in the BWW suspended spermatozoa vis a vis those suspended in native seminal plasma.

Seasonal variations in Sertoli and germ cell marker enzymes in testis of rhesus and bonnet monkeys.

Variations in specific activities of the marker enzymes of Sertoli and germ cells during breeding (November-December) and non-breeding (May-June) seasons were investigated in rhesus and bonnet monkeys maintained under laboratory conditions. The marker enzymes selected for testicular cells were-Sertoli cells: beta-glucuronidase, gamma-glutamyl transpeptidase; pre-meiotic germ cells: glucose 6-phosphate dehydrogenase, malate dehydrogenase, alpha-glycerophosphate dehydrogenase; mature germ cells: LDH-X, sorbitol dehydrogenase. Results have indicated significant seasonal variation in marker enzymes only in rhesus testis. Marker enzymes of Sertoli cell increased while those of germ cell decreased significantly during non-breeding season. Marker enzymes of mature germ cells were affected much more drastically than those of the pre-meiotic germ cells.

Renin-like activity in the rat epididymis.

'Whole' cauda epididymal homogenate and individual components of the cauda epididymis viz, cauda epididymal cells, epididymal plasma and sperm cells, as well as the blood plasma of the rat were screened for renin-like activity by in vivo method. Enzyme activity was high in 'whole' cauda epididymal homogenate, but very low in blood plasma. Evaluation of individual components of the epididymis revealed a relatively high enzyme activity in the epididymal cells but low activity in epididymal plasma and epididymal sperm cells. The high activity in epididymal cells was not affected by efferent duct ligation for 8 weeks and bilateral nephrectomy. Like most other epididymal functions, renin-like activity in the epididymis was androgen dependent. It was concluded that cauda epididymis of the rat contains the enzyme renin, whose activities may be predominantly intracellular.

Especies reactivas de oxígeno en el espermatozoide / Oxygen reactive species in the spermatozoa

Los radicales libres de oxígeno son especies químicas de este elemento formadas mediante reacciones enzimáticas y no enzimáticas y han sido implicados en muchos procesos patológicos y fisiológicos. Las especies químicas de oxígeno más reactivas son el peróxido de hidrógeno y los radicales libres anión superóxido e hidróxilo, siendo este último el más reactivo. El espermatozoide de mamíferos, incluyendo el del humano producen radicales libres de oxígeno y peróxido de hidrógeno; sin embargo esta última célula normalmente posee mecanismos enzimáticos para protegerse del posible daño que pudiera causarle estos agentes tóxicos. A pesar de ello, el espermatozoide es bastante suceptible al "estrés oxidativo", probablemente debido a su alto contenido de ácidos grasos insaturados. Este hecho puede tener importancia en la patología de ciertos tipos de infertilidad masculina como lo es la oligozoospermia, ya que se ha demostrado que en estos casos, se presenta una producción incontrolada de especies reactivas de oxígeno por parte del espermatozoide

Autoimmune-like activity of sperm specific LDH: a pathophysiological and electron microscopic study of atrophied testis and epididymis.

Hyper-immunization of male mice with human LDH-C4 evoked autoimmune reactions illustrated by the loss of LDH activity, associated histopathological changes in testes and epididymis and induction of sterility in mice. This was substantiated by the altered morphology of sperm mitochondria and plasma membrane, and by reduced number of cytoplasmic droplets as observed by electron microscopy. However, the presence of lymphoblasts and other lymphoid cells in testes indicated that the testicular damage is accentuated by activated T lymphocytes. It is concluded that immunization with human LDH-C4 produces lesions in mouse testis and epididymis, which are similar to experimentally induced autoimmune orchitis.

Effects of immunization with sperm specific lactate dehydrogenase with & without muramyl dipeptide as adjuvant.

The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

Phosphoinositide specific phospholipase C activity of goat spermatozoa in transit from the caput to the cauda epididymis.

Goat sperm lysate from cauda epididymis was incubated in the presence of [14C]phosphatidyl-choline, -ethanolamine, -inositol and diphosphatidyl-glycerol. The release of [14C]diacylglycerol from only phosphatidyl inositol confirmed the presence of phosphatidyl inositol specific phospholipase C. The enzyme activity was linear up to 1 hr of incubation at a sperm concentration of 1-10 x 10(9). It had pH optimum of 7.5 in a broad range of pH activity profile (pH 6-9). Maximum activity was observed at 8 mM calcium ion concentration. EDTA and EGTA (5 mM) did not inhibit the activity completely. A comparative study on spermatozoa and fluid from caput, corpus and cauda epididymis revealed 6.5-fold increase of activity in spermatozoa and a 4-fold decrease in case of fluid during the epididymal transit. However, the total protein content remained unchanged in fluid and decreased up to the extent of 2.4-fold in spermatozoa during this process. Thirty five percent of the caudal sperm activity was soluble and the rest was associated with head (44%), mid-piece (10%) and tail (10%).

Solubilizacion de la membrana del espermatozoide humano con n-dodecil sarcosinato de sodio: I.accion del detergente / Solubilization of the membrane human spermatozoa: I. action of the detergent

Espermatozoides humanos fueron lavados con PBS y tratados com n-dodecil sarcosinato de sodio (Sarkosyl). El insoluble remanente estaba compuesto por cabezas, cuyas colas fueron cortadas a la altura del cuello o del segmento intermedio y cuyas membranas no sufrieron mayores danos, estando el acrosoma intacto y por pequenos corpusculos que podrian ser acrosomas aislados. Los resultados indican que la accion del detergente es de dos tipos claramente diferenciados: a)diseccion del espermatozoide con separacion de la cola y b)disolucion del mismo dejando el acrosoma intacto. El residuo conserva actividad inmunologica e inmunobiologica