Mechanism of action of L-arginine on the vitality of spermatozoa is primarily through increased biosynthesis of nitric oxide

The ability of sperm to fertilize the egg is primarily dependent on sperm motility and membrane integrity. Nitric oxide (NO) plays a decisive role in regulating multiple functions within the male reproductive system. The aim of the present study is to determine the mechanism by which L-arginine confers a protective action on spermatozoa obtained from the goat epididymis. NO is synthesized from L-arginine by the enzyme nitric oxide-synthase (NOS) present in spermatozoa. A possible participation of NO and NOS in arginine action has been suggested.     

Inhibition of in vitro capacitation of hamster spermatozoa by nitric oxide synthase inhibitors.

In an attempt to understand the role of nitric oxide(NO) in sperm capacitation, in the present study, hamster spermatozoa were used to evaluate the effects of NO on motility, viability, hyperactivation, capacitation and protein tyrosine and serine phosphorylation using specific inhibitors of nitric oxide synthase (NOS); namely L-NAME (N-nito-L-aginine methyl ester) and 7-Ni (7-nitroindazole). The results indicated that L-NAME inhibits sperm motility, hyperactivation and acrosome reaction where as 7-Ni inhibits only hyperactivation and acrosome reaction thus implying that NOS inhibitors exhibit subtle differences with respect to their effects on sperm functions. This study also provides evidence that NOS inhibitors inhibit sperm capacitation by their ability to modulate protein tyrosine phosphorylation. However, the inhibitors had no effect on the protein serine phosphorylation of hamster spermatozoa during capacitation. Thus, these results indicate that NO is required     

Photoperiod-dependent effects of neuronal nitric oxide synthase inhibition on aggression in Siberian hamsters

Many nontropical species undergo physiological and behavioral adaptations in response to seasonal changes in photoperiod, or day length. In most rodent species, short winter photoperiods reduce testosterone concentrations, which provoke gonadal regression and reduce testosterone-dependent behaviors such as mating and aggression. Seasonally-breeding Siberian hamsters, however, are paradoxically more aggressive in short-days, despite much reduced reproductive activity and testosterone concentrations. Nitric oxide (NO) signaling has been proposed as part of an alternate mechanism underlying this phenomenon. A reduction in neuronal nitric oxide synthase (nNOS), the enzyme responsible for synthesizing NO in the brain, is associated with increased aggression in male short-day hamsters. In the present study, we hypothesized that pharmacological inhibition of nNOS would increase aggressive behavior in long days, but not in short days because nNOS is already reduced. Adult male Siberian hamsters were housed in either long (LD 16:8h) or short (LD 8:16h) photoperiods for 8weeks, then treated with either the selective nNOS inhibitor, 3-bromo-7-nitroindazole (3BrN) or oil vehicle, and subsequently tested for aggression in a resident-intruder test. Treatment with 3BrN increased attack frequency and duration in long days, but had no effect in short days. Short days also reduced testosterone concentrations, without any effect of treatment. These data provide further evidence linking reduced nNOS to elevated short-day aggression and support a role for NO signaling in this phenomenon.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

European hamsters (Cricetus cricetus) show a transient phase of insensitivity to long photoperiods after gonadal regression

Annual rhythms of body weight and reproduction in the European hamster (Cricetus cricetus) are the result of an interaction between seasonal changes in day length (photoperiod) and seasonal changes in the responsiveness of animals to these photoperiods. The present study demonstrates that under natural conditions European hamsters are not able to perceive long photoperiods (i.e., a 16L:8D cycle) before mid-November. This is an important difference to other hamster species, in which regrowth of the gonads can be stimulated by exposure to long photoperiods at any stage of gonadal regression. The experiments also demonstrate the existence of an annual phase of sensitivity to long photoperiods that starts around mid-November and extends until March/April. During this phase of sensitivity, exposure to a long photoperiod (16L:8D) induced gonadal regrowth within 3 wk. Additional experiments with an accelerated photoperiodic lighting regimen indicated that a photoperiod of approximately 13 h is necessary to stimulate gonadal regrowth. Under natural light conditions in Stuttgart (48.46 degrees N), a photoperiod of 13 h is reached by the beginning of April, which fits well with the finding that the majority of animals kept under a natural light:dark cycle had well-developed gonads by the end of April. Nevertheless, these animals showed a rather variable timing of gonadal regrowth, ranging from early January to late April. This is most likely the result of two processes: first, an endogenous mechanism (photorefractoriness) that induces gonadal recrudescence without any photoperiodic information while the animals are still in their hibernation burrows, and second, a direct stimulatory effect of long photoperiods.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Ultrastructural and biochemical seasonal changes in epididymal corpus and cauda of viscacha (Lagostomus maximus maximus)

The reproductive and adaptative behavior of wild rodents is synchronized primarily by the photoperiod. The viscacha, a South American rodent of nocturnal habits and seasonal reproduction is photoperiod‐dependent and its reproductive behavior is regulated by the retinohypothalamic‐pituitary pineal axis. Adult males exhibit an annual reproductive cycle with periods of maximum gonadal activity (summer‐early autumn) and gonadal regression (winter). The corpus and the cauda, the most sensitive segments of the epididymis to changes induced by the photoperiod, were analyzed using electron microscopy and enzymatic biochemistry. During gonadal regression, principal and clear cells showed signs of involution with respect to the activity period. These were characterized by more irregular nuclei, smaller cytoplasms, large vacuoles, altered mitochondria, and glycogen deposits. All cellular populations of the epididymal epithelium in regression presented abundant lysosome‐like dense bodies during the active period. In addition, we measured the activity of four acid glycosidases in the cauda epididymis along the reproductive cycle. N‐acetyl‐β‐D‐glucosaminidase (NAG), an enzyme that degrades endocytosed substances from the epididymal lumen, increased significantly during gonadal regression relative to the active period. These results demonstrate that the viscacha epididymis exhibits significant ultrastructural and biochemical changes during the reproductive cycle. We demonstrate that during regression, melatonin secretion in viscacha increases. This study shows that the epididymal epithelium is reduced. Thus, we postulate that the changes observed in the epididymis are modulated by pineal melatonin. Despite these changes, the epididymis might maintain a microenvironment suitable for the survival of stored spermatozoa. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Acquisition of sperm motility and its maintenance during storage in the lizard, Lacerta vivipara

Lizard spermatozoa, which are non-motile in the testis, develop the ability to swim as they pass along the excurrent duct. The addition of caffeine, a phosphodiesterase inhibitor, induced forward motility in spermatozoa from the caput epididymidis and increased the velocity of spermatozoa from the distal part of the epididymis. Caffeine had no effect on the motility of testicular spermatozoa. This suggests that sperm motility in this species is cyclic AMP-dependent but this factor alone is not sufficient to induce testicular sperm motility. In samples from the distal region of the epididymis, sperm motility was maximal in April just after the breeding season and then decreased significantly during the following months. A parallel can be drawn between these data and the levels of testosterone in the plasma. In the lizard, as in mammals, the epididymis may play an important role in the maturation of spermatozoa.     

Evolution of the flagellar waveform of ram spermatozoa in relation to the degree of epididymal maturation.

Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum.     

小鼠附睾头精子获得与卵子质膜融合能力的物质基础研究

随着精子在附睾中的转运,它们与卵子质膜的融合能力逐渐增加。怩证明２附睾体和附睾尾的精子均具有相当高的膜融合能力,而附睾头中的精了奶少能与卵子质膜融合,这是还说明附睾头中的精子不具备与云透明带卵子融合的物质条件呢？利用附睾结扎留并延长体外获能时间,可使附睾头远端精子的融合能力明显地提高;在精子培养液中加入ＡＴＰ,并延长精卵共培养时间,也可使一少部分附睾头近端的精子获得与卵子质膜融合的能力。这表明附睾     

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways

The effect of nitric oxide (NO*) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05-100 microM), a NO* donor. The participation of NO* was confirmed by the use of scavengers, i.e. methylene blue (50,100 microM) and hemoglobin (20-40 microg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NA) in concentrations ranging from 1 to 500 microM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO*-induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 microM; bisindolylmaleimide I, 0.1 microM and genistein, 3 microM). The role of hydrogen peroxide or superoxide anion in NO*-induced capacitation was evaluated by incubation with catalase (20-100 microg/ml) or superoxide dismutase (SOD, 0.05-0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 microM SNP treatment (31 +/- 5.15%) were similar to those of heparin treated samples (33 +/- 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO*- scavengers (P <0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 +/- 0.71, 12.75 +/- 1.41, 9.00 +/- 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO* may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO* acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

Measurement of the motility of rat spermatozoa collected by micropuncture from the testis and from different regions along the epididymis.

Spermatozoa from the testis and various regions along the epididymis of the rat were collected by micropuncture and their motility after dilution was estimated over a 15-min period by using a Quantimet image analyser. The motility of sermatozoa from the rete testis and seminiferous tubules was too low to be measured. The estimate of motility of spermatozoa from the proximal caput epididymidis was much lower than that of spermatozoa from the other regions. Spermatozoa from the distal part of the caput showed sustained motility for 15 min, whereas those from the caudal region and ductus deferens, although active initially, became less active during this period.     

The development of signal transduction pathways during epididymal maturation is calcium dependent

Capacitation has been correlated with the activation of a cAMP-PKA-dependent signaling pathway leading to protein tyrosine phosphorylation. The ability to exhibit this response to cAMP matures during epididymal maturation in concert with the ability of the spermatozoa to capacitate. In this study, we have addressed the mechanisms by which spermatozoa gain the potential to activate this signaling pathway during epididymal maturation. In a modified Tyrode's medium containing 1.7 mM calcium, caput spermatozoa had significantly higher [Ca2+]i than caudal cells and could not tyrosine phosphorylate in response to cAMP. However, in calcium-depleted medium both caput and caudal cells could exhibit a cAMP-dependent phosphorylation response. The inhibitory effect of calcium on tyrosine phosphorylation was also observed in caudal spermatozoa using thapsigargin, a Ca(2+)-ATPase inhibitor that increased [Ca2+]i and precipitated a corresponding decrease in phosphotyrosine expression. We also demonstrate that despite the activation of tyrosine phosphorylation in caput spermatozoa, these cells remain nonfunctional in terms of motility, sperm-egg recognition and acrosomal exocytosis. These results demonstrate that the signaling pathway leading to tyrosine phosphorylation in mouse spermatozoa is negatively regulated by [Ca2+]i, and that maturation mechanisms that control [Ca2+]i within the spermatozoon are critically important during epididymal transit.     

The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency

In our previous studies, we showed that a finasteride-induced DHT deficiency may cause changes in the morphology of the seminiferous epithelium without any morphological alteration of the epididymis. In this study, we demonstrated the constitutive immunoexpression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of Wistar rats treated with finasteride for 28 days (the duration of two cycles of the seminiferous epithelium) and 56 days (the duration of one spermatogenesis). We noted that a 56-day finasteride treatment mainly caused a decrease in the level of circulating DHT, as well as a statistically insignificant decrease in the level of T. The hormone deficiency also led to a change in the iNOS immnoexpression in the testis and epididymis of the finasteride-treated rats. In vitro, DHT did not modify NO production by the epithelial cells of the caput epididymis even when stimulated with LPS and IFNγ, but it did give rise to an increase in NO production by the epithelial cells of the cauda epididymis without the stimulation. DHT did not have a statistically significant influence on estradiol production by cultured, LPS- and IFNγ-stimulated epithelial cells from the caput and cauda epididymis. In conclusion, our data clearly indicates that a finasterideinduced DHT deficiency intensifies the constitutive expression of iNOS in most rat testicular and epididymal cells, so it can be expected that the expression of inducible nitric oxide synthase (iNOS) could be regulated by DHT. On the other hand, the profile of the circulating DHT and T levels strongly suggests that the regulation of constitutive iNOS expression is complex and needs more detailed study.     

植物体内一氧化氮合成途径研究进展

一氧化氮(NO)作为一种气体信号分子,在植物生理过程中发挥重要作用,它参与调节植物的生长、发育及对外界环境的应激反应.植物体内主要通过酶催化途径和非酶催化途径合成NO.酶催化途径合成NO的主要酶包括一氧化氮合酶(nitric oxide synthase,NOS)和硝酸还原酶(nitrate reductase,NR),以及在某些植物的特定组织或器官或在特殊环境条件下存在的一氧化氮氧化还原酶(nitric oxide oxidoreductase,Ni-NOR)和黄嘌呤氧化还原酶(xanthine oxidoreductase,XOR).非酶催化合成途径主要是在酸性和还原剂存在条件下将亚硝酸盐还原成NO.该文主要结合研究方法,综述了植物体内NO合成途径的研究进展,为植物体内NO信号的作用机理的深入研究提供信息资料.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

Motility of undiluted bull epididymal spermatozoa collected by micropuncture

Motility of whole undiluted semen collected from different regions of the bull epididymis by micropuncture was determined by examining a droplet under paraffin oil. Bull caudal spermatozoa showed vigorous motility in undiluted semen. This motility was less in samples collected from nearer the testis: samples from the distal caput showed weak but detectable motility while those from the proximal and mid-caput were completely quiescent. Motility of spermatozoa from the distal caput and the proximal corpus was markedly increased after incubation at 34 or 37 degrees C for 1 h, but was depressed by incubation at 25 degrees C. Similar but smaller effects were observed with spermatozoa collected from the mid-corpus and the mid-cauda, except that motility of spermatozoa from the mid-corpus was reduced after incubation for 1 h at 37 degrees C. The inhibitory effect of low temperature was completely reversible. Incubation of caudal spermatozoa under anaerobic conditions produced partial and reversible inhibition of sperm motility. The results suggested that bull epididymal spermatozoa may not be completely quiescent in their native environment as previously assumed.     

Role of Nitric Oxide in the Progression of Pneumoconiosis

Conflicting evidence has been reported as to whether nitric oxide (NO) possesses anti-inflammatory or inflammatory properties. Data are presented indicating that in vitro or in vivo exposure to selected occupational dusts, i.e., crystalline silica, organic dust contaminated with endotoxin, or asbestos, results in upregulation of inducible nitric oxide synthase (iNOS) and the production of NO by alveolar macrophages and pulmonary epithelial cells. Nitric oxide production is associated temporally and anatomically with pulmonary damage, inflammation, and disease progression in response to occupational dusts. Blockage of inducible nitric oxide synthase by administration of NOS inhibitors or in iNOS knockout mice decreases the magnitude of injury and inflammation following in vivo exposure to silica, endotoxin, or asbestos. Therefore, NO may play an important role in the initiation and progression of pneumoconiosis.     

Nitric oxide pathway in lower metazoans

The presence of nitric oxide (NO) pathway has been well demonstrated in the main invertebrate groups, showing parallel findings on the role of NO in vertebrates and invertebrates. Noteworthy is the example of the role played by the nitrergic pathway in the sensorial functions, mainly in olfactory-like systems. On the other hand, the emerging molecular information about NOSs from lower metazoans (Porifera, cnidarians up to higher invertebrates) suggests that NO pathways might represent examples of a parallel evolution of the NOS prototypes in different animal lineages. Nevertheless, increasing evidence suggests that NO is one of the earliest and most widespread signaling molecules in living organisms.Here, we attempt to provide a survey of current knowledge of the synthesis and possible roles of NO and the related signaling pathway in lower metazoans (i.e., Porifera and Cnidaria), two phyla forming a crucial bridge spanning the evolutionary gap between the protozoans and higher metazoans. From the literature data here reported, it emerges that future research on the biological roles of NO in basal metazoans is likely to be very important for understanding the evolution of signaling systems.