低温冷冻对精子膜功能的影响

目的评价低温冷冻对精子膜功能的影响,为冻精的临床利用提供理论指导。方法用精子尾部低渗肿胀试验(hypoosmotic swelling test,HOST),比较冷冻前后及不同冷冻方法间精子尾部的低渗肿胀率,以评估低温冷冻对精子膜功能的影响。结果精尾低渗肿胀率在冷冻前为63．9±10．4％,经速冻法冷冻后为17．3±8．3％,经缓冻法冷冻后为 27．7±11．4％,任意二者间比较结果均有显著差异(P<0．01)。结论低温冷冻破坏了精子膜的功能完整性;降温速度越快,精子膜的功能完整性下降越显著。在冷冻前后进行精子尾部低渗肿胀试验,具有重要的临床意义。     

The effect of colchicine treatment on sperm production and function: a review

Colchicine is used for the treatment of various diseases including gouty arthritis, familial Mediterranean fever (FMF) and Behcet's disease. As a modulator of the microtubules at the cytoskeleton level, it arrests cell division at metaphase and inhibits microtubular- dependent cell motility. Controversy exists as to the adverse effect of colchicine on sperm production and function in healthy subjects as well as in gout, FMF and Behcet's patients. Sperm analysis shows a spectrum of pathology, from oligo- and azoospermia to normospermia with disturbances in sperm motility. These inconsistent sperm pathologies can be explained in part by the variability of the pathophysiology of the underlying disease. Thus, it seems that colchicine by itself may not have a significant direct adverse effect on sperm production and function.      

The effect of the replacement of calcium by strontium on excitation-contraction coupling in frog skeletal muscle

1. In frog skeletal muscle strontium can replace calcium in potassium contractures for 5 hr, though it is less effective than Ca. Sr can restore the responsiveness to K after it had been lost in the presence of Mn.2. Muscles refractory to caffeine following repeated exposure to it in the absence of Ca, recover in part following addition of Sr.3. The uptake of (85)Sr was increased during mechanical activity, whereas the uptake of (58)Co was not changed. Resting uptake of (58)Co was 3-4 times greater than that of (85)Sr.4. Sr fully activated the myofibrillar adenosine triphosphatase (ATP-ase), though its affinity was about 30 times less than Ca.5. The sarcoplasmic reticulum took up Sr, though less effectively than Ca.     

Regulators of sperm function: A role for diacylglycerol in human sperm acrosomal exocytosis

Using human spermatozoa stimulated with either progesteroneor the Ca²⁺ ionophore A23187 to undergo acrosomal exocytosis,we have investigated potential pathways for generation of diacylglycerol(DAG) and have examined the possibility that DAG plays an importantrole in the exocytotic response. Both treatments resulted inrapid and considerable generation of DAG, followed by a limitedrise in phosphatidic acid (PA). Further experiments indicatedthat phospholipase C (PLC) activity is important in this generationof DAG, but phospholipase D activity probably is not. In addition,polyphosphoinositide-specific phosphoinositidase C activationand hydrolysis of phosphatidylinositol 4, 5-bisphosphate appearsto be a necessary prerequisite for activation of the PLC pathway.Finally the DAG formed appears to be important in acrosomalexocytosis: (i) blocking DAG metabolism with a DAG kinase inhibitorresulted in both increased endogenous levels of DAG and a significantlyincreased exocytotic response in stimulated cells and (ii) exogenousDAG induced exocytosis in capacitated spermatozoa whereas PAdid not. Taken together, these results suggest that DAG playsa key role in events leading to membrance fusion during humansperm acrosomal exocytosis stimulated by natural agonists. acrosome reaction/diacylglycerol/human spermatozoa/phospholipase C/phospholipase D     

Regulators of sperm function: Effect of gastrin-releasing peptide on sperm functions

Male infertility can be related to defects in motility, capacitation,acrosome reaction, binding and penetration of the zona pellucida.While different in-vitro techniques (such as micromanipulationwhich is complicated and expensive) are available for the treatmentof male infertility, several pharmacological agents have beenshown to increase fertilizing capacity under accurate experimentalconditions. Gastrin-releasing peptide (GRP, the mammalian homologueof the amphibian skin peptide bombesin) is present in the reproductivetract and expressed by the pregnant ovine endometrium priorto attachment and throughout the pregnancy. A bombesin-likepeptide resulting from alternate splicing of the GRP gene intestis has been detected in primates. In this study, we havetested the ability of GRP to enhance human sperm functions suchas motility, capacitation, zona binding and acrosome reaction.Analysis of sperm motility was performed with the ATS 20 computer-assistedsemen analysis (CASA) system. Zona binding was analysed usingintact human unfertilized oocytes and selective labelling ofspermatozoa with two fluorochromes. Our results did not showany positive effect of GRP on these parameters under our experimentalconditions. However, when GRP at the concentration of 100 nMwas added after ionophore treatment, the percentage of reactedcells increased significantly (P <0.05) compared with situationswhere each agent was used alone. This led us to suppose thatthe role of bombesin in the different stages of fertilizationmight not exclude other unknown factors. acrosome reaction/gastrin-releasing peptide (GRP)/human male infertility/sperm motility/zona pellucida binding     

Varicocele: effect on sperm functions

Despite the numerous studies published over the past decade, the role of varicocele in male infertility is still controversial. Although more frequent in infertile men, its influence on sperm production or function has not, as yet, been determined. Moreover, the exact mechanism of varicocele action is not clear. We have surveyed the literature, the correlation of varicocele to sperm parameters and to sperm function tests, such as binding capacity, hypo-osmotic swelling test, presence of reactive oxygen species, and in particular, the correlation to fertility potential. Almost every subject examined had contradictory results. Larger control studies may possibly elucidate and clarify the cases in which varicocele is associated to sperm function, and where treatment may improve fertility.     

The effect of nitric oxide on sperm cell function and embryo development

PROBLEM: Nitric oxide (NO) has been known to have multifunctional roles both in the male and female reproductive systems. We investigated the effects of sodium nitroprusside (SNP)-dependent NO release on sperm cell function and embryo development to elucidate the mechanisms of action of NO. METHOD OF STUDY: Semen samples from 20 healthy men were processed by the swim-up method. Sperm motility, hyperactivation, and acrosome reaction were examined following incubation with various concentrations of SNP. The concentration of 10 nM to 1 mM was used for sperm motility and hyperactivation measurement and 1 microM to 1 mM for examining the effect on acrosome reaction. Embryo development to blastocyst stage was assessed using 100 nM to 1 mM of SNP added before transferring the mouse embryos into the culture medium. Finally, to understand the mechanism of action of NO, changes in embryo development were examined after zygotes were treated with various concentrations ranging up to 1 mM of 8-bromo-cGMP, an analog of cGMP. RESULTS: Both sperm motility and hyperactivation were significantly reduced at 100 microM and 1 mM concentrations of SNP after 6 hr of incubation. After 24 hr of incubation, they were greatly decreased with all, except the 10 nM concentration of SNP. The percentage of acrosomal-reacted spermatozoa was increased with the increasing concentration of SNP following incubation with 10 microM and 1 mM of SNP. Embryo development was arrested since the two-cell embryonic stage with all except the 100 nM concentration of SNP, and inhibited by 200 microM of SNP regardless of SNP treatment stage. However, embryo development was not influenced by 8-bromo-cGMP. CONCLUSIONS: We concluded that SNP-inhibited sperm cell function and embryo development in a dose- and time-dependent manner, and the inhibitory effect on embryo development, may not be a stage-specific treatment mediated via a cGMP-independent pathway. This result suggests that NO may be enough to affect the fecundity potential in vivo.     

The effect of strontium administration on bones and teeth of rats maintained on diets with different calcium contents

Summary In experiments on young rats with different levels of calcium intake it was found that rachitic changes in the bones and teeth developed when the diet contained very small amounts of calcium even though vitamin D was present in the food. When strontium was added to these diets more pronounced rickets developed. In animals receiving diets with a calcium content sufficient to prevent rickets, and also when the calcium intake was optimal, the addition of strontium resulted in rachitic changes. By increasing the calcium level of the diet with the strontium level kept constant these changes could be mitigated. Addition of strontium to the diet significantly depressed the blood calcium value in rats receiving low calcium diets when compared with controls on the same calcium intake. The blood phosphate remained unchanged.Animals with a low calcium intake and with strontium added to the diet, and which were given injections of calcium gluconate during the last 9 days of a 3 week period, exhibited healing rickets. In these animals the blood calcium level was significantly increased.The influence of addition of strontium to the diet on the blood calcium level is a fundamental factor in the development of strontium rickets but other effects have also to be considered, such as the influence of strontium on local factors in bone remodelling.

---

Der Einfluß von Strontium auf Knochen und Zähne von Ratten bei verschiedenen Kostformen mit unterschiedlichem Calciumgehalt

Zusammenfassung Junge Ratten zeigen bei calciumarmer Kost, selbst wenn Vitamin D in der Nahrung enthalten ist, am Skelet wie an den Zähnen rachitische Veränderungen. Wird der Nahrung noch Strontium beigegeben, so entwickelt sich eine sehr schwere Rachitis. Es kommt zu rachitischen Veränderungen selbst dann, wenn der Calciumgehalt der Diät genügend ist, um eine Rachitis zu verhindern. Bei weiterer Steigerung der Calciumzusätze verwischen sich die rachitischen Veränderungen. Strontiumzusätze zu calciumarmen Kostformen führen zu einer markanten Senkung des Serumcalciumspiegels. Dagegen bleiben die Blutphosphatwerte unverändert. Erhalten die Ratten bei einer calciumarmen Kost mit Strontiumzusätzen während eines dreiwöchigen Versuches mindestens während 9 Tagen Calciumgluconat-Injektionen, so zeigt das Skelet rachitische Veränderungen in Heilung. Bei diesen Versuchstieren ist der Blutcalciumspiegel signifikant erhöht. Die Senkung des Blutcalciumspiegels durch Strontiumzusätze zur Nahrung ist für die Entwicklung von Rachitis von entscheidender Bedeutung. Darüber hinaus muß doch auch mit anderen Einwirkungen des Strontiums gerechnet werden, beispielsweise auf örtliche Faktoren die den Knochenumbau beeinflussen.

     

Assessment of sperm function for IVF

Recent advances in our ability to understand and manipulatethe fundamental mechanisms regulating human sperm function haveled to the development of a new generation of diagnostic techniques,designed to give objective data on the functional competenceof human spermatozoa. These techniques have proved to be ofvalue in predicting the ability of patients' spermatozoa tofertilize the human ovum in vitro and in the evaluation of techniquesand reagents of relevance to the therapeutic application ofIVF, including sperm preparation protocols and reagents forthe artificial enhancement of sperm function. Moreover the useof sperm function tests has shed light on the biochemical natureof the lesions pre sent in the spermatozoa of subfertile males,with particular emphasis on the role played by reactive oxygenspecies. As a result of such studies we are now in a positionto adopt a more rational approach to the development of modifiedIVF protocols for the treatment of male infertility.     

The fate of sperm after vasectomy in the hamster

Young adult male hamsters were subjected to bilateral vasectomy. The reproductive tracts were studied by light and electron microscopy at intervals up to 1 year after the operation. Sperm continued to be produced, since testicular alterations were focal. Spermatic granulomas were associated with the excurrent ducts of all animals 5 months or 1 year after vasectomy and with those of one of four hamsters 2 weeks after the operation. Phagocytosis of sperm in the lumina of the efferent ducts and proximal parts of the epididymis, and disintegration of membranous components of intraluminal sperm occurred in approximately three-fourths of the animals studied 5 months or more after vasectomy. The results indicate that after vasectomy in the hamster sperm are disposed of by phagocytosis in spermatic granulomas, intraluminal phagocytosis, and dissolution in the lumen of the male ducts, although the latter process may be incomplete.     

The effect of calcium on the mechanical behaviour of aorta media elastin and collagen

Mechanical tests were conducted on rings of pig thoracic aorta that had been treated with calcium ions to evaluate their effect on aorta media collagen and elastin. There was an increase in stiffness just before rupture and a decrease in storage energy when calcium was introduced into intact rings, but there was no significant difference in mechanical properties when it was introduced into the elastin framework of the media. The calcium was able to bind to the intact aorta media far more readily than to the elastin alone, suggesting that the collagen and muscle framework were far more susceptible to the introduction of calcium than elastin. The calcium content rose along the length of the aorta, as did the collagen content, whilst the elastin content fell, which was also reflected in the change in mechanical properties along the aorta.     

Effects of progesterone on sperm function: mechanisms of action

Progesterone stimulates sperm functions, e.g. hyperactivation, acrosome reaction, binding to oocyte zona pellucida and penetration rate into the hamster oocyte. The physiological relevance of these effects has been shown using female genital tract fluids which modulate sperm function according to their progesterone content. Progesterone interacts with specific sperm binding sites that, unlike the classic nuclear receptors, are located on the plasma membrane of the spermatozoon. Binding studies have revealed the presence of two classes of progesterone receptors in the human spermatozoon, one class has an elevated affinity constant (nanomolar) and is specific for progesterone, whereas the other class has an affinity constant in the micromolar range and binds equally well other hydroxylated progesterone derivatives. Following exposure to progesterone, the main event is a rapid (within seconds) increase of the intracellular free calcium concentration, followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor. Progesterone also modulates sperm function by stimulating a trypsin-like proteolytic activity, the biosynthesis of polyamine (putrescine and spermidine), phospholipase A2 activity and protein tyrosine kinase activity in the sperm cell. Recent studies have shown that chloride ion efflux is vital for progesterone to promote the acrosome reaction. This effect is achieved by interaction with a sperm membrane receptor which resembles the neuronal GABA(A) receptor. Accordingly, GABA(A) receptors have been found in the spermatozoon plasma membrane and GABA stimulates hyperactivation and promotes the acrosome reaction.     

Characteristics of a short incubation model of scrapie in the golden hamster.

Repeated passage of the "Chandler" strain of scrapie in female golden hamsters using the intracerebral route of inoculation reduces the minimum incubation period to 60 days, about half of the minimum incubation period so far found in any of the mouse models of scrapie. The infectivity titres in brain in the clinical stage of the disease are considerably higher (greater than 8-0 -log10 LD50 i.c. units/0-05 g) than those found in mouse scrapie. The biological characteristics of this model of hamster scrapie are reported, including the effects on incubation period of route of inoculation, dose of agent, sex of hamster, ambient temperature (hibernation) and splenectomy. Some general and specific applications of this experimental model of scrapie are discussed.     

Comparative growth of Eikenella corrodens on 15 media in three atmospheres of incubation.

The abundance of growth and size of colonies of 10 isolates of Eikenella corrodens were studied using 15 commonly available media in aerobic, 5% CO2, and anaerobic atmospheres of incubation. Chocolate agar was the best medium for overall growth in all three atmospheres. Growth on Mueller-Hinton agar supplemented with 5% blood and on Brucella agar supplemented with 5% blood was equivalent to that on chocolate agar in 5% CO2 and anaerobic environments but not in an aerobic atmosphere. Strains of E. corrodens did not grow on the selective media studied. Growth on media without blood or hemin supplements occurred in anaerobic but not in aerobic or 5% CO2 environments, and was consistently poorer than growth on the same media with these supplements.     

The effect of calcium on the mechanical behaviour of aorta media elastin and collagen.

Mechanical tests were conducted on rings of pig thoracic aorta that had been treated with calcium ions to evaluate their effect on aorta media collagen and elastin. There was an increase in stiffness just before rupture and a decrease in storage energy when calcium was introduced into intact rings, but there was no significant difference in mechanical properties when it was introduced into the elastin framework of the media. The calcium was able to bind to the intact aorta media far more readily than to the elastin alone, suggesting that the collagen and muscle framework were far more susceptible to the introduction of calcium than elastin. The calcium content rose along the length of the aorta, as did the collagen content, whilst the elastin content fell, which was also reflected in the change in mechanical properties along the aorta.     

Effect of intracellular injection of calcium and strontium on cell communication in heart.

1. The influence of Ca and Sr on the electrical coupling of canine Purkinje cells was investigated by injecting the ions electrophoretically into the cytoplasm. 2. It was found that the intracellular injection produced electrical uncoupling which was spontaneously reversed. 3. No change in resting potential of the cell adjacent to the injection site was found except in those fibres not completely healed. 4. The input resistance of the injected cell increased concomitantly with the establishment of the electrical uncoupling. 5. Caffeine (6mM), added to the extracellular fluid, reduced the rate of spontaneous recoupling. Reduction of temperature of the Tyrode solution had the same effect. 6. The abolition of cell communication produced by Ca injection seems to indicate that the ion plays an important role in the control of junctional conductance in heart fibres.     

Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay

The effect of anti-sperm antibodies (ASA) on the rate of acrosome reactions (AR) during "in vitro" capacitation of human sperm used for the hamster egg penetration assay (HEPA) was assessed. Motile sperm suspensions from donors were exposed to several sera and seminal plasma with sperm head-directed ASA, then they were washed and capacitated "in vitro." After capacitation, the proportion of acrosome-reacted viable sperm was assessed by staining with Fluoresceinated Pisum Sativum Agglutinin and supravital stain Hoechst 33258. ASA of any immunoglobulin class did not significantly affect either the AR rate, or the hamster egg penetration rate. In conclusion, interference of ASA on spontaneous AR rate during "in vitro" capacitation can not be advocated as an explanation of the impairment of the interaction of human sperm with egg or its vestments, which have been reported in several studies.