Effects of gonadotropin-releasing hormone agonist administered in microparticles on sperm quality and quantity,and plasma sex steroid levels in northern pike

Artificial reproduction of northern pike Esox lucius is impeded by the likelihood of obtaining only a small volume of sperm of inconsistent quality. A controlled-release hormone delivery system has the potential to enhance sperm production while avoiding multiple injections The objective of this study was to investigate the effects of mammalian gonadotropin-releasing hormone agonist (mGnRHa) incorporated into poly(lactic-co-glycolic acid) (PLGA) microparticles on milt production, spermatozoon characteristics, and secretion of 17β-estradiol (E2), 11-keto testosterone (11-KT), and testosterone in northern pike. Fish were divided into four groups and injected with 2 mg/kg BW carp pituitary extract (CPE), 20 µg/kg BW mGnRHa in PLGA microparticles, or 20 µg/kg BW mGnRHa plus 20 mg/kg BW metoclopramide (MET) in PLGA microparticles (PLGA + MET), along with a control group injected with 1 ml/kg 0.9% NaCl. At 48 h postinjection, the volume of milt produced was significantly greater in groups treated with CPE and PLGA + MET than in other groups. At 96 h postinjection, all hormone-treated groups exhibited significantly higher spermatozoon average velocity than recorded in the control group. Spermatozoon motility was significantly increased (P < 0.05) in the CPE and PLGA groups compared to baseline values. All treated groups showed significantly lower levels of 11-KT after the hormone injection compared to baseline values and to controls. Plasma testosterone levels increased in all hormone-treated groups. The use of PLGA microparticles, with or without metoclopramide, is suitable for use as a carrier of hormone treatments to regulate spermiation in mature northern pike.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

Na+、K+、Mg2+、Ca2+和葡萄糖溶液作为授精-激活介质对中华鲟精子受精率的影响

以不同浓度的NaCl、KCl、MgCl2、CaCl2溶液和葡萄糖溶液作为授精介质,研究了中华鲟(Acipenser sinensis)的受精效果.结果显示,适量的阳离子和葡萄糖作为激活授精介质时中华鲟卵受精率都有所提高.在实验设置浓度范围内25 mmol/L NaCI溶液、0.1 mmol/L KCl溶液、1 mmol/L MgCl2溶液、1 mmol/LCaCh溶液和50 mmol/L葡萄糖溶液浓度下,受精率分别可达到最高值,依次为87.72%、86.82%、82.24%、89.76%、80.92%.随着实验浓度继续增加,受精率反而呈下降趋势.结果显示,作为人工配制的中华鲟精子授精一激活介质,最适NaCI溶液浓度在25 mmol/L附近,最适葡萄糖溶液浓度在25 mmol/L附近,最适KCI溶液浓度≤0.1 mmol/L,最适MgCl2溶液浓度≤1 mol/L,最适CaCh溶液浓度≤1 mmol/L.     

Characterization of a cell surface glycoprotein associated with maturation of rat spermatozoa

The principal galactose oxidase/NaB[³H]₄-labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane-associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised. Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O-glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the M_r = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important componetn in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin-like structure indicates this may be a member of a new class of membrane proteins. © 1994 Wiley-Liss, Inc.     

In vitro maturation of the potential for movement of carp spermatozoa

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.     

Production of motile acrosome-reacted mouse sperm with nanomolar concentration of calcium ionophore A23187

A method to generate a population of motile, acrosome-reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55–60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40–65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the <10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187-induced acrosome-reacted sperm ws similar to that of the acrosome-reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome-reacted sperm were able to penetrate zone-free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome-reacted sperm. © 1994 Wiley-Liss, Inc.     

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20β-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 μg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.     

n-6/n-3 多不饱和脂肪酸营养失衡对小鼠精子发生的影响

目的:探讨n-6/n-3多不饱和脂肪酸营养失衡对小鼠精子发生的影响。方法:健康的30只C57/B6雄鼠随机分为对照组(CON)、高脂组(HF)、花生四烯酸组(HF+AA)。喂食16周,做合笼实验并记录致孕率,通过精子动力分析仪检测小鼠精子活力和数量的变化,用Elisa试剂盒测血清中睾酮和甘油三酯水平。通过病理组织染色观察小鼠睾丸组织的形态学变化。利用Realtime-PCR的方法检测小鼠睾丸中Dazl基因表达水平的变化。结果:高脂组、花生四烯酸组与对照组相比精子活力[(16±0.01;12.33±2.83 vs72.2±12.73)%,P0.001],精子数量[(7.5±1.13;6±0.14 vs 13.87±0.35)million/m L,P0.001],致孕率[(28.57;14.29 vs 78.57)%,P0.001]及血清睾酮含量[(0.35±0.14;0.27±0.07 vs 3.51±0.7)ng/m L,P0.001]均显著性降低。高脂组、花生四烯酸组与对照组相比,血清中甘油三酯的含量显著增高[(0.74±0.04;0.74±0.04 vs 0.45±0.04)mmol/L,P0.001]。病理组织染色观察到花生四烯酸组小鼠睾丸组织出现了明显异形,曲细精管内部的初级精母细胞明显缺失,管腔中从初级精母细胞到精子的发生过程出现了变异,精子的数量也显著性下降。参与精子生成过程中的减数分裂前的有丝分裂增殖期、精原细胞的发育等过程的Dazl基因在高脂组和花生四烯酸组小鼠睾丸中的表达量与对照组相比显著降低(0.87±0.05;0.65±0.03 vs 1.07±0.04,P0.05)。结论:膳食中n-3/n-6多不饱和脂肪酸失衡会导致雄鼠精子发生发育的障碍     

Secretion patterns and effect of prostate-derived granules on the sperm acrosome reaction of rabbit buck

There is increasing evidence that the particulate fraction of seminal plasma plays an important role in reproduction of several mammalian species. However, the origin and role of these granules in the physiology of rabbit spermatozoa is partially unknown. The aim of this study was to investigate the implication of prostate gland in the production and secretion of granules into the rabbit semen and the role of prostate-derived granules in the sperm acrosome reaction. Light and electron microscopy of the prostate gland showed that the anterior and middle tracts of the prostate (namely the proprostate and prostate, respectively) are chiefly implicated in the secretion of granules of different size: smaller granules (SG; 0.5 μm) and large granules (LG; 4 μm). Two major patterns of secretion were identified, based on electron microscope views: storage granules (large granules) seem to empty inner smaller granules directly into the duct by exocytosis, or the storage vesicle itself is released in toto into the ducts (diacytosis). In vitro experiments using granules from vasectomized rabbits, to exclude testicular origin of granules, showed that granules reduce the acrosome reaction of Percoll-selected spermatozoa, independently of the size. Interestingly, spermatozoa incubated with heat-treated granules showed a higher sperm acrosome reaction rate, suggesting a potential role of granule-derived proteins in this process. Inhibition of the acrosome reaction is a crucial event in rabbit reproduction; ejaculated spermatozoa have to wait for a long time (8-16 h) for egg availability in the female tract after mating. Taking together, our results demonstrate that prostate granules secreted either by exocytosis or diacytosis can preserve spermatozoa fertilizing ability, by preventing sperm acrosome reaction. The type of granule-derived proteins or other macromolecules implicated in this process should be further investigated.     

Effect of exogenous nitric oxide on sperm motility in vitro

Background

Nitric oxide (NO) has been shown to be important in sperm function, and the concentration of NO appears to determine these effects. Studies have demonstrated both positive and negative effects of NO on sperm function, but have not been able to provide a clear link between NO concentration and the extent of exposure to NO. To study the relationship between nitric oxide and sperm capacitation in vitro, and to provide a theoretical basis for the use of NO-related preparations in improving sperm motility for in vitro fertilization, we investigated the effects of NO concentration and time duration at these concentrations on in vitro sperm capacitation in both normal and abnormal sperm groups. We manipulated NO concentrations and the time duration of these concentrations using sodium nitroprusside (an NO donor) and NG-monomethyl-L-argenine (an NO synthase inhibitor).

Results

Compared to the normal sperm group, the abnormal sperm group had a longer basal time to reach the appropriate concentration of NO (p < 0.001), and the duration of time at this concentration was longer for the abnormal sperm group (p < 0.001). Both the basal time and the duration of time were significantly correlated with sperm viability and percentage of progressive sperm (p < 0.001). The experimental group had a significantly higher percentage of progressive sperm than the control group (p < 0.001).

Conclusions

We hypothesize that there is a certain regularity to both NO concentration and its duration of time in regards to sperm capacitation, and that an adequate duration of time at the appropriate NO concentration is beneficial to sperm motility.     

Motility and fertility of cryopreserved semen in Persian sturgeon,Acipenser persicus,stored for 30–60 min after thawing

We investigated the effect of storage times of frozen–thawed Persian sturgeon (Acipenser persicus) semen on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30, and 60 min at 4 °C post-thawing. Frozen thawed semen analyzed immediately after thawing had similar quality characteristics as fresh semen. For cryopreserved semen stored for 30 min after thawing the characteristics did not differ to fresh semen and cryopreserved semen. For cryopreserved semen stored for 60 min a significant decline in the parameters was observed. Fertilization and hatching rates were not affected by storage times of maximally 30 min of storage.     

Effects of gossypol on the motility of mammalian sperm

The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.     

The effect of cooling rate on the survival of cryopreserved bull,ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines

Spermatozoa from three species, bovine, ovine, and porcine, were frozen using standard techniques in two controlled-rate cooling machines, a commercial instrument and a custom-built device. Ice crystallisation was induced mechanically by touching the straws with a pre-cooled rod. The sperm samples were stored 24h, and then thawed rapidly and evaluated for motility, viability, and acrosomal integrity in the membrane-intact population. The custom-built controlled-rate cooling machine proved significantly better at all cooling rates for all species. This was particularly evident for the ram and the boar spermatozoa. In general, -30 or -50 degrees C/min were better than -1 degrees C/min, with a slight advantage being evident for -30 degrees C/min. However, this became very apparent for boar spermatozoa. It is clear that the higher cooling rates are necessary for successful freezing of spermatozoa from these species, and that careful control of the cooling rate is essential for maximal recovery of viable and functional cells. This is best achieved when the cooling profile is controlled from within a dummy sample.     

Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley-Liss, Inc.