Localization of the chaperone proteins GRP78 and HSP60 on the luminal surface of bovine oviduct epithelial cells and their association with spermatozoa

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.     

鱼类精子活力研究进展

鱼类精子在精巢和精浆中一般不活动,只有当精子被排到体外并被外界环境的溶液稀释后才能活动．鱼类精子活力受渗透压、离子、ｐＨ 值、温度及ＣＯ２ 等因子的调节和影响, 不同的鱼类其精子活力有不同的调节方式;外界因子对鱼类精子活力的影响, 是通过影响ｃＡＭＰ－ＡＴＰ－Ｍｇ２＋ 系统来影响鞭毛的活动而实现的． 精子活力的评价指标主要有：精子激活后的运动时间、精子激活比例、精子运动速度及精子鞭毛摆动频率等． 大多数鱼类的精子,其活动能力是在生殖管道中获得的．     

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.     

Application of seminal plasma in sex-sorting and sperm cryopreservation

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.     

Movement of turritella spermatozoa: direction of propagation and chirality of flagellar bends.

The marine snail, Turritella communis, produces two types of spermatozoa, named apyrene and eupyrene. Eupyrene spermatozoa are usually paired, but unpaired ones are involved in fertilization. Movements of these spermatozoa were analyzed using a video camera with a high-speed shutter. The eupyrene spermatozoa usually swim with the head foremost but are able to swim flagellum foremost. A reversal of the direction of their swimming was found to be the result of a change in the direction of flagellar bend propagation, which changed with calcium concentration. Reversal of the direction of bend propagation was accompanied by a reversal of direction of the rotational movement of the spermatozoa around their long axis, suggesting that the bending waves keep the sense of their three-dimensional form. The swimming speed of apyrene spermatozoa in natural seawater was about one-eighth of that of the eupyrene ones and remained almost constant in highly viscous medium.The swimming speed of conjugated eupyrene spermatozoa was the same as that of unpaired spermatozoa over a wide viscosity range (<3,000 cP). No advantage of swimming by two spermatozoa could be detected in Turritella spermatozoa.     

The role of small molecules in sperm capacitation

Mammalian spermatozoa released into an appropriate environment in vitro can capacitate but then may undergo spontaneous acrosome reactions. Since successful sperm interaction with the zona pellucida of an unfertilized oocyte requires an intact sperm plasma membrane, spontaneous acrosome loss is biologically undesirable because it renders spermatozoa non-fertilizing. Several small molecules (fertilization promoting peptide [FPP], adenosine, calcitonin and adrenaline), found in various body fluids including seminal plasma, have been shown to regulate capacitation in vitro. They initially accelerate capacitation but then inhibit spontaneous acrosome loss, allowing spermatozoa to maintain their fertilizing potential. Specific receptors for all these molecules are present on mammalian spermatozoa and their activation by the appropriate ligands leads to modulation of membrane-associated adenylyl cyclase activity and production of cAMP, stimulating cAMP production in uncapacitated cells and inhibiting it in capacitated cells. Boar spermatozoa have been shown to respond in vitro to adenosine and FPP, suggesting that the addition of these molecules to sperm samples used for artificial insemination could be beneficial in helping spermatozoa maintain fertilizing potential until they reach their target.     

Interstrain competition amongst mouse spermatozoa inseminated in various proportions, as affected by the genotype of the Y chromosome

Nonagouti (KP X C57BL)F1 hybrid females were artificially inseminated with a mixture of spermatozoa from males of the KE (nonagouti) and CBA (agouti) strains and the genotype of young was estimated by fur pigmentation. When KE and CBA spermatozoa mixed in the ratios of 1:1, 2:1 and 4:1 were inseminated after ovulation, 87%, 56% and 29% of progeny, respectively, were sired by CBA males, i.e. proportions of CBA progeny were significantly higher than ratios of CBA spermatozoa in the mixture. The surplus of CBA progeny was significantly less in females inseminated before ovulation, which may suggest that more rapid capacitation of CBA spermatozoa is partly responsible for their competitive advantage. In preparations from oviducal flushings of females killed 2-3 h after insemination, CBA spermatozoa (recognized by their shape) were found in similar proportions as in the inseminated mixture. There was therefore no evidence of their preferential selection at the uterotubal junction. No competitive advantage of CBA spermatozoa occurred when they were inseminated with spermatozoa from males of the KE.CBA strain, congenic with KE but with the Y chromosome derived from the CBA strain. This indicates that genetic factors linked with the Y chromosome may influence competitive ability of spermatozoa.     

Interaction of male and female germ cells of two mammalian species in culture.

When viewed by scanning electron microscopy, the heads of mouse spermatozoa are smaller than those of the hamster. The vitelline microvilli of hamster eggs are longer than those of the mouse egg. Both these factors may contribute to the enhanced interaction of mouse spermatozoa and hamster eggs. Treating unfertilized mouse eggs with Newcastle disease virus causes the viteline microvilli to elongate, thus improving the interaction between mouse eggs and hamster spermatozoa.     

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.     

Genetic and non-genetic parameters of several characteristics of production and semen quality in young bucks

The objective of this study was to evaluate genetic and non-genetic factors influencing characteristics of young buck semen production using a multivariate model that takes into account the longitudinal structure of data. Data were collected from 1989 to 2002 at two French A.I. centres. The data corresponded to 13151 and 9206 ejaculates of 758 Alpine and 535 Saanen bucks respectively, collected at the beginning of the first breeding season (September-December). The semen volume, the total number of spermatozoa, the concentration, the motility score of spermatozoa after freezing and the percentage of motile spermatozoa after freezing were registered for each ejaculate. Within-breed heritabilities and repeatabilities were estimated using a multivariate animal model using a power spatial covariance structure for environmental effect. For all characteristics and the two breeds, the main source of variation was the year-month interaction that interacted with the centre. We observed a decrease in years of motility score after freezing. Age and frequency of collection had a significant effect on semen volume and number of spermatozoa for both breeds, and on concentration of spermatozoa for the Alpine breed. No effect of these factors was found on the characteristics observed after freezing. Heritabilities for concentration, number of spermatozoa, semen volume, motility score after freezing and percentage of motile spermatozoa after freezing per ejaculate were respectively, 0.32, 0.15, 0.25, 0.12 and 0.05 for the Saanen breed and 0.34, 0.25, 0.29, 0.17 and 0.03 for the Alpine breed. Genetic correlations between volume and number of spermatozoa were respectively, 0.74 for the Alpine breed and 0.86 for the Saanen breed. Further study is required to compare the semen characteristics of young bucks with their mature production.     

The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors

Mammalian spermatozoa must undergo capacitation before acquiring the ability to fertilize the oocyte. This process is believed to be initiated following the release of surface-associated decapacitation factors that are elaborated by both the epididymis and the male accessory organs. Herein, we report the identification of a number of proteins that are actively released from the surface of mouse spermatozoa during capacitation in vitro. As anticipated, the addition of these factors back to suspensions of mouse spermatozoa was shown to suppress several correlates of the capacitation process. Specifically, they induced a significant, dose-dependent inhibition of the ability of spermatozoa to undergo a progesterone-induced acrosome reaction and to bind to the zona pellucida in vitro. Inhibition of these functions was associated with the suppression of tyrosine phosphorylation in the sperm plasma membrane but had no effect on the phosphorylation of internal proteins in either the sperm head or tail. This inhibitory activity was attributed to a subset of the isolated proteins compromising at least four putative decapacitation factors. These proteins were identified via tandem-mass spectrometry amino acid sequence analysis as plasma membrane fatty acid binding protein, cysteine-rich secretory protein 1 (CRISP1), phosphatidylethanolamine binding protein 1 (PBP), and an unnamed protein product that we have termed decapacitation factor 10 (DF10). Of these proteins, PBP was identified as a primary candidate for a decapacitation factor.     

Motility of fish spermatozoa: from external signaling to flagella response

For successful fertilization, spermatozoa must access, bind, and penetrate an egg, processes for which activation of spermatozoa motility is a prerequisite. Fish spermatozoa are stored in seminal plasma where they are immotile during transit through the genital tract of most externally fertilizing teleosts and chondrosteans. Under natural conditions, motility is induced immediately following release of spermatozoa from the male genital tract into the aqueous environment. The nature of an external trigger for the initiation of motility is highly dependent on the aquatic environment (fresh or salt water) and the species’ reproductive behavior. Triggering signals include osmotic pressure, ionic and gaseous components of external media and, in some cases, egg-derived substances. Extensive study of environmental factors influencing fish spermatozoa motility has led to the proposal of several mechanisms of activation in freshwater and marine fish. However, the signal transduction pathways initiated by these mechanisms remain clear. This review presents the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoa plasma membrane via the external membrane components, ion channels, and aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dyneins and regulation of their activity; and (4) pathways supplying energy for flagellar motility.     

Immunodetectable galactosyltransferase is associated only with human spermatozoa of high buoyant density

Human ejaculated spermatozoa are heterogeneous and can be separated into two distinct populations according to their respective buoyant densities. In order to investigate the functional differences between these two types of spermatozoa, we have searched for the presence of galactosyltransferase. A Western blot of sperm proteins following their electrophoresis was probed with an anti-galactosyltransferase serum revealing that this enzyme is present in human spermatozoa. Furthermore, galactosyltransferase is detectable only in those proteins isolated from the head of high density spermatozoa. These results suggest that ejaculated spermatozoa consist of two populations that are functionally different.     

Effects of temperature on the immobilization and the initiation of motility of spermatozoa in the male reproductive tract of the domestic fowl, Gallus domesticus

1. The motility of undiluted fowl spermatozoa taken from testis, epididymis and ductus deferens was negligible at 40 degrees C, around the normal avian body temperature. 2. The immobilization was not permanent and motility was restored by decreasing the temperature to 30 degrees C or by suspending in a NaCl/TES buffer with 2 mM Ca2+, 2 mM HCO3- or 10% seminal plasma at 40 degrees C. 3. Demembranated spermatozoa taken from testis, epididymis and ductus deferens were also immotile at 40 degrees C. However, these spermatozoa were restored the motility at 30 degrees C except testicular spermatozoa. 4. These results suggest that the capacity of movement of fowl spermatozoa can be readily obtained from testis, but that these spermatozoa are immotile due to temperature-dependent immobilization in the male reproductive tract. 5. Furthermore, it is possible that changes in environmental temperature at ejaculation are one of the important exogenous physiological factors of the initiation of fowl sperm motility.     

Calcium alters capacitation and progressive motility of uterine spermatozoa from +/+ and congenic tw32/+ mice.

The importance of calcium-dependent sperm processes for fertilization in vitro is well known, but their interaction with sperm transport in vivo is not yet clear. To determine whether exposure to calcium alters sperm physiology after incubation in the uterus, spermatozoa from +/+ mice were incubated in medium with 1.7 mM calcium prior to artificial insemination (AI). Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium before AI. When recovered from the uterus 60 min post-AI, neither prior exposure to calcium nor genotype affected numbers of spermatozoa, or percentage of motile or acrosome-reacted spermatozoa. However, significantly more calcium-treated spermatozoa were capacitated and significantly fewer were progressively motile than spermatozoa preincubated without calcium. In addition, significantly fewer spermatozoa from tw32/+ mice than from +/+ mice were progressively motile. These results suggest that uterine sperm physiology is changed by prior exposure of sperm to calcium. Since the level of progressive motility of spermatozoa recovered from the uterus was correlated with their ability to reach the oviduct (as determined in a previous study), these data support the hypothesis that progressive motility of uterine spermatozoa is important for passage to the oviduct and fertility.     

Viability of ram testicular spermatozoa following cryopreservation in rete testis fluid

Ram testicular spermatozoa, collected continuously from the cannulated testis, were frozen in rete testis fluid in straws using the cryoprotective agents egg yolk and glycerol. The effect of cryopreservation on the viability of the spermatozoa was assessed by studying their metabolism, morphology, ultrastructure, and radioiodination patterns. Freeze-thawing significantly depressed the respiration rate and glycolytic activity of testicular spermatozoa. Morphologically, there was little evidence of cryodamage in frozen-thawed testicular spermatozoa. Except for some slightly corrugated acrosomes and a more loosely attached plasma membrane over the sperm head, frozen-thawed testicular spermatozoa were indistinguishable from nonfrozen control spermatozoa. Surface radioiodination of frozen-thawed testicular spermatozoa was highly selective and resulted in a labeling pattern similar to that of the nonfrozen controls. In contrast, the radiolabeling pattern of frozen-thawed electroejaculated spermatozoa was characterized by high background radioactivity and low selectivity. These results confirm previous suggestions that testicular spermatozoa have a greater low-temperature tolerance than do ejaculated spermatozoa and indicate that cryopreservation of immature testicular spermatozoa in rete testis fluid with added egg yolk and glycerol may be a useful approach to extend the availability of these cells.     

Inhibition of the metabolism of ram spermatozoa by derivatives of α-chlorohydrin

The effects of the male antifertility agent, α-chlorohydrin, six of its derivatives, and glycidol were studied on the metabolism of washed ram spermatozoa in vitro with fructose as substrate. The α-chlorohydrin derivatives were the amino, the phosphorylated, and four glycol-bridge (ketal) compounds. All compounds except glycidol, in a concentration between 0.1 and 100 mM, reduced the aerobic glycolsis and/or oxidation of fructose. However, there was not a high correlation between the ability of these compounds to inhibit the metabolism of ram spermatozoa in vitro and their antifertility activity when administered to male rats. Other factors are clearly involved in their antifertility activity, eg, the concentration of the compounds in the epididymis and their conversion of either more or less spermicidal compounds in the body.     

Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa

Human spermatozoa are characterized by poor functionality and abundant DNA damage that collude to generate the high incidences of male infertility and miscarriage seen in our species. Although apoptosis has been suggested as a possible cause of poor sperm quality, the ability of these cells to enter an apoptotic state and the factors that might trigger such an event are unresolved. In the present study we provide evidence that the commitment of these cells to apoptosis is negatively regulated by PI3K (phosphoinositide 3-kinase)/AKT. If PI3K activity is inhibited, then spermatozoa default to an apoptotic cascade characterized by rapid motility loss, mitochondrial reactive oxygen species generation, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolization and oxidative DNA damage. However, the specialized physical architecture of spermatozoa subsequently prevents endonucleases activated during this process from penetrating the sperm nucleus and cleaving the DNA. As a result, DNA fragmentation does not occur as a direct result of apoptosis in spermatozoa as it does in somatic cells, even though oxidative DNA adducts can clearly be detected. We propose that this unusual truncated apoptotic cascade prepares spermatozoa for silent phagocytosis within the female tract and prevents DNA-damaged spermatozoa from participating in fertilization.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.