Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm. 相似文献
In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit. 相似文献
The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO2 in air and heparin.
In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface. 相似文献
Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation. 相似文献
Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[57Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H+ production as measured by amino[14C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H2 receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/106 cells per h under basal conditions and 14.3 fmol in response to histamine. 相似文献
Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt2 cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca2+, or replacing Ca2+ with Ba2+ or Mg2+, when nonhyperactivated motility was maintained, but Sr2+, like Ca2+, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca2+ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca2+ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca2+) suggesting that glucose acts on a Ca2+ — primed system. Removal from high ionic strength medium, chelation of Ca2+ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility. 相似文献
The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites. 相似文献
The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0–24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates. 相似文献