Immunoprecipitation of lipid transfer protein activity by an antibody against human plasma lipid transfer protein-I

Two lipid transfer proteins, designated lipid transfer protein-I (Mr 69 000) and lipid transfer protein-II (Mr 55 000), each of which facilitates the transfer of radiolabelled cholesteryl ester, triacylglycerol and phosphatidylcholine between plasma lipoproteins, were purified from human plasma. Immunoglobulin G was prepared from goat antiserum to human lipid transfer protein-I (i.e., anti-human LTP-I IgG). The progressive addition of anti-human LTP-I IgG to buffered solutions containing either a highly purified mixture of human lipid transfer protein-I and lipid transfer protein-II, or highly purified rabbit lipid transfer protein (Abbey, M., Calvert, G.D. and Barter, P.J. (1984) Biochim. Biophys. Acta 793, 471-480) resulted in specific immunoprecipitation and the removal of increasing amounts, up to 100%, of cholesteryl ester, triacylglycerol and phosphatidylcholine transfer activities. However, similar precipitation studies on human and rabbit lipoprotein-free plasma resulted in the progressive removal of all cholesteryl ester and triacylglycerol transfer activities but only 30% (human) or 20% (rabbit) of phosphatidylcholine transfer activity. In all cases more anti-human LTP-I IgG was required to precipitate rabbit lipid transfer activity than human lipid transfer activity. These results suggest that lipid transfer protein-I and lipid transfer protein-II have antigenic sites in common, allowing precipitation of both proteins by specific antibody to lipid transfer protein-I. Most plasma phosphatidylcholine transfer activity is mediated by a protein (or proteins) other than lipid transfer protein-I and lipid transfer protein-II. In lipoprotein-free plasma all cholesteryl ester and triacylglycerol transfer activity, and some phosphatidylcholine transfer activity, is mediated by lipid transfer protein-I (or lipid transfer protein-I and an antigenically similar protein, lipid transfer protein-II.     

Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? cytochemical studies

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.     

Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation

The majority of published studies have reported the use of commercial heparin to capacitate bovine sperm. However, heparin is not present in the female genital tract fluids. In this study, we purified large amounts of glycosaminoglycans (GAGs) from bovine follicular fluid (FF), characterized them and determined their potential to capacitate sperm. FF-GAGs were isolated by protease digestion, lipid extraction, and by different precipitation conditions and then purified by ion exchange chromatography. Two GAGs, heparan sulfate and chondroitin sulfate B, were present in FF. To determine the capacitation potential of FF-GAGs, bovine ejaculated sperm were incubated 5 hr with or without 12 or 24 microg/ml of each of the FF-GAG fractions or with heparin (12 microg/ml). The purified FF-GAGs and heparin did not stimulate sperm acrosome reaction (AR), but stimulated sperm capacitation. Fractions 1 and 2 (heparan sulfate) were more active to promote capacitation (stimulated up to 3.2-fold) than fractions 3 and 4 (mostly chondroitin sulfate B). Fractions 3 and 4 stimulated capacitation two times more than the control (without FF-GAGs or heparin). When the heparan sulfate impurity was removed from fractions 3 and 4 by acid hydrolysis, the capacitation-promoting activity associated with these fractions did not change significantly. When 24 microg/ml of fraction 1 or 2 were used, the percentage of sperm capacitation observed was similar to the capacitation with 12 microg/ml of heparin. Our results also indicated that the FF-GAGs interact strongly with the BSP proteins. Therefore, it is concluded that heparan sulfate is the GAG that is the most potent capacitating factor present in bovine FF.     

Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro.

The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.     

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

Isolation and characterization of a phospholipid transfer protein (LTP-II) from human plasma

In this report we have described the purification of a human plasma phospholipid transfer protein, designated LTP-II, which displayed the following characteristics: i) facilitated both the exchange and net mass transfer of lipoprotein phospholipids; ii) did not facilitate the transfer of lipoprotein cholesteryl esters (CE) or triglycerides (TG); iii) was not recognized by antibody to the human cholesteryl ester transfer protein (LTP-I); iv) showed no amino acid sequence homology to the cholesteryl ester transfer protein (LTP-I); v) has an apparent molecular weight (Mr) of 70,000 off Sephacryl S200, and 69,000 off sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); vi) has an apparent isoelectric point of 5.0 by chromatofocusing; and vii) when added to an incubation mixture of VLDL, HDL3, and the human plasma cholesteryl ester transfer protein (LTP-I), enhanced the observed transfer of cholesteryl esters from HDL3 to VLDL, even though LTP-II has no intrinsic cholesteryl ester transfer activity of its own. These results show that this phospholipid transfer protein is unique from the human plasma cholesteryl ester transfer protein, and may play an important role in human lipoprotein lipid metabolism.     

Regulated vectorial secretion of cholesteryl ester transfer protein (LTP-I) by the CaCo-2 model of human enterocyte epithelium

We have investigated the human CaCo-2 enterocyte model for secretion of the plasma cholesteryl ester transfer protein, LTP-I. CaCo-2 cells secrete a cholesteryl ester transfer protein which possesses molecular identity with plasma LTP-I, demonstrated by anti-LTP-I immunoblot analysis and immunoinhibition of all cell-secreted cholesteryl ester transfer activity. When CaCo-2 are cultured on permeable membranes, cholesteryl ester transfer activity is detected only in the lower culture compartment. Thus, CaCo-2 vectorially sort and secrete LTP-I, as well as the intestinal apolipoproteins, from the basolateral cellular domain. Over a 24-h period, CaCo-2 secrete cholesteryl ester transfer activity in a time-dependent manner, at approximately twice the rate of HepG2. Furthermore, CaCo-2 enterocytes, but not HepG2 hepatocytes, regulate LTP-I secretion in response to fatty acid concentration in the culture medium. Based on these observations, we speculate that the intestine may be the principal regulated source of human plasma LTP-I.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Effect of seminal phospholipid-binding proteins and follicular fluid on bovine sperm capacitation.

Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% +/- 0.8% and 18.2% +/- 2.8%, respectively, control, 9.0% +/- 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% +/- 0.4% and 30.9% +/- 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% +/- 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation.     

Decrease in order of human sperm lipids during capacitation

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of the sperm sterols, cholesterol and desmosterol, is an obligatory step in the capacitation of human sperm. Because sterols can increase the order of membrane phospholipids, it has been suggested that the importance of sterol loss is that it decreases membrane lipid order. The present study tested the hypotheses that sterol loss decreases sperm membrane lipid order during capacitation and that lipid disorder is a sufficient stimulus for capacitation. Steady-state fluorescence anisotropy of the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, decreased during capacitation, indicating a decrease in lipid order. The decrease was dependent on the loss of sperm sterols, suggesting that it reflected diminished sterol-mediated phospholipid ordering. However, the lipid-fluidizing agents, benzyl alcohol and 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, did not cause sperm capacitation or overcome inhibition by cholesterol. In summary, loss of sperm sterols caused a significant decline in lipid order during capacitation; however, decreased bulk lipid order was not sufficient to trigger the subsequent events that complete capacitation.     

Purification and characterization of a human plasma cholesteryl ester transfer protein

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.     

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M_r > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M_r 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human lgG. A polyclonal antibody raised against the purified protein and anti-human lgG were both able to suppress the acrosome reactioninducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human lgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies. © 1994 Wiley-Liss, Inc.     

Regulation of LTP-I secretion from human monocyte-derived macrophages by differentiation and cholesterol accumulation in vitro

Human macrophages in vitro synthesize and secrete the cholesteryl ester (CE) transfer protein, LTP-I. The effect of differentiation of monocyte-to-macrophage on the synthesis and secretion of LTP-I cholesteryl ester transfer activity was investigated. One marker of macrophage differentiation is expression of the 'scavenger' receptor, which mediates macrophage uptake and degradation of acetylated low-density lipoprotein. Monocytes secreted very little detectable CE transfer activity in the first 24 h following cell isolation. Both CE transfer activity and scavenger receptor activity increased with time in culture. Thus, although circulating monocytes probably do not secrete CE transfer activity, tissue macrophages such as hepatic Kupffer cells may contribute to plasma CE transfer activity. Resident macrophages of the arterial wall are derived from circulating monocytes which enter the vessel wall where they differentiate into macrophages. Such macrophages are the principal source of lipid-laden foam cells of the atherosclerotic plaque. Cholesterol accumulation results when uptake of lipoprotein cholesterol overwhelms the capacity of macrophages to excrete cholesterol. Since LTP-I is postulated to function in reverse cholesterol transport, the effect on LTP-I secretion of loading macrophages with cholesterol was determined after exposure of macrophages to acetylated-LDL or free cholesterol (FC). Cholesterol loading by both these maneuvers resulted in dose-dependent increases in macrophage secretion of CE transfer activity, and there was a significant positive correlation between CE transfer activity secreted and accumulation of CE. Thus, LTP-I may function at the cellular level in maintenance of lipid homeostasis: macrophage LTP-I secretion may be a protective mechanism in response to excess cholesterol accumulation in resident macrophages of the arterial wall.     

A new role of anandamide in human sperm: Focus on metabolism

The endocannabinoid system and the presence of CB1 receptor (CB1‐R) target of the anandamide were identified in human sperm, however the anandamide action in this context needs to be further elucidated. At this purpose we analyzed the effects of anandamide on human sperm capacitation and motility. Afterwards, we focused on lipid and glucose sperm metabolism and also investigated the interrelationship between anandamide and insulin secretion by sperm. By intracellular free Ca²⁺ content assay and proteins tyrosine phosphorylation, we evidenced that anandamide did not induce capacitation process and a negative effect was obtained on sperm motility. The blockage of CB1‐R by the specific antagonist SR141716 increased both capacitation and sperm motility suggesting an involvement of the CB1‐R in the acquisition of sperm fertilizing activity. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that anandamide exerts a lipogenetic effect on human sperm lipid metabolism. Concerning the glucose metabolism, anandamide increases GSK3 phosphorylation indicating that it is involved in the accumulation of energy substrates. G6PDH activity was not affected by anandamide. Interestingly, AEA is involved in insulin secretion by sperm. As insulin had been demonstrated to be an autocrine factor that triggers capacitation, the endocannabinoid might be inserted in the signaling cascade that induces this process. Altogether these findings highlight a pivotal involvement of the CB1‐R in the control of sperm energy homeostasis and propose a new site of action for endocannabinoids in the control of energy metabolism. J. Cell. Physiol. 221: 147–153, 2009. © 2009 Wiley‐Liss, Inc     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro

1. Evidence has been provided for the transfer of phosphatidyl[14C]choline and [3H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa. 5. Rates of [14C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.     

Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction

The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as capacitation. We demonstrate here that in vitro capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-beta-cyclodextrin, Ca(2+), or NaHCO(3), components that are all required for capacitation, prevented actin polymerization as well as capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO(3), cAMP, epidermal growth factor, H(2)O(2), and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm capacitation, whereas F-actin breakdown occurs before the acrosome reaction.     

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.