The lysophospholipase of human spermatozoa was purified to homogeneity by sequential ion-exchange, gel filtration, and hydrophobic chromatography. The final preparation exhibited a single protein band on SDS-PAGE. The molecular mass of the enzyme was estimated to be 51 kDa by SDS-PAGE and 52 kDa by gel filtration. The optimal pH of this enzyme is 8.0. Polyclonal antibodies against lysophospholipase were prepared by placing the enzyme adsorbed on nitrocellulose directly into the spleen of rabbits. These antibodies were purified by protein A-agarose and by affigel-lysophospholipase chromatography. The purified antibodies and enzyme were used to study the possible role of lysophospholipase in the acrosome reaction. The addition of these antibodies led to an increase in the acrosome reaction, thus suggesting that inhibition of lysophospholipase produces a higher lysophosphatidylcholine concentration and results in an acrosome reaction level similar to that obtained by the calcium ionophore A23187. Immunofluorescence localization of the enzyme indicated that the enzyme is located on the head of spermatozoa. The purified sperm lysophospholipase and its specific antibodies represent important tools for the study of the regulation of this enzyme in reproductive processes. Furthermore, the study of this enzyme will allow evaluation of the mechanisms underlying the acrosome reaction.

The distribution of lysophosphatidylcholine, lyso-platelet-activating factor and platelet-activating factor (PAF) was studied in human plasma and in follicular and peritoneal fluid. In plasma, peritoneal and follicular fluids, 51%, 87% and 89%, respectively, of the total lipids were found in the protein fraction (the density > 1.21 fraction). Two forms of lysophospholipids were identified in this fraction: one of high affinity and one of low affinity for albumin. The metabolism of PAF in human follicular fluid, peritoneal fluid and plasma was also investigated. PAF-acetylhydrolase activity was found in both peritoneal and follicular fluids which induced a time-dependent hydrolysis of [3H]PAF. The half-life of PAF was estimated to be 7-12 min in plasma, 15-25 min in peritoneal fluid and approximately 2 h in follicular fluid. PAF-acetylhydrolase activity in embryo culture media supplemented with 10% serum was markedly inhibited by addition of commercial serum albumin. When 25 g albumin l-1 was added, 22% of [3H]PAF was hydrolysed h-1 compared with 72% in media without albumin. The concentrations of lysophosphatidylcholine measured in plasma, in follicular and peritoneal fluids were 252, 286 and 53 mumol l-1, respectively. The distribution of these lysophospholipids and the metabolism of PAF in the female genital tract fluids reported in the present study provide evidence for the involvement of these biologically active lipid mediators in a variety of reproductive processes including sperm-egg interactions and embryonic development.

The properties and origin of platelet-activating factor acetylhydrolase- (PAF-AH)-like activity in the male reproductive tract were investigated. Seminal plasma (SP) and serum were obtained from normal donors and infertile patients while prostatic fluid (PF), seminal vesicle fluid (SVF) and vas deferens fluid were collected at autopsy. PAF-AH-like activity was found in all fluids tested. The specific activity of the enzyme in SP and PF was twice that of PAF-AH in serum and 15-fold higher than that in SVF and vas deferens fluid. In SP, PAF-AH-like activity was Ca(++)-independent, acid and heat labile, stable to freezing, not inhibited by phosphatidylcholine, but was inhibited by 10 mM disopropylfluorophosphate (DFP) and 13 mM phenylmethylsulfonylfluoride (PMSF). These data indicate that the properties of the enzyme in SP are similar to those reported for PAF-AH in serum. The variation in specific activity of PAF-AH in reproductive tract fluids suggest that there are either activators of PAF-AH in SP or inhibitors in one or several of the other reproductive tract fluids.

Objectives: To determine the effect of platelet-activating factor (PAF), the PAF derivative lyso-PAF, and lysophosphatidylcholine on in vitro sperm motility and to determine the role of albumin in this interaction.Design: Washed human spermatozoa were exposed to a range of PAF, lyso-PAF, or lysophosphatidylcholine concentrations, supplemented with different albumin concentrations, and the effect on sperm motion was quantified with a computer-assisted motion analysis. The metabolism of these compounds by spermatozoa was also assessed.Setting: University research laboratory.Patients, Participants: Semen samples were obtained from donors and patients attending an infertility clinic.Interventions: Human spermatozoa were incubated with PAF, lyso-PAF, or lysophosphatidylcholine at 10(-11) to 6 X 10(-4) M, with 0% to 1.2% albumin, and motility was evaluated at different time periods from 5 to 240 minutes. Tritiated PAF, lyso-PAF, or lysophosphatidylcholine was incubated with spermatozoa, and the metabolites were separated and quantified by thin-layer chromatography (TLC).Main Outcome Measures: Sperm motion characteristics, including the percentage of motile spermatozoa, velocity, and linearity, and sperm viability were determined. The metabolism of PAF, lyso-PAF, and lysophosphatidylcholine by spermatozoa was also studied.Results: Fifty micromolar of PAF and 100 muM lyso-PAF, supplemented with 0.3% albumin, increased sperm linear velocity by 41% +/- 5% (+/- SEM) and 44% +/- 5% and curvilinear velocity by 17% +/- 3% and 21 +/- 3%, respectively. Lysophosphatidylcholine had a similar effect but only at 22-degrees-C and not 37-degrees-C. In the absence of albumin, neither PAF, lyso-PAF, or lysophosphatidylcholine induced increases in sperm motion. Lysophosphatidylcholine and lyso-PAF are not detectably metabolized by spermatozoa, whereas 12.5% +/- 1.2% of PAF is hydrolyzed to lyso-PAF in 1 hour.Conclusion: Platelet-activating factor, lyso-PAF, and lysophosphatidylcholine independently stimulate sperm linear and curvilinear velocity. This action requires albumin. These compounds may be of use in the treatment of asthenozoospermic males.

OBJECTIVE:To search for the presence of platelet-activating factor (PAF) acetylhydrolase activity in human seminal plasma.DESIGN:Experimental.SETTING:Reproductive laboratory in a university-affiliated hospital research center.PARTICIPANTS:Human male volunteers were selected on the basis of apparent normal health.RESULTS:Human seminal plasma contained significant levels of PAF-acetylhydrolase activity. Enzymatic hydrolysis of PAF displayed typical kinetics and was a calcium-independent process. Platelet-activating factor acetylhydrolase in seminal plasma was associated with a very high-density lipoprotein fraction. Enzymatic activity was independent of sperm count and motility.CONCLUSION:Human seminal plasma contains significant levels of PAF acetylhydrolase. This enzyme may be an important factor in the regulation of PAF concentration and activity.

We investigated the endocrine function of a patient with Klinefelter syndrome in which the extra chromosome was an isochromosome Xq. This man was azoospermic but with normal secondary sex characteristics; smallness of the testes was the only abnormal physical finding. High follicle-stimulating hormone (FSH, 70 mIU/mL) and moderately elevated luteinizing hormone (LH, 40 mIU/mL) were found; the FSH and LH response to LH-RH was exaggerated. Androgen and estrogen levels were normal. The insulin test (measure of glycemia, growth hormone, and corticol) and the test with TRH (measure of TSH and prolactin) gave normal results. We conclude that the presence of additional long arms of the X chromosome is sufficient to cause Klinefelter syndrome and that the presence of two extra Xq does not intensify the degree of androgenic insufficiency.

Using an antiserum raised against hamster oviductal zona pellucida, we observed specific immunogenic components of the reproductive tract on the zonae of oviductal eggs and in oviductal fluid. Results of immunohistochemical studies suggested that these oviductal components may originate from epithelial cells of the isthmus and, to a lesser extent, of the ampulla and fimbria. The oviductal immunogenic components have also been observed within the bursal cavity, which contains the ovary. These observations suggest that these oviductal components may play an important role in the first steps of the hamster reproductive process.

Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.

ABSTRACT: The presence of zona pellucida antibodies was sought in a group of ten women with infertility of unknown cause. The reaction of these sera with ova was compared to the reaction obtained with a control antizona pellucida serum produced by the immunization of a rabbit with 8,000 solubilized hamster zonae pellucidae. This antiserum reached a maximum titer of 1 in 256 as measured by an immunoprecipitation assay and of 1 in 1,024 as measured by indirect immunofluorescence. It is tissue‐specific but is not species‐specific.With the indirect immunofluorescence technique on ova obtained from different species, the sera of eight of the ten patients gave a positive reaction with at least one type of test ova: three patients gave a positive reaction with hamster ova, six with porcine ova, and two with monkey ova. One infertile woman presented a positive reaction on hamster ova and another on pig ova as detected with all three types of γ‐globulins. Two other patients presented a positive reaction with the anti‐IgG and ‐IgM on porcine ova. The titer of the reaction never rose above one in four in any of the positive cases.Adsorption of the sera with fresh erythrocytes from human, hamster, and porcine blood did not change the titer or the intensity of the fluorescent reaction on ova. This indicates that the positive reactions are not due to nonspecific heteroagglutinins. Adsorption of the sera with formalin‐fixed erythrocytes from human and porcine blood completely abolished the fluorescent reaction. However, the latter treatment, applied to a dihydrotestosterone antiserum and a β‐hCG antiserum also greatly diminished the binding of hormones to these antisera; fresh erythrocytes did not adsorb the antibodies in these two antisera. This demonstrates that formalin‐fixed erythrocytes are not suited for adsorption of sera before testing for specific autoantibodies to the zona pellucida in infertile women.None of the sera from the infertile women gave a positive reaction with human pre‐ovulatory or post‐ovulatory ova or with frozen sections of human ovary. None of the sera produced an inhibition of in vitro fertilization of hamster ova. On the contrary, the control zona pellucida antiserum completely blocked in vitro fertilization. Furthermore, in a group of pregnant women, 2 of 11 exhibited a positive reaction with hamster ova while 6 of 28 showed a positive reaction with porcine ova.These results suggest the presence of low titer zona pellucida antibodies in 8 of the 10 infertile women. The importance of these antibodies in their infertility remains to be established.

The origin of the zona pellucida in follicles at different stages of maturation was studied in the hamster and human by indirect immunofluorescence microscopy using a specific anti-hamster zona pellucida serum. The earliest detection of the zona pellucida material occurred in the ooplasm of oocytes in primordial follicles. In primary follicles, the fluorescence was localized at the periphery of the oocyte. In secondary and in mature follicles a deposition of fluorescent material was visualized on the inner and outer regions of the zona pellucida. Follicular cells, other than the oocyte, failed to exhibit a fluorescent reaction with the antizona pellucida serum. It is concluded that the oocyte is an important source of the antigenic material of the zona pellucida of hamster and human ova.