Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro.

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl2 and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl2 revealed that plasma membrane was removed only from the acrosomal region and remained predominantly intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl2 lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg2+, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca2+ (5 mM) to the capacitated spermatozoa induced approximately 78 +/- 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 +/- 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg2+, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.

Isolation of a stable apical segment of the guinea pig sperm acrosome.

The large apical segments of guinea pig sperm acrosomes were mechanically separated from the spermatozoa and subsequently isolated by density gradient centrifugation. The isolated acrosomal caps were very stable and maintained their crescent morphology when suspended in sucrose-based medium buffered at pH 5.6, with or without the acrosin inhibitor p-aminobenzamidine (pAB). Examination under the electron microscope showed that the acrosomal caps were free of plasma membrane and were bound by an outer acrosomal membrane which was discontinuous. Enzymatic analysis after lysis of the caps indicated that acrosin and hyaluronidase were present with high specific activity, while only a trace amount of acid phosphatase activity and no arylsulphatase, phospholipase A2, or phospholipase C activities were present. Significant particulate acrosin activity, but only trace amounts of soluble acrosin activity, could be detected in the isolated acrosomal caps if assayed immediately after isolation in the absence of pAB. However, soluble acrosin activity of high specific activity was obtained after the acrosomal caps were extracted by 10% glycerol buffered at low pH (pH 3.0). The new procedures provide a means to isolate and purify guinea pig sperm apical acrosomal segments rapidly.

Pregnancy from in vitro fertilization of human eggs after separation of motile spermatozoa by density gradient centrifugation.

The spermatozoa of infertile men, previously found to have poor fertilizing capacity in an in vitro system, were fractionated on discontinuous gradients of Percoll (Pharmacia Laboratories, Piscataway, NJ) and were used for insemination of human oocytes in an in vitro fertilization and embryo replacement program. Three of the five patients in the study had established ongoing pregnancies, including one patient with a triplet pregnancy from four transferred embryos. However, the other two patients continued to have poor fertilizing capacity with their husband's Percoll-separated spermatozoa.

The immunoglobulin class of antispermatozoal antibodies in serum.

The aim of this study was to determine the immunoglobulin class of circulating antisperm antibody using a technique called the indirect immunobead test (IBT). In the IBT sperm bound antibodies are detected using polyacrylamide beads coated with rabbit antihuman immunoglobulin classes IgG, IgA, and IgM. Of the 20 infertile men with serum immobilizins, 100% were found to be positive for sperm-bound IgG, 50% positive for IgA, and 0% positive for IgM, using the IBT. Similarly, 20 infertile females with serum immobilizins showed 95% positivity for IgG, 60% for IgA, and 15% for IgM. Thus there was a good correspondence between the presence of serum immobilizins as determined by the sperm immobilization test (SIT) and the IBT. This study provides data that indicates that IgG and IgA are the two major immunoglobulin classes of sperm antibody in male and female immune sera as detected by a simple, sensitive immunological technique, the serum IBT.

Detection of antispermatozoal antibodies of IgA class in cervical mucus.

A simple procedure for detection of antisperm antibodies of IgA class in human cervical mucus is described and the results of its application to samples from 102 patients are presented. The results suggest that the IgA immunobead test (IgA-IBT) is a specific and clinically useful test for sperm antibodies. There was a strong correlation between the IgA-IBT and the presence of complement-dependent sperm immobilization in serum (Spearman's, r = 0.92, p less than 0.001). Positive IgA-IBT results occurred only in mucus samples that showed poor penetration by normal sperm. An added advantage of the IgA-IBT is that both the immunoglobulin class and the site of binding to the sperm surface can be determined simultaneously.