Dynamics of a human seminal vesicle specific protein.

The present paper is concerned with the temporal alterations and tissue localization of a seminal antigen secreted by the human seminal vesicle. This antigen is recognized by antibody MHS-5, which is one of a set produced in mice by immunization with human sperm. The respective clone produced an antibody of the IgG1 subtype, which reacted with seminal fluid from over 400 normal donors and 21 semen samples from vasectomized men. Incubation of seminal vesicle secretion with either prostatic fluid or prostate specific antigen (PSA) resulted in degradation on the antigen. The experiments showed that MHS-5 antigen is a substrate for the serine protease PSA: Immunohistochemical studies suggested that MHS-5 is a "sperm-coating" antigen and is exclusively synthesized and secreted by the seminal vesicle.

Electron microscopic immunolocalization of seminal vesicle-specific antigen in human seminal vesicle.

Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.

The influence of vasovasostomy on antisperm antibodies in rats.

Serum antisperm antibodies were studied in Sprague-Dawley rats after vasectomy and vasovasostomy. Animals received a bilateral vasectomy, a vasectomy followed 3 mo later by vasovasostomy, or sham operations. Blood samples were obtained at 1, 3, 4, and 7 mo, and antisperm antibodies were assayed by an enzyme-linked immunosorbent assay. After vasectomy reversal was performed at 3 mo, antisperm antibodies were significantly higher in rats in the vasovasostomy group at 4 mo than in animals that had a persisting vasectomy or sham operations. At 7 mo, the antisperm antibody level for the vasovasostomy group was approximately double that for the vasectomized rats. Spermatic granulomas occurred in 76% of rats after vasovasostomy. Antisperm antibody levels were higher in vasovasostomized animals with granulomas than in those lacking granulomas. The results suggest that vasovasostomy may stimulate an antibody response to sperm rather than lead to a reduced response, as was anticipated upon removal of the obstruction. Spermatic granulomas may serve as sires for continued antigenic challenge. The observed increase in antisperm antibodies after vasovasostomy in Sprague-Dawley rats may be related to their relatively low immunologic responsiveness to vasectomy, with vasovasostomy serving as a second major immunologic challenge, aided by the formation of an additional granuloma. In the more responsive Lewis strain, we previously observed a rise in antisperm antibodies after the initial vasectomy, with no further increase after vasovasostomy.

Testicular alterations are linked to the presence of elevated antisperm antibodies in Sprague-Dawley rats after vasectomy and vasovasostomy.

The relationship between alterations in testicular histology and antisperm antibodies was studied after vasectomy and vasovasostomy in Sprague-Dawley rats, which are immunologically relatively non-responsive to vasectomy. Testes were prepared for histologic study at intervals up to seven months after vasectomy, vasectomy followed three months later by vasovasostomy, or sham operations. Antisperm antibodies were assessed with an ELISA. Testicular alterations, which were observed in a minority of animals after vasovasostomy, consisted mainly of depletion of germ cells. Mean serum antisperm antibody levels were greater for animals with altered testes than for rats with normal testicular histology. In addition, the proportion of rats that showed a positive antisperm antibody response was greater among animals with testicular changes than among those with unaltered testes. When the present results on Sprague-Dawley rats were compared with previous findings on the highly responsive Lewis strain, it was evident that the incidence of testicular changes and the proportion of positive antibody responders were greater in the Lewis strain. However, elevated antisperm antibodies and testicular alterations appeared to be more tightly linked in the less responsive Sprague-Dawley rats.

Effects of granuloma formation at site of vasovasostomy.

Spermatic granulomas forming after vasectomy reversal have been thought to mechanically compromise the anastomosis. We have studied the physiologic effects of vasectomy and vasovasostomy in rats. Following delayed microsurgical vasovasostomy, fluid flow through the anastomosis and cauda epididymidal hydrostatic pressure are not significantly different in tracts that form, or do not form, sperm granulomas at the anastomosis. Given a properly performed microsurgical vasectomy reversal, a sperm granuloma arising from a small leak does not appear to mechanically compromise the anastomosis in the rat. Fertility after vasovasostomy is not significantly reduced in rats with granulomas.

The relation between antisperm antibodies and testicular alterations after vasectomy and vasovasostomy in Lewis rats.

The relationship between antisperm antibodies as determined by enzyme-linked immunosorbent assay (ELISA) and the occurrence of alterations in testicular weight and histology was studied following vasectomy in Lewis rats. The effects of vasovasostomy on antisperm antibody levels were also examined. At 1, 3, and 4 months after vasectomy, the mean absorbance values in an ELISA for sera from animals with altered testes was significantly greater than that from animals lacking testicular alterations. However, animals showing positive antisperm antibody responses were represented both in the group with testicular alterations and among those that lacked testicular damage. Levels of antisperm antibody in both vasectomy and vasovasostomy groups significantly exceeded that for sham-operated animals, but the level of antisperm antibodies in vasovasostomized animals with positive responses was similar to vasectomized animals one and four months after reanastomosis. It is suggested that persistence of antisperm antibodies or testicular alterations, or both, may play roles in limiting the restoration of fertility after vasovasostomy.

Temporal appearance of antisperm autoantibodies in Lewis rats following vasectomy.

An indirect enzyme-linked immunosorbent assay (ELISA) was employed to monitor antisperm autoantibodies in 16 Lewis rats for up to 36 weeks following vasectomy. This assay was capable of discriminating all prevasectomy from postvasectomy sera at a 1:16 dilution. Weekly serum samples were obtained for the first 13 weeks and bimonthly samples thereafter. Half of the animals developed a positive antisperm autoantibody response by the end of the first postoperative week. By the end of the second week, 81% of the animals had positive responses. The greatest proportion (88%) of animals having a positive response over the course of the study was found at the end of the seventh postoperative week and the highest mean absorbance value for all 16 animals was observed at this time. Only 25% of the animals had positive responses for antisperm autoantibody at the end of the 35th week of the study. These findings indicate that circulating antisperm autoantibodies arise in the Lewis rat earlier than has been generally appreciated. The time course is similar to that of antibody titers to infectious agents or arising from inoculation of rats with spermatozoa. These findings on autoantibody levels in the Lewis rat are compared with the dynamics of antisperm autoantibody formation in man.

The influence of vasovasostomy on testicular alterations after vasectomy in Lewis rats.

The occurrence of alterations in testicular weight and morphology after vasectomy and vasectomy reversal by vasovasostomy was studied in Lewis rats. Animals were studied 3, 4, and 7 months after bilateral vasectomy or a vasectomy followed 3 months later by vasovasostomy. Other rats served as sham-operated controls. The weights of the testes in vasectomy and vasovasostomy animals fell into two groups-small testes weighing less than 0.88 g and normal-sized testes of 1.2 g or more. When the extent of testicular alterations was estimated in sections for light microscopy by use of a semiquantitative testicular biopsy score count (TBSC), the morphology of the testes corresponded closely to the testis weight (r = .94), small testes having correspondingly low TBSC scores. In severely altered small testes, the seminiferous tubules were narrower than in sham-operated rats, and numbers of germ cells were greatly depleted. Many tubules contained only Sertoli cells and spermatogonia, although spermatocytes were present in a minority of tubules. A few seminiferous tubules contained multinucleate spermatids. Electron microscopy of severely altered tubules revealed closely apposed processes of Sertoli cells, which contained filaments, microtubules, and endoplasmic reticulum. In contrast, testes with normal weight in vasectomy and vasovasostomy groups resembled those of the sham-operated animals. Comparison of distributions of testicular biopsy score counts demonstrated differences between vasectomy and vasovasostomy groups as time after operation increased. At the 3-4-month intervals, approximately one-third of the testes were severely altered in both vasectomy and vasovasostomy groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The incidence of spermatic granulomas and their relation to testis weight after vasectomy and vasovasostomy in Lewis rats.

The occurrence of spermatic granulomas of the vas deferens was studied in Lewis rats at intervals up to 7 months after vasectomy or vasectomy followed 3 months later by vasovasostomy. The incidence of granuloma progressed with time to involve one or both tracts in 100% of vasectomized rats. In addition, the majority of animals developed new granulomas after vasovasostomy, even though fluid flow through the reconnected vas deferens was demonstrated in vitro. When individual tracts were analyzed, the weight of the testis was related to ipsilateral spermatic granuloma formation in both vasectomy and vasovasostomy groups at 3 and 4 months after initial operation. Testes were small in the absence of a granuloma but similar to those of sham-operated rats if a granuloma was present. The possible protective effect of spermatic granuloma formation on the testis is discussed.

An enzyme-linked immunosorbent assay for measuring antisperm autoantibodies following vasectomy in Lewis rats.

An indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm auto-antibodies in the Lewis rat following vasectomy. The assay system was validated by employing prevasectomy sera and postvasectomy antisera, previously demonstrated to contain antisperm antibodies by indirect immunofluorescence. A standardized ELISA protocol was developed employing 10(5) sperm per microtiter plate well and sucrose-polyvinylpyrrolidone as a postcoat stabilizer solution. The ELISA was shown to yield significant detectable antibody at dilutions of 1/512 or greater in the most reactive sera. A standard for scoring positive titers was adopted: 1.96 standard deviations above the mean of the preimmune value. Using the criterion, 88% of 7-week postvasectomy samples could be discriminated from preimmune samples at a 1:16 dilution, which was adopted for subsequent assays. The ELISA demonstrated 73% and 91% reproducibility for an intraassay analysis of single prevasectomy and postvasectomy serum samples (7 weeks postvasectomy) tested in 160 determinations on a standard sperm pool. When this single antigen pool was employed in 35 determinations at 0, 1, and 4 weeks in an interassay study, 56% and 70% reproducibility was found for pre- and postvasectomy sera respectively. A correlation (r = 0.75) was made between a single absorbance value and the endpoint titer of the same sera, which indicated that single absorbance values could be used to predict serum titer and single dilutions could be used for general screening of a large number of samples. The ELISA described provides a rapid, sensitive, and reliable method that discriminated between samples taken before and after vasectomy.

Type-A retrovirus particles in heterohybridomas: precaution for human monoclonal antibody purification.

Fine structural features of the murine myeloma MHFP-1 and two heterohybridomas secreting human IgM monoclonal antibody were examined. Intracisternal type-A retrovirus particles were found in both MHFP-1 and the heterohydribomas constructed by fusing MHFP-1 and human peripheral blood lymphocytes. The implications of this finding for the purification of human monoclonal antibody for therapeutic applications is discussed.