FGF-2 dependent angiogenesis is a latent phenotype in basic fibroblast growth factor transgenic mice.

Overexpression of basic fibroblast growth factor (FGF-2) in transgenic (TgFGF2) mice results in a chondrodysplasia as the principle phenotype. Here we report a second phenotype in TgFGF2 mice that was previously undetected: A predisposition to angiogenic reactions with subsequent amplified angiogenesis that are both FGF-2 dependent. We used subcutaneous injection of extracellular matrix as an angiogenic assay. The matrix formed vascularized cysts in the TgFGF2 group after seven days, whereas the non-transgenic (NTg) group developed avascular cysts. Cysts from the TgFGF2 group contained 3-7X more hemoglobin (Hb), two-fold more vonWillebrand Factor (VWF), and 150X more FGF-2 than cysts from the NTg control group. Significant angiogenic reactions occurred only in the TgFGF2 group that express FGF-2 from the transgene. The TgFGF2 mice, therefore, constitute a unique experimental system to study FGF-2 dependent angiogenesis because they have no spontaneous or inherent vascular defects, but provision of an angiogenic substrate results in an amplified angiogenic response. In addition, we report development of an ELISA for VWF that provides a sensitive, quantitative assay for angiogenesis.

The juxtamembrane, cytosolic region of the epidermal growth factor receptor is involved in association with alpha-subunit of Gs.

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha. However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha. However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.

Activation of Gsalpha by the epidermal growth factor receptor involves phosphorylation.

Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving Gsalpha and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B. (1990) J. Biol. Chem. 265, 21317-21322; Nair, B. G., and Patel, T. B. (1993) Biochem. Pharmacol. 46, 1239-1245). Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates Gsalpha and activates this protein. Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha. As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation. However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha. In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha. Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase.

Localization of epidermal growth factor receptors in first- and third-trimester human placentas.

Studies to date have demonstrated epidermal growth factor (EGF) receptors primarily on the outer plasma membrane of the human placental syncytiotrophoblasts facing maternal blood and to a lesser extent on the cytotrophoblast stem cells. In the present studies, first- and third-trimester human placental tissues were immunostained with monoclonal antibodies (MAb) to the EGF binding domain of the human EGF receptor or to the activated (tyrosine-phosphorylated) human EGF receptor. Cytotrophoblasts, syncytiotrophoblasts, and fetal connective tissue cells in first-trimester tissues immunostained with both MAb, with the notable exception of the absence of staining of activated EGF receptor over cytotrophoblast plasma membranes. In contrast, staining of third-trimester placentas with either MAb yielded little to no staining of either trophoblast cell layer but intense staining of fetal connective tissue cells. Staining for EGF receptors over cytotrophoblasts in the first trimester is consistent with the hypothesis that maternal EGF or TGF-alpha derived from the endometrium or placenta may be the mitogen responsible for cytotrophoblast cell division and that the receptors localized to the syncytiotrophoblast are involved in EGF regulation of differentiated function. The absence of heavy staining of activated EGF receptor on trophoblast plasma membranes in third-trimester placentas is consistent with down-regulation of EGF receptor activity.

Ontogeny of the epidermal growth factor receptor during development of the fetal bovine mesonephros and associated organs of the urogenital tract.

A primary cell culture system was developed to study the epidermal growth factor (EGF) receptor in the fetal bovine mesonephros and its urogenital derivatives. Radioreceptor assays demonstrated EGF binding as early as Day 37 in mesonephric cells and in cells derived from the fetal reproductive ducts, gonads, and metanephros--all mesonephric derivatives. Immunocytochemical studies revealed that EGF receptors were localized in the ductal and tubular epithelium of these urogenital organs. EGF induced DNA synthesis and tyrosine phosphorylation in the bovine mesonephric cells, suggesting that EGF receptors detected in these cells were functional. In addition, transforming growth factor alpha, the putative fetal ligand for the EGF receptor, was found to specifically compete for EGF binding to the receptor, as well as to induce DNA synthesis in a manner similar to that of EGF. Estrogen did not regulate EGF receptors or specifically bind to bovine mesonephric cells. The development of estrogen receptors in the bovine species occurs markedly later than that of the EGF receptor, in contrast to the mouse, where both receptors are observed at very early stages of reproductive tract development.

Vasectomy, serum assays, and coronary heart disease symptoms and risk factors.

We compared three serum assays (two antisperm antibody assays and one assay for circulating immune complexes) and a number of CHD-related variables in 69 vasectomized (V) and 126 non-vasectomized (NV) participants in the Portland Center for the Multiple Risk Factor Intervention Trial. Significant differences between the V and NV men were found in sperm agglutination (SA) and sperm immobilization (SI) titers, as well as in several CHD risk factors, symptoms, and treatments; men in the V group had higher titers for SA and SI, smoked more, and had lower diastolic and systolic blood pressure than men in the NV group. Differences between V and NV in SA and SI activity remained even after we controlled for any effects that CHD risk factors, symptoms, and treatments may have had on the serum assays. Antibody development tended to decrease with age-at-vasectomy and increase with time-post-vasectomy. In the case of SA the antibodies clearly increased with time-post-vasectomy.

PIP: A comparative study of 69 vasectomized and 126 nonvasectomized men enrolled in the Portland (Oregon, US) Center for the Multiple Risk Factor Intervention Trial evaluated vasectomy as a risk factor for cardiovascular disease. In animal studies, atherosclerosis development has been linked to circulating anti-sperm antibodies and immune complexes formed in response to sperm breakdown products released in the body after vasectomy. Vasectomized men smoked more and had lower diastolic and systolic blood pressure than men in the control group. As expected, both sperm immobilization and sperm agglutination assays were significantly higher among vasectomized men than controls; 29.4% of vasectomized men compared with only 2.5% of nonvasectomized men had sperm immobilization values of 0.3 or less, while 54.1% of vasectomized men compared with 12.5% of nonvasectomized men had sperm agglutination values of 20.0 or above. These significant differences persisted even when a variety of coronary heart disease risk factors and treatments were controlled. Multivariate analysis showed that antibody development tended to decrease with age at vasectomy and increase with time since vasectomy. In the case of sperm agglutination, the antibodies clearly increased with time since vasectomy.

Effect of fucoidin on human sperm-zona pellucida interactions.

The authors recently reported that fucoidin (a polymer of predominantly L-fucose sulfate) produced a strong, significant, and dose-dependent inhibition of sperm-zona binding under hemizona assay conditions. The current studies were designed to evaluate the mechanisms underlying this inhibitory activity. Using computerized semen analysis, the monoclonal anti-sperm antibody T-6 and indirect immunofluorescence technique, and the fura-2 indicator, no significant impact of fucoidin on sperm motion parameters, on the spontaneous acrosome reaction, or its prerequisite, the increase in calcium influx, were observed. Subsequently, a mild acid hydrolysis of the fucoidin molecule was performed, followed by sizing hydrolysates in a Biogel P2 column and separating fractions (n = 6). All fucoidin fragments significantly inhibited tight binding of human sperm to human zona pellucida under hemizona assay conditions (range, 56%-94%) when tested individually. These results provide further evidence that the effect of fucoidin is produced by a receptor-ligand association.

Hemizona assay (HZA) demonstrates effects of characterized mouse antihuman sperm antibodies on sperm zona binding.

Characterized antihuman sperm monoclonal antibodies from mice were evaluated using the hemizona assay (HZA) to determine whether sperm:zona binding was effected. The seven monoclonal antibodies were characterized using human sperm in agglutination, immobilization, and penetration assays. Semen was provided by four fertile men and used in the HZA to determine if the presence of a monoclonal antibody would affect tight binding of the sperm to the zona pellucida. Pre-incubation of MA-14 for 1 h with the sperm induced a 33-54% reduction of the number of tightly bound sperm. This antibody reacts to an antigen located on the acrosome and midpiece. Experiments in which there was no pre-incubation of the antibody with sperm, resulted in no significant reduction in the number of sperm bound in the HZA. These findings suggest that an anti-human sperm antibody produced in mice can modulate sperm:zona binding. Reduction in zona binding could indicate a cause of immune-related infertility and this test may be useful in selecting an antigen for contraceptive vaccine development.

Evaluation of human sperm-zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens.

Characterized WHO monoclonal antibodies (MAbs) to human sperm antigens were evaluated as to whether they inhibited sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the 26 MAbs tested, only one inhibited zona binding. The whole sperm-specific MAb inhibited zona binding by 70%. The MAb also caused strong agglutination. Two procedures, Sephadex column chromatography and papain digestion, were used to determine whether agglutination or steric hindrance was a factor in the capability of MAb to inhibit zona binding. However, inhibition remained comparable to previous results. The MAb did not prevent capacitation, nor calcium influx and the resulting increase in hyperactivated motility and acrosome reaction. Since its inhibitory influence is not due to agglutination factors, steric hindrance or prevention of normal pre-fertilization maturation, the MAb may be blocking a portion of the zona binding receptor and may be useful in elucidating sperm antigens important to sperm-egg interaction. The approach used in this study allows definition of sperm surface antigens involved in zona pellucida binding.

PIP: This paper reports on a study in which 26 monoclonal antibodies (MAbs) to human sperm were evaluated for their inhibition characteristics in preventing sperm-zona pellucida tight binding. Evaluation was performed using the hemizona assay (HZA) technique. Of the 26 MAbs tested, only 1 (3.9%) inhibited sperm binding to the zona pellucida. The whole sperm-specific MAb did reduce zona binding by 70%. The MAb also caused strong agglutination, but did not prevent capacitation nor calcium influx. Data were statistically evaluated using either the Student's t-test, the Chi-square analysis, or the two-way analysis of variance. Two-way analysis of variance showed a significant decrease in sperm-zona tight binding in the presence of the MAb. Motion parameters of sperm exposed to the MAb were not significantly different from untreated controls.

Studies on the in vitro spermicidal activity of synthetic magainins.

Two synthetic magainins A and G are shown to have spermicidal activity. Transmission electron microscopic micrographs show that both magainins alter the plasma membranes of sperm and that these actions are rapid. Further studies will better delineate the contraceptive potential of synthetic magainins.

PIP: 2 synthetic magainins--A and G-- have been shown, through transmission electron microscopy, to have spermicidal activity. Magainins, a class of peptides isolated from the skin of the African clawed frog. I have been demonstrated to have wide spectrum in vitro antimicrobial activity. In this study, semen sample collected from 7 healthy volunteers were diluted with magainins A and G and then examined for spermiostatic activity. Sperm diluted only with saline were used as a control. Sperm assays indicated the potency of magainin A to be significantly greater than that of magainin G. Magainin A demonstrated spermiostatic activity at concentrations of 0.024-0.095 mg/mL compared to concentrations of 0.095-0.380 mg/mL for magainin G. A similar pattern was identified in the concentration of peptide required to inhibit sperm motility. Magainin A inhibited sperm motility when diluted in seminal plasma at concentrations of 0.77-1.54 mg/mL, while 1.54-3.08 mg/mL of magainin G were required to produce this effect. In the transmission electron microscopic studies, magainin-treated sperm cells incubated with either peptide consistently demonstrated denudation of the outer plasma membrane and partial disappearance of the acrosome, while sperm incubated in saline remained unaltered. It is hypothesized that magainins exert their spermicidal activity by disrupting the outer plasma membrane. The contraceptive potential of synthetic magainins should be explored through animal studies that measure the in vivo effects of seminal fluid and vaginal secretions on magainin activity and the effects of these agents on vaginal and cervical mucosa.

Antisperm antibodies to sperm surface antigens in women with genital tract infection.

Antisperm antibodies to sperm surface antigens in nulligravid women with primary upper genital tract infections were measured by the sperm mixed agglutination reaction assay. As many as 56% of women with a primary episode of pelvic inflammatory disease had antisperm antibodies. In addition, 69% of those women with no history of genital tract infection but with laparoscopic evidence of past pelvic infection had significant levels of circulating antisperm antibodies. Electroimmunoblots of sperm preparations probed with the sera of women who had either known or presumed upper genital tract infection revealed a uniformly recognized 69 kd antigen. In contrast, women with circulating antisperm antibodies before primary upper genital tract infection recognized up to five distinct sperm antigen determinants of 27, 54, 131, 146, and 174 kd. It is a distinct possibility that genital tract infections may lead to immunopotentiation of antisperm antibodies that could affect fertility.

Sperm preparation affects reactivity of monoclonal antibodies.

The polyacrylamide gel electrophoresis (SDS-PAGE) patterns of extracts prepared from thrice-washed human sperm were compared with extracts of swim-up and non-rise sperm. For sperm extraction sodium deoxycholate (DOC), octylphenoxypolyethoxyethanol (NP-40), dithiothreitol (DTT), lithium 3,5-diiodosalicylate (LIS), or 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) were used. A pellet of pooled thrice-washed semen was divided and extracted with these detergents. Similar gel electrophoresis profiles, as revealed by silver staining, were observed with the LIS, DOC, NP-40 and CHAPS extracts, whereas DTT extracts lacked the 150 and 200 kDa bands. Different thrice-washed semen pools varied in their protein pattern, probably due to the presence of sloughed germinal cells, white blood cells and sloughed cells from the reproductive tract. Swim-up sperm extracts had a different protein profile from that of thrice-washed pools but pools of swim-up sperm had a more constant protein pattern than those of thrice-washed semen pellets. Monoclonal antisperm antibodies did not always recognize antigens of the same molecular weights in western blots when extracts of swim-up sperm versus thrice-washed sperm were compared. In immunoblot studies with monoclonals (produced against thrice-washed sperm, cloned five times, reactive by ELISA and sperm function tests), 4/12 reacted with 0.3% NP-40 extracts of thrice-washed sperm and 5/12 with 0.3% NP-40 extract of swim-up sperm. These findings suggest that definition of antigens by monoclonal antisperm antibodies will be influenced by the nature of sperm extract employed.

Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody.

A monoclonal antibody, T-6, useful for detecting acrosome-reacted sperm based on an immunofluorescent assay, was employed to evaluate acrosomal status of human sperm that were tightly bound to hemisected human zonae pellucidae (hemizona assay). Over 90% of the bound sperm evaluated exhibited immunofluorescent patterns indicative of acrosome reaction. This staining method for evaluating the acrosomal status of sperm bound to the zona pellucida may enable definition of a group of male infertility patients heretofore not recognized.

Serum hormone levels affect sperm function.

If hormone levels affect sperm motility during capacitation, then the serum added to samples prepared for artificial insemination could affect sperm fertilizability. We investigated various sperm functional movement characteristics (percent motile, progressive velocity, linearity, beat cross frequency, lateral head displacement, longevity, and hyperactivation) in specimens incubated with women's sera, as well as exogenous hormone preparations of estradiol (E2) and progesterone (P). Early follicular phase serum (low E2 and low P) maintained motility and longevity. Sperm in E2-treated medium exhibited higher progressive velocity, linear motility, and longevity. In contrast, the percent motility decreased as sperm exhibited hyperactivated motility with P.

Characteristics of monoclonal antibodies reactive with human sperm and seminal plasma.

The characteristics of monoclonal antibodies developed against human spermatozoa are described. Out of 10 monoclonal antibodies 9 did not react in ELISA with human RBC, WBC, platelets, Raji cells nor mouse sperm. Four monoclonal antibodies reacted with monkey sperm and all 10 reacted with human seminal plasma. Monoclonal antibodies showed differential reactivity with pre- and post-capacitated sperm. Four monoclonal antibodies were able to agglutinate sperm whereas none of these were positive in sperm-immobilization assay. Interestingly, two monoclonal antibodies (MA-46 and MA-50) were able to block the attachment of pre-capacitated sperm to zona denuded hamster oocytes. MA-46 and MA-50 recognized in immunoblot spermatozoa antigens having apparent molecular weights of 14 and 20 K Da and greater than 200 K Da respectively. The monoclonal antibodies reported in this study will be useful in further delineating the spermatozoa antigens involved in regulation of fertility.

Production of large amounts of mouse polyclonal antisera.

A simple and inexpensive method for producing larger volumes of mouse polyclonal antisera is described. Groups of BALB/c mice were immunized with different human sperm antigens classified according to molecular weight. Mice were immunized twice with the antigen, twice pristane pretreated, and then injected with a non-antibody-secreting myeloma cell line to induce ascites formation. Antibody activity of the ascites fluid approximated that of the serum. Thus, one mouse can provide about six times the amount of antibody-containing fluid usually obtained after conventional immunization. This method also may be applicable for ascites fluid production in nude mice.

Distribution of T cell subsets in follicular fluid.

Examination of follicular fluid (FF) from in vitro fertilization patients revealed a significant difference in concentrations of lymphocytes and T cell subpopulations with increased oocyte maturation. A total of 111 follicles containing 82 oocytes were aspirated from 10 patients undergoing laparoscopic oocyte retrieval. FF from 61.3% of the follicles was classified as clear and 38.7% as bloody, based on gross and microscopic appearance. A mean of 1.78 X 10(6) lymphocytes/ml was obtained from peripheral blood (PB) as compared to 2.14 X 10(5) and 2.79 X 10(5) lymphocytes/ml for clear and bloody FF, respectively. There were 6.3 X 10(5) T4 and 3.7 X 10(5) T8 lymphocytes in PB, resulting in a T4/T8 ratio of 1.72, which is not significantly different from that of the general population. The mean concentration of FF T4 and T8 lymphocytes decreased with increased oocyte maturation; the T8 reduction was statistically significant (P less than 0.05). The proportion of T4 to T8 lymphocytes in FF remained unchanged and was unaffected by maturity of the oocyte. Although estradiol (E2) did not vary with oocyte maturity, progesterone (P) increased and E2/P decreased. There was no correlation between E2 or P levels and distribution of T cells. Fertilization rates were higher in more mature oocytes, but there was no correlation between fertilization and E2, P, E2/P, or T cell subpopulations. It remains to be determined what factors result in the decrease in lymphocytes with increased oocyte maturity and the observed difference in FF T4/T8 compared to PB.

Rectal infusion of semen results in transient elevation of blood prostaglandins.

Repeated semen deposition in the gut may be linked to the development of viral infections in homosexual men. Other investigators have suggested that rectal insemination may diminish immune responsiveness. We approximated conditions of human insemination by infusing 2 ml of pooled human seminal plasma (SP) into the rectum and/or vagina of rhesus monkeys. This resulted in increased blood plasma concentrations of the bicycloderivative of prostaglandin E (PGEM-II) which reached peak concentrations 2 h after rectal SP instillation in seven of eight test monkeys, but not the controls. The rate of PGE2 diffusion appeared to occur more rapidly across vaginal than rectal mucosa. Suppression of peripheral cellular immune functions was not demonstrated after the single exposure of this study, although persistent and repeat exposures could lead to local or generalized suppression of host defense mechanisms. Absorption of PGE's from the gut may be a cofactor in the development of sexually transmitted viral diseases.

In vitro immune absorption of antisperm antibodies with immunobead-rise, immunomagnetic, and immunocolumn separation techniques.

Fourteen men with a mean duration of infertility greater than 3 years who had significant sperm immobilizing or sperm-agglutinating antibodies were studied. All patients had greater than 20% IgG or IgA immunobinding to sperm in their seminal plasma and 7 had immunobinding levels of greater than 50%. Sperm from these men were less able to penetrate an overlaying buffer layer than sperm from a fertile control. Addition of immunobeads to the specimen was of little use, because few motile sperm could swim into the overlaying buffer; retained immunobeads were noted in the buffer layer of 18-hour capacitated specimens. Magnetic isolation of antibody-coated sperm from antibody-free sperm avoids potential damage to fragile sperm through centrifugation. Viable spermatozoa were isolated from magnetite-complexed spermatozoa, but the motility of the isolated spermatozoa deteriorated rapidly during the subsequent capacitation period. Passage of diluted ejaculate through a column of dextran beads for antisperm antibody processing (ASAP) was associated with superior sperm quality and fertilizing potential. The use of ASAP resulted in good sperm velocity and linearity and improved sperm function, as measured with the hamster egg penetration test. Sperm from men with immunologically mediated infertility can be processed through the ASAP and used for artificial insemination of their partners or in an in vitro fertilization program.