Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.     

Solubilization of porcine zonae pellucidae by trypsin and pronase

The porcine zona pellucida was dissolved with difficulty by trypsin in isotonic solution, whereas it was efficiently dissolved by pronase. A structural change of the zona was induced in hypotonic solution, resulting in acceleration of dissolution by these proteases. The solubilization rate of three families (PZP1-3) of zona protein by both enzymes was analyzed by HPLC. In hypotonic solution, PZP1 was solubilized first, followed by PZP2; and PZP3 was then finally released. In isotonic solution, PZP1 and PZP2 were also solubilized faster than PZP3, which was almost completely resistant to trypsin, showing that the solubilization of the zona depended on that of PZP3. Noticeably, high molecular weight products were produced as the proteolytic hydrolysis proceeded in PBS. Circular dichroic spectra and electrophoretograms of the tryptic hydrolysates showed that the zona may have a regular supramolecular structure.     

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Inhibition of fertility in mice by passive immunization with antibodies to isolated zonae pellucidae.

A chromatographically purified preparation of gamma globulin produced against isolated zonae pellucidae of mice was used to immunize mice. A single 2.5 mg dose totally inhibited fertility for a minimum of 11 days.     

Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zonae pellucidae

Cynomolgus monkeys (Macaca fascicularis) were immunized with porcine zonae pellucidae to assess the possible antifertility effects of the zona antibodies. Serum antibody titers were evaluated utilizing a rapid solid-phase radioimmunoassay. Six of twelve monkeys conceived 6 to 10 wk after vaccination. All monkeys reached maximal antiserum titers by the time of conception, although the six animals that did not conceive had considerably lower antibody titers. Further pregnancies did not occur until antibody level had declined markedly, 8 mo after last immunization. The menses of all but one of the remaining six monkeys were interrupted intermittently. Also, the usual midcycle elevated estradiol levels were absent for several cycles. Both menses and midcycle estradiol peaks were reestablished in all but one monkey 3 to 5 mo after the last booster was given. Two monkeys conceived when serum antibody levels dropped to one fourth of maximal, but both had a still birth. Histological observations showed accumulation of luteal tissue and massive atresia of small follicles at the end of the study (18 mo). We conclude that through heteroimmunization with porcine zona pellucida monkeys can become infertile and that this condition is reversible. Because the zona preparation used in this study appeared to contain traces of nonzona material, it was not possible to determine whether the menstrual irregularities and oocyte atresia that we observed were owing to immunological effects on the zona itself or to the production of antibodies against other ovarian components.     

Production of monoclonal antibodies to antigens of specific mouse T-suppressors

F1(MSU X WAG) rats were immunized with anti B6 BALB/c specific suppressor T cells (SSTC), purified by absorption/elution technique, with the following fusion of splenocytes to NS-I myeloma cell line. Hybrids were screened for their ability to affect SSTC, cytotoxic T lymphocytes (CTL) and producers of macrophage migration inhibition factor (MIF-producers) all triggered by in vivo priming with allogeneic cells. Two hybridoma cell lines--C1 and C4 inactivated SSTC by approximately 50%, leaving CTL and MIF-producers intact. C4 were also active in vivo, if injected as ascitic fluid from nu/nu mice, though to a lesser extent than in vitro.     

Fractionation of glycoproteins from porcine zonae pellucidae into three families by high-performance liquid chromatography

The glycoproteins of porcine zonae pellucidae were fractionated into three families (PZP1-3) by high-performance liquid chromatography on a gel filtration column. Their molecular weights were estimated to be 164K for PZP1, 99K for PZP2, and 55K for PZP3; and they were present in an approximate weight ratio of protein moiety of 2:3:18, respectively. Differences in carbohydrate composition position between them were found, although their amino acid compositions were similar to each other. CD spectra showed that the three families had an almost identical secondary structure of the helical form in the presence of SDS. In PBS without SDS, they had different secondary structures, predominantly in the beta-form, and probably different tertiary structures.     

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Use of monoclonal antibodies to follow the phylogenic distribution of human renal antigens

Nine human differentiation antigens have been defined by monoclonal antibodies (M. Abs) developed from mice immunized with embryonic kidney cells (mesonephros or metanephros of 7 week-developmental ages). Their spatial and temporal distributions during human kidney organization were previously studied [3]. In this paper we have attempted to follow by immunofluorescence their phylogenic location, from fish to mammals. Six of them recognized the same structures as in humans: proximal convoluted tubules (PCT) (EG9.11, EG19.6, E116.1), glomerular basement membrane (GBM) (EG14.1) and extracellular matrix (EK8.1, EK17.1). However, staining was limited to certain mammals. EK17.1 has been characterized as an anti-fibronectin. These antibodies revealed the same histological structures in the human mesonephros and metanephros. The three other antibodies revealed epitopes appearing earlier in evolution and whose histological distribution varied according to species. These antibodies stained different structures in the mesonephros and metanephros. Thus, the staining particularities observed during human renal ontogenesis were found again in the phylogenetical study.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Use of specific polyclonal antibodies to detect heterogeneous lipases from Geotrichum candidum.

Geotrichum candidum CMICC 335426 was previously shown to produce two lipases termed lipase A and lipase B, lipase B being highly specific for hydrolysis of esters of cis-delta 9 fatty acids. We now describe the isolation of polyclonal antibodies specific for lipase A and lipase B. These antibodies were used in Western blotting techniques to detect the appearance of the lipases during the course of the fermentation of G. candidum CMICC 335426. A and B were found to be produced simultaneously in the extracellular medium at the start of the growth phase. The two lipases were always present at similar levels in the medium. The specific antibodies were then used to detect the presence of A- and B-like lipases in crude lipase samples from other strains of G. candidum. The lipases were found at different levels in all these samples, and the specificities of the crude lipases varied significantly from one strain to another. Differences in specificity could therefore be explained by different levels of specific (B-type) and non-specific (A-type) lipases in the medium. This was verified by purifying A- and B-type lipases from the G. candidum strain ATCC 34614.     

Removal of swine zonae pellucidae with sequential exposures to pronase and acidified medium

Exposure to acidified PBS (pH 3) for 60 sec removed swine zonae pellucidae from 70.4% of 27 swine morulae, and 73.7% of these formed blastocysts in culture. Further investigations revealed that treating embryos with 0.5% pronase and acidified PBS (pH 3) for 30 sec each was more effective. Zonae were removed in 90.6% of 85 embryos (four-cell to morulae) treated. A total of 76.9% of 65 zona-free embryos and 81.6% of 38 untreated embryos formed blastocysts (P > 0.05). An additional 57 untreated and 49 zona-free embryos (morulae to blastocysts) were transferred to seven recipient sows. Four sows returned to estrus (18 to 27 days), but three others were pregnant when slaughtered at 38 to 42 days. One pregnant sow had received a combination of five zona-free and six untreated embryos and demonstrated the potential for further development of treated embryos in vivo.