Anthony G. Sacco

Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: The ZPA, ZPB and ZPC gene families

Full length zona pellucida cDNAs from cat, dog and pig that are homologous to the ZP2/rc75 genes from mouse, human and rabbit, a full length zona pellucida cDNA from cat and a gene and full length cDNA from human that are homologous to the rc55/ZP3 alpha genes from rabbit and pig, and full length zona pellucida cDNAs from cat, cow, dog, pig and rabbit that are homologous to the ZP3 genes from mouse, hamster, human and marmoset have been cloned and characterized. The members of these gene families are herein referred to as ZPA, ZPB and ZPC genes to avoid the confusion that currently exists in the zona pellucida of nomenclature. This report is the first to describe the presence all three major zona pellucida genes within individual mammalian species. Within the ZPA, ZPB and ZPC gene families, the DNA and deduced amino acid sequences are highly homologous to each other, and are most homologous between members of the same order within the class mammalia. These results imply that all or most mammalian species express the ZPA, ZPB and ZPC proteins, which form the zona pellucida layer surrounding the oocyte.

Immunologic infertility: I. Cervical mucus antibodies and postcoital test

SAA and SIA were assayed in the sera of 172 infertile couples and in the CM of the women and 18 control subjects. Complete infertility evaluation was performed in all subjects except the controls. Results of these studies were correlated with PCTs. Sperm antibodies were detected in the CM of 25.6% of infertile women and in the sera of 12.7% of female and 6.4% of male subjects. No sperm antibodies were found in the CM of controls. Sperm antibodies were present exclusively in sera of 8.1% of women and in the CM of 20.9%. In only 4.7% both serum and CM contained antibodies. A significantly higher incidence of sperm antibodies was found in the sera of women and in CM and sera of all couples with unexplained infertility compared to those with explained infertility. The presence and the number of liver spermatozoa in CM collected for PCTs were found to be significantly related to the existence of SAA and SIA in CM and serum. Stepwise multiple regression analysis indicated that SAA or SIA antibodies in CM and SAA in female serum have a significant negative correlation with endocervical PCT, whereas sperm motility has a positive correlation. These factors, together, accounted for 24.5% of variance. These data indicate that the appearance of SAA and SIA in CM is not related to their presence or titer in blood serum and that the PCT is a reliable screening method when sperm antibodies are suspected to exist in CM and to a lesser extent in serum.

Association of sperm receptor activity with a purified pig zona antigen (PPZA)

Three types of data are presented which suggest that sperm receptor activity is associated with a 58,000 Mr acidic glycoprotein (PPZA) isolated from pig zonae: (1) boar sperm fail to bind to pig zonae pretreated with univalent antibody to PPZA; (2) boar sperm pretreated with PPZA are inhibited in binding to intact pig zonae; (3) binding studies utilizing boar sperm and radiolabeled PPZA indicate an inhibition of radiolabeled PPZA binding to sperm by non-labeled PPZA in a dose-related manner. Collectively these data suggest that, at least in the pig system, the contraceptive action of zona antibodies is accomplished by a specific immunologic reaction between antibody and the zona sperm receptor site rather than to a non-specific blockage of this receptor.

Anti-zona pellucida activity in human sera.

One hundred and twenty-five human sera from male and female fertile and infertile subjects were tested by fluorescent techniques against porcine and human zonae for autoantibodies to zona pellucida. A high incidence of positive sera was found in all groups examined, suggestive that all sera from females demonstrating such activity may not represent cases of autoimmunity to a specific zona component.

Effect of varying dosages and adjuvants on antibody response in squirrel monkeys (Saimiri sciureus) immunized with the porcine zona pellucida Mr = 55,000 glycoprotein (ZP3).

ABSTRACT: Using the porcine zona pellucida Mr = 55,000 (ZP3) glycoprotein as immunogen, the effect of varying ZP3 dosages (50 μg, 25 μg, 5 μg) or adjuvants (200 μg ZP3 with either Freunds, Al[OH]3, or a muramyl dipeptide analog [MDP] on antibody response was evaluated in female squirrel monkeys. Although the dosage range investigated did result in dose‐dependent titration profiles, antisera from all dosage groups demonstrated in vitro contraceptive potential. MDP was as effective an adjuvant as Freunds in eliciting high titers of antibodies demonstrating contraceptive potential. Al(OH)3 was not an effective adjuvant in this system. Fertility data, utilizing those monkeys in the dosage study, suggested that an RIA titer >40% binding was necessary to prevent pregnancy. We conclude that in the squirrel monkey system, a 50 μg dosage of ZP3 should be adequate for inhibiting fertility and that MDP would serve as a satisfactory alternative adjuvant for Freunds.

Follicular fluid concentrations of thiopental and thiamylal during laparoscopy for oocyte retrieval

Because access into ovarian tissue of drugs used during anesthesia may be potentially harmful to the oocyte and/or follicular structure, we measured concentrations of thiopental (n = 15) and thiamylal (n = 9) in follicular fluid (FF) aspirates of 24 patients who underwent laparoscopic oocyte retrieval. In both groups, measurable amounts of the respective drug were found in all FF aspirates. Within individual patients, plasma concentrations of both drugs declined during the period of sampling between initial and final follicular aspiration. The mean plasma drug concentration was 7.99 +/- 3.97 micrograms/ml in the thiamylal group and 4.13 +/- 0.90 micrograms/ml in the thiopental group. Mean drug concentrations in FF were similar in both groups (thiopental 1.62 +/- 0.61 micrograms/ml; thiamylal 1.67 +/- 0.83 micrograms/ml). The mean FF/plasma concentration ratio during the sampling period was greater in the thiopental group (0.41 +/- 0.19) as compared with the thiamylal group (0.22 +/- 0.14). Several steps in the clinical management of these patients can be taken to reduce exposure of oocytes to drugs used during anesthesia.

Human spermatozoal FA-1 binds with ZP3 of porcine zona pellucida

Experiments were carried out to study the binding of the major porcine zona pellucida glycoprotein ZP3 (Mr = 55,000) with human FA-1, a fertilization-related antigen isolated from human male germ cell plasma membranes and with GA-1, a germ cell antigen isolated from murine male germ cell plasma membranes. FA-1 showed a dose-dependent binding with ZP3 in an enzyme-linked immunosorbent assay (ELISA) and by the Western blot procedure. FA-1 also bioneutralized the sperm ligand activity of ZP3 in a boar sperm/porcine zona pellucida attachment assay. In contrast, GA-1 displayed no reactivity for ZP3 using any of these procedures. These results indicate that FA-1 but not GA-1 has zona pellucida binding activity in the porcine system.

Proteins of Human Vaginal Fluid

Proteins of vaginal fluid from normal and hysterectomized women were identified and compared with those of cervical mucus. Albumin, alpha1-antitrypsin, alpha2-haptoglobin, alpha2-macroglobulin, beta-lipoprotein, orosomucoid, ceruloplasmin, gamma chains, gamma G. K. (Bence Jones), and immunoglobulins G, A, and M were present in both vaginal fluid and cervical mucus. Fibrin and C-reactive protein were not found in the vaginal fluid but were identified in cervical mucus. alpha2-Haptoglobin, alpha2-macroglobulin, beta-lipoprotein, orosomucoid, and immunoglobulin M were absent in the vaginal fluid of hysterectomized women, indicating that their presence in the vaginal secretion of normal women may be due to contamination from cervical mucus.

Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay

OBJECTIVE To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. DESIGN In vitro study. SETTING Academic medical center. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Plasmid relaxation. RESULT(S) Hams F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. CONCLUSION(S) In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Effects of oocyte exposure to local anesthetics on in vitro fertilization and embryo development in the mouse

The effect on fertilization and development of local anesthetics routinely used during ultrasound-guided oocyte retrieval in women undergoing in vitro fertilization was examined in a mouse in vitro fertilization system. Mouse oocytes were exposed in vitro to lidocaine, chloroprocaine, and bupivacaine at concentrations of 0 (control), 0.01, 0.1, 1.0, 10.0, 100.0 micrograms/mL for 30 min, washed, and then inseminated. In vitro oocyte fertilization at 24 and 48 h and embryo development at 72 h were determined. Bupivacaine adversely affected mouse in vitro fertilization and embryo development only at the highest exposure concentration, 100 micrograms/mL, while lidocaine and chloroprocaine produced adverse effects at concentrations as low as 1.0 and 0.1 microgram/mL, respectively. Furthermore, an adverse dose-related effect on fertilization and embryo development was shown for lidocaine and chloroprocaine, but not for bupivacaine. These data demonstrate that the local anesthetics, lidocaine (L), chloroprocaine (C), and bupivacaine (B), adversely affect mouse in vitro fertilization and embryo development in the order of C greater than L greater than B.