William C. Buhi

Secreted proteins of the oviduct.

During late follicular development and estrus, the mammalian oviduct undergoes specific physiological and biochemical modifications which contribute to an optimization of the microenvironment for fertilization and early cleavage-stage embryonic development. These changes appear to be hormonally regulated by ovarian steroids, most importantly, estrogen. The hundreds of macromolecules found within the oviductal lumen are contributed by selective serum transudation and active biosynthesis and secretion from nonciliated epithelial cells. Recent studies have indicated temporal and regional (infundibulum, ampulla and isthmus) differences in steady-state levels of specific mRNAs and in de novo protein synthesis and secretion by the oviduct. One protein synthesized de novo, the estrogen-dependent oviductal secretory glycoprotein (OSP), has been shown to be unique to the oviduct and is conserved across a number of mammalian species. This protein associates with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos. OSPs have been shown to enhance sperm binding and penetration in oocytes and may regulate development in early preimplantation embryos. Other regulatory molecules, protease inhibitors, growth factors, cytokines, binding proteins, enzymes and immunoglobulins have been identified in the oviductal microenvironment. The identification and potential roles for oviduct-secreted proteins will be reviewed and discussed. Current research focuses on continued identification and characterization of specific oviductal proteins and a determination of the molecular basis of their interactions with the oocyte, sperm or embryo.

Effects of the Porcine Oviduct-Specific Glycoprotein on Fertilization, Polyspermy, and Embryonic Development In Vitro

Abstract This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0–100 μg/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0–100 μg/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0–100 μg/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0–50 μg/ml had no effect on sperm penetration rates; however, compared with the control, 100 μg/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10–100 μg/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24–29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19–34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.

Oviduct-Specific Glycoprotein Modulates Sperm-Zona Binding and Improves Efficiency of Porcine Fertilization In Vitro

Abstract Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 μg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 μg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 μg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

Respiratory distress syndrome with mature lecithin/sphingomyelin ratios: Diabetes mellitus and low Apgar scores

A detailed study was made of 150 women delivered of their infants within 72 hours of an amniocentesis where the lecithin/sphingomyelin (L/S) ratio was 2.0 or greater. There were nine neonates with respiratory distress (6.0 per cent). There were two neonatal deaths, both due to severe congenital heart disease. A mature amniotic fluid L/S ratio predicts a newborn infant who will not have respiratory distress syndrome (RDS) in most pregnancies. There is a significantly increased risk of RDS in neonates with a mature L/S ratio if the mother has insulin-dependent diabetes or if there is a resulting low Apgar score. The method of delivery (cesarean section or vaginal) does not affect the frequency of RDS where the L/S ratio is 2.0 or more.

Effects of blood or meconium on the determination of the amniotic fluid lecithin/sphingomyelin ratio

Increasing quantities of maternal and fetal serum were added to amniotic fluids with orignal high or low lecithin/sphingomyelin (L/S) ratios. It was found that serum contains an L/S ratio of approximately 1.31 to 1.46 and therefore its addition lowered high L/S ratios and raised low L/S ratios. The addition of meconium to amniotic fluid decreased the L/S ratio.

The effects of norgestrel on carbohydrate and lipid metabolism over one year.

Carbohydrate and lipid metabolism were studied with the use of a three-hour oral glucose tolerance test in 71 women before and after one year of daily oral norgestrel, 0.075 mg., treatment. There was no significant change in weight or fasting plasma triglyceride and cholesterol levels. There was a significant elevation of both the blood glucose and plasma insulin levels after one year of treatment. This was true for both the fasting and the glucose-stimulated values. Whereas all of the individual glucose tolerance curves at the pretreatment control test were normal by selection, 15.5 per cent of the curves were borderline abnormal to abnormal at the one-year test. The significance of these metabolic alterations is discussed.

Fetal movements and ultrasound: Effect of maternal intravenous glucose administration

Fetal movements were directly visualized with a real-time ultrasound unit, and they were recorded after the random intravenous administration of either glucose or saline solution to the mother. A significant increase in fetal activity was seen 1 hour after the administration of glucose.

Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils

Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1–0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.

The acute effects of ritodrine infusion on maternal metabolism: Measurements of levels of glucose, insulin, glucagon, triglycerides, cholesterol, placental lactogen, and chorionic gonadotropin

Twenty-nine women in premature labor were randomly assigned to a ritodrine (N = 14) or placebo (N = 15) treatment group. Thirteen serial blood samples were drawn during the first 12 hours of therapy by intravenous drug infusion and they were analyzed for a variety of metabolic substances. There was a significant increase in the blood glucose level in the ritodrine group after one hour and this persisted for the 12 hours of intravenous drug treatment. Plasma insulin levels similarly did not increase in the placebo but significantly rose in the ritodrine group by 30 minutes, peaked at 2 1/2 hours, and remained elevated throughout the infusion. There were no significant differences between levels of plasma glucagon, cholesterol triglyceride, human placental lactogen, or human chorionic gonadotropin in the two treatment groups. Ritodrine caused significant maternal and fetal tachycardia. Its use in women with carbohydrate abnormalities should be monitored carefully. The increased glucose levels may lead to an increased fetal weight.

Pituitary and ovarian responsiveness to a graded gonadotropin releasing factor stimulation test in women using a low-estrogen or a regular type of oral contraceptive☆

47 volunteer women were investigated to assess hypothalamic-pititary-ovarian functions under different types of contraception. 20 women were on mechanical contraception; 17 were on SOC (standard oral contraception) with 50 mg of estrogen; 10 were on LEOC (low-estrogen oral contraceptive) with 35 mg of ethnyl estradiol. Measurement of basal blood levels of LH (luteinizing hormone), FSH (follicle stimulating hormone), prolactin, estradiol progesterone, testosterone, and dihydrotestosterone were made. Lower concentrations were found in the SOC group. In addition, intravenous gonadotropin-releasing factor (GNRF) was administered in increasing quantities, and the same blood analyses repeated. The SOC group had a significantly suppressed response for both LHand FSH. The LEOC group had no suppression of FSH response, and LH response was suppressed only after maximal GNRF stimulation. These results suggest that pituitary gonadotropin suppression is dose-related, and that the low-estrogen type of oral contraceptives have potentially fewer adverse effects.