Fertilization in vitro of rhesus oocytes has been performed in order to provide a model for the study of primate embryonic development. Rhesus monkeys were superstimulated by daily injections

The bicarbonate: CO2 (HCO3-:CO2) concentration dependencies of hamster sperm motility, spontaneous acrosome reactions, and zona penetration (used to assay the zona-induced acrosome reaction) were examined. A cross-over experimental design was used to segregate effects on early stages of capacitation, spanning the first 5 h of incubation, from those on acrosome reactions and zona penetration during the last 1 h. After 5 h, HCO3-:CO2 concentrations were increased, decreased, or kept the same for 1 h. Compared to no HCO3-:CO2, as little as 2.9 mM: 0.6% HCO3-:CO2 increased the sperm motility index (MI) by 2.7-3.6 times. When HCO3-:CO2 was continuously present, both progressive and hyperactivated motility were stimulated by HCO3-:CO2 in a dose-dependent manner by 3-4 h, well before completion of capacitation. Stimulation of acrosome reactions or zona penetration, by addition of HCO3-:CO2 to sperm for 1 h late in capacitation, depended mainly on levels of HCO3-:CO2 present earlier in capacitation. When 25 mM: 5% HCO3-:CO2 was added only at 5 h, responses were significantly lower than with sperm treated continuously with the same concentration of HCO3-:CO2, being 2.5 times lower for MI, 2 times lower for acrosome reactions, and 6.3 times lower for zona penetration. In contrast, decreasing HCO3-:CO2 to suboptimal levels after 5 h did not decrease any 6-h sperm responses significantly. The average maximal and one-half maximal preincubation HCO3- concentrations for all responses were 34.2 +/- 1.0 and 9.2 +/- 0.3 mM, respectively. Zona penetration and hyperactivation were highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific aims of this study were to compare relationships between size of intact antral follicles and meiotic competence, diameters, and chromatin configurations of germinal vesicle (GV) oocytes in non-gonadotropin-stimulated rhesus monkeys. Intact antral follicles were dissected from excised ovaries of nine normally cycling monkeys in the follicular phase of the menstrual cycle and of two acyclic monkeys. Follicles were classified according to diameter: I (200-450 microns), II (451-700 microns), III (701-1000 microns) and IV (> 1000 microns). Cumulus-enclosed oocytes were released from follicles and either measured (diameter) and fixed immediately or cultured for 48 h in modified CMRL medium containing 0.5 micrograms/ml ovine FSH, 10 micrograms/ml ovine LH, and 10% bovine calf serum. Following Hoechst staining, three distinct patterns of chromatin organization (GV1-3) were identified in GV oocytes according to the degree of association with the nucleolar periphery (encapsulation or "rimming"). In antral follicles > 450 microns in diameter, perinucleolar encapsulation (GV3) of oocytes before culture and meiotic maturation (metaphase II) of oocytes after culture increased (p < 0.01) with antral follicle growth in a graded fashion. While 56.3% of oocytes from large (> 1000 microns) follicles completed maturation, few (9.3%) oocytes from small (200-450 microns) follicles were competent to mature in vitro. At 0 h of culture, class IV follicles contained a greater (p < 0.01) proportion of GV3 oocytes and a smaller (p < 0.01) proportion of GV1 oocytes than classes I, II, and III follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Previously, we found oviductal eggs to be significantly more penetrable and fertilizable in vitro than ovulated eggs collected from the ovarian bursa, while bursal eggs were comparable to mature (unovulated) follicular eggs. Incubation of follicular eggs with a soluble eluate of oviductal egg cumulus complexes (COF) increased sperm penetration: the activity was macromolecular, was destroyed at 56 degrees C, and was produced in the oviduct. We now report purification of this oviductal factor that enhances penetration of follicular eggs and have identified it as oviductin (OVN). Oviducts, 1-1.5 h post-LH from eCG-primed females, were homogenized and the cytosolic fraction was chromatographed on a Helix pomatia lectin affinity column; specific proteins were eluted with 0.2 M N-acetyl-D-galactosamine. Fractions were monitored by dot-blot assay using as the primary antibody monoclonal antibody (mAb) 1C4 against OVN. Proteins were resolved by one-dimensional SDS-gel electrophoresis, followed by electrotransfer and immunostaining of Western blots. OVN fractions were indexed to COF by quantitative dot-blot assay, and activity was bioassayed by penetration of follicular eggs within 1 h of coincubation with precapacitated sperm +/- factors: COF and BSA (high and low controls, respectively) and fractions from the lectin-isolated peak. The mean penetration rates for three isolations were 17 +/- 4.0a, 51.7 +/- 5.0b, and 49 +/- 2.7b% for BSA, COF, and column fractions, respectively (p < or = 0.05). Purified OVN bound to follicular zonae during culture. Acrosome-intact sperm heads bound OVN during 30 min of incubation both before (t = 0 h) and after capacitation (t = 5.5 h) (visualized by indirect immunofluorescence).(ABSTRACT TRUNCATED AT 250 WORDS)