Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo.

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 +/- 0.011 mM and 129.3 +/- 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 +/- 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 +/- 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture.

Responses of gametes to the oviductal environment.

Spermatozoa and oocytes are separately formed in highly specialized biological compartments (testes and epididymis, ovarian follicle), then deposited communally in the oviduct in an environment designed to facilitate their final maturation, promote their union, and nurture the resultant zygotes and early embryos. The mammalian oviduct undergoes hormonally-mediated cyclical modifications that climax during the periovulatory period thus ensuring production of the specialized environment required for the gametes. While the number of potential bioeffectors present in the oviduct is immense, there are four classes of modulators that are of particular note for the 'capacitation' or maturation of both the spermatozoa and the eggs or early embryos: beta-amino acids, bicarbonate ion, progesterone, and oviductins. Only the oviductins are unique to the oviduct, while the other three are present either at higher concentrations than in other tissues or arrive within the oviductal milieu coordinately with the gametes. For spermatozoa, beta-amino acids, bicarbonate ion, and progesterone work interactively to promote motility, capacitation, and the acrosome reaction, while oviductins facilitate capacitation and species-specific zona pellucida recognition and adhesion. For embryonic development, progesterone works indirectly by promoting a permissive oviductal environment, bicarbonate ion is required for cleavage, and beta-amino acids, acting as organic osmolytes, membrane stabilizers, and/or antioxidants, are facilitatory. Oviductins adhere to the zona pellucida of the ovulated egg thereby increasing sperm adhesion and speed of sperm penetration. Oviductins in the perivitelline space or endocytosed by the pre-implantation embryo may regulate differentiation during the morula to blastocyst transition. The roles of these mediators and their mechanisms of action for the gametes and early embryos are reviewed and discussed.

Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst.

The behavior of golden hamster blastocysts was studied in vitro by continuous time-lapse videomicrography and computer imaging, during and immediately following escape from the zona pellucida. This study revealed numerous small cytoplasmic trophectoderm projections (TEPs) approximately 18 microns long that penetrated the zona pellucida both radically and tangentially and appeared to be actively involved in zona escape in vitro. After escape from their zonae, some blastocysts moved across the culture dish by an endogenous means of locomotion, most likely involving activity of the small TEPs. Several hours after zona escape, embryos expressed large TEPs up to 46 microns long that moved in an undulating manner and showed rapid cycles of extension and retraction; the timing of their appearance suggested that these TEPs are normally involved in attachment to the uterine epithelium. Embryos fixed in utero, during the developmental interval between zona loss and embryo attachment, exhibited large TEPs similar in morphology to those expressed by cultured blastocysts. These observations document for the first time that mammalian blastocysts are capable of endogenous locomotion, confirm TEPs as components of normal blastocyst activity, reveal that there are two kinds of TEPs that differ temporally and morphologically, and extend earlier reports of TEP activity in guinea-pig embryos to the hamster.

Progesterone concentrations in serum, follicular fluid, and oviductal fluid of the golden hamster during the periovulatory period.

In vitro, progesterone induces capacitation, hyperactivated motility, and acrosome reactions in sperm, possibly acting through a non-genomic plasma membrane receptor, but neither progesterone functions nor concentration within the mammalian oviduct during the periovulatory period is well characterized. The objectives of this study were to determine the physiological concentrations of progesterone in serum, follicular fluid, and oviductal fluid of golden hamsters during the period of the reproductive cycle in which capacitation and fertilization occur. Fluids were collected from different groups of animals for each of four time points: the normal time for mating (5-6 h post-LH surge [post-LH]), early in capacitation (8-9 h post-LH), immediately preovulation (11-12 h post-LH), and after ovulation near the beginning of fertilization (14-15 h post-LH). Oviductal fluid was collected by 1-h cannulation followed by cardiac puncture for serum collection and follicle aspiration for follicular fluid. Within each time period, the three types of fluid differed significantly in progesterone concentration. Over time, concentrations of progesterone did not change in either serum (range: 5.64-12.85 ng/ml) or follicular fluid (range: 4.2-7.4 micrograms/ml), but the concentration of progesterone in oviductal fluid decreased from 175.06 ng/ml at the first period to 44.01 ng/ml at the fourth (p < 0.05), while the volume of oviductal secretions collected by the same sampling procedure increased from 1.6 to 2.9 microliters (p < 0.05). With this information concerning in vivo concentrations of progesterone during capacitation and fertilization, the physiological role of progesterone in sperm-egg interactions can be addressed.

Effects of progesterone on in vitro sperm capacitation and egg penetration in the golden hamster.

Progesterone (P) is postulated to have a physiological role in vivo during sperm capacitation and/or during sperm-egg interaction as a cofactor for induction of the acrosome reaction. Effects on in vitro fertilization were tested by adding P (0.01-2 micrograms/ml) to sperm during capacitation (4-6 h) or after capacitation during sperm-egg interaction for 1 h. Additionally, to test for acceleration of the onset of capacitation by P, eggs were inseminated with sperm that were incubated with P for 3.5 and 5 h. Neither the solvent dimethyl sulfoxide nor P affected the motility or vigor of treated sperm. The effects of P on egg penetration were dependent on the dose, the temporal sequence of its addition to sperm (i.e., before or after capacitation), and the duration of sperm exposure. Capacitation of sperm with 20 ng/ml P for 5 h, but not 3.5 h, increased the percentage of eggs penetrated by sperm over controls (52.65 +/- 4.01% vs. 39.30 +/- 5.18%, p < 0.05). But P (0.01-2 micrograms/ml), either added after capacitation (4.5 h) during sperm-egg coincubation (1 h) only, or present (1 micrograms/ml) throughout capacitation (4-6 h) and sperm-egg coincubation (1 h), did not increase egg penetration over control levels. In conclusion, the best results occurred with a protocol that mimicked in vivo conditions of capacitation time (5 h) and preovulatory oviductal levels of P (20 ng/ml).

Identification of a sperm penetration factor in the oviduct of the golden hamster.

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)

Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction-inducing factor of the ovulated egg-cumulus complex.

Three sources of hamster periovulatory fluids (+/- heat inactivation at 56 degrees C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (+/- heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zona-bound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as evidenced by dissolution times in beta-mercaptoethanol (beta-MEOH), were not "harder" than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: 1) the oviductal lectin-binding antigen (ZPO or oviductin), 2) an additional heat-labile component, and 3) the heat-stable ARIF for free-swimming sperm.

Effects of gonadotropins upon the incidence and kinetics of meiotic maturation of macaque oocytes in vitro.

The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 micrograms/ml human follicle-stimulating hormone (hFSH), (2) 10 micrograms/ml human luteinizing hormone (hLH), (3) 1.0 microgram/ml hFSH and 10 micrograms/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P > 0.05) among treatments. The time to GVB was accelerated (P < 0.05) by gonadotropins (hFSH: 10.8 +/- 1.7 hr; hLH: 10.1 +/- 1.8 hr; both gonadotropins: 8.8 +/- 1.1 hr) when compared to controls (17.4 +/- 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P > 0.05) among treatments (hFSH: 32.3 +/- 1.2 hr; hLH: 35.1 +/- 1.4 hr; both gonadotropins: 35.2 +/- 1.3 hr; no gonadotropins: 34.1 +/- 1.2 hr). The mean interval to extrusion of PB1 was 34.1 +/- 0.6 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in morphology, sperm penetration and fertilization of ovulated hamster eggs induced by oviductal exposure.

In the human, mature eggs at the pre-ovulatory follicular stage placed into the oviduct via gamete intra-Fallopian transfer (GIFT) establish more implantations and pregnancies than do those for standard in-vitro fertilization and embryo transfer (IVF). Previous studies in the hamster have shown that mature follicular eggs are less readily penetrated by spermatozoa than oviductal eggs. This study examines whether ovulation or pre-fertilization exposure to the oviduct per se affects sperm penetration and fertilization of mature ova. Three types of eggs were used: pre-ovulatory, follicular [12 h post-human chorionic gonadotrophin (HCG), 1-1.5 h pre-ovulation], and ovulated (bursal and oviductal, both 15 +/- 0.5 h post-HCG). Bursal eggs were obtained by ligating the infundibulum on one or both sides of the tract. The morphological changes in eggs due to oviductal exposure were quantified using computerized image analysis. Cumulus-free follicular and bursal eggs were significantly less penetrated than oviductal eggs 1 h post-insemination (36, 39 and 62%, respectively). Cumulus-intact oviductal compared to bursal eggs, paired within females, were fertilized at a significantly higher rate (4 h post-insemination; 89 and 58%, respectively). Fresh oviductal and bursal eggs had equivalent cell diameters (79 microns) and zona thickness (15-15.8 microns), but oviductal compared with bursal eggs had larger zonae (119 and 116 microns, respectively) and perivitelline volumes (107 and 47 pl). Oviductal, but not bursal, zonae had the oviductal glycoprotein, oviductin, bound to them. We conclude that prefertilization oviductal exposure and not ovulation or time post-HCG alters the morphology and fertilizability of eggs.

Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys.

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)

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.

Bicarbonate: carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reactions.

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)

Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey.

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.

Addition of hypotaurine can reactivate immotile golden hamster spermatozoa.

Hamster epididymal spermatozoa became virtually immotile following washing and dilution in chemically defined medium (TLP-PVA). The sperm motility factors (penicillamine, hypotaurine, and epinephrine: PHE) were examined for their ability to reactivate immotile sperm. Sperm could be reactivated by addition of PHE at 1 h of incubation. Hypotaurine alone was capable of reactivating sperm motility, but epinephrine and penicillamine together were not. However, overall sperm motility and percentage of motile sperm during incubation were higher when PHE components were used in combination than when hypotaurine was used alone. Addition of hypotaurine to immotile sperm suspensions could be postponed for up to 6 h with subsequent recovery of sperm motility, although the degree of recovery of motility declined progressively with each hour that addition of hypotaurine was delayed. The rescuing effect of hypotaurine was due to an increase both in the percentage of motile sperm and in the quality (grade) of sperm motility. The data show that hypotaurine is required for expression of sperm motility in the hamster, and support the concept that the loss of hypotaurine from sperm following washing and dilution is responsible for the sperm-immobilizing effect of these procedures. Additionally, the data demonstrate that hamster sperm can remain viable for several hours after becoming immotile, and that many of the immotile sperm are capable of being reactivated.

A quantitative assay for capacitation: evaluation of multiple sperm penetration through the zona pellucida of salt-stored hamster eggs.

The endpoint for sperm capacitation occurs when spermatozoa become able to penetrate intact zonae pellucidae of unfertilized homologous eggs. Activation of eggs stimulated by sperm fusion or by gamete aging initiates changes in zonae pellucidae that bar further sperm entry. This zona block mechanism reduces the usefulness of eggs as indicators for sperm capacitation. Egg storage in concentrated salt solutions destroys the zona block mechanism while retaining the biological sperm receptor/activator functions of the zona pellucida [Yanagimachi et al., Fertil Steril 31:562-574, 1979]. We have developed techniques for the quantitative assay of capacitation using multiple sperm penetration into zonae and have evaluated the sperm-response characteristics of hamster eggs stored in a modified salt solution (STOR). Sperm, preincubated in capacitating or noncapacitating treatments (CAP or NOCAP, respectively), were coincubated with unfertilized and fertilized STOR-treated zonae for 4 hr. Then zonae were stripped of externally bound sperm and the sperm heads in the perivitelline space (PVS) were fluorescently labeled with Hoechst dye 33342. Entry of CAP sperm into the PVS of STOR-treated unfertilized eggs was highly correlated with sperm concentration (Rw = .859) and was log linear from 1 X 10(3)-2 X 10(5) CAP sperm/ml. At 2 X 10(5) CAP sperm/ml, the mean number of PVS sperm was 63.5 and the maximum observed was 158. NOCAP sperm very rarely penetrated unfertilized zonae (2 sperm into 75 eggs). Few CAP sperm entered the PVS of STOR-treated fertilized eggs (two sperm into 54 eggs at 2 X 10(5) sperm/ml) unless the sperm concentration was raised to high levels. Differences between replicates were due to male but not female (ie, egg) differences. We conclude that 1) STOR-treated unfertilized zonae can be used to accurately quantitate differences between sperm capacitation and/or fertility states and 2) the availability of large numbers of homogeneous "shelf-stable" zonae will make it feasible to perform hamster sperm capacitation bioassays on a large scale.

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators.

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.

Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer.

The birth of a rhesus monkey resulting from in vitro fertilization is reported. Oocytes recovered at laparoscopy from five gonadotropin-stimulated donors were inseminated in vitro with sperm preincubated with caffeine and dibutyryl cyclic AMP. After insemination, oocytes were cultured for 33-46 hr. Twenty-two embryos were transferred nonsurgically into 11 recipient females. One recipient showed signs of implantation but did not carry to term. A second female became pregnant after receiving one 4-cell and one 6-cell embryo fertilized in vitro. The subsequent course of early pregnancy and embryonic and fetal development were characteristic of normal singleton pregnancies. A healthy term male infant was delivered by Cesarean section 176 days after fertilization. This birth has validated our procedures for in vitro fertilization of rhesus monkey gametes and provides an experimental model for studies of early embryonic development in primates.