Effect of pre-freezing conditions on semen cryopreservation in rhesus monkeys.

Although sperm cryopreservation has been studied in at least 17 non-human primate species, systematic factor optimization for any single species is lacking. Gene banking of non-human primate sperm is still in its infancy. The objective of the present study was to initiate a systematic approach to optimize the process of sperm cryopreservation for rhesus macaques, specifically, factors related to pre-freezing conditions (e.g., straw freezing position, sperm concentration, sperm washing, equilibration methods, and equilibration time periods). Straw position had no effect on post-thaw motility (P=0.193). Sperm concentration was tested in a range from 5 x 10(6)mL(-1) to 5 x 10(8)mL(-1); post-thaw motility of sperm samples frozen at 5 x 10(7)cell mL(-1) (51.0+/-10.6%; mean+/-S.D.) and 5 x 10(8)cell mL(-1) (48.1+/-7.3%) were higher than samples frozen at 5 x 10(6)cells mL(-1) (33.0+/-12.0%, P=0.003). Comparison of motility immediately after thawing between samples with (51.2+/-6.2%) and without washing (53.9+/-6.8%) revealed no differences (P>0.05). However, washing improved sperm forward progression within 1h after thawing, whereas unwashed sperm retained higher post-thaw motility and progression during extended incubation (4h) after thawing (P<0.05). Equilibration methods (with or without pre-cooling) made no difference on post-thaw motility (P>0.05), and the most effective equilibration time was the duration required for samples to acclimate to 4 degrees C prior to freezing. Evaluation and optimization of these pre-freezing conditions will help to minimize sources of injury, maximize survival, and contribute to the development of an optimized cryopreservation protocol for rhesus macaque sperm.

Adenylate cyclase in the primate corpus luteum during chorionic gonadotropin treatment simulating early pregnancy: homologous versus heterologous desensitization.

Stimulation of the primate corpus luteum by endogenous CG in early pregnancy or by exogenous CG in simulated conditions is transient despite continued exposure to this luteotropic hormone. The transitory response to CG is not due to the down-regulation of gonadotropin receptors. The current studies were designed to determine if the transient response involves a postreceptor lesion at the membrane level, i.e. the loss of CG receptor activation of adenylate cyclase. Nonpregnant female rhesus monkeys received increasing doses of hCG for up to 10 days beginning near the typical time of implantation (9 days post-LH surge) to simulate early pregnancy. Corpora lutea were removed at specific intervals after the onset of hCG treatment, luteal homogenates were prepared, and adenylate cyclase activity was assessed by the conversion of [alpha-32P]ATP to [32P] cAMP. Basal activity of adenylate cyclase was unchanged throughout the in vivo hCG treatment interval. Nonhormonal activators, such as forskolin (100 microM) and 5'-guanylylimidodiphosphate (50 microM) stimulated (P less than 0.05) adenylate cyclase to a similar extent (greater than 10-fold the control level) throughout hCG treatment. On day 0, both gonadotropins (hCG and human LH; 250 nM) and prostaglandins (PGE2 and PGI2; 500 nM) stimulated cAMP production (approximately 3-fold the control level; P less than 0.05). The responses of adenylate cyclase to PGE2 and PGI2 did not diminish throughout the in vivo hCG treatment. In contrast, exposure to hCG for 3 days reduced the sensitivity of adenylate cyclase to gonadotropin. Moreover, adenylate cyclase in luteal tissue after 6-10 days of treatment was insensitive to hCG. The loss of gonadotropin sensitivity of adenylate cyclase by 6 days of hCG treatment correlated with the decline in circulating progesterone levels. These results demonstrate that 1) the gonadotropin-responsive adenylate cyclase of the macaque corpus luteum is also stimulated by paracrine factors, notably PGs of the E and I series; and 2) CG exposure stimulating early pregnancy conditions leads to homologous, not heterologous, desensitization of the adenylate cyclase system. We hypothesize that homologous desensitization of the adenylate cyclase system is an important mechanism leading to the transient response of the primate corpus luteum to CG in early pregnancy.

Molecular target of endocrine disruption in human luteinizing granulosa cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin: inhibition of estradiol secretion due to decreased 17alpha-hydroxylase/17,20-lyase cytochrome P450 expression.

Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.

Chronic exposure of the developing corpus luteum in monkeys to chorionic gonadotropin: persistent progesterone production despite desensitization of adenylate cyclase.

The transient steroidogenic response of the macaque corpus luteum to chronic human CG (hCG) treatment beginning on days 9-10 of the luteal phase (i.e. stimulated early pregnancy) is associated with decreased numbers and affinity of available receptors for gonadotropin and homologous desensitization of adenylate cyclase. This study determined if similar changes in the receptor-adenylate cyclase system accompany the persistent steroidogenic response which occurs when hCG treatment begins earlier in the luteal phase. Female rhesus monkeys received increasing doses of hCG (15 up to 5760 LU) twice daily beginning 5-6 days after the midcycle LH surge. The levels of circulating progesterone increased (P less than 0.05) within 24 h of initial hCG exposure and did not decrease throughout the 10-day regimen. The corpus luteum was removed after 0 (n = 8), 6 (n = 4), or 10 (n = 4) days of hCG treatment. Whereas the numbers of available [125I]hCG binding sites in luteal particulates remained unchanged by 10 days of hCG exposure, the dissociation constant (Kd) for gonadotropin binding was greater than at day 0 (6.17 +/- 1.41 vs. 0.91 +/- 0.06 X 10(-10) M, P less than 0.05). Since the number of binding sites occupied by injected hCG increased with treatment (7.81 +/- 1.55 fmol/mg wet wt at day 10), the total number (available + occupied) of gonadotropin receptors was 3-fold greater (P less than 0.05) at day 10 than at day 0. Adenylate cyclase activity in luteal homogenates, assessed by conversion of [alpha-32P]ATP to [32P]cAMP, was stimulated on day 0 by hCG (2.7 +/- 0.7 X control, at 250 nM hCG), prostaglandin E2 (2.5 + 0.5 X control, at 0.5 mM), and prostaglandin I2 (2.3 +/- 0.5 X control at 0.5 mM) as well as forskolin (100 microM) and 5'-guanylyl-imidodiphosphate (50 microM). In contrast, cAMP production by day 6 of treatment was insensitive to hCG, but remained responsive to prostaglandin E2, prostaglandin I2, and nonhormonal activators. We conclude that CG treatment in the early luteal phase did not prevent the development of gonadotropin receptors to levels typically observed in the functional corpus luteum of the menstrual cycle. Also, many changes in the gonadotropin receptor-adenylate cyclase system in macaque luteal tissue were similar after CG treatment beginning on days 5-6 or days 9-10 of the luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Simulation of human luteal endocrine function with granulosa lutein cell culture.

Human granulosa cells collected from in vitro fertilization have previously been cultured to provide a system to simulate the granulosa lutein cells of the corpus luteum. In most of these systems, the cultures have been relatively short term, and attempts to simulate the normal pattern of hormone production observed during the luteal phase of the cycle have not been reported. Additionally, the hormone relaxin has generally been absent from the endocrine analysis of these systems. In this report, methods were used that supported secretion of ovarian steroids and relaxin that mimics the profiles of these hormones in vivo. This system was used to observe the endocrine responses of the granulosa lutein cells to three different protocols of CG administration designed to mimic the normal luteal phase, early pregnancy, and early pregnancy followed by pregnancy loss. The normal luteal phase was simulated by a constant baseline (0.02 IU/mL) CG model to simulate a nonconceptive cycle (baseline). The second model was baseline CG until day 8 of culture, followed by daily doubling from days 9-17 to simulate an early pregnancy (rescue-plateau). CG concentrations were then held constant from days 17-20 (5.12 IU/mL). A third model (rescue-drop) was used that was identical to the early pregnancy model except that on day 17 CG was returned to baseline concentrations (0.02 IU/mL) to simulate an early pregnancy loss. Baseline CG stimulation resulted in profiles of estrogen, progesterone, and relaxin secretion in culture that were closely related to secretory profiles previously reported in serum during the nonconceptive luteal phase. The timing of appearance of relaxin secretion and later declines in steroid and relaxin secretion paralleled that observed in serum. In the CG rescue protocols, ovarian steroids rose in response to daily doubling of CG and fell when CG either plateaued or fell. Relaxin did not show an increase in response to increasing CG, but its secretion did not drop when CG concentrations plateaued or dropped. This cell culture system model mimics the profile of ovarian steroids and relaxin seen in serum during the nonconceptive luteal phase, although the relative magnitude of the hormones was not the same as seen in vivo. It was also used to investigate responses to luteal rescue protocols designed to simulate early pregnancy and pregnancy loss. This culture system may be useful to study differences in endocrine response in granulosa cells collected from different patients and to provide information of clinical relevance. This culture system provides a model to study luteal function and its response to different protocols of luteal rescue and thus may provide insight into early pregnancy and pregnancy loss.

Luteal function following ovarian stimulation in rhesus monkeys for in vitro fertilization: atypical response to human chorionic gonadotropin treatment simulating early pregnancy.

This study determined if corpora lutea of hyperstimulated cycles in rhesus monkeys could be "rescued" by the pregnancy signal, chorionic gonadotropin (CG), given at the typical time of implantation. At menses, female monkeys received human follicle-stimulating hormone (hFSH, 60 IU, days 1 to 6) followed by human menopausal gonadotropin (hMG, 60 IU hFSH/60 IU luteinizing hormone [hLH], days 7 to 9). On day 10, human chorionic gonadotropin (hCG) was given to mimic the LH surge. Nine days later, a regimen of daily increasing doses of hCG (15 to 360 IU twice a day) was initiated to simulate rescue of the corpus luteum in early pregnancy. Serum levels of progesterone (P) increased through day 5 of the luteal phase but then declined. Circulating levels of bioactive LH were significantly less on days 7 to 9 of the luteal phase than at this stage in the natural cycle. The hCG regimen extended (P less than 0.05) the luteal phase in five of six animals. The hCG treatment elicited a persistent increase (P less than 0.05) in circulating P levels, rather than a transient rise typical of normal or simulated pregnancy in natural cycles. The authors conclude that (1) corpora lutea of hyperstimulated cycles can respond to CG, but (2) there are differences in luteal function during both the luteal phase and simulated early pregnancy that may be due to inadequate luteal development or the abnormal gonadotropin milieu existing after ovulation or both.

Immunization of monkeys with recombinant complimentary deoxyribonucleic acid expressed zona pellucida proteins.

OBJECTIVE: To evaluate the effects of immunization with zona pellucida (ZP) proteins produced by recombinant complementary DNA (cDNA) technology for the elicitation and antibodies that inhibit sperm binding without altering ovarian function in the nonhuman primate. DESIGN: Controlled nonhuman primate study. SETTING: Controlled environment with individual housing of monkeys in facility approved by National Institutes of Health (NIH) guidelines. PARTICIPANTS: Monkeys housed and treated according to NIH regulations. INTERVENTIONS: Monkeys immunized and boosted at regular intervals with ZP proteins produced using recombinant cDNA techniques. MAIN OUTCOME MEASURE: Urinary estrogen, P, serum antibody levels, sperm-ZP binding, and ovarian morphology. RESULTS: Monkeys immunized with a recombinant rabbit 75-kd ZP protein expressed from a partial cDNA in the pEX bacteria expression system produce antibodies that interfere with ovarian follicular development and ovarian cyclicity. On the contrary, monkeys immunized with a recombinant rabbit 55-kd ZP protein develop antibodies that inhibit homologous sperm binding but do not affect ovarian follicular development or subsequent ovarian hormonal cyclicity. CONCLUSION: Monkey antibodies to the rabbit 75-kd ZP recombinant protein can be generated that inhibit ovarian cyclicity as desired for animal sterilization vaccines. Antibodies to the 55-kd ZP recombinant protein inhibit homologous monkey sperm binding to the ZP without altering ovarian endocrine function or morphology as is desired for human immunocontraception.

Effects of glucose and other energy substrates on the hyperactivated motility of macaque sperm and the zona pellucida-induced acrosome reaction.

Energy sources in sperm capacitation media have various effects on mammalian sperm and are required for stimulation of hyperactivated motility and/or acrosome reactions in some species. The present experiments were performed to investigate the energy substrate requirement for these two functions of macaque sperm. Semen from six cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with Biggers, Whitten, and Whittingham media. In one set of experiments, sperm were incubated in the complete capacitation medium or in medium without glucose. In another set of experiments, the complete medium was used for comparison with medium containing no energy substrates. The absence of glucose did not affect survival of sperm during a 6-hour incubation period; however, removal of all energy substrates resulted in a decrease in percent motility by 3 hours. Sperm were incubated for 1 hour prior to evaluation of sperm motility by computer-aided sperm analysis and sperm-zona binding experiments. During the last 30 minutes of incubation, half of the aliquots of sperm suspensions were treated with activators (ACT; caffeine and dbcAMP, 1 mM each). As previously reported, when sperm were incubated in complete medium and treated with ACT there were changes in sperm movements that are consistent with hyperactivation. Similar or greater changes were observed in sperm that were incubated prior to treatment with ACT in glucose-free medium or in medium without any energy substrates. Whether sperm were incubated in complex medium or in glucose-free medium, sperm binding to zonae was enhanced when the sperm were treated with ACT.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.

Hyaluronic acid enhances the zona pellucida-induced acrosome reaction of macaque sperm.

Hyaluronic acid (HA) not only surrounds the zona pellucida as part of the cumulus matrix but also is present throughout the zona pellucida and the perivitelline space of many mammalian oocytes. However, most in vitro techniques to study sperm-oocyte interaction eliminate HA from the oocyte through enzymatic digestion and/or do not expose sperm to HA prior to zona pellucida binding. This study explores the effect of preincubation of sperm or oocytes with HA on sperm-zona pellucida binding and subsequent acrosome reaction of bound sperm. Cynomolgus macaque semen was washed. Incubated, chemically capacitated with dibutryl cyclic adenosine monophosphate (dbcAMP) and caffeine, and sperm-zona pellucida binding assays were performed. In one experiment, sperm were pretreated with HA (100 micrograms/ml) during the last 10 minutes of the 30-minute period for chemical capacitation. In another experiment, only the oocytes were preincubated in the media containing HA. In the third experiment, gametes were exposed to HA for 10 minutes after sperm had been allowed to bind to zonae pellucida. The preincubation of either sperm or zonae pellucida with HA enhanced the percentage of bound sperm that were acrosome reacted. However, HA did not affect the number of sperm bound to zonae pellucida. When sperm already bound to the zonae pellucida were exposed to HA, there was no increase in the percentage of bound acrosome-reacted sperm. The HA enhancement of the acrosome reaction of sperm bound to the zona pellucida shown in this study required less than 1 minute of sperm exposure to HA-treated zonae during the zona binding process. However, this enhancement was observed only if the HA exposure preceded sperm-zona binding. This result suggests that HA is interacting with the sperm surface, possibly via a receptor, at the time of initiation of the acrosome reaction. It is unlikely that the effects noted in the current experiments were the result of motility retention or improvement because the full enhancement of the acrosome reaction was observed when only the oocytes were pretreated with HA.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) modulates function of human luteinizing granulosa cells via cAMP signaling and early reduction of glucose transporting activity.

This study examined the changes in cellular glucose uptake, cAMP-dependent protein kinase (PKA), and progesterone production induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human luteinizing granulosa cells (LGCs) in culture. The role of Ah receptor on TCDD-mediated toxicity in human LGCs was investigated. Treatment of human LGCs with TCDD produced a time- and dose-dependent decrease in the cellular uptake of glucose. The Vmax and the K(m) of glucose transport were decreased by TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the portion of glucose transport activity that is sensitive to TCDD. Pretreatment of the cells with the Ah receptor blockers 4,7-phenanthroline and alpha-naphthoflavone antagonised the effect of TCDD on 3H-Me-glucose uptake. Structure-activity relationship studies with TCDD and three dioxin congeners revealed a rank order for their potency in the inhibition of glucose transport as follows: TCDD > 1,2,3,7,8-PCDD > 1,2,4,7,8-PCDD > 2,7-DCDD. Such a rank order is consistent with the previously determined biological activity of TCDD and the other dioxin congeners. Treatment of cells for 48 h with 10 nM TCDD substantially reduced PKA and progesterone production. The inhibitory effect of TCDD on progesterone production was more pronounced in the presence of insulin (10 micrograms/mL) and D-glucose (13.3 mM). However, cytochalasin B abolished the effect of TCDD on progesterone production. Forskolin (adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progesterone production but it did not affect the action of TCDD on PKA activity. A relationship between glucose transporting activity and progesterone production in human LGCs treated with TCDD is indicated by several lines of evidence: a) cytochalasin B downregulated glucose transporting activity and progesterone production, b) insulin plus D-glucose downregulated glucose uptake and amplified the negative effect of TCDD on progesterone production, and c) forskolin abolished the negative effect of TCDD on glucose transporting activity and on progesterone production. From the present data we conclude that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in human steroid-producing cells and that effect of TCDD on steroid production is mediated through the cAMP-dependent protein kinase.

Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.

Gender differences in the mechanism of dioxin toxicity in rodents and in nonhuman primates.

This study examined the differences in mechanisms of toxicity when adipose cells from males and females were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Glucose uptake by adipose tissue in vitro was decreased significantly in male guinea pigs within 1 d of intraperitoneal injection of TCDD, but there was no significant effect in females, even at 28 d after treatment. A similar difference between male and female guinea pigs was detected in the effect of TCDD on lipoprotein lipase (LPL) activity, except that a significant decrease in LPL activity was observed 28 d after treatment. Experiments with adipose tissue explants from untreated guinea pigs and macaques revealed similar gender differences in the effect of TCDD in vitro on glucose uptake and LPL activity. Both time-course studies and dose-response studies with TCDD in vitro confirmed the greater sensitivity of male tissues to TCDD toxicity. TCDD induced lipid peroxidation in the adipose tissues of male guinea pigs, while it had no effect in females. 3H-TCDD binding affinity studies in adipose explant tissues showed that tissues from male guinea pigs and monkeys had a higher binding capacity for TCDD than female tissues. TCDD induced a significant reduction in nuclear protein phosphorylation and an increase in cytosolic protein phosphorylation in adipose tissue from male guinea pigs; the effects in female tissues were opposite: nuclear protein phosphorylation increased and cytosolic protein phosphorylation decreased. In a cell-free system in the absence of the nucleus, adipose tissues from male guinea pigs and monkeys responded to TCDD with a rapid stimulation of tyrosine kinase activity but female tissues from both species had a significantly lower and slower response. TCDD induced the DNA binding of AP-1 in adipose tissues of male guinea pigs, but in female tissues TCDD reduced the DNA binding of AP-1. In summary, the results of this study demonstrate gender differences in the response of nonreproductive cells to TCDD. Some of these differences involve different mechanisms of toxicity in both the cytoplasmic and nuclear compartments of the cell.

Improved collection and developmental competence of immature macaque oocytes.

Methods previously described to aspirate immature oocytes from ovaries of macaques result in approximately half the oocytes being stripped of cumulus cells. Here, we describe modifications of the needle aspiration assembly that yield much higher percentages of cumulus-intact oocytes when used with an ultrasound-guided method for oocyte recovery in monkeys. Sealing of the needle assembly appears to stabilize vacuum pressure at the needle tip and prevents air from entering the tubing. Reduction of the vacuum pressure from -100 to -20 kPa resulted in a significant decrease of denuded oocytes from over 50% to fewer than 10%. This was accompanied by a significant increase in the percentage of oocytes that developed into blastocysts after in vitro fertilization. Reduction of the aspiration pressure below -20 kPa significantly reduced the total number of oocytes recovered. We concluded that these modifications represent the best compromise to collect the largest number of cumulus-intact oocyte complexes from macaques.

Regulation of granulosa cell proliferation and EGF-like ligands during the periovulatory interval in monkeys.

BACKGROUND: This study seeks to clarify cell cycle dynamics of granulosa cells following hCG and elucidate the expression of epidermal growth factor (EGF)-like ligands during luteinization. METHODS: Granulosa cells were obtained from rhesus macaques undergoing controlled ovarian stimulation protocols before or after an ovulatory hCG bolus. Cell cycle characteristics were determined by flow cytometry and levels of EGF receptor (EGFR), amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC) mRNAs were measured by real-time RT-PCR. RESULTS: The proportion of cells in S-phase was 7.5% prior to hCG and did not decline until 24 h after hCG (3.1%). EGFR protein and BTC mRNA did not change following hCG, whereas AREG and EREG mRNA increased starting at 3 and 12 h post-hCG, respectively, and remained elevated thereafter. CONCLUSIONS: Cell cycle transit of macaque granulosa cells does not change until 24 h after an ovulatory stimulus, whereas the EGF-like ligands EREG and AREG are increased rapidly. This suggests that luteinizing granulosa cells are refractory to mitogenic stimulation by EGFR ligands.

2,3,7,8-tetrachlorodibenzo-p-dioxin induces CYP1B1 expression in human luteinized granulosa cells.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive toxicant in multiple species; however, mechanisms and direct ovarian effects are poorly understood. DNA microarrays were used to characterize gene expression profiles of human luteinized granulosa cells (HLGCs) exposed to TCDD in primary cultures. Exposure to 10 nM TCDD for 24 h induced a significant increase in CYP1B1, while few other genes responded. TaqMan PCR and Western immunoblotting demonstrated that induction was dose-dependent. Additionally, the microsomal form of catechol-O-methyltransferase (COMT) was highly expressed in HLGCs, along with only fractional amounts of the soluble form. This is the first report of CYP1B1 and COMT expression, and CYP1B1 induction, in cells from the human ovary. The role of CYP1B1 in the oxidative metabolism of estrogens and potential generation of DNA adducts in the ovary may have significant consequences for oocyte quality, corpus luteum function, and ovarian carcinogenesis.

The macaque model for in vitro fertilization: superovulation techniques and ultrasound-guided follicular aspiration.

Prior methods for macaque in vitro fertilization (IVF) have incorporated laparoscopy and/or laparotomy as the primary means for oocyte recovery. Sonographic techniques, as used with human IVF, have been applied to the macaque, both for monitoring the response to hyperstimulation and for follicular aspiration prior to ovulation. Pergonal (hMG) was administered for 7 or 8 days beginning on cycle day 1 or 2 or for 6 days beginning on cycle day 3. This was followed by Pregnyl (hCG) prior to follicular aspiration. The quality of oocytes recovered from the 6-day treatment group was considerably better than those treated for greater than or equal to 7 days. It was concluded that ultrasound can provide a reliable means for documenting the response to ovarian stimulation and the successful transabdominal aspiration of multiple follicles.

Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization-embryo transfer success.

We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle. There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied. As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point. It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro. All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies. A limited number of cycles from donor/recipient cycles did not show similar results. Steroid concentrations were not predictive of conception. These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles. This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.

Macaque sperm fusion with zona-free hamster oocytes.

Semen from six cynomolgus macaques was washed twice by centrifugation in BWW media and resuspended in 1:2 (vol:vol) of Tes-Tris-buffered eggyolk extract (EXT) diluent and BWW (EXT-BWW). Caffeine and dbcAMP were added to induce capacitation. Sperm were treated with calcium ionophore, A23187, followed by the addition of EXT to recover motility, then washed into BWW. The percent motile sperm was similar in ionophore-treated and control sperm suspensions, but motility of treated sperm decreased significantly by 20 min after treatment. The percentage of viable, acrosome-reacted sperm was 56.3% following ionophore treatment versus 4.0% in control suspensions. When ionophore-treated sperm were coincubated with zona-free hamster oocytes, 51% of oocytes had evidence of sperm fusion and no oocytes were penetrated after incubation with control sperm. These results are consistent with the hypothesis that macaque sperm are capable of fusion with zona-free hamster oocytes if sperm are viable and acrosome-reacted.

Recombinant human gonadotropins for macaque superovulation: repeated stimulations and post-treatment pregnancies.

This report summarizes data from the superovulation and ultrasound-guided follicular aspiration of 40 female rhesus monkeys (Macaca mulatta) with recombinant human gonadotropins. Of the animals treated, 12 were stimulated for only one cycle, either because of a poor response to the hormones or due to ectopic ovarian position precluding ease of access via ultrasound. The majority of animals were stimulated for a minimum of 3 cycles and 3 females continued to respond for a minimum of 8 and a maximum of 10 cycles. For those animals with repeated stimulation cycles, the number of follicles developed during each of the stimulation protocols remained relatively comparable. Of the animals mated since cessation of treatment, 70% conceived. There was no difference between the conception rate in this subset of animals and the rest of the macaque breeding colony. These data indicate that participation in these studies does not impact on the reproductive potential of female rhesus monkeys.