SPID#: 35 Experiments were conducted to compare the ovarian stimulatory responses and the numbers and quality of oocytes/IVF embryos following multiple injections of five chimpanzees with FSH and LH. Each of five chimpanzees was subjected to three cycles of gonadotropin treatments. In the first two cycles (experiment 1) each animal was given twice daily injections of 300 i.u hFSH for 7 days and 300 i.u. hMG for 7 days. In the last cycle each animal received half the dose of gonadotropin stimulatory treatment, i.e., 150 i.u. of hFSH and 150 i.u. of hCG for 7 day each. On day 15 animals were injected with 5000i.u. hCG to induce meiotic maturation. Estradiol 17( (E2) hormone levels, perineal swelling (scored on a 13 point scale), and ultrasound were all used to assess and monitor follicular development. Oocytes were recovered at laparoscopy. Follicular immature oocytes were cultured for IVM in CMRL supplemented with 15% homologous serum and (5 (g FSH, 10 (g LH, 1(g E2 and 5/5/5 (g.(g.ng /m Insulin / Transferrin / Selenium) at 37C for up to 48 h. IVF was in Earle's medium supplemented with 10% donor serum using semen from males of proven fertility. Cleavage rate and development to 8-cell stage after 72 h were the end points. Data were analyzed by least square analysis of variance. The mean laparoscopic oocyte recovery rates were 36.4 and 43.9% for experiment 1 and experiment 2 respectively. Chimpanzees receiving lower doses of hFSH and hLH had higher estrogen level, significantly higher numbers of large follicles and of oocytes recovered. There was no difference in the number of embryos produced by IVF. These results suggest that some instances of low response to gonadotropin stimulation can be corrected by reducing, not increasing, the dose of gonadotropin used. Meiotic status and embryonic development after IVF were used to assess 2 follicular superstimulation protocols in 12 monkeys ( 8 rhesus and 4 cynomolgus). Cumulus-enclosed oocytes (CEO) were recovered from superstimulated ovaries after 1) multiple hFSH (540 IU) and hLH (180 IU) injections followed by laparoscopic follicular aspiration (N = 6); and 2) a single SC injection of eCG (PMSG, Sigma; 1000 IU) and hLH (30 IU) followed by ovariectomy (N = 6). Although the proportion of viable MII oocytes recovered from the FSH/LH group was higher than from the eCG group, there was no difference (P2, P > 0.72) in the proportion of oocytes undergoing IVF and embryo development . Germinal vesicle (GV) CEO isolated from eCG, FSH/LH and 2 untreated controls were cultured for IVM in CMRL supplemented with 15% homologous serum and (5 :g FSH, 10 :g LH, 1:g E2 and 5/5/5 :g.:g.ng /ml Insulin/Transferrin/Selenium) at 37EC for 48 h. A significantly higher proportion of GV oocytes isolated from the hormonally treated animals matured and fertilized in vitro than those from unstimulated. No difference in IVM/IVF was noted between the eCG and FSH/LH treatment groups. When combined, in vivo MII oocytes produced more embryos than IVM oocytes (P2, P< 0.012). Although fewer mature oocytes were obtained using single injection of eCG and hCG combined, high quality MII oocytes were produced that were capable of forming embryos comparable to the commonly used multiple injections of FSH/LH. Unfertilized mature and immature rhesus monkey oocytes were assessed for morphological changes and osmotically induced volumetric changes following exposure to different concentrations of Ethylene glycol (ETOH), propylene glycol (PROH) and glycerol. The concentrations used were 0.0, 0.75 M, 1.5 M, and 2.0 M. In each experiment a single oocyte was transferred to clean cavity slide and viewed on a TV monitor hooked to a VCR. Throughout the procedure of adding and removing the CPA, specific time dependent volume changes were observed and recorded by videomicroscopy. Information has been obtained on the response of monkey oocytes to the three CPAs. We investigated the response of macaque oocytes to glycerol, and cooling in the presence of glycerol to 00C, in order to gain an overall understanding of the physical response of oocytes to cryopreservation. Microflourescence histochemical techniques were used to assess the state of the F-actin microfilament system of rhesus monkey oocytes after exposure to, and equilibration with, different concentrations of glycerol at 230C and 00C. Our results showed that the F-actin organization around the cortex and in the transzona pellucida foot processes was destroyed by exposure to 1.0 or 2.0 M glycerol at 230C. However, those effects were substantially reduced by exposure to glycerol at 00C.
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