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Production of monkey embryos from in vivo and in vitro matured oocytes
362
Theriogenology PRODUCTION OF MONKEY EMBRYOS FROM IN VIVO AND IN VITRO MATURED CCCMES A.I. Younis, K.G. Gould, S. Phythyon, and J. Johnson Division of Reproductive Biology, Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30322
Meiotic status and embryonic development after IVF were used to assess 2 follicular superstimulation protocols in 12 monkeys ( 8 rhesus and 4 cynomolgus). Cumulusenclosed oocytes (CEO) were recovered from superstimulated ovaries after 1) multiple hFSH (540 IU) and hLH (180 IV) injections followed by laparoscopic follicular aspiration (N = 8); and 2) a single SC injection of eCG (PMSG, Sigma; 1000 IU) and hLH (30 IU) followed by ovariectomy (N = 6). In vivo matured oocytes were IVF immediately in Earte’s medium supplemented with 10% donor serum, heparin (10 us/ml), and dbcAMP (1 mM). A modified CMRL-1066 medium plus 5% homologous serum was used for embryo culture. The ovarian response to these 2 stimulation protocols and the embryonic development of in vivo Metaphase II (MII) oocytes are shown in Table 1. Table 1. In vivo effects of FSH/LH and eCG on maturation and IVF of oocytes Treat
CEO
MII (%)
2- Cell/lVF (“?)
FSWLH
125
63 (50%)”
24133 (72.7)
8-l 6-Cell (%) 12l33 \:?.I)
Other& 21
27 (16%)b 1O/l 5 (66.7) 50 PMSG 169 6/l 5 (40.0) T ‘sgenerate, polyspermic; a,b: Significantly different @, PcO.00001). Although the proportion of viable MII oocytes recovered from the FSH/LH group were higher than from the eCG ,.3up, there was no differences (f, P > 0.72) on proportion of oocytes undergoing IVF and embryo development. Germinal vesicle (GV) CEO isolated from eCG, FSWLH and 2 untreated controls were cultured for IVM in CMRL supplemented with 15% homologous serum and (5 ug FSH, 10 ug LH, lug E, and 5/5/5 ug.pg.ng /ml InsuliruTransferrin/Selenium) at 37°C for 48 h. Results are given in Table 2. Table 2. IVM and IVF of oocvtes from control, FSWLH, or eCG stimulated monkeys Treat
CEO
MII (%)
PPNflVF (%)
8-16-Cell (%)
Others
None
50
11 (22%)8
4110 (40)a
200 (20) *
39
PMSG
61
25 (41 %)b
13125 (52)b
8/25 (32)b
36
FSH/LH 32 17 (53.l)b 9/l 7 (52.9)b 6/i 7 (35.3)b 15 a,b values within columns with different superscripts are significant (P
Theriogenology PRODUCTION OF MONKEY EMBRYOS FROM IN VIVO AND IN VITRO MATURED CCCMES A.I. Younis, K.G. Gould, S. Phythyon, and J. Johnson Division of Reproductive Biology, Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30322
Meiotic status and embryonic development after IVF were used to assess 2 follicular superstimulation protocols in 12 monkeys ( 8 rhesus and 4 cynomolgus). Cumulusenclosed oocytes (CEO) were recovered from superstimulated ovaries after 1) multiple hFSH (540 IU) and hLH (180 IV) injections followed by laparoscopic follicular aspiration (N = 8); and 2) a single SC injection of eCG (PMSG, Sigma; 1000 IU) and hLH (30 IU) followed by ovariectomy (N = 6). In vivo matured oocytes were IVF immediately in Earte’s medium supplemented with 10% donor serum, heparin (10 us/ml), and dbcAMP (1 mM). A modified CMRL-1066 medium plus 5% homologous serum was used for embryo culture. The ovarian response to these 2 stimulation protocols and the embryonic development of in vivo Metaphase II (MII) oocytes are shown in Table 1. Table 1. In vivo effects of FSH/LH and eCG on maturation and IVF of oocytes Treat
CEO
MII (%)
2- Cell/lVF (“?)
FSWLH
125
63 (50%)”
24133 (72.7)
8-l 6-Cell (%) 12l33 \:?.I)
Other& 21
27 (16%)b 1O/l 5 (66.7) 50 PMSG 169 6/l 5 (40.0) T ‘sgenerate, polyspermic; a,b: Significantly different @, PcO.00001). Although the proportion of viable MII oocytes recovered from the FSH/LH group were higher than from the eCG ,.3up, there was no differences (f, P > 0.72) on proportion of oocytes undergoing IVF and embryo development. Germinal vesicle (GV) CEO isolated from eCG, FSWLH and 2 untreated controls were cultured for IVM in CMRL supplemented with 15% homologous serum and (5 ug FSH, 10 ug LH, lug E, and 5/5/5 ug.pg.ng /ml InsuliruTransferrin/Selenium) at 37°C for 48 h. Results are given in Table 2. Table 2. IVM and IVF of oocvtes from control, FSWLH, or eCG stimulated monkeys Treat
CEO
MII (%)
PPNflVF (%)
8-16-Cell (%)
Others
None
50
11 (22%)8
4110 (40)a
200 (20) *
39
PMSG
61
25 (41 %)b
13125 (52)b
8/25 (32)b
36
FSH/LH 32 17 (53.l)b 9/l 7 (52.9)b 6/i 7 (35.3)b 15 a,b values within columns with different superscripts are significant (P