1 Division of Reproductive Sciences, Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006 and 2 Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA
![]() |
Abstract |
---|
Key words: granulosa cell/macaque/periovulatory interval/steroid synthesis/steroidogenic enzymes
![]() |
Introduction |
---|
In humans and Old World monkeys, ovulation generally occurs 3638 h following the onset of the LH surge (Weick et al., 1973; Hoff et al., 1983
). Unlike in many non-primate species, circulating oestrogen concentrations do not drop immediately following the onset of the LH surge in primates, but rather remain elevated for approximately 24 h. However, progesterone concentrations begin to rise after the surge in both non-primate and primate species, supporting a common function for this hormone during the periovulatory interval (Weick et al., 1973
; Smith et al., 1975
; Hoff et al., 1983
; Park and Mayo, 1991
; Sirois, 1994
). The onset of progesterone synthesis and the increase in progesterone receptor expression following the ovulatory stimulus (e.g. Iwai et al., 1990
; Park and Mayo, 1991
) argues for a direct, possibly early, role for progesterone in ovulation and luteinization, although the time-frame and mechanism by which progesterone exerts this influence is not known.
While limited studies in primates have examined the production of steroids following an endogenous surge of LH (e.g. Weick et al., 1973; Mori et al., 1978
; Hoff et al., 1983
), none has yet controlled the timing of the ovulatory stimulus. The use of ovarian stimulation cycles (Wolf et al., 1989
) provides multiple large antral follicles for study and permits analysis at precise time points after administering the ovulatory gonadotrophin bolus. In addition, the data are relevant to clinical in-vitro fertilization (IVF) settings. The present study was designed to elucidate the dynamics of steroid synthesis in the primate follicle during the periovulatory interval both in vivo and in vitro using granulosa cells aspirated before, and 12, 24, or 36 h following an ovulatory stimulus of human chorionic gonadotrophin (HCG).
![]() |
Materials and methods |
---|
Daily blood samples were obtained from unanaesthetized animals by saphenous venipuncture from the beginning of gonadotrophin treatment. The blood sample from the day of follicle aspiration was collected just prior to surgery. Serum oestradiol and progesterone concentrations were determined using specific radioimmunoassays (Wolf et al., 1990), and follicular growth was monitored using serum steroid concentrations and ultrasonography performed on days 67 of stimulation (Zelinski-Wooten et al., 1997
). Blood samples were collected until 5 days after follicle aspiration to verify the presence of typical concentrations of serum progesterone (i.e. functional corpora lutea) during the luteal phase. Serum concentrations of bioactive LH were determined for the 3 days prior to and including the day of follicle aspiration using an in-vitro mouse Leydig cell bioassay (Ellinwood and Resko, 1980
) to confirm the absence of an endogenous LH surge.
In order to better evaluate the changes in circulating steroids during the early periovulatory interval, serum samples from rhesus monkeys undergoing ovarian stimulation (Chandrasekher et al., 1994) were assayed for progesterone and 17ß-oestradiol concentrations by radioimmunoassay following a 1000 IU bolus of urinary HCG. Serum samples were collected before, or 0.5, 1, 2, 4, 6, 8, 12, or 24 h following administration of HCG.
![]() |
Granulosa cell preparation |
---|
![]() |
Granulosa cell incubation |
---|
![]() |
Follicular fluid radioimmunoassay |
---|
![]() |
Statistical analysis |
---|
![]() |
Results |
---|
![]() |
Serum steroids |
---|
|
|
![]() |
Follicular fluid steroids |
---|
|
![]() |
In-vitro steroidogenesis |
---|
|
|
![]() |
17ß-oestradiol |
---|
|
![]() |
Discussion |
---|
Administration of an ovulatory stimulus resulted in a rapid and sustained rise in progesterone concentrations in serum and follicular fluid. In-vitro studies on granulosa cells isolated from follicular aspirates suggest that these cells are a likely source of the increased progesterone production following HCG. While the mechanism for the enhanced steroidogenesis remains unknown, recent reports in rodent and primates species suggest that several key components in the pathway leading to progesterone synthesis increase upon exposure to a gonadotrophin bolus, notably P450 side-chain cleavage (P450scc) (Doody et al., 1990; Ronen-Furhmann et al., 1998), steroidogenic acute regulatory protein or StAR (Pollack et al., 1997
), and LDL receptor (Golos and Strauss, 1987
). In order to better understand the changes in enzyme activity and substrate utilization that occur within the primate follicle throughout the periovulatory interval, macaque granulosa cells aspirated at specific times following the administration of HCG were incubated for 2 h with several steroidogenic precursors. The addition of pregnenolone as a substrate for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased progesterone production above basal values at all time points examined, indicating that macaque granulosa cells possess significant amounts of 3ß-HSD activity before, as well as following, the ovulatory stimulus (Sasano et al., 1990
). This strongly suggests that the rise in progesterone following the ovulatory stimulus is not due to an increase in 3ß-HSD activity. Interestingly, mRNA encoding 3ß-HSD decreased in bovine granulosa cells following the ovulatory stimulus (Voss and Fortune, 1993
), supporting a minor role for this enzyme in the periovulatory rise of progesterone. Species differences exist with regards to the expression of 3ß-HSD in preovulatory granulosa cells: in pig and sheep, 3ß-HSD is localized only in the theca, while rat, cow and human express 3ß-HSD in both cell layers (Sasano et al., 1990
; Voss and Fortune 1993
; Conley et al., 1994
, 1995
; Donath et al., 1997
; Garrett and Guthrie, 1997
). The localization of 3ß-HSD in granulosa cells in the primate preovulatory follicle raises the possibility for coupling androstenedione and oestrogen synthesis together, for example, to increase the efficiency of preovulatory oestrogen synthesis (Conley and Bird, 1997
).
The ability of isolated granulosa cells to utilize the soluble analogue 25-hydroxycholesterol was used as a marker of P450scc activity (i.e. cholesterol to pregnenolone). This compound readily enters the mitochondria without facilitation (Toaff et al., 1982), and hence the amount of progesterone produced should not be limited by cholesterol transport. Granulosa cells obtained prior to HCG had little P450scc activity, which is consistent with the idea that the theca cell is the primary site of cholesterol conversion to pregnenolone in the preovulatory follicle (Sasano et al., 1989
). Within 12 h of HCG, granulosa cells acquire P450scc activity and this, along with existing 3ß-HSD activity, allows these cells to produce progesterone (Conley and Bird, 1997
). Therefore, it does not appear that the mid-cycle gonadotrophin surge induces granulosa cell 3ß-HSD activity; rather the early periovulatory acquisition of P450scc activity by granulosa cells is an essential feature of the rise in progesterone associated with luteinization in the primate follicle.
Granulosa cells were unable to metabolize either LDL or cholesterol in vitro to produce progesterone above basal values at any time following HCG administration. There are reports that LDL-receptor mRNA in human granulosa cells is increased after 1 h of HCG exposure in vitro (Soto et al., 1984; Golos and Strauss, 1987
), while macaque granulosa cells collected 27 h after the ovulatory HCG bolus increased progesterone synthesis in response to LDL within 24 h of culture (Brannian et al., 1992
; Brannian and Stouffer, 1993
). Thus, granulosa cell LDL usage in vitro increases following the gonadotrophin surge in primates via a LDL receptor-mediated mechanism. However, follicular fluid from both natural and stimulated menstrual cycles contains very low concentrations of LDL as compared with those of high-density lipoprotein, even 36 h after an ovulatory stimulus (Simpson et al., 1980
; Enk et al., 1986
), while preovulatory granulosa cells are laden with cholesterol esters (Endersen et al., 1990
). The lack of usage of LDL and cholesterol by periovulatory granulosa cells in the current study supports the notion that esterified cholesterol is the primary steroid precursor at least until very late in the periovulatory interval, at which time LDL may become an important steroidogenic substrate.
In contrast to progesterone, both androstenedione and oestrogen concentrations in serum and follicular fluid increased in a transient fashion, with the greatest concentration 12 h following HCG. Despite the fact that oestrogen concentrations declined following the administration of an ovulatory stimulus, metabolism of androstenedione to 17ß-oestradiol by granulosa cells in vitro remained very high throughout the periovulatory interval. Consistent with previous data, the decrease in oestrogens 24 h following HCG administration was not due to a decrease in aromatase activity, but rather a reduction in aromatizable androgens (Hillier et al., 1984; Tamura et al., 1992
; Foldesi et al., 1998
). This pattern may reflect an initial increase in steroidogenesis, perhaps through increased P450scc, with a subsequent decrease in the ratio of P450c17:3ß-HSD (Conley and Bird, 1997
), although a general decline in total steroids was observed 1236 h post-HCG.
In the current study, granulosa cells aspirated prior to the ovulatory stimulus were able to respond to HCG exposure in vitro with a 14-fold increase in progesterone synthesis, while cells obtained at any time point following the ovulatory stimulus were not gonadotrophin responsive. The ability of gonadotrophins to support granulosa cell steroidogenesis in vitro has been established, although the responses are generally blunted in cells obtained following the ovulatory stimulus (e.g. Zelinski-Wooten et al., 1997). Zelinski-Wooten et al. (1997) have shown that the half-life for 1000 IU recombinant HCG is 16 h; therefore appreciable concentrations of bioactive HCG remain 36 h following injection. By 24 h post-HCG, the existing LH receptor pathways may have been occupied by HCG administered in vivo, possibly accounting for the high basal progesterone production in vitro. Alternatively, the down-regulation of LH receptor or desensitization of the LH receptorcyclase system may account for the decrease in steroid production 24 h post-HCG via a transient reduction in several key steroidogenic enzymes (e.g. Richards et al., 1976
; Jaaskelainen et al., 1980
; LaPolt et al., 1990
; Segaloff et al., 1990
; Voss and Fortune, 1993
). Thus, late periovulatory/luteal steroidogenesis may be linked to the reacquisition or resensitization of LH receptors.
Although it is evident that hormonally stimulated monkeys have higher concentrations of serum progesterone and oestrogen than naturally cycling monkeys or women (Weick et al., 1973; Hoff et al., 1983
), the profile of serum steroids during the periovulatory interval is similar to natural cycles. In all cases, progesterone increases rapidly following the ovulatory stimulus, either plateaus or declines between 1224 h, and begins to rise near the predicted time of follicle rupture. Oestrogen remains constant for approximately 12 h after the ovulatory stimulus before beginning to decline (Hoff et al., 1983
), while serum androstenedione concentrations increase within 12 h of the onset of the LH surge (Hoff et al., 1983
). These data, plus evidence that follicles will ovulate in a timely manner (e.g. Hibbert et al., 1996
), support the idea that ovarian stimulation of monkeys results in follicles that are steroidogenically similar to follicles of the natural cycle (Weick et al., 1973
; Hoff et al., 1983
; Zelinski-Wooten et al., 1997
). Thus, monkeys undergoing ovarian stimulation should prove useful models for further studies on the dynamics and regulation of periovulatory events in primates.
For example, a local role for progesterone in ovulation and luteinization has been postulated in both primate and rodent species (e.g. Lydon et al., 1995; Hibbert et al., 1996
). Data from the current study demonstrate that progesterone increases within 30 min of the ovulatory stimulus, and remains at very high concentrations in the follicular fluid until the time of ovulation. In addition, progesterone receptor expression increases early in the periovulatory interval (Iwai et al., 1990
; Park and Mayo, 1991
; Natraj and Richards, 1993
), for example, within 12 h of the HCG bolus during ovarian stimulation cycles in monkeys (Chaffin et al., 1998
). Further studies are warranted to examine progesterone action, beginning early in the periovulatory interval in monkeys.
In summary, we have utilized rhesus monkeys undergoing hormonally controlled ovarian stimulation to examine changes in steroidogenesis that occur during the 3638 h periovulatory interval. Progesterone increased rapidly following the administration of an ovulatory bolus of HCG, and remained elevated throughout the periovulatory interval. Androstenedione and oestrogen concentrations peaked 12 h following HCG administration, and thereafter declined. In-vitro studies on isolated granulosa cells collected before or after HCG demonstrate that a rise in P450scc activity drives the increase in progesterone from intracellular stores of esterified cholesterol. Collectively, these data argue for a locally mediated, early periovulatory role for the progesterone signal, perhaps to initiate or maintain the cascade of events resulting in ovulation and luteinization.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
Brannian, J.D., Shigi, S.M. and Stouffer, R.L. (1992) Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles. Biol. Reprod., 47, 355360.[Abstract]
Brännström, M. and Janson, P. O. (1989) Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary. Biol. Reprod., 40, 11701178.[Abstract]
Chaffin, C. L., Hess, D. L. and Stouffer, R. L. (1998) Dynamics of progesterone (P) and progesterone receptor (PR) expression in the primate follicle during the periovulatory interval. 80th Annual Meeting of the Endocrine Society, Abst. #P1-306, p. 184.
Chandrasekher, Y. A., Hutchison, J. S., Zelinksi-Wooten, M. B. et al. (1994) Initiation of periovulatory events in primate follicles using recombinant and native human luteinizing hormone to mimic the midcycle gonadotropin surge. J. Clin. Endocrinol. Metabol., 79, 298306.[Abstract]
Christenson, L. K., and Stouffer, R. L. (1997) Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles. J. Clin. Endocrinol. Metabol., 82, 21352142.
Conley, A.J., Howard, H.J., Slanger, W.D. et al. (1994) Steroidogenesis in the preovulatory porcine follicle. Biol. Reprod., 51, 655661.[Abstract]
Conley, A.J., Kaminski, M.A., Dubowsky, S.A. et al. (1995) Immunohistochemical localization of 3ß-hydroxysteroid dehydrogenase and P450 17-hydroxylase during follicular and luteal development in pigs, sheep, and cows. Biol. Reprod., 52, 10811094.
Conley, A.J. and Bird, I.M. (1997) The role of cytochrome P450 17-hydroxylase and 3ß-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the
5 and
4 pathways of steroidogenesis in mammals. Biol. Reprod., 56, 789799.[ISI][Medline]
Donath, J., Michna, H. and Nishino, Y. (1997) The antiovulatory effect of the antiprogestin onapristone could be related to down-regulation of intraovarian progesterone (receptors). J. Steroid Biochem. Molec. Biol., 62, 107118.[ISI][Medline]
Doody, K.J., Lorence, M.C., Mason, J.I. et al. (1990) Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle. J. Clin. Endocrinol. Metabol., 70, 10411045.[Abstract]
Ellinwood, W.E. and Resko, J.A. (1980) Sex differences in biologically active and immunoreactive gonadotropins in the fetal circulation of rhesus monkeys. Endocrinology, 107, 902907.[Abstract]
Endersen, M.J., Haug, E., Åbyholm, T. et al. (1990) The source of cholesterol for progesterone synthesis in cultured preovulatory human granulosa cells. Acta Endocrinol., 123, 359364.[ISI][Medline]
Enk, L., Crona, N., Olsson, J.-H. et al. (1986) Lipids, apolipoproteins and steroids in serum and in fluid from stimulated and non-stimulated human ovarian follicles. Acta Endocrinol., 111, 558562.[ISI][Medline]
Fanjul, L.F., Ruiz de Galarreta, C.M. and Hsueh, A.J.W. (1983) Progestin augmentation of gonadotropin-stimulated progesterone production by cultured rat granulosa cells. Endocrinology, 112, 405407.[Abstract]
Foldesi, I., Breckwoldt, M., and Neulen, J. (1998) Oestradiol production by luteinized human granulosa cells: Evidence of the stimulatory action of recombinant human follicle stimulating hormone. Hum. Reprod., 13, 14551460.[Abstract]
Garrett, W.M. and Guthrie, H.D. (1997) Steroidogenic enzyme expression during preovulatory follicle maturation in pigs. Biol. Reprod., 56, 14241431.[Abstract]
Golos, T.G. and Strauss, J.F. III. (1987) Regulation of low density lipoprotein receptor gene expression in cultured human granulosa cells: Roles of human chorionic gonadotropin, 8-bromo-3',5'-cyclic adenosine monophosphate, and protein synthesis. Molec. Endocrinol., 1, 321326.[Abstract]
Hibbert, M.L., Stouffer, R.L., Wolf, D.P. et al. (1996) Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates. Proc. Natl Acad. Sci. USA, 93, 18971901.
Hillier, S.G., Wickings, E.J., Afnan, M. et al. (1984) Granulosa cell steroidogenesis before in vitro fertilization. Biol. Reprod., 31, 679686.[Abstract]
Hoff, J.D., Quigley, M.E. and Yen, S.S.C. (1983) Hormonal dynamics at midcycle: A re-evaluation. J. Clin. Endocrinol. Metabol., 57, 792796.[Abstract]
Iwai, T., Nanbu, Y., Iwai, M. et al. (1990) Immunohistochemical localization of oestrogen receptors and progesterone receptors in the human ovary throughout the menstrual cycle. Virchows Arch. A Pathol. Anat. Histopathol., 417, 369375.[ISI][Medline]
Iwamasa, J., Shibata, S., Tanaka, N. et al. (1992) The relationship between ovarian progesterone and proteolytic activity during ovulation in the gonadotropin-treated immature rat. Biol. Reprod., 46, 309313.[Abstract]
Jaaskelainen, K., Hyvonen, T. and Rajaniemi, H. (1980) Human choriogonadotropin-induced desensitization of granulosa-cell adenylate cyclase to gonadotropins and loss of LH/hCG receptor. Molec. Cell. Endocrinol., 20, 145156.[ISI][Medline]
LaPolt, P.S., Oikawa, M., Jia, X.C. et al. (1990) Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology, 126, 32773279.[Abstract]
Lioutas, C., Einspanier, A., Kascheike, B. et al. (1997) An autocrine progesterone positive feedback loop mediates oxytocin upregulation in bovine granulosa cells during luteinization. Endocrinology, 138, 50595062.
Lipner, H. and Wendelken, L. (1971) Inhibition of ovulation by inhibition of steroidogenesis in immature rats. Proc. Soc. Exp. Biol. Med., 136, 11411145.
Loutradis, D., Bletsa, R., Aravantinos, L. et al. (1991) Preovulatory effects of the progesterone antagonist mifepristone (RU486) in mice. Hum. Reprod., 6, 12381240.[Abstract]
Lydon, J.P., DeMayo, F.J., Funk, C.R. et al. (1995) Mice lacking the progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev., 9, 22662228.[Abstract]
Morgan, A., Keeble, S.C., London, S.N. et al. (1994) Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells. Fert. Steril., 61, 949955.[ISI][Medline]
Mori, T., Fujita, Y., Suzuki, A. et al. (1978) Functional and structural relationships in steroidogenesis in vitro by human ovarian follicles during maturation and ovulation. J. Clin. Endocrinol. Metabol., 47, 955966.[Abstract]
Natraj, U. and Richards, J.S. (1993) Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology, 133, 761769.[Abstract]
Park, O.-K. and Mayo KE. (1991) Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Molec. Endocrinol., 5, 967978.[Abstract]
Pollack, S.E., Furth, E.E., Kallen, C.B. et al. (1997) Localization of the steroidogenic acute regulatory protein in human tissues. J. Clin. Endocrinol. Metabol., 82, 42434251.
Richards, J.S., Ireland, J.J., Rao, M.C. et al. (1976) Ovarian follicular development in the rat: Hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone. Endocrinology, 99, 15621569.[Abstract]
Ronen-Fuhrmann, T., Timberg, R., King, S.R. et al. (1998) Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary. Endocrinology, 139, 303315.
Sanders, S.L. and Stouffer, R.L. (1995) Gonadotropin- and lipoprotein-supported progesterone production by primate luteal cell types in culture. Endocrine, 3, 169175.[ISI]
Sasano, H., Okamoto, M., Mason, J.I. et al. (1989) Immunolocalization of aromatase, 17-hydroxylase and side-chain cleavage cytochromes P-450 in the human ovary. J. Reprod. Fertil., 85, 163169.[Abstract]
Sasano, H., Mori, T., Sasano, N. et al. (1990) Immunolocalization of 3ß-hydroxysteroid dehydrogenase in human ovary. J. Reprod. Fertil., 89, 743751.[Abstract]
Segaloff, D.L., Wang, H., and Richards, J.S. (1990) Hormonal regulation of luteinizing hormone/chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization. Molec. Endocrinol., 4, 18561865.[Abstract]
Simpson, E.R., Rochelle, D.B., Carr, B.R. et al. (1980) Plasma lipoproteins in follicular fluid of human ovaries. J. Clin. Endocrinol. Metabol., 51, 14691471.[Abstract]
Sirois, J. (1994) Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo. Endocrinology, 135, 841848.[Abstract]
Smith, M.S., Freeman, M.E., and Neill, J.D. (1975) The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: Prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology, 96, 219226.[Abstract]
Soto, E., Silavin, S.L., Tureck, R.W. et al. (1984) Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate: The effect of low density lipoprotein. J. Clin. Endocrinol. Metabol., 58, 831837.[Abstract]
Tamura, T., Kitawaki, J., Yamamoto, T. et al. (1992) Immunohistochemical localization of 17-hydroxylase/C17-20 lyase and aromatase P-450 in the human ovary during the menstrual cycle. J. Endocrinol., 135, 589595.[Abstract]
Toaff, M.E., Schleyer, H., and Strauss, J.F. III. (1982) Metabolism of 25-hydroxycholesterol by rat luteal mitochondria and dispersed cells. Endocrinology, 111, 17851790.[Abstract]
VandeVoort, C.A., Baughman, W.L. and Stouffer, R.L. (1989) Comparison of different regimens of human gonadotropins for superovulation of rhesus monkeys: Ovulatory response and subsequent luteal function. J. In Vitro Fertil. Embryo Transfer, 6, 8591.[ISI][Medline]
Voss, A.K. and Fortune, J.E. (1993) Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3ß-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge. Endocrinology, 132, 888894.[Abstract]
Weick, R.F., Dierschke, D.J., Karsch, F.J. et al. (1973) Periovulatory time courses of circulating gonadotropic ovarian hormones in the rhesus monkey. Endocrinology, 93, 11401147.[ISI][Medline]
Wolf, D.P., VandeVoort, C.A., Meyer-Haas, G.R. et al. (1989) In vitro fertilization and embryo transfer in the rhesus monkey. Biol. Reprod., 41, 335346.[Abstract]
Wolf, D.P., Thomson, J.A., Zelinski-Wooten, M.B. et al. (1990) In vitro fertilization-embryo transfer in nonhuman primates: The technique and its applications. Mol. Reprod. Dev., 27, 261280.[ISI][Medline]
Zelinski-Wooten, M.B., Hess, D.L., Wolf, D.P. et al. (1994) Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fert. Steril., 61, 11471155.[ISI][Medline]
Zelinski-Wooten, M.B., Hutchison, J.S., Trinchard-Lugan, I. et al. (1997) Initiation of periovulatory events in gonadotrophin-stimulated macaques with varying doses of recombinant human chorionic gonadotrophin. Hum. Reprod., 12, 18771885.[Abstract]
Submitted on August 21, 1998; accepted on November 30, 1998.