Mol. Cell s, Vol. 4, pp. 39-44 Temporal Changes in Ovarian Gonadotropin-Releasing Hormone mRNA Levels by Gonadotropins in the Rat Sung Ho Lee, Eun-Seob Song, Sun Kyeong Yu, Changmee Kim, Dae Kee Lee, Wan Sung Choi l and Kyungjin Kim* Department of Molecular Biofogy and SRC for Cell Differentiation, Seoul National University, Seoul 150-742, Korea; IDepartment of Anatomy, College of Medicine, Gyeongsanf; National University, Chinju 660-280, Korea (Recei*. cd on December 29, 1993) The present study examines whether gonadotropins are involved in the regulation of ovarian GnRH gene expression and how ovarian GnRH gene expression temporalJy correlates with alterations in hypothalamic GnRH, pituitary LH~ gene expression in respons to gonadotropins. Hypothalamic and ovarian GnRH mRNA and pituitary LH~ mRNA levels were determined by respective RNA-blot hybridizations, and ovarian GnRH and estradiol contents and serum LH levels were measured by respective radioimmunoassays. Three animal models such as 1) PMSG-treated, 2) PMSG and heG-treated immature rats and 3) proestrous stage of adult rats were used. Immature rats (25-days old) were administered with PMSG (10 iu) at 10:00 hand 48 h later with heG (10 iu) to induce ovulation. In the PMSG-injected model, hypothalamic GnRH mRNA levels were markedly augmented about 9-fold at 50 h, and pituitary LH mRNA 3-fold at 52 h after PMSG administration. Serum LH levels were increased to the preovulatory surge levels at 56 h, and ovarian GnRH mRNA levels were augmented 4-fold at 60 h after PMSG injection. Administration of heG also induced a marked enhancement in ovarian GnRH mRNA levels in comparison to the values shown in both intact and PMSGtreated rats at 52 hand 54 h, respectively. In the proestrous stage of normal adult rats, pituitary LH~ mRNA levels were peaked at 16:00 h. The preovulatory LH surge was evident at 4 h before increment in ovarian GnRH mRNA levels as shown in PMSG-treated rats. The present study clearly showed the sequential increase in hypothalamic GnRH mRNA, pituitary LH~ mRNA and ovarian GnRH mRNA levels, indicating that ovarian GnRH may play a possible role in the control of follicular maturation and the ovulation process. T he mammalian ovary is a highly heterogenous o rgan composed of many functionally different cells including granulosa, theca, interstitial and luteal cells, and oocyte. There is an unique spatial and temporal expression of a specific set of genes that orchestrates cell growth and diffe rentiation of these cell types which are regulated by hormones in the hypothalamic-pituitary-ovarian axis (H all et af., 1991). Numerous neuropeptides and growth factors are known to act as intraovarian mediators. For instance, GnRH exerts inhibitory and/or stimulatory effects on intraovarian function (Sha rper, 1982; H sueh et al., 1983). GnRH regulates the fo rmation of gonadotropin receptor (H sueh et al., 1983), and increases the biosynthesis of progesterone in the ovary (D avis et af., 1988). G nRH also stimulates maturation of oocytes (Hillensjo and LeM aire, 1980; D ekel et af. , 1985), and ovulation (Ekholm et af., 1981). G nRH or GnRH-like peptide is evidently present in the ovary of various species including cows, sheep, * To whom correspondence should be add ressed. hu mans and rats (Ying et aI., 1981; Aten et af., 1986; Ireland el al., 1988). Existence of the amplified GnRH transcript in the rat ovary was recently demontrated by a reverse transcription-polymerase chain reaction method (O ikawa et al., 1990; G oubach et aI., 1992; Bauer-Dantoi n ef at., 1993). Our laboratory also demonstrated the c, i\ tence and localization of G nRH transcript and ih g:e ne products in the ovary using in situ hybridiza ti () n histochemistry and immunohistochemistry (Park , 'f al., 1988; Choi et aI., 1990). It is, however, yet unk nown whether the ovarian G nRH gene expression is p hysiologically regulated by gonadotropins, although it can be hypothesized that the regulation of ovarian G nRH may be under the influence of hypothalamic-pitui tary-ovarian hormonal axis. The present study examines whether gonadotropins The abbreviations used are: PM SG, r regnant mare serum gonadotropins; hCG, human chorion ic gonadotropins; MOPS, 3-(n-morpholino)propanesul fo nic acid; CV, coefficients of variation; LSD, least significa nce difference; tPA, tissue plasminogen activator. © 1994 The Korean Society of Molecular Biology 40 Regulation of Ovarian GnRH Mol. Cells are involved in the regulation of ovarian GnRH gene expression and how ovarian GnRH gene expression temporally correlates with alterations in hypothalamic GnRH and pituitary LHa gene expression in response to gonadotropins. Since administration of pregnant mare serum gonadotropins (PMSG) to immature female rats resulted in the stimulation of follicular maturation and endogenous preovulatory-like LH surge (Bahr and Ben-Jonathan, 1981), the immature female rats administered with PMSG and/or human chorionic gonadotropins (hCG) were used as a model system in the present study. Materials and Methods Animals Immature (25 days old) and adult female (2-3 months old) Sprague-Dawley rats (Seoul National University .-}nimal Breeding Center, Seoul, Korea) were allowed ad libitum to water and food and maintained under 14 h light, 10 h dark conditions (light on 06:00). Twenty five-day-old rats were ip administered with either 10 iu PMSG (Sigma) alone or 10 iu PMSG + 10 iu hCG (Sigma). hCG was administrated 48 h after PMSG. In the adult rats, the estrous cycle was monitored by a daily vaginal smear procedure and those rats showing at least two consecutive 4-day cycles were used in the present study. After decapitation, ovaries, pituitaries and hypothalamic tissues were removed and stored at 70 °c until use. Serum was also collected from trunk blood and kept at -20 °c prior to assay. Preparation of the rat GnRH and LH {3 gene probes GnRH antisense RNA transcript or GnRH oligomer were used as a hybridization probe. GnRH cDNA clone inserted into plasmid pGEM4 (a gift from Dr. Kelly Mayo, Northwestern University, Evanston, Ill.) was linearized and transcribed using SP6 RNA polymerase to specific activity (1.0 X 109 cpm) (Seong et ai., 1993). GnRH oligomer (3 '-CG/GTC/ GTG/ACC/AGG/ATNCCC/AAC/GCG/GGA-5'; 29 mer) complementary to the sequence of the rat GnRH mRNA coding for amino acids 1 to 9 of decapeptide (Adelman et ai., 1986) was 5' end-labeled with y-32P-ATP (SA.; 3,000 Ci/mmole, NEN) to specific activity (2.l X 108 cpm/50 pmole) (Lee et al., 1990). Rat LH cDNA probe (a gift from Dr. J. L. Roberts, Mt. Sinai, New York) was labeled by a random primer labelling method (Feinberg and Vogelstein, 1984). Total RNA preparation and RNA analysis Total RNAs from hypothalami, pituitaries and ovaries were extracted by an acid guanidium-phenol-chloroform method (Chomczynski and Sacchi, 1987). GnRH mRNA levels in hypothalami and ovarian tissues were determined by Northern blot analysis or slot blot hybridization as described previously (Kim et al., 1989; Lee et al., 1990; Seong et al., 1993). For Northern blot analysis, total RNAs (20 J..1g of each group) were dissolved and denatured in 50% formamide, 7.4% formaldehyde, 20 mM 3-(n-morpholino) propanesulfonic acid (MOPS), 5 mM sodium acetate and 1 mM EDTA at 60 °c. RNA was then fractionated by size using electrophoresis on 1.2% agarose gel containing 6.4% formaldehyde and 20 mM MOPS (Sam brook et ai., 1989) and transferred to Nytran membrane (Schleicher & Schuell; 0.22 J..11Tl pore size). For slot blot hybridization, RNAs (10 J..1g of each group) were solubilized in buffer consisting of 7.4% formaldehyde and 6X SSe. Nytran fUters were prehybridized with 10 ml hybridization buffer for 3 h at room temperature in a heat sealable plastic bag (Kapak) followed by hybridization at 62 °c overnight with a gentle shaking. Hybridization buffer for GnRH RNA blot hybridization consists of 2X SSC, 2X Denhardt's solution, 150 J..1g/mI of denatured salmon sperm DNA and 200 J..1g/ml of yeast tRNA (Lee et ai., 1990). Following hybridization, Nytran membranes were washed with 2X SSC at 62 t for 5 min and then three times with 2 X SSC at 52 t for 30 min each. LH mRNA levels in pituitary were determined by RNA blot hybridization at 42 °c for 16 h. The details for hybridization procedure are described elsewhere (Tepper and Roberts, 1984). The fLlters were auto radiographed with X-ray fLlm (Kodak, X-Omat RP fUm) and the density of bands was quantitated by densitometric scanning (Transdyne General Corp.). Nytran membrane was rehybridized with an 18S rONA control probe. GnRH and LHa mRNA levels were normalized by 18S RNA control values and expressed as an arbitrary unit. Radioimmunoassays for GnRH. LH and 17 fJestradiol GnRH concentrations in ovarian extracts were measured in duplicate by a RIA procedure using Chen-Ramirez anti-GnRH antiserum (CRR-II-B-72) at a final dilution of 1 : 20,000 (Kim et at., 1989). Ovarian tissues were homogenized in 600 J..1l of O.l N HC1, neutralized with 10 N NaOH and centrifuged at 15,000 X g for 20 min at 4 °c. Synthetic GnRH (Sigma) was used as radioiodination and a reference standards. The sensitivity at 80% binding was about 0.5 pg/tube. The intraand interassay coefficients of variation (CV) were about 6 and 7%, respectively. Serum LH levels were measured by a double antibody RIA method using reagents supplied by the NIADDK rLH-I-5 and rLH-RD-2 were used as radioiodination and a reference standard, respectively. Anti-LH antiserum (antirLH-S-6) was used at a final dilution of 1 : 1,000,000. The detection limit for the LH assay was 20 pg/tube and the intraand interassay CV were 5.9 and 8.9%, respectively. The ovarian concentrations of 17~-estra- diol were determined using reagents supplied by the WHO Matched Reagent Program. Labeled ligand was [2, 4, 6, 7, 16, 17-3HJ-estradiol (3.73 X 1011 dpm/ mole, NEN). The intraand interassay CV were 9.6 and 4.1 %, respectively. Vol. 4 ( 1994) Sung Ho Lee el 01. 41 Statistical analysis C hanges in G nRH a nd LH~ mRNA and serum LH levels were analyzed by a one way analysis of va riance (ANOY A). F isher's least significance difference (LSD) test was used for post-hoc comparison with P < 0.05 requi red for statistical significance. Student's t-test was also employed fo r a nalysis o f the differences in hypothalamic and ovarian GnRH contents between the control and PMSG-treated groups. ::;; :! ~ 6 ~ :z: 0: E 7 X 0: C 2 <> 0 ~ ~ I ~ ... ~ 0 * * * .. :5 4 ::;; -< ~ .! 3 < :z: 0: E x 2 0: C <> 0 t . .r 5 0 52 54 o Conlrol ~ PWSC o Control E7ZJ PWSC-Injecled (n3) CJ Control ~ PWSC Injecled (n"3) ,.I ). ,..x. 56 56 60 ~ ~ ~ ~ ~ ~ Time (h) after PMSG injection Figure 1. Effeet of PMSG on hypothalamic GnRH mRNA (upper panel), pituita ry LH~ mRNA (middle panel) and ovarian GnRH mRNA (lower panel) levels in the control and PMSG-treated rats. mRNA levels are expressed as an arbitrary unit (A.U.) and bar represents the mean (± S.E. ) of repeated experiments (n=3). *, vs the control and other time points (P<O.Ol ). Results Effect oj PMSG on hypothalamic GnRH, pituitary LHp and ovarian GnRH mRNA levels The validation of GnRH RNA blot hybridization was previously well documented (Kim et al. , 1989; Lee et al., 1990; Seong et al., 1993). The sizes of GnRH a nd LH~ mRNA were approximately 0.6 kb (Kim et al., 1989) and 0.72 kb (Tepper and Roberts, 1984) as shown previously. The time course changes in hypothalamic GnRH, pituitary LH~ and ovari an GnRH mRNA levels were examined at 2 h intervals from 50 h to 60 h a fter PMSG treatment. Fifty hours following PMSG administration, hypothalamic GnRH mRNA levels we re marked ly augmented. Hypothalamic G nRH mRNA levels we re then dra matically decl ined to the control value a nd remained uncha nged until 60 h (Fig. 1, upper p anel). Pituitary LH~ mRNA levels were significa ntly increased 2-fold a t 52 h after PMSG injection and then returned to the basal levels (Fig. 1, middle panel). Note that the peak of hypothalamic GnRH mRNA levels was 2 h earlier than that of pituitary LH~ mRNA levels. Ovarian GnRH mRNA levels did not increase until 58 h after PMSG S 10 ;:l ... ., UJ • 0--<) Contro l ______ pwsc 52 56 6 0 64 68 72 Time (h) after PMSG injection Figure 2. Serum LH levels of control and PMSG-injected ra ts. Each point represents the mean S.E. (n=611). *, vs other time points (P <O.05). Table 1. Effects of PMSG on ovarian GnRH and estradiol contents Time(h) after GnRH content Estradiol content injection control PMSG control PMSG 24 ND 29.8± II ND 136.6± 19.0 48 20.8 ± 3.1 64.9± 1.7" 19.5± 4.9 181.2± 38.3" 56 8.5± 1.7 67.6± 7.5" l8.0± 3.9 287.8± 41.9" 72 2.6± 0.6 4.2± 0.9 20.0± 3.2 111.7 ± 28.5" GnRH and estradiol contents represent the mean ± S.E. (pg/pair of ova ries and pmol/pair of ovaries, respectively). Experiments were repeated six to eight times. ND; not determined. "PMSG vs control: significantly (P<0.05) diffe rent. 42 Regulation of Ovarian GnRH Mol. Cells Injection. Ovarian GnRH mRNA levels were signifi cantly higher by 6-fold than the control values at 60 h following PMSG administration (Fig. 1, lower panel). Serum LH levels started to increase and reached a peak at 56 h and then declined to the control levels. Serum LH levels in the controls remained unchanged throughout the period examined (Fig. 2). Effect oj PMSG on ovarian GnRH and estradiol contents Table 1 shows the stimulatory effects of PMSG on ovarian GnRH and estradiol contents. A similar pattern of both GnRH and estradiol contents was observed with maximal values at 56 h after PMSG injection. While ovarian GnRH contents in the control group were endogenously fluctuated with a high level at 24 h, ovarian GnRH contents began to increase at 24 h, reached a peak at 56 h, and then returned to nadir levels at 72 h. Estradiol contents rose IS-fo ld and then rapidly decreased. Effect oj PMSG and hCG on hypothalamic GnRH, pituitary LHp and ovarian GnRH mRNA levels As shown in Figure 1, hypothalamic GnRH mRNA levels were markedly increased at 50 h after PMSG injection when compared to the control values. However, this increment was not observed at 50 hand even 52 h after administration of hCG to PMSG-pri med rats (Fig. 3, upper panel). Similarly, an increase in pituitary LH~ mRNA levels at 52 h post-PMSG injection was not observed in PMSG and hCG-injected rats (Fig. 3, middle panel). These data indicated that the animals had not been affected by the endogenous LH surge and negative effects of hCG on hypothalamic-pituitary neural circuitry. On the other hand, administration of hCG to PMSG-pretreated immature rats significantly enhanced ovarian GnRH mRNA levels 4-fold at 2 h and about 7-fold at 4 h after hCGadministration (Fig. 3, lower panel). Temporal changes in pituitary LHp mRNA, serum LH and ovarian GnRH mRNA levels during the proestrous stage in normal cycling rats The time course changes in LH~ mRNA levels during the proestrous stage were then examined. The pituitary LH~ mRNA levels were increased about 2-fold at 16:00 h and then returned to the basal levels (Fig. 4, upper panel). At the same time, serum LH levels were significantly elevated by 16:00 h in the proestrous stage and then gradually decreased to the basal levels (Fig. 4, middle panel). Ovarian GnRH mRNA levels, however, were markedly increased at 14:00 h as compared to those at 10:00 h, and remained at similar values until 18:00 h with a peak at 20:00 h (Fig. 4, lower panel). Likewise, in the PMSG-injected immature rats, serum LH levels were elevated at 16:00 h, 4 h before the peak of ovarian GnRH gene expression. ;:> ~ ] .. z '" E -c " 0 ~ i ;;- ;:; ~ ] < z '" E .. ::; r - ;:; ~ ] .. Z '" E 0: • c " ; .~ 2 0 2 I D ~ ~ D ~ ~ ~o Conlrol PIISG PIISG/ h CG ~o CONTROL PIISG P IISG/ hCG o Conlrol ~ PIISG ~ PIISG/ h CG ~2 ~2 ~O ~2 Time (h) after PMSG injection Figure 3. Effects of PMSG and hCG on hypothalamic GnRH mRNA (upper panel), pituitary LHB mRNA (middle panel) and ovarian GnRH mRNA (lower panel) levels. Fourth-eight hours after PMSG injection, animals were administered with saline and 10 iu hCG, then sacrificed at indicated times. Bar represents the mean (± S.E.) of repeated experiments (n = 3). *, vs the control and PMSG-injected. group (P<O.OI). Discussion The present study provides evidence that there are temporal relationships between hypothalamic Gn RH, pituitary LH~ and ovarian GnRH gene expression in PMSG-treated rats. These data indicate that ovarian GnRH gene expression appears to be under the infVol. 4 (1994) Sung Ho Lee el af. 43 « z a:: 8 1 50 8 40 "-.,. c 30 :5 8 ;:l 20 ... ., CfJ 10 0 ::i ~ -.; :> ~ 2 -<: z a:: S ::t: a:: c '-' C as .;: '" :> 0 0 10 14 16 18 20 22 • n ro ,.-, 10 14 16 18 20 22 . ,..-. ,.r- ,.r11 10 14 16 18 20 22 Time (h) of Proesh*us Figure 4. Temporal changes in pituitary LH~ mRNA (upper panel), serum LH (middle panel) and ovarian GnRH mRNA levels (lower panel) during proestrous stage in normal cycling rats. Pituitary LH~ and ovarian GnRH mRNA levels are expressed as an arbitrary units (pituitary LH~ mRNA level at 10:00 h and ovarian GnRH mRNA level at 14:00 h were set to 1.0 A.U). Bar indicates the mean S.E. of repeated experiments (n =4). *, vs other time points (P± 0.01). luence of classical hypothalamic-pituitary-ovarian hormonal axis in the rat. In the endocrine axis, activation of hormonal genes, such as hypothalamic GnRH, pituitary LH and ovarian GnRH was sequentially occurred in a well-ordered manner. The changes in ovarian GnRH and estradiol contents by PMSG also showed a similar pattern, indicating that ovarian GnRH is involved in ovarian steroidogenesis. It can be hypothesized that activation of ovarian GnRH gene expression in preovulatory follicles may require the endogenous LH surge. Administration of hCG to PMSG-treated rats suppressed hypothalamic GnRH and pituitary LH~ mRNA levels which were thought to be responsible for inducing the LH surge. It appears that there is a negative short-loop feedback of LH, which inhibited hypothalamic GnRH activity. During the proestrous stage of normal rats, ovarian GnRH production was significantly enhanced at 4 h after the preovulatory LH surge (Fig. 4). These results support the above notion. Hypothalamic GnRH is transported to pituitary gonadotropes through a specialized portal vessel and sti mulates the release of gonadotropins (FSH and LH) . Gonadotropins then regulate the production of steroids which modulate ovarian functions, such as production of intracellular proteins, cell proliferation and activation of steroidogenic enzymes. Several intraovarian regulators including GnRH can affect local cellular response and directly respond to gonadotropins (Tonetta and diZerega, 1989). Indeed, PMSG and/or hCG can stimulate ovarian inhibin (Davis et al., 1988), thymosin (Hall et al., 1991) and tissue plasminogen activator (tPA) (Ny et al., 1987; O'Connell et al., 1987). It has been known that PMSG is capable of inducing LH/hCG receptors in granulosa cells of preovulatory follicles (Richards et aI., 1989), and hCG exerts the LH-like effects on enhancing GnRH production in granulosa cells. The present study suggests that FSH may act as a primer and LH as a stimulator in ovarian GnRH gene expression. However, the possibility that FSH may have a direct stimulatory effect on GnRH production can not be excluded. Special notice has also taken of the regulatory mechanism(s) of oocyte maturation and ovulation. Since the preovulatory LH surge-like response is elicited by GnRH and its agonist (Hillensjo and LeMaire, 1980; Oekel et al., 1983), and, more importantly, the GnRH receptor is present in rat oocyte membrane (Oekel et al., 1988), it is possible to presume that there may be some common mechanism shared by GnRH and LH in the rat ovary (Oekel et al., 1985). Several lines of evidence suggest that tPA plays an important role in gonadotropins-induced ovulation process (Beers, 1975; Reich et al., 1985). Gonadotropins stimulate tPA activity and the activity of this protease is temporally correlated with ovulation. GnRH is able to increase tPA activity and mRNA levels in cultured rat granulosa cells (Ny et al., 1987). It appears then that an increase in tPA may represent a common pathway in the mechanism of ovulation induced by LH/hCG or GnRH. Indeed, in the present study, hCG directly enhanced ovarian GnRH synthesis before ovulation. It is possible to presume that LH/hCG may increase ovarian GnRH which, in tum, may induce tPA and eventually ovulation. Further studies are necessary for elucidation of the precise molecular mechanism of ovulation. 44 Regulation of Ovarian GnRH Mol. Cells Acknowledgment This work was supported by research grants from KOSEF through the Research Center for Cell Differentiation (92-2-2) and the Ministry of Education to K Kim. References Adelman, J. P., Mason, A J., Hayflick, J. S., and Seeburg, P. H . (1986) Proc. Natl. Acad. Sci. USA 83, 179183 Aten, R F , Williams, A T , and Behman, H. R (\ 986) Endocrinology 118, 961-967 Bahr, 1., and Ben-Jonathan, N . (1981) Endocrinology 108, 1815-1820 Bauer-Dantoin, A D., Hollenberg, A N., and Jameson, 1. L. (1993) The 75th Annu. Meeting Endocrinology (Las Vegas) Abst. No. 564 Beers, W. H. (1975) Cell 6, 379-386 Choi, W. S., Lee, S. H., Kim, H. S., Cho, S. S., Namkung, Y , Yoon, Y-D., Paik, S. H., and Cho, W. K (1990) Korean J Zool. 33, 435-445 Chomczynski, P ., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 Davis, R S., Burger, H. G., and Robertson, D. M. (1988) Endocrinology 123, 2399-2407 Dekel, N., Sherizly, I., Tsafriri, A, and Naor, Z. (1983) BioI. Reprod. 28, 161-166 Dekel, N., Sherizly, I., Phillips, D . M., Nimrod, A , Zilberstein, M ., and Naor, Z . (1985) J Reprod. Fertil. 75, 461-466 Dekel, N ., Lewysohn, 0., Ayalon, D., and Hazum, E. (1988) Endocrinology 123, 1205-1207 Ekholm, C, Hillensjo, T , and Isaksson, O. (1981) Endocrinology 108, 2022-2024 Feinberg, A P., and Vogelstein, B. (1984) Anal. Biochern. 137, 266-267 Goubach, S., Bond, C T , Adelman, 1. P., Misra, V , Hynes, M. F , Schultz, G. A, and Murply, B. D. (1992) Endocrinology 130, 3098-3100 Hall, A K , Aten, .R , and Behrman, H. R (1991) Endocrinology 128, 951-957 Hillensjo. T.. and LeMaire, W. 1. (1980) Nature 287, 145-146 Hsueh, A 1. W., Jones, P. B. C , Adashi, E. Y , Chang, C , Zhuang, L., and Welsh, T H. (\983) Reprod. Fertil. 69, 325-342 Ireland, J. 1., Aten R F , and Behrman, H. R (1988) BioI. Reprod. 38, 544-550 Kim, K, Lee, B. 1., Park, Y, and Cho, W. K (1989) Mol. Brain Res. 6, 151-158 Lee, B. 1., Kim, K , and Cho, W. K (\ 990) Mol. Brain Res. 8, 185191 Ny, T., Liu, Y-X., Ohlsson, M., Jones, P . B. C , and Hsueh, A 1. W. (1987) J BioI. Chern. 262, 1179011793 O'Connell, M., Canipari, R , and Strickland, S. (1987) J Bioi. Chern. 262, 2339-2344 Oikawa, M., Dargan, TN., and Hsueh, A J. W. (1990) Endocrinology 127, 2350-2356 Park, Y , Lee, B. J., Kim, K , and Cho, W. K (1988) The 8th Int'l Congress Endocrinology (Kyoto, Japan) Abst. No. 3049 Reich, R , Miskin, R , and Tsafriri, A (1985) Endocrinology 116, 516-521 Richards, J . S., Jahnsen, T., Hedin, L., Lifka, J ., Ratoosh, S., Durica, J. M., and Golding, N . (1989) Recent Prog. Horm. Res. 43, 231-270 Sambrook, 1., Fritsch, E. F, and Maniatis, T (1989) Molecular Clonning, 2nd Ed, Cold Spring Harbor Laboratory Press Seong, 1. Y , Lee, Y K , Lee, C C , and Kim, K (1993) Neuroendocrinology 58, 234-239 Sharpe, R M. (1982) J Reprod. Fert. 64, 517-527 Tepper, M . A, and Roberts, 1. L. (1984) Endocrinology 115, 385-391 Tonetta, S. A, and diZerega, G. S. (1989) Endocr. Rev. 10, 205-229 Ying, S. Y, Ling, N., Bohlen, P., and Guillemin, R (1981) Endocrinology 108, 1206-