J. E. FORTUNE,2 R. N. WISSLER,3

Transcription

1 BIOLOGY OF REPRODUTION 35, 9-99 (986) Prolactin Modulates Steroidogenesis by Rat Granulosa ells: II. Effects on Estvadiol J. E. FORTUNE, R. N. WISSLER,3 and S. E. VINENT Section of Physiology in the Division of Biological Sciences Women s Studies Program in the ollege of Arts and Sciences and Department of Physiology in the ollege of Veterinary Medicine ornell University Ithaca, Ne York 4853 ABSTRAT The effects of prolactin on secretion of estradiol by rat granulosa cells obtained at to stages of differentiation and cultured under various conditions ere determined. Relatively undifferentiated granulosa cells ere obtained from immature, diethylstilbestrol-treated, hypophysectomied (HPX) rats and cultured in serumfree medium or ith % serum. More highly differentiated granulosa cells ere obtained on the morning of proestrous from the preovulatory follicles of immature rats in hich an estrous cycle had been induced ith 4 JU pregnant mare s serum gonadotropin; these cells ere cultured in medium containing % serum. ells ere cultured for 3 days ith graded doses of prolactin (,.,.,, or pg/ml) alone or in combination ith follicle-stimulating hormone (FSH; 3 ng/ml), testosterone (.5 pm), or FSH + testosterone. In control cultures (no prolactin) the relatively undifferentiated granulosa cells from HPX rats secreted negligible quantities of estradiol except hen both FSH and testosterone ere supplied. Prolactin alone or in combination ith FSH or testosterone bad no effect on estradiol secretion, but prolactin in combination ith FSH + testosterone significantly decreased secretion in a dose-dependent fashion. This set of prolactin treatments as applied in both serum-free medium and medium containing % serum, ith similar results under both culture conditions. The inhibitory effects of prolactin appeared to be reversible if cells ere cultured ith prolactin for only day, but ere not reversed ifcells ere cultured ith prolactin for or 3 days. Prolactin inhibited secretion of estradiol by cultures that had been exposed initially to FSH + testosterone (to induce aromatase), but the prior exposure to FSH increased the lag time before the effects of prolactin ere evident. ells from proestrous rats secreted only small amounts of estradiol in medium alone or ith FSH. Either FSH or FSH #{47} testosterone greatly increased estradiol secretion on the first days of culture, but only FSH #{47} testosterone maintained aromatase activity on the third day of culture. Prolactin alone or in combination ith FSH had no effect on estradiol secretion. On the third day of culture, prolactin significantly increased estradiol secretion in the presence of testosterone alone, but decreased secretion hen both FSH and testosterone ere present. In summary, under various culture conditions and at to different stages of differentiation, prolactin inhibited FSH-stimulated secretion of estradiol. The data suggest that prolactin inhibits secretion of estradiol by rat granulosa cells by inhibiting the induction of aromatase by FSH. INTRODUTION Lactational anestrous/amenorrhea is a common phenomenon among mammalian species. Suckling and the accompanying hyperprolactinemia suppress Accepted December, 985. Received February 4, 983. This study as supported by NIH grant HD Reprint requests: J. E. Fortune, 83 Veterinary Research Toer, ornell University, Ithaca, NY 4853 NSF predoctoral fello. ovarian activity, but the degree and duration of ovarian inhibition vary among species (revieed by McNeilly et al., 98). It has been postulated that prolactin suppresses follicular development by acting at the level of the hypothalamus and/or pituitary to depress circulating gonadotropins. In omen, levels of follicle-stimulating hormone (FSH) are normal but luteiniing hormone (LH) concentrations are lo during the early postpartum period. Later, LH increases to normal basal levels due to decreases in the suckling stimulus and in circulating prolactin; hoever, follicular development is still abnormal and 9 Donloaded from on December 7

2 PROLATIN MODULATES ESTRADIOL SERETION 93 deficient luteal phases are common (McNeilly et al., 98). Hence, it seems possible that iirolactin may suppress follicular development indirectly via effects at the level of the hypothalamus/pituitary and directly by acting on one or more aspects of follicular development and function. Estradiol secretion is the hallmark of developing preovulatory follicles, and estradiol exerts positive feedback on follicular development by promoting mitosis of granulosa cells and acting ith FSH to increase the number of LH receptors (Richards, 98). Therefore, if prolactin inhibited secretion of estradiol, this could explain its inhibitory effects on ovarian activity. Data from several laboratories suggest that in rats prolactin acts directly to reduce follicular production of estradiol and aromatase activity (Uilenbroek et al., 98; Tsai-Morris et al., 983; Uilenbroek and van der Linden, 984; Kalison et al., 985). In rodents, estradiol production is believed to require the interaction of the to types of follicular cells (Falck, 959), ith theca cells producing androgen for aromatiation to estradiol by granulosa cells (Makris and Ryan, 975; Fortune and Armstrong, 977, 978). Therefore, either theca or granulosa cells are potential targets for prolactin, if it acts on the follicle to suppress estradiol secretion. Hoever, granulosa cells appear to be more likely targets for prolactin, since results of binding studies and histochemical localiations indicate that rat granulosa cells have receptors for prolactin (Midgley, 973; Richards and Williams, 976; Wang et al., 979; Dunaif et al., 98), hile theca cells do not appear to bind prolactin (Dunaif et al., 98). Interest in direct effects of prolactin on steroidogenesis by granulosa cells has blossomed recently, and a multitude of effects has been observed. Prolactin is reported to depress aromatiation by granulosa cells from immature rats primed ith estrogen (Dorrington and Gore-Langton, 98, 98), hypophysectomied, immature rats primed ith estrogen and FSH in vivo (Wang et al., 98), and proestrous rats (Wang and han, 98). In contrast, various effects of prolactin on secretion of progesterone by granulosa cells have been observed. Prolactin is reported to inhibit production of progesterone by murine follicles (McNatty et al., 976) and human granulosa cells (McNatty et al., 974) and to stimulate secretion of progesterone by granulosa cells from humans (McNatty et al., 974) and rats (Wang et al., 98; Wang and han, 98). Veldhuis et al. (98) have reproted that prolactin inhibited progesterone secretion by porcine granulosa cells from small follicles, but stimulated secretion by cells from large follicles. Veldhuis and Hammond (98) have shon that estradiol and prolactin interact synergistically to depress production of progesterone by granulosa cells from small porcine follicles early in the culture period and to stimulate production later in the culture period. The actions of prolactin on steroidogenesis by granulosa cells in vitro appear to be complex and dependent pn the dose of prolactin, stage of differentiation of the granulosa cells, and the culture conditions. In this study, e have attempted to elucidate further the effects of prolactin on secretion of estradiol and progesterone by granulosa cells. We applied a range of doses of prolactin, both alone and in combination ith other hormones knon to affect production of estradiol and progesterone, to rat granulosa cells obtained at to different stages of differentiation and cultured under various conditions. This paper presents the results obtained for estradiol production, and the preceding companion paper contains results for progesterone secretion (Fortune and Vincent, 986). MATERIALS AND METHODS Experimental Models and Isolation and ulture of Granulosa ells Granulosa cells at to different stages of development ere obtained by using to different rat models. ) The ovaries of hypophysectomied (HPX) rats treated ith diethylstilbestrol (DES) yielded large numbers of relatively undifferentiated granulosa cells that ere not under the influence of endogenous gonadotropins at the time they ere isolated. ) Intact, immature rats ere injected ith 4 IU pregnant mare s serum gonadotropin (PMSG) on Day 8 of life to induce a normal estrous cycle; the granulosa cells isolated from the - largest follicles on the morning of proestrous ere relatively ell differentiated. Details on these to experimental models and on the isolation and culture of the granulosa cells are given in the preceding companion paper (Fortune and Vincent, 986). Briefly, granulosa cells ere isolated from ovaries of HPX rats and from individual, preovulatory follicles of proestrous rats and cultured (, cells in 5 pl medium) in medium alone (Eagle s minimum essential medium, MEM) or ith Donloaded from on December 7

3 94 FORTUNE ET AL. testosterone (.5 pm), FSH (NIH-FSH-S8; 3 ng/ ml), testosterone + FSH, or various concentrations of prolactin (NIH-P-S-;,.,., and pg/ ml) alone or in combination ith the other treatments. In some experiments, culture media contained % fetal bovine serum. Treatments ere replicated ithin experiments and experiments ere repeated ith cells from at least to groups of animals. Media ere collected and replaced daily for 3-6 days. Media ere froen for later steroid analyses. Radio immu noassays and Statistics Media samples ere assayed ithout extraction for estradiol-73 by radioimmunoassay as described previously (Fortune and Armstrong, 977). The estradiolantiserum (GDN-S44) as kindly provided by Dr. Gordon Nisender and is highly specific for estradiol- 73 (Korenman et al., 974). Data ere expressed per, cells and subjected to analysis of variance. Specific comparisons ere made using Duncan s multiple range test. Data ere transformed to logarithms hen heterogeneity of variance as present. RESULTS Effects of Prolactin on Estradiol Secretion by Granulosa ells from HPX Rats ultured in Defined Medium Effects of prolactin. Granulosa cells secreted estradiol only hen culture media included both FSH and testosterone (Fig. A). These results, shoing that FSH stimulates estradiol secretion under these conditions, are similar to those reported previously (Dorrington et al., 975; Erickson and Hsueh, 978). Prolactin alone (.,.,, or pg/ml) had no effect on production of estradiol (data not shon), nor did the same doses of prolactin in combination ith testosterone or ith FSH modify secretion of estradiol (data not shon), i.e., production as negligible, as shon in Figure A for testosterone and FSH alone. Hoever, prolactin added to cultures containing FSH + testosterone inhibited accumulation of estradiol in a dose-dependent manner (Fig. ). During the second and third days of culture all except the loest dose of prolactin significantly inhibited accumulation of estradiol in the medium, and both and pg/mi ere maximally effective (p<.5). Reversibility of prolactin effects on estradiol secretion. Granulosa cells ere cultured ith prolactin (,. or pg/mi) and FSH + testosterone for, or 3 days. After this initial culture period in the presence of prolactin, cultures ere rinsed ith ml MEM and cultured for an additional 3 days ith FSH + testosterone alone. When cells ere cultured ith prolactin for or 3 days, the suppressive effects of prolactin ere not reversed during the subsequent 3 days of culture in the absence of prolactin (Fig. 3, middle and loer panels). When cells ere exposed to prolactin for only day, the inhibitory effects of prolactin ere attenuated during the first days of culture ithout prolactin; by the third day after the removal of prolactin there as no difference (p>.5) in secretion of estradiol by cells exposed to prolactin compared to secretion by control cells (Fig. 3, upper panel). Therefore, the effects of prolactin appeared to be reversible if the cells ere exposed for + 4) U I ĪiJ :: -J (I) IjJ A. HPX.O B. HPX (% SERUM) 3 DAYS OF ULTURE FIG.. Estradiol secretion (ng/, cells ± SEM; n 6 cultures) by granulosa cells from HPX rats cultured in defined medium (panel A) or medium containing % serum (panel B). ells ere cultured in medium alone (), ith FSH (F; 3 ng/ml), testosterone (T;.5 pm) or FSH + testosterone (F + T). F+T Donloaded from on December 7

4 PROLATIN MODULATES ESTRADIOL SERETION 95 cr)+ Q. 5 pg ug. ug in cultures containing prolactin (p<.). Therefore, prolactin inhibited secretion of estradiol by cells in hich aromatase had already been induced by FSH, but longer prior exposure to FSH seemed to increase the lag time before the effects of prolactin ere evident. () -Jo ug pg Effects of Prolactin on Estradiol Secretion by Granulosa ells from HPX Rats ultured ith Fetal Bovine Serum a cr) c /)- DAYS OF ULTURE We had observed that adding % fetal bovine serum to the culture medium radically changed the effects of prolactin on secretion of progesterone by granulosa cells from HPX rats (see preceding companion paper, Fortune and Vincent, 986). To determine if serum has a similar effect on secretion of estradiol, FIG.. Estradiol secretion (ng/, cells ± SEM; n = 6 cultures) by granulosa cells from HPX rats cultured ith FSH (3 nglml), testosterone (.5 M) and graded doses of prolactin (,.,., or pg/ml) in defined medium. only one day, but a longer exposure caused an inhibition that as not reversed ithin 3 days. Effects of delayed addition ofprolactin to granulosa cell cultures. The results presented in Figure indicated that prolactin inhibited either the aromatiation of testosterone or the induction of aromatase by FSH. To determine if cells in hich aromatase had already been induced ere as susceptible to the inhibitory effects of prolactin, cells ere cultured ith FSH + testosterone for, or 3 days. After this initial culture period, prolactin, at doses that had been inhibitory hen added at the beginning of culture (. and pg/ml), as included in the culture medium for an additional 3 days (i.e., cells ere cultured for a total of 4, 5 or 6 days). When prolactin as added after day of culture ith FSH + testosterone, its inhibitory effects ere even greater than hen prolactin as added at the beginning of the culture period (Fig. 4, upper panel, vs. Fig. ). When the addition of prolactin as delayed until 48 h of culture (Fig. 4, middle panel), its effects appeared similar to those hen prolactin as added at Time. In contrast, addition of prolactin at 7 h had no apparent effect on estradiol secretion for the next 4 h (Fig. 4, loer panel). Over the subsequent 48 h (Days 4-6) secretion of estradiol by control cultures decreased, but the decrease as significantly greater /) + a) ci).) ) /) -J (I) DAYS OF ULTURE FIG. 3. Estradiol secretion (ng/, cells ± SEM; n = 8 cultures) by granulosa cells from HPX rats cultured in defined medium ith FSH (3 ng/ml) and testosterone (.5 pm) for the duration of culture and ith prolactin (PRL;. or pg/mi) for the first, or 3 days of culture. Donloaded from on December 7

5 96 FORTUNE ET AL. /) 4,) ci) ), -j,) 3 4 DAYS OF ULTURE FIG. 4. Estradiol secretion (ng/, cells ± SEM; n = 8 cultures) by granulosa cells from HPX rats cultured in defined medium ith FSH (3 ng/ml) and testosterone (.5 sm) for the duration of culture and ith prolactin (PRL;. or l.lg/ml) for the last 3 days of culture. since the maximally effective dose as tenfold loer, but maximal inhibition as 4%, as it as in the absence of serum (Fig. vs. Fig. 5). When granulosa cells ere cultured ith % serum and prolactin alone (.- pg/mi), prolactin + testosterone, or prolactin + FSH, secretion of estradiol as similar (data not shon) to that observed in control, testosterone, or FSH medium (see Fig. B). Effects of Prolactin on Granulosa ells from Proestrous Follicles Granulosa cells from proestrous follicles ere alays cultured in media containing % fetal bovine serum since these cells, in contrast to those from HPX rats treated ith DES, attach poorly in serum-free medium. When granulosa cells ere cultured in control medium or ith FSH, estradiol secretion as belo the sensitivity of the radioimmunoassay (Fig. 6). With the addition of testosterone, estradiol as maintained at detectable but steadily declining levels throughout 3 days of culture. Testosterone and FSH together ere as effective as testosterone alone on the first days of culture, but increased secretion of estradiol about sevenfold on the third day, as cells from HPX rats ere cultured in media containing % fetal bovine serum. Figure lb shos estradiol secretion in cultures containing % serum and FSH and/or testosterone. As in the absence of serum (Fig. A), only cultures containing FSH and testosterone produced measurable quantities of estradiol. Hoever, on the second and third days of culture, estradiol secretion as 3- to 4-fold loer in the presence of serum than in its absence. In the presence of serum, prolactin inhibited secretion of estradiol in cultures containing FSH + testosterone, but the dose-response curve as altered (Fig. 5). The loest dose of prolactin (. pg/mi) as not significantly different at any time from controls cultured ithout prolactin (p>.5). Hoever, on the third day of culture,. pg/ml as maximally effective in suppressing accumulation of estradiol (p<.ol) and the effects of this dose ere not significantly different (p>.5) from those of higher doses ( and pg/ml). Therefore, in the presence of serum, granulosa cells ere more sensitive to prolactin.8 -. I W. + Wa, -Jo - o.6 ) #{76}.. W,. iig PRL 3 DAYS OF ULTURE FIG. 5. Estradiol secretion (ng/, cells ± SEM; n = 6 cultures) by granulosa cells from HPX rats cultured ith FSH (3 ng/ml), testosterone (.5 pm), and graded doses of prolactin (PRL;,.,., or pg/mi) in medium containing % fetal bovine serum. Donloaded from on December 7

6 PROLATIN MODULATES ESTRADIOL SERETION FSH in vivo secrete estradiol early in the culture period in the presence of androgen alone, but secretion declines to lo levels unless FSH is also present (Fig. 6; Erickson and Hsueh, 978; Fortune and.5 Armstrong, 978). These findings have suggested that,,, -8 <.- ILl..5 I. FSH induces aromatase and that, at least in rodents, levels of aromatase are maintained only if FSH is continually present to induce more enyme. The presence of serum in culture media diminished, but did not abolish, the effects of FSH on estradiol secretion (Fig. B). Hence, in this study the secretion of estradiol in the absence of added prolactin as at the levels expected on the basis of our on and other studies. F T F+l FIG. 6. Estradiol secretion (ng/, cells ± SEM, n = 6 cultures) by granulosa cells from proestrous rats cultured in medium containing % serum. ells ere cultured in medium alone (), ith FSH (F; 3 ng/ml), testosterone (T;.5 pm), or FSH and testosterone (F + T). A. PrI + T + FSH compared to cultures treated ith testosterone,) (p<o.ol). Granulosa cells ere cultured ith various + concentrations of prolactin (,.,., or DAY DAY3 pg/rnl) either alone or in combination ith FSH, testosterone, or FSH + testosterone. Prolactin alone or in combination ith FSH had no effect on estradiol secretion (data not shon), i.e., it as negligible, as in the absence of prolactin (Fig. 6). When prolactin as administered in combination ith FSH + testosterone, it had no significant effect on the first, or second days of culture, but, at doses of. pg/mi or greater, prolactin suppressed accumulation of estradiol on the third day of culture to about 5% of the levels in cultures ith no prolactin (p<.o; Fig. 7A). Therefore, it appears that prolactin inhibited the actions of FSH in maintaining aromatase activity. Paradoxically, hen cells ere treated ith prolactin and testosterone, prolactin had no effect on estradiol Ui Ui (I) -J 4 3 B. Pri + I secretion until the third day of culture hen the to highest doses ( and pg/mi) significantly increased estradiol accumulation (p<.; Fig. 7B). Ui DAV DAY3 DISUSSION Aromatiation by rat granulosa cells requires FSH to induce aromatase and an androgen substrate. When granulosa cells have not been stimulated by FSH in vivo, they secrete only negligible quantities of estradiol in vitro unless both FSH and an androgen precursor are provided (Fig. ; Dorrington et al., 975). In contrast, granulosa cells that have been stimulated by ONENTRATION OF PROLATIN (ug/mi) FIG. 7. Estradiolsecretion(ng/,cells± SEM; n = cultures) by granulosa cells from proestrous rats cultured ith A) FSH (3 ng/ ml), testosterone (T;.5 pm) and graded doses of prolactin (PrI;,.,., or pg/mi) or B) testosterone (T;.5 pm) and graded doses of prolactin (PrI;.,., or pg/mi). Donloaded from on December 7

7 98 FORTUNE ET AL. Over a range of doses, prolactin alone or prolactin in combination ith FSH had no effect on estradiol secretion. Therefore, prolactin, as one might expect, had no effect on the ability of granulosa cells to synthesie androgen precursor; the requirement for exogenous androgen as unchanged. Prolactin in combination ith testosterone had no effect on the secretion of estradiol by granulosa cells in hich aromatase had not been induced (i.e., cells from HPX rats cultured ith or ithout serum). It is not surprising that prolactin did not alter the requirement for FSH as the inducer of aromatase. In contrast, in cultures of granulosa cells from proestrous rats in hich aromatase had been induced by FSH in vivo, the to highest doses of prolactin increased secretion of estradiol on Day 3 of culture above the negligible levels observed ith testosterone alone or in combination ith the to loer doses of prolactin. This suggests that prolactin may have some protective effect on existing aromatase. Hoever, it is difficult to kno if this effect is physiologically important. This result is in contrast to a report by Wang and hang (98); in their experiments, the secretion of estradiol by granulosa cells from proestrous rats as depressed by prolactin in the presence of androgen later in the culture period (Days -4 and 4-6). Hoever, their results ere also different from ours in that estradiol secretion continued to increase in the presence of androgen alone, hile e have alays found that in the absence of FSH, estradiol levels decline to very lo or nondetectable levels by the third day of culture (Figs. 6, 7A; Fortune and Armstrong, 978). In all three experimental systems used in these studies, prolactin inhibited the actions of FSH in stimulating aromatiation of exogenous androgen to estradiol (Figs., 5, 7A). This is consistent ith other recent reports (Wang et al., 98; Dorrington and Gore-Langron, 98, 98; Wang and han, 98). In the current study, the presence of % serum in the culture medium appeared to enhance the sensitivity of granulosa cells to the inhibitory effects of prolactin (Figs. 5 and 7A vs. Fig. ). Hoever, stage of differentiation did not seem to affect sensitivity to prolactin if cells ere cultured under the same conditions (i.e., % serum; Fig. 5 vs. Fig. 7A). When granulosa cells from HPX rats ere cultured ith FSH and androgen for, or 3 days and then exposed to prolactin, prolactin still inhibited estradiol secretion (Fig. 4). Hoever, the longer the initial period of culture in the absence of prolactin, the more delayed ere the inhibitory effects. This suggests that the prolactin acts on the induction of ne aromatase by FSH rather than interfering ith the functioning of existing aromatase. This conclusion is also consistent ith the effects of prolactin observed ith cells from proestrous rats. The third day of culture as the only day hen FSH + testosterone increased secretion of estradiol above levels observed ith testosterone alone, presumably because FSH increased aromatase levels (Fig. 6). This as also the only day hen prolactin inhibited estradiol secretion (Fig. 7A). Experiments by Dorringron and Gore-Langton (98) have indicated that prolactin probably interferes ith the induction of aromatase by acting distal to the formation of cyclic AMP. Reversibility of the inhibitory effects of prolactin appears to depend on the length of exposure to prolactin since cells eventually overcame the effects of exposure to prolactin for day, but not exposure for or 3 days (Fig. 3). In contrast, Dorrington and Gore-Langton (98) have reported that the inhibitory effects of a 4-h exposure to g prolactin/ml on granulosa cells from intact, immature rats primed ith DES had been reversed 33 h after removal of prolactin. Various studies have suggested that changes in stage of granulosa cell differentiation, dose of prolactin, and culture conditions can all reverse, at least in some situations, the effects of prolactin on steroid production. Hoever, these dichotomous reports have come primarily from studies of prolactin s effects on progesterone secretion. The studies focusing on proiactin s effects on estradiol secretion have all suggested that prolactin inhibits the induction of aromatase by FSH in vitro. These inhibitory effects of prolactin in vitro are consistent ith recent reports that elevated prolactin in vivo can suppress the ability of rat follicles to produce estradiol in vitro in the absence of any depression in plasma LH (Uilenbroek et al., 98; Uilenbroek and van der Linden, 984). In addition, McNatty (979) has reported that in humans, concentrations of prolactin ere lo in antral follicles hen plasma prolactin as lo, but ere significantly elevated hen plasma prolactin as high. High concentrations of prolactin in follicular fluid ere accompanied by lo levels of FSH and estradiol in the fluid and by reduced numbers of granulosa cells. Taken together, these studies in vitro and in vivo provide support for a direct inhibitory effect of prolactin on follicular secretion of estradiol. Donloaded from on December 7

8 PROLATIN MODULATES ESTRADIOL SERETION 99 In summary, the results presented here sho that prolactin inhibits estradiol secretion by granulosa cells obtained at to different stages of differentiation and cultured under various conditions. In contrast, secretion of progesterone by the same cultures as usually increased, but sometimes inhibited, by prolactin, depending on culture conditions (see preceding companion paper, Fortune and Vincent, 986). Therefore, prolactin suppressed estradiol secretion under conditions in hich it enhanced progesterone secretion, as ell as under conditions in hich it depressed progesterone secretion. The effects of prolactin on secretion of estradiol appear to be exerted primarily via inhibition of the induction of aromatase by FSH and to be irreversible if exposure to prolactin is longer than day. Since production of estradiol is crucial to successful follicular development, these inhibitory effects of prolactin on estradiol secretion by isolated granulosa cells lend support to the idea that direct effects of prolactin on the ovary may account, at least in part, for the suppression of follicular development that occurs during hyperprolactinemic states. AKNOWLEDGMENTS We are grateful to Dr. G. D. Nisender for supplying estradiol antiserum, to the National Pituitary Agency for gifts of ovine prolactin and FSH, and to H. Dormady for typing the manuscript. REFERENES Dorrington J, Gore-Langton RE, 98. Prolactin inhibits oestrogen synthesis in the ovary. Nature 9:6- Dorrington JH, Gore-Langton RE, 98. Antigonadal action of prolactin; further studies on the mechanism of inhibition of folliclestimulating hormone-induced aromatase activity in rat granulosa cell cultures. Endocrinology :7-7 Dorrington JH, Moon YS, Armstrong DI, 975. Estradiol-73 biosynthesis in cultured granulosa cells from hypophysectomied immature rats; stimulation by follicle-stimulating hormone. Endocrinology 97:38-3 Dunaif AE, immerman EA, Friesen HG, Frant AG, 98. Intracellular localiation of prolactin receptor and prolactin in the rat ovary by iinmunocytochemistry. Endocrinology :465-7 Erickson GF, Hsueh AJW, 978. Stimulation of aromatase activity by follicle stimulating hormone in rat granulosa cells in vivo and in vitro. Endocrinology :75-8 Falck B, 959. Site of production of oestrogen in rat ovary as studied in micro-transplants. Acts Physiol Scand, Vol. 47, Suppl. 63 Fortune JE, Armstrong DT, 977. Androgen production by theca and granulosa isolated from proestrous rat follicles. Endocrinology : Fortune JE, Armstrong DT, 978. Hormonal control of 7j3-estradiol biosynthesis in proestrous rat follicles: estradiol production by isolated theca versus granulosa. Endocrinology :7-35 Fortune JE, Vincent SE, 986. Prolactin modulates steroidogenesis by rat granulosa cells: I. Effects on progesterone. Biol Reprod 35: 84-9 Kalison B, WarshaML, Gibori G, 985. ontrasting effects of prolactin on luteal and follicular steroidogenesis. J Endocrinol 4:4-5 Korenman SG, Stevens RH, arpenter LA, Robb M, Nisender GD, Sherman BM, 974. Estradiol radioimmunoassay ithout chromatography: procedure, validatiop and normal values. J lin Endocrinol Metab 34:78- Makris A, Ryan KJ, 975. Progesterone, androstenedione, testosterone, estrone, and estradiol synthesis in hamster ovarian follicle cells. Endocrinology 96:694-7 McNatty K?, 979. Relationship beteen plasma prolactin and the endocrine microenvironment of the developing human antral follicle. Fertjl Steril 3: McNatty K?, Neal F, Baker TG, 976. Effect of prolactin on the production of progesterone by mouse ovaries in vitro. J Reprod Fertil 47:55-56 McNatty KP, Saers RS, McNeilIy AS, 974. A possible role for prolactin in control of steroid secretion by the human Graafian follicle. Nature 5: McNeiIly AS, Glasier A, Jonassen J, Hoie PW, 98. Evidence for direct inhibition of ovarian function by prolactin. J Reprod Fertil 65: Midgley AR Jr, 973. Autoradiographic analysis of gonadotropm binding to rat ovarian tissue sections. Advances in Exp Med & Biol 36: Richards JS, 98. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiological Revies 6:5-89 Richards JS, Williams JJ, 976. Luteal cell receptor content for prolactin (PRL) and luteiniing hormone (LH): regulation by LH and PRL. Endocrinology 99:57-8 Tsai-Morris H, Ghosh M, Hirschfield AN, Wise PM, Brodie AMH, 983. Inhibition of ovarian aromatase by prolactin in vivo. Biol Reprod 9:34-46 Uilenbroek JThJ, van der Linden R, 984. Effects of prolactin on follicular oestradiol production in the rat. J Endocrinol : 45-5 Uilenbroek JThJ, van der Schoot P, den Besten D, Lankhorst RR, 98. A possible direct effect of prolactin on follicular activity. Biol Reprod 7:9-5 Veldhuis JD, Hammond JM, 98. Oestrogens regulate divergent effects of prolactin in the ovary. Nature 84: 6-64 Veldhuis JD, Klase F, Hammond JM, 98. Divigerent effects of prolactin upon steroidogenesis by porcine granulosa cells in vitro: influence of cytodifferentiation. Endocrinology 7:4-46 Wang, han V, 98. Divergent effects of prolactin on estrogen and progesterone production by granulosa cells of rat Graafian follicles. Endocrinology :85-93 Wang, Hsueh AJW, Erickson GF, 979. Induction of functional prolactin receptors by follicle-stimulating hormone in rat granubaa cells in vivo and in vitro. J Biol hem 54: Wang, Hsueh AJW, Erickson GF, 98.?rolactin inhibition of estrogen production by cultured rat granulosa cells. Mol ell Endocrinol :35-44 Donloaded from on December 7