Effect of prolactin on the steroidogenic response of rat luteal cells

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Molecular and Cellular Endocrinology, 36 (1984) 187-194 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01168

Effect of prolactin on the steroidogenic response of rat luteal cells Marta Tesone

*, Ruth G. Ladenheim,

Violeta A. Chiauzzi and Eduardo

H. Charreau

Insrituto de Biologia y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, and Departamento de Quimica Biohgica, Fact&ad de Ciencias Exactasy Natwales, Universidad de Buenos Aires (Argentina) (Received 16 January 1984; accepted 14 March 1984)

Keywordr:

luteal cells; prolactin; progesterone and CAMP production; LH receptors.

Summary The role of prolactin on some ovarian functions was studied in collagenase-dispersed luteal cells obtained from PMSG/hCG-primed rats. The in vitro effect of ovine prolactin (oPr1) on luteal cell function was assayed. This hormone produced a dose-dependent increase of progesterone production and an additive effect on hCG stimulation. oPr1 had no effect on CAMP production. Chronic effects of prolactin were studied in sulpiride (S), bromocriptine (Br) and oPrl-treated rats. Serum levels of prolactin were significantly higher in S-treated animals whereas Br administration rendered undetectable values. Serum progesterone was reduced in Br-treated animals and LH levels were similar in all groups studied. In vitro studies demonstrated a marked reduction of hCG stimulation of progesterone and CAMP production by luteal cells from hypoprolactinemic animals, while a significant increase was observed in hyperprolactinemic states. oPr1 and S treatment significantly increased ovarian LH binding sites while a reduction was observed in Br-treated rats. These data suggest that luteal cell function is regulated by circulating levels of prolactin and that this hormone has some direct effect on the steroidogenic process.

The luteotropic action of prolactin in the rat has been clearly established (Astwood, 1941; Evans et al., 1941; Macdonald et al., 1970). The classical works of Armstrong and co-workers (1969) describe an increase in the ability of LH to stimulate progesterone secretion in corpora lutea of prolactin-treated rats. Luteal function in hypophysectomized rats can be maintained by endogenous prolactin from pituitary gland implants (Everett, 1956; Macdonald et al., 1973) or by exogenous prolactin (Macdonald et al., 1973; Takayama and Greenwald, 1973). Low prolactin levels obtained by ergot alkaloids (Wuttke et al., 1971; Clemens et al., 1974) interfere with luteal function, interrupting early pregnancy and pseudopregnancy * To whom correspondence should be addressed. 0303-7207/84/$03.00

(Shelesnyak, 1955), while hyperprolactinemia induced in immature female rats by chronic treatment with sulpiride results in advancement of the onset of puberty (Advis and Ojeda, 1978). Prolactin appears to provide the principal luteotropic stimulus for lengthening progesterone secretion from day 2 through day 7 of rat pregnancy (Smith et al., 1975; Diiler and Wuttke, 1974) while LH appears to be essential between days 8 and 12 of pregnancy. It is well established that prolactin can induce the formation of LH receptors in the ovary of pseudopregnant rats. LH in the absence of prolactin induces the formation of corpora lutea which have reduced capability of progesterone production, but if prolactin is present during the early luteinization process, corpora lutea develop an en-

0 1984 Elsevier Scientific Publishers Ireland, Ltd.

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hanced ability to bind LH and to produce progesterone (Grinwich et al., 1976; Holt et al., 1976). These facts are well correlated with the increased capacity of the luteal adenylyl cyclase to respond to LH stimulation after prolactin treatment (Day et al., 1980). On the other hand, acute ovarian stimulation by prolactin has been investigated with different results depending on the experimental model used (Huang and Pearlman, 1962; Marsh et al., 1966; Veldhuis et al., 1980). Accordingly, the present experiments were designed to determine the in vitro effects of prolactin in progesterone and CAMP production by isolated luteal cells. In addition, chronic effects of prolactin were studied in hyper- or hypoprolactinemic states. For this purpose, sulpiride (S), bromocriptine (Br) and ovine prolactin (oPr1) were administered to immature PMSG/hCG-primed rats. In each experimental group LH receptors and progesterone and CAMP production by luteal cells were measured, as were serum levels of Prl, LH and progesterone. Part of the results has been presented in abstract form elsewhere (Tesone et al., 1981). Materials and methods Materials Carrier-free “‘1 (100 mCi/ml) was purchased from New England Nuclear (Boston, MA) or the Radiochemical Centre (Amersham, U.K.). Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (Endocoriona) used for pseudopregnancy induction and highly purified hCG (11500 U/mg) were kindly donated by Dr. Eduardo Passeron from Laboratories Elea (Buenos Aires). [1,2,6,7-3H(N)]progesterone (98.3 Ci/ mmole) and [2,8- 3H]adenosine-3’,5’-cyclic phosphate (cyclic AMP, 34.6 Ci/mmole) were purchased from New England Nuclear. Progesterone cyclic AMP and bovine serum albumin, fraction V (BSA) were from Sigma Chemical Co. (St Louis, MO), medium 199 was from Difco Laboratories (Detroit, MI). Collagenase type 4176 CLS II 49 A 087 (153 U/mg) was from Worthington Biochemical Co. (Freehold, NJ). Human LH (hLH) (31079) was from the Hormone and Isotope Laboratory, Aker Hospital (Oslo, Norway) and oPr1 (NIH-PS-14) was kindly

supplied by NIAMDD (Bethesda, MD). According to the specifications of NIAMDD, the LH activity of oPr1 measured by the ovarian ascorbic acid depletion assay was less than 0.005 NIH-LHSl units/mg. In addition, the absence of LH activity of the oPr1 NIH-PS-14 preparation was demonstrated using a sensitive radioligand-receptor assay for LH determination (Catt et al., 1974). This fact was also confirmed by the inability of oPr1 to stimulate in vitro testosterone production by isolated Leydig cells. Other reagents and chemicals were of analytical grade. Animals Female Wistar rats, 25-30 days old, fed Purina rat chow ad libitum and kept in an air-conditioned, light-controlled environment (lights on from 0700 to 1900 h) were used throughout. The animals were injected with PMSG (25 II-J/rat) and 48 h later with hCG (50 IU/rat). Beginning 72 h after the PMSG injection, oPr1 (freshly dissolved in saline solution at pH 10) was administered by injection twice daily at a dose of 1 mg/kg body weight (BW)/day. Bromocriptine (Br) (Sandoz, Basel, Switzerland) was prepared by dissolving equal quantities of Br and tartaric acid in ethanol-saline (30 : 70, v/v). It was given with the same schedule as oPr1 at a dose of 3.0 mg/kg BW/day. Sulpiride (S) (Vipral, Roemmers, Buenos Aires, Argentina) was diluted with saline solution and S.C. injections were administered twice daily at a dose of 30 mg/kg BW/day. Control animals received vehicle only. The different treatments took place during the 7 days between hCG administration and sacrifice. A separate group of rats was primed with PMSG/hCG only and used in studies on in vitro effects of Prl on luteal cells. All animals were sacrificed 10 days after PMSG injection and pseudopregnant state was assessed by visual inspection of the ovaries at the sacrifice and further by optical microscopy (see below). The animals were killed by decapitation without anesthesia and trunk blood was collected for hormone determinations. Blood was allowed to clot and serum stored at -2O’C until assays were performed. Preparation of collagenase-dispersed Luteal cells from pseudopregnant

luteal cells rats were iso-

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lated as described previously (Tesone et al., 1983). In brief, ovaries which had been placed in medium 199 containing 0.1% BSA were finely minced and washed to remove blood cells. The minced tissue was suspended in medium 199 containing 2 mg/ml of collagenase, using 1 ml per ovary. The tubes were shaken at 37°C for 30 min. The supernatant containing the cells was withdrawn. The cell suspension was passed through Nytex into a plastic bottle on ice. The residual particles were washed with medium until the supernatant was clear. The combined supernatants were centrifuged at 250 X g for 5 min at 4°C. The cell pellet was resuspended in the same medium at a ratio of 1 ml per ovary and counted in a Levy ultraplane counting chamber after being stained with methylene blue. The effectiveness of the treatment with PMSG/hCG was verified by a yield of approximately 99% for luteal cells. The viability of the cells was in the 90% range as determined by trypan blue staining. Final dilutions were made in medium 199 to give lo6 or 10’ cells/ml. Production of cyclic AMP andprogesterone by luteal cells Isolated luteal cells from pseudopregnant rats were incubated under constant shaking with increasing concentrations of ovine prolactin (oPr1) (0.1-1000 ng/incubation), hCG (0.1-1000 ng/ incubation) or hCG (0.1-1000 ng/incubation) plus 10 ng oPr1 in medium 199 containing 0.1 mM 1-methyl-3-isobutylxanthine (MIX). Incubations were carried out at 34°C under an atmosphere of 95% 0,: 5% CO, for 3 h. Longer incubation periods (up to 4 h) were tested but did not produce significantly different results from those obtained after 3 h of incubation. Each incubation had 2 x lo6 cells in a final volume of 2.2 ml. 1 ml aliquots of the incubates were heated at 100°C for 15 min and stored at -20°C until determination of total cyclic AMP by means of a protein binding competition assay (Brown et al., 1971). Other 1 ml aliquots were centrifuged at 800 X g for 10 min at 4°C and the supernatants kept frozen at - 20°C for determination of progesterone by RIA (see below). Isolated luteal cells from pseudopregnant rats treated with S, oPr1, Br or saline were incubated with 10F9 M of hCG medium 199 containing 0.1

mM MIX using the same conditions above.

as described

Membrane preparation for binding assays Ovaries from pseudopregnant animals were minced and homogenized at 0°C in phosphatebuffered saline (PBS), pH 7.4, at a ratio of 1 : 6 (w/v) using an Ultra-Turrax homogenizer (IKAWerk). The homogenates were filtered through nylon cloth (Nitex 50) and centrifuged at 12000 x g for 20 min at 2-4°C. The resulting membrane pellet was resuspended in the same volume of PBS and used for binding studies. Appropriate aliquots were taken for protein determination by the method of Lowry et al. (1951). Preparation of iodinated hormones ‘251-labelled hLH was prepared using the lactoperoxidase technique according to Thorell and Johansson (1971), except that the reaction was allowed to proceed in 0.5 M phosphate buffer, pH 7.4, for 2 min. Prior to use, hormones were further purified by chromatography on Ultrogel ACA-54 (LKB, Sweden) columns. Experiments to determine precise specific activity calculations were performed using a self-displacement assay as described by Calvo et al. (1983). In each case, never less than 40% of the radiolabelled hormone was bound to an excess of reference membrane preparations. Specific activities of typical preparations of [‘*‘I]hLH averaged 55 &i/pg. Binding assays The binding activity of the membrane-rich fraction of ovary homogenates was studied as follows. Aliquots of the membrane suspension (400-600 pg protein) were incubated in PBS medium with increasing concentrations of [‘*‘I]hLH in a final volume of 0.25 ml for 16 h at 20°C. Non-specific binding was assessed in parallel incubates containing 5 pg of unlabelled hormone. The reaction was stopped by addition of 2 ml of cold PBS and the tubes were centrifuged at 12000 X g for 20 min at 4°C. The pellets were washed twice with the same medium and the radioactivity in the final pellets was determined using a Beckman Auto-Gamma 4000 spectrometer with 66% efficiency. Specific binding data were obtained using Scatchard analysis (Scatchard, 1949).

190

Other methods Serum progesterone radioimmunoassay was performed using a specific anti-4-pregnene-3,20dione 3-CM0 : BSA antiserum kindly supplied by Drs. Pirke and Doerr (Max-Planck-Institut, Miinchen, F.R.G.) following a modification of the method of Abraham et al. (1971). Under our conditions, recoveries averaged 90%; the within-assay and between-assay variations were 8.0% and 14.2% respectively. Progesterone production by luteal cells was evaluated by the same RIA procedure performed directly in suitable dilutions (1 : 25, v/v) of the incubation media (without ether extraction). The scintillation fluid used for measurements of radioactivity in the cyclic AMP determinations contained 30% Triton X-100. RIA for serum Prl and LH was performed using a double antibody technique with kits provided by the NIAMDD Rat Pituitary Hormone and the results were Distribution Program, expressed as ng/ml in terms of the rat pituitary PRL-RP-1 or LH-RP-1 respectively. All results are given as the mean + SEM. Statistical comparisons were made either by Student’s l-test or by the t-test for unpaired values with Dunnett’s correction for multiple comparisons (Li, 1964). Experiments were repeated at least 3 times. Results The in vitro progesterone and CAMP production in response to oPr1 was studied by incubation of dispersed luteal cells from pseudopregnant rats for 3 h with increasing concentrations of oPr1, hCG or different concentrations of hCG plus 10 ng oPr1. Data were expressed as the percentage of progesterone or CAMP stimulation over basal values obtained in the absence of hormones. The CAMP response is shown in Fig. 1. Cyclic AMP levels reach a maximum with lo-” M hCG; however, oPr1 neither stimulated the cyclic AMP formation nor enhanced the hCG response. Progesterone production is shown in Fig. 2. oPr1 maximally stimulated the progesterone response by luteal cells when it was added to the incubation medium at concentrations greater than lo-’ M, while hCG produced maxima! stimulation at lo-” M. On the other hand, oPr1 had an additive effect when it was added to the incubates with hCG at

STIMULANT

(ng/incub)

Fig. 1. Effect of increasing doses of hCG (open bars), oPrl (hatched bars) or hCG plus 10 ng oPrl (closed bars) on the production of cyclic AMP by isolated luteal cells. Cells were incubated with the hormones in the presence of 0.1 mM MIX at 34°C under an atmosphere of 95% 0, : 5% CO, for 3 h. After incubation, 1 ml aliquots were heated at 100°C for 15 min and stored at - 20°C until determination of total cyclic AMP. Each bar represents the mean* SEM of triplicate determinations of 3 different experiments. Basal values were 11.3 f 1.4 pmoles/2 X lo6 cells.

concentrations that produce submaximal stimulation. Higher doses of oPr1 (more than 10 ng) in the combined experiment were not used since Prl stimulation itself could superpose with Prl enhancement of hCG stimulation. On the other hand, the maximal response cannot be exceeded, even in the presence of high amounts of both hormones. Table 1 shows the effects of oPrl, S and Br administration on ovarian weight and circulating levels of progesterone, Prl and LH. Weights of ovaries from the different groups did not differ significantly from one another. Serum levels of progesterone were reduced in Br-treated animals

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Fig. 2. Effect of increasing doses of hCG (open bars), oPrl (hatched bars) or hCG plus 10 ng oPrl (closed bars) on progesterone production in isolated luteal cells. Cells were incubated under the same conditions as described in the legend to Fig. 1. 1 ml of the incubates was centrifuged at 800X g for 10 min at 4°C and the supernatant kept frozen at - 20°C until determination of progesterone by radioimmunoassay. Each bar represents the meanf SEM of triplicate determinations of 3 different experiments. Statistical comparisons were made in relation to the control values (* P < 0.01). Basal values were 10.3 + 1 ng/2 X lo6 cells.

while neither oPr1 nor S treatment produced any detectable change. As was expected, S significantly enhanced serum Prl levels, whereas Br produced no detectable Prl. Serum LH levels were similar to those seen in control animals in all groups studied. The effect of oPr1, S and Br treatment on in vitro hCG-stimulated progesterone and CAMP production was evaluated in collagenase-dispersed luteal cells prepared from ovaries of PMSG/ hCG-primed rats. Under these conditions, a marked reduction in the ability of hCG to stimulate progesterone and CAMP production was found in the Br-treated animals, while a significant in-

TABLE EFFECT LEVELS

Fig. 3. Effect of different treatments on CAMP and progesterone production by luteal cells from immature PMSG/ hCG-primed rats. Animals were injected twice daily as follows: C, control, vehicle; P, ovine prolactin, 1 mg/kg BW; S, sulpiride, 30 mg/kg BW; Br, bromocriptine, 3 mg/kg BW. Treatments started at the 3rd day after PMSG administration and continued for 7 days. Aliquots of each cell suspension containing 2 X lo6 luteal cells were incubated with hCG (10m9 M) in the presence of MIX (lOA M) at 34°C for 3 h. Progesterone and CAMP production were measured as described under Materials and Methods. Data are expressed as the percentage of progesterone or CAMP stimulation compared to increase over basal values obtained in the absence of hCG. Progesterone basal values were: C=8.0*0.2, S=15.6*0.5, P=18.9&-1.0, Br = 9.7kO.4 ng/2 X IO6 cells. CAMP basal values did not differ among the experimental groups. Mean value was 10.2& 2.1 pmoles/2 x lo6cells. Each bar is the mean f SEM. Asterisks indicate significant differences (* P -c0.001, ** P < 0.05 by Dunnett’s multiple r-test) from the control.

crease was observed after oPr1 or S administration (Fig. 3). The possible direct action of S and Br on the luteal cell response was assayed under in vitro conditions. Neither drug, within the range of 0.03-3 pg/ml incubation, had any effect on basal

1 OF S, oPrl AND Br TREATMENTS IN IMMATURE PSEUDOPREGNANT Ovarian

Control (saline) (16) Sulpiride (30 mg/kg BW) (13) Prolactin (1 mg/kg BW) (13) Bromocriptine (3 mg/kg BW) (16)

ON OVARIAN RATS weight

WEIGHT

AND

SERUM

PROGESTERONE,

LH AND

Progesterone

LH

Prl

(mg)

(ng/ml)

@g/ml)

(ng/ml)

65+4 65*6 61k5 60&5

68.3* 7 76.1 f 11 55.7* 4 5.5* 0.8 *

10.6 f 3.2 14.2 + 1.5 11.4k1.6 6.9 f 2.1

11.6+1 47.8 k 3 * -

Prl

Undetectable

Numbers in parentheses indicate the number of animals per group, and values are shown as mean f SE. Animals were sacrificed after 7 days of treatment, ovaries were weighed and serum processed as described in Materials and Methods. * indicates significant differences (P < 0.001) compared to control by Dunnett’s multiple r-test.

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300.

0 k

200.

F \ a E z I

100.

$ :

C

S

P

Br

Fig. 4. Effect of different treatments on [“‘l]hLH binding to membrane-rich fraction of ovaries from immature PMSG/ hCG-primed rats (see legend to Fig. 3 for details). Each bar represents the mean+ SEM of the number of LH receptors obtained from Scatchard analysis. The experiment was repeated 3 times and asterisks indicate significant difference (* P < 0.01, ** P < 0.05) compared to the control by Dunnett’s multiple t-test.

CAMP or progesterone production after incubation for 3 h in the absence of stimulatory hormones (data not shown). Fig. 4 shows the effect of oPr1, S and Br treatments on hLH binding sites in ovarian particulate fractions. A significant increase was found in hLH binding sites after Prl and S treatment (60% and 95% respectively) while a marked reduction (4-fold) was observed in the animals treated with Br. The Scatchard analysis of these data did not show a significant difference in the K, among the groups (mean value among groups is K, = 3.8 * 0.3 X lo9 M-‘). Figs. 3 and 4 show also that sulpiride treatment is more efficient than oPr1 injections, since the last one is a heterologous hormone, probably less active and with a shorter half-life than the endogenous hormone. Discussion The results of our investigations clearly demonstrated that prolactin peripheral levels regulate cell

sensitivity to LH/hCG stimulation. Hyperprolactinemic states increased the in vitro LH/hCGstimulated luteal progesterone and CAMP production, while the opposite effect was observed under hypoprolactinemic conditions. The changes seen in LH receptor levels could be, at least in part, responsible for the changes in luteal cell responsiveness to LH/hCG stimulation. Moreover, the present results indicate that in pseudopregnant rats, specific hLH binding to ovarian membranes is regulated by Prl circulating levels, with hyperprolactinemia increasing and hypoprolactinemia reducing this binding activity. These findings are in accordance with the reported regulation of LH receptors by Prl (Holt et al., 1976; Richards and Williams, 1976). On the other hand, it has been suggested that the increase in LH receptor and progesterone production could occur as independent events both mediated by the luteotrophic action of Prl (Holt et al., 1976). This hypothesis is supported by our findings with regard to the direct effect of Prl on progesterone production by luteal cells from pseudopregnant rats. Interestingly, some reports are compatible with the possibility that LH receptor is not the limiting factor for luteal cell production of progesterone. These include the inability of antiserum against LH to decrease serum progesterone between days 3 and 6 of pregnancy (Rothchild et al., 1974) and the apparent failure of exogenous LH to stimulate an increase in serum progesterone during the same period of pregnancy (Ichikawa et al., 1975). Veldhuis et al. (1980) demonstrated that Prl exerts a direct inhibitory effect in granulosa cells from small, immature follicles of non-cyclic pigs. In contrast, granulosa cells isolated from large, ‘mature’ follicles associated with regressing corpora lutea of cycling sows exhibit a stimulatory response to Prl in vitro. In spite of the different experimental model used, the stimulatory effect of Prl on luteal cells reported here is in accordance with that of Veldhuis et al. obtained in ‘mature’ follicles. Prl could then be acting by providing luteotrophic support to ovarian cells, as Crisp (1977) demonstrated in rat granulosa cells in culture. In this work, only Prl and hPL but not FSH, rGH or hLH were able to bring about complete luteinization (as observed by electron microscopy)

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and enhanced progesterone secretion. In this study, dibutyryl CAMP failed to mimic the enhancement of progesterone secretion seen when rPr1 or hPL was present, suggesting that another messenger may be involved. In this context, our results indicate that Prl stimulates the in vitro progesterone production without any detectable rise in CAMP levels. This dissociation between CAMP formation and progesterone production could be a consequence of an activation of specific enzymes in the pathway of progesterone by-passing an intermediate elevation of CAMP levels. Another possibility is that the known effects of Prl result from a promotion of general cellular metabolism, and progesterone production would be reflecting the physiological response of luteal cells, although in this case such a rapid effect should not be expected. An action of Prl upon the accessibility of endogenous progesterone precursors to the biosynthetic enzymes cannot be ruled out. The low basal values could be attributable to a conversion of progesterone to 20c+dihydroprogesterone, which is being tested at present. Experiments are in progress to determine the precise intracellular mechanism of prolactin action in luteal cells. The experimental model used in this study included treatments of PMSG/ hCG-primed rats with oPr1 and two neuropharmacological agents (bromocriptine and sulpiride). Br, a specific inhibitor of prolactin secretion, is a dopaminergic agonist which blocks primarily prolactin release (Lu et al., 1971) but also its synthesis (Weinstein et al., 1981). On the other hand, the mechanism of enhancement of prolactin secretion by S is not yet clear. It has been reported that although S has no effect on newly synthesized prolactin in vitro, this drug significantly overcomes the inhibitory effect of dopamine upon prolactin secretion (MacLeod and Roby, 1977), presumably through an interaction with dopamine receptor. That our results are not the consequence of a direct action of S or Br on the ovary is indicated by the close similarity between the changes observed with S or oPr1 treatment and by the unaltered luteal cell response under in vitro conditions in the presence of these drugs. It is also important to point out that serum LH levels remained at control values in all experimental groups; this fact provides evidence in support of the view that the results obtained from

luteal cells of S, oPr1 or Br-treated rats are a consequence of the changes in serum Prl levels. In summary, our data suggest that luteal cell function is regulated by the circulating levels of prolactin and that this hormone has some direct action on the steroidogenic process. However, knowledge of the precise role of prolactin in luteal tissue will require further investigation. Acknowledgements This work was supported by a grant from the Secretaria de Estdo de Ciencia y Tecnologia (SECyT) (9726) the Consejo National de Investigaciones Cientificas y Tecnicas (CONICET) and the University of Buenos Aires. The authors are grateful to Dr. E. Passeron from Laboratorios Elea. We acknowledge the technical assistance of Mrs. Ana Rosa de la Camara. We are very grateful to Dr. Laura McMurry for her revision of the manuscript. References Abraham, G.E., Swerdloff, R., Tulchinsky, D. and Odell, W.D. (1971) J. Clin. Endocrinol. Metab. 32, 619-624. Advis, J.P. and Ojeda, S.R. (1978) Endocrinology 103, 924-935. Armstrong, D.T., Miller, L.S. and Knudsen, K.A. (1969) Endocrinology 85, 393-401. Astwood, E.B. (1941) Endocrinology 28, 309-314. Brown, B.L., Albano, J.D.M., Ekins, R.P. and Sgherzi, A.M. (1971) B&hem. J. 121, 561-562. Calvo, J.C., Radicella, J.P. and Charreau, E.H. (1983) Biochem. J. 212, 259-264. Catt, K.J., Tsuruhara, T., Mendelson, C., Ketelslegers, J.M. and Dufau, M.L. (1974) In: Current Topics in Molecular Endocrinology, Eds: M.L. Dufau and A.R. Means (Plenum Press, New York) pp. l-30. Clemens, J.A., Chaar, C.J., Smalstig, E.B., Bach, N.J. and Komfeld, E.C. (1974) Endocrinology 94, 1171-1180. Crisp, T.M. (1977) Endocrinology 101, 1286-1297. Day, S.L., Kirchick, H.J. and Bimbaumer, L. (1980) Endocrinology 106, 1265-1269. Dbler, K.D. and Wuttke, W. (1974) Endocrinology 94, 1595-1600. Evans, H.M., Simpson, M.E., Lyons, W.R. and Turpeinen, K. (1941) Endocrinology 28, 933-945. Everett, J.W. (1956) Endocrinology 58, 786-796. Grinwich, D.L., Hichens, M. and Behrman, H.R. (1976) Biol. Reprod. 14,212-218. Holt, J.A., Richards, J.S., Midgley, A.R. and Reichert, L.E. (1976) Endocrinology 98,1005-1013. Huang, W.Y. and Pearlman, W.H. (1962) J. Biol. Chem. 237, 1060-1065.

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Ichikawa, S., Sawada, T., Nakamura, Y. and Morioka, H. (1975) Endocrinology 94, 1615-1620. Li, C.C. (1964) In: Introduction to Experimental Statistics (McGraw Hill, New York). Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Lu, K.H., Koch, Y. and Meites, J. (1971) Endocrinology 89, 229-233. Macdonald, G.J., Tashjian Jr., A.H. and Greep, R.O. (1970) Biol. Reprod. 2, 202-208. Macdonald, G.J., Moudgal, N.R., Madhwa Raj, H.G. and Greep, R.O. (1973) Proc. Sot. Exp. Biol. Med. 144,923-926. MacLeod, R.M. and Roby, C. (1977) J. Endocrinol. 72, 273-277. Madhwa Raj, H.G. and Moudgal, N.R. (1970) Endocrinology 86, 874-889. Marsh, J.M., Telegdy, G. and Savard, K. (1966) Nature (London) 212, 950-952. Richards, J.S. and Williams, J.J. (1976) Endocrinology 99, 1571-1581. Rothchild, I., Pepe, G.J and Mirishige, W.K. (1974) Endocrinology 95, 280-288.

Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Shelesnyak, M.C. (1955) Am. J. Physiol. 180, 47-49. Smith, M.S., Freeman, M.E. and Neill, J.D. (1975) Endocrinology 96,219-226. Takayama, M. and Greenwald, G.S. (1973) J. Endocrinol. 56, 421-429. Tesone, M., Chiauzi, V.A., Oliveira-Filho, R.M. and Charreau, E.H. (1981) Medicina 41, 755-756 (abstract). Tesone, M., Ladenheim, R.G., Oliveira-Filho, R.M., Chiauzzi, V.A., Foglia, V.G. and Charreau, E.H. (1983) Proc. Sot. Exp. Biol. Med. 174, 123-131. Thorell, J.I. and Johansson, B.G. (1971) Biochim. Biophys. Acta 251, 363-369. Veldhuis, J.D., Klase, P. and Hammond, J.M. (1980) Endocrinology 107, 42-46. Weinstein, D., Shenker, J.G., Gloger, I., Slonim, J.H., De Groot, N., Hochberg, A.A. and Folman, R. (1981) FEBS Lett. 126, 29-32. Wuttke, W., Cassell, E. and Meites, J. (1971) Endocrinology 88, 737-741.