Effect of Progesterone In Vitro on Luteinizing Hormone Production in Hen Pituitary Cells Pretreated with Estrogen1

PHYSIOLOGY AND REPRODUCTION Effect of Progesterone In Vitro on Luteinizing Hormone Production in Hen Pituitary Cells Pretreated with Estrogen1 MITSUO KAWASHIMA, AIICHIROU UKAI, MICHIHARU KAMIYOSHI, and KATUfflDE TANAKA 2 Department of Poultry and Animal Sciences, Gifu University, Gifu 501-11, Japan

1992 Poultry Science 71:1215-1220

INTRODUCTION Progesterone (P4) stimulates the release of luteinizing hormone (LH) from the anterior lobe of pituitary of the hen (Wilson and Sharp, 1975) and induces ovulation (Fraps and Dury, 1943). The site of action of P4 for the release of LH is thought to be the hypothalamus or other sites of the central nervous system (Ralph and Fraps, 1960). Besides the central nervous system, the pituitary itself is also the site of action of P4, because the presence of a P4 receptor has been reported (Kawashima et al, 1978, 1979a). In the laying hen pituitary, the number of the P4 receptor binding sites changes during the ovulatory cycle (Kawashima et al., 1979b), but no change is found in the nonlaying hen throughout a 24-h period (Kawashima et al., 1981). When isolated pituitary cells of the laying hen were

Received for publication November 18, 1991. Accepted for publication March 5, 1992. Supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (Number 03454097). To whom correspondence should be addressed.

challenged with P4 in vitro, increased cellular LH was found only in cells having a large number of P4 receptor binding sites (Kawashima et al., 1982). Receptors for both estrogen (Kawashima et al., 1987) and androgen (Kawashima et al, 1989) are also present in the hen pituitary, suggesting that the pituitary is also a site of action for both estrogen and androgen. Administration of estradiol-17P (E2) to the nonlaying hen in vivo (Kawashima et al, 1979c) or incubation of isolated pituitary cells of the nonlaying hen with E2 (1 ng) in vitro for 2 h (Kawashima et al, 1992) causes an increase in the number of P4 receptor binding sites. The present experiments were conducted to investigate the effect of P4 on the cellular LH content and LH release in isolated pituitary cells pretreated with E2 in vitro.

MATERIALS AND METHODS Animals and Tissues White Leghorn hens, 14 to 22 mo of age and weighing 1.7 to 2.3 kg, were used. The hens had been kept under a light regime of 14 h light and 10 h darkness with feed and

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ABSTRACT Isolated cells from the pituitary gland of nonlaying hens were preincubated in vitro with or without estradiol-17p (E2), and then incubated with or without progesterone (P4), and luteinizing hormone (LH) in the cells and media was measured by a homologous radioimmunoassay. When the cells were preincubated with E2 for 2 h and then incubated with P4 for 4 h, cellular LH was increased, but LH in the medium remained unchanged. The increase in cellular LH in response to P4 was dose-dependent. Protein synthesis inhibitors (cycloheximide, actinomycin-D, and a-amanitin) blocked the response of the cells to P4. The results suggest that LH production in estrogen-primed pituitary cells of the hen was stimulated by P4 through a protein-synthesizing pathway. (Key words: luteinizing hormone, progesterone, estradiol-17|5, pituitary cell, protein synthesis inhibitor)

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adjusted to 1 x 10 7 cells/mL by dilution with Eagle's MEM-RRA. The viability of the cells was tested by trypan blue staining (McLimans et al, 1957) and was found to be more than 98%. Each cell suspension (.5 mL: 5 x 10 6 cells) was placed in a polystyrene tube (16 x 125 xamf to which .5 mL Eagle's MEM-RRA, with or without 1.0 ng E2,4 was added. Tubes (three tubes per treatment) were preincubated at 40.5 C for 2 h under 95% O2 and 5% CO2 in a shaking bath (120 cycles per min). After the preincubation, the cells were collected by centrifugation (100 x g, 5 min), washed once with Eagle's MEM-RRA, and suspended in 1 mL Eagle's MEM-RRA containing or not containing: 1) .1,1.0, and Isolation and Incubation 10 ng P4,4 2) 1.0 and 10 ng P4, E2, of Pituitary Cells testosterone (T),4 or 5a-dihydrotestoster4 Pituitary cells were isolated and in- one (DHT),- 3) various amounts (.05 to 12.8 cubated in vitro using a previously reported ng) of P4; or 4) 10 ng P4 with or without 10 method (Kawashima et al, 1982,1992) with ug of a protein synthesis inhibitor (cyclo4 4 a slight modification. Cells were dispersed heximide, actinomycin-D, or a-aman4 3 (40 min, 37 C) with .2% collagenase, . 1 % itin ). Different concentrations of the hyaluronidase, 4 and .001% deoxyribo- steroid solutions containing .01% ethanol n u c l e a s e 4 in C a + + - a n d M g + + - f r e e were prepared from a stock solution (100 Romanoffs avian Ringer's salt solution Hg/mL ethanol) by diluting with incuba(CMF-RRA, p H 7.4; Romanoff, 1943) con- tion medium to a desired concentration. taining .25% BSA (Fraction V) 4 and 25 mM The tubes were then incubated again at 40.5 HEPES (N-2-hydroxyethylpiperazine-N'- C for 2, 4, or 8 h. After the incubation, the 2-ethanesulfonic acid). 5 Cells were collected tubes were immediately cooled in an ice by centrifugation (100 X g, 5 min) and bath, and media and cells were collected washed twice with CMF-RRA. The washed separately following centrifugation (100 xg, cells were suspended in Eagle's minimal 5 min, 4 C). The media were stored at -20 C essential medium (MEM)-RRA. The MEM- until LH assay. The cells were washed twice RRA was prepared from Eagle's MEM with RRA and suspended in 1 mL RRA. The amino acids and vitamins medium "Nis- suspension was immediately frozen in a sui" 6 that had been balanced with RRA bath containing dry ice and acetone, and containing 25 mM HEPES, .25% BSA, and was then thawed in a warm bath (30 C). 1 % pullet sera. The pullet sera was obtained After freezing and thawing, which were from immature pullets (10 wk of age), and repeated twice, the suspension was centriwas inactivated by heat treatment (56 C, 30 fuged (1,000 x g, 15 min, 4 C) and the min) and treated by the dextran-coated supernatant fluid (cell extract) was stored at charcoal (DCC). The concentration of E2 -20 C until LH assay. and P4 in the sera after the DCC treatment was undetectable by RIA. The concentra- Assay for Luteinizing Hormone tion of the cells in the suspension was Immunoreactive LH in the medium and determined by a hemacytometer, and was cellular extract was measured by the same method as reported earlier (Kawashima et al, 1982) using a homologous RIA system ^ a k o Pure Chemical Ltd., Japan. for avian LH of Hattori and Wakabayashi ^igma Chemical Co., St. Louis, MO 63178-9916. (1979). For radioiodination and standard, a TMacalai Tesque, Inc., Kyoto, Japan. highly purified preparation of chicken LH TMissui Pharmaceutical Co., Ltd., Tokyo, Japan. 7 [CP-2(PS), Gunma] was used. The chicken Falcon Plastics, Lincoln Park, NJ 07035.

water provided for ad libitum consumption. Hens that had not laid an egg for 1 wk, after a 3-day deprivation of feed and water, were used. All hens were decapitated (150 to 300 birds in each experiment), and the anterior lobe of the pituitary (7 to 10 mg per bird) was excised. Blood was collected from the clavian artery and plasma was obtained. In all of the hens necropsied, the ovarian weight was less than 8 g and the oviduct weight was less than 15 g. Plasma concentrations of P4 and E2 were less than 98 and 78 p g / m L , respectively, as determined by radioimmunoassay (RIA; Shodono et al., 1975).

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PROGESTERONE EFFECT IN PITUITARY CELLS 1

2

6

TABLE 1. Amounts of luteinizing hormone (LH) in isolated pituitary cells (5 x 10 cells/mL per tube) and in medium after a 2-h preincubation with or without estradiol-17p (E2) (1.0 ng per tube) and then a 2, 4, or 8 h incubation with or without progesterone (P4) Treatment Preincubation Incubation

Cell

2-h Incubation Medium

Cell

4-h Incubation Medium

Cell

8-h Incubation Medium

(ng/10 6 cells) Vehicle Vehicle 36.0 ± 1.9 10.2 ± .7 29.5 ± 1.8C 15.7 ± 1.1 27.6 ± 1.6B 20.0 ± 1.1 Vehicle P 4 (.1 ng) 38.6 ± 1.2 105 ± .5 33.0 ± I T 0 16.7 ± .7 29.0 ± 2.2B 18.7 ± .9 Vehicle P 4 (1.0 ng) 37.2 ± 1.7 8.9 ± .5 36.9 ± 2.1 c 15.4 ± .7 30.6 ± 1.1BA 19.7 ± .6 C BA Vehicle P 4 (10 ng) 38.8 ± 1.4 9.0 ± 5 35.8 ± 1.9 16.0 ± .7 31.1 ± 1.9 22.4 ±1.5 E2 Vehicle 37.9 ± 22 8.6 ± .6 28.3 ± 1.8C 17.5 ± .8 29.0 ± \2B 22.3 ± 1.5 35.9 ± 1.9 8.4 ± .4 33.4 ± 2.9C 16.8 ± .6 30.4 ± 1.2BA 185 ± 1.8 P 4 U ng) E2 B BA E2 P 4 (1.0 ng) 33.2 ± 2.9 8.1 ± .6 53.9 ± 5.3 17.0 ± .7 36.4 ± 1.3 22.2 ± 1.8 E2 P 4 (10 ng) 36.0 ±1.2 9.1 ± .3 80.0 ± 4.4A 16.5 ± 1.1 38.3 ± 2.2A 23.2 ± 2.0 A_c Means in the same column with no common superscripts are significantly different (P < .01). I^termined by a homologous radioimmunoassay. Obtained from pooled anterior pituitaries of 300 nonlaying hens. The amount of cellular LH before preincubation was 56.5 ± 2.8 (SEM) ng/10 6 cells. ''Mean ± SEM of five replicate determinations. 3

Statistical Analyses To assess the significance of difference between two means, the Student's t test was used. For the comparison among more than two groups, analysis of variance was used to determine treatment difference and the Newman-Keuls test was used to separate means (Snedecor, 1956).

(Table 2). The increase in intracellular LH appeared to be dose-dependent on P4 added, from .4 to 6.4 ng/mL (Figure 1). No further increase in LH was observed when P4 concentrations were over 6.4 ng/ mL. When the cells were preincubated with E2 and then incubated for 4 h with 10 ng P4 plus cycloheximide, actinomycin-D, or a-amanitin, no increase in intracellular LH was found (Table 3).

^

60

C;

40

RESULTS There was no increase in intracellular or medium LH when the cells were preincubated without E2 (Table 1). However, when the cells were preincubated with E2 and then incubated for 4 h with P4 (1.0 or 10 ng), intracellular LH was increased significantly but medium LH remained unchanged. The increase in the intracellular LH was also observed when the incubation was made for 8 h with 10 ng P4. A similar incubation with other steroid (E2, T, or DHT) did not cause an increase in either intracellular LH or medium LH

i-H ttfSSSSSSSSSSSSSSSSSSSSs

20 u

.05 .1

.2

.4

.8

1.6 3.2 6.4 12.8

Progesterone (ng/mL)

FIGURE 1. Amount of luteinizing hormone (LH) in cells when isolated pituitary cells were preincubated for 2 h with estradiol-17P (1.0 ng/mL per tube) and then incubated for 4 h with or without various amounts of progesterone. The amount of cellular LH was measured by a homologous radioimmunoassay. Each point represents the mean of triplicate determinations and the vertical bars represent SEM. The shaded area represents the range of control values. *, **, ***Significantly different from control at 5%, 1%, and .1% level (f test), respectively.

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LH preparation and antisera against chicken LH were kindly provided by Y. Hasegawa, Department of Obstetrics and Gynecology, Gunma University, Gunma, Japan. The intra- and interassay coefficients of variation were 7.8 and 10.8%, respectively, from eight assays.

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KAWASHIMA ET AL.

TABLE 2. Amounts of luteinizing hormone 1 (LH) in isolated pituitary cells 2 (5 x 10* cells/mL per tube) and in medium after a 2-h preincubation with or without estradiol-17f$ (E2) (1.0 ng per tube), and then a 4-h incubation with or without progesterone (P4), E2, testosterone (T), or 5a-dihydrotestosterone (DHT) (1.0 and 10 ng per tube) Treatment Preincubation

Incubation

Cell

Medium (ng/10 6 cells)

Vehicle Vehicle P 4 (1.0 ng) P 4 (10 ng) E 2 (1.0 ng) E 2 (10 ng) T (1.0 ng) T (10 ng) DHT (1.0 ng) DHT (10 ng)

33.6 33.0 48.1 72.6 34.0 35.9 31.3 30.7 31.8 34.8

± ± ± ± ± ± ± ± ± ±

2.9 3 0 \2C 3.4B 5.7 A 1.9C 1.3C 1.8C 2.4 C 2.8 C 2.1c

15.7 15.7 16.9 16.4 17.2 18.1 14.5 16.5 16.7 15.8

± ± ± ± ± ± ± ± ± ±

1.1 .8 1.1 1.2 1.2 1.7 1.3 1.0 .8 1.3

A_c

M e a n s in the same column with no common superscripts are significantly different (P < .01). ^Determined by a homologous radioimmunoassay. O b t a i n e d from pooled pituitaries of 150 nonlaying hens. The amount of cellular LH before preincubation was 56.4 ± 2.4 (SEM) n g / 1 0 6 cells. ^ l e a n ± SEM of triplicate determinations.

DISCUSSION The amount of LH in the pituitary cells was found to increase after being preincubated with E2 and then incubated with P4 for 4 h (Table 1). The increase may be d u e to a direct action of E2 and P4 on the pituitary cells, because receptors for both hormones are present in the pituitary cells (Kawashima et ah, 1978, 1979a, 1987). The difference in response to P4 stimulation between the estrogen-primed and unprimed cells may be due to the difference in the number of P4 receptor binding sites. It has been reported that the number of P4 receptor binding sites in the pituitary of the nonlaying hen increases after E2 treatment in vivo (Kawashima et ah, 1979c) and in vitro (Kawashima et ah, 1992). In the 8-h incubation with 10 ng of P4 after the preincubation with E2, the results in the cellular LH were similar to those obtained in the 4-h incubation with P4. However, the degree of the increase in the cellular LH was greater in the 4-h incubation than in the 8-h incubation. Although the reason for the difference is unknown, some immunochemical changes of LH might occur inside of the cells during the period after 4 h of incubation. The failure of the cellular LH to increase at 8 h of incubation in spite of an

apparent increase at 4 h of incubation has been also found in the cells of laying hens at 18-h before ovulation (Kawashima et ah, 1982). In the incubation of the cells of the laying hen, the medium LH was found to be increased when LH releasing hormone (LHRH) was added at 4 h of the incubation with P4. It seems likely that the increased cellular LH found in the present study is capable to be released if a proper agent such as LHRH is present. In contrast to the 4-h incubation, a 2-h incubation produced no increased cellular LH (Table 1). Because a 2-h incubation with E2 causes an increase in the number of P4 receptor binding sites (Kawashima et al., 1992), the production of LH may require more than 2 h after P4 receptor binding. A similar increase in the amount of cellular LH following 4 h incubation of pituitary cells has been found also in the laying hen but only in those pituitaries possessing a relatively large number of P4 receptor binding sites at a specific time during the ovulatory cycle (Kawashima et ah, 1982). When the pituitary cells were preincubated with E2 and then incubated for 4 h with P4, intracellular LH gradually increased with the amount of P4 added,

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Vehicle E2 E2 E2 E2 E2 E2 E2 E2 Eg

PROGESTERONE EFFECT IN PITUITARY CELLS

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TABLE 3. Amounts of luteinizing hormone1 (LH) in isolated pituitary cells2 (5 x 106 cells/mL per tube) and in medium after a 2-h preincubation with or without estradiol-17f} (E2) (1.0 ng per tube), and then a 4-h incubation with or without progesterone (P4) (10 ng per tube) and cycloheximide (CYCL), actinomycin-D (Act-D), or o-amanitin (10 |Xg per tube)

Preincubation

Treatment Incubation

Cell

Medium (ng/106 cells)

Vehicle + vehicle Vehicle + vehicle Vehicle + CYCL Vehicle + Act-D Vehicle + ct-amanitin P 4 + vehicle P 4 + CYCL P 4 + Act-D P 4 + q-amanitin

36.9 ± 40.8 ± 41.1 ± 42.9 ± 37.0 ± 75.6 ± 39.5 ± 33.4 ± 34.6 ±

3.63B Z9 B 2.1 B 2.6B 1.4B 62A 3.9B 3.5B 2.3B

18.0 ±1.6 17.5 ±1.4 16.8 ± 1 5 19.6 ±1.4 19.9 ± 1.6 19.0 ± .9 16.1 ± .9 21.0 ± 1.9 19.4 ± 1.5

A/B

Means in the same column with no common superscripts are significantly different (P < .01). Determined by a homologous radioimmunoassay. Obtained from pooled pituitaries of 150 nonlaying hens. The amount of cellular LH before preincubation was 61.4 ± 2.5 (SEM) ng/10 6 cells. Mean ± SEM of triplicate determinations.

up to 6.4 ng, indicating a dose-dependent response (Figure 1). Because .2 ng (.6 nM) of P4 is already over the level of the binding affinity of the pituitary P4 receptor [dissociation constant (Kd) = .2 to .5 nM; Kawashima et ah, 1978, 1979a], the effect of P4 would have been manifested. The effect of P 4 on the amount of cellular LH was not observed when the pituitary cells were incubated in the presence of the protein synthesis inhibitors, cycloheximide, actinomycin-D, or
acid into a protein (Baulieu et ah, 1972) have been found to occur .5 to 3 h after steroid treatment, suggesting that steroids affect gene expression on early protein synthesis as proposed by Baulieu et ah (1972). The results of the present experiments suggest that P4 stimulates the production of LH in the pituitary through a proteinsynthesizing pathway. Estrogen priming is important for the action of P4.

REFERENCES Apfelbaum, M. E., 1983. Influence of gonadal steroids on the time-course of release and synthesis of luteinizing hormone, follicle-stimulating hormone and prolactin from rat pituitary glands in vitro. J. Endocrinol. 96:171-179. Baulieu, E. E., A. Alberga, C. Raynaud-Jammet, and C. R. Wira, 1972. New look at the very early steps of oestrogen action in uterus. Nature 236: 236-238. Fraps, R. M., and A. Dury, 1943. Occurrence of premature ovulation in the domestic fowl following administration of progesterone. Proc. Soc. Exp. Biol. Med 52346-^49. Hamilton, T. H., C. C. Widnell, and J. R. Tata, 1965. Sequential stimulations by oestrogen of nuclear RNA synthesis and DNA-dependent RNA polymerase activities in rat uterus. Biochim. Biophys. Acta 108:168-172. Hattori, M., and K. Wakabayashi, 1979. Isoelectric focusing and gel filtration studies on the heterogeneity of avian pituitary luteinizing hor-

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KAWASHIMA ET AL. duction and release of LH and FSH. Gen. Comp. Endocrinol. 48:362-371. Kawashima, M., A. Ukai, M. Kamiyoshi, and K. Tanaka, 1992. Enhancement of progesterone receptor bindings by estradiol in pituitary cells of the hen. Poultry Sci. 71:352-356. Liu, T. C , and G. L. Jackson, 1977. Effect of in vivo treatment with estrogen on luteinizing hormone synthesis and release by rat pituitaries in vitro. Endocrinology 100:1294-1302. McLimans, W. P., E. V. Davis, F. L. Glover, and G. W. Rake, 1957. The submerged culture of mammalian cells: The spinner culture. J. Immunol. 79: 428-433. Ralph, C. L., and R. M. Fraps, 1960. Induction of ovulation in the hen by injection of progesterone into the brain. Endocrinology 66:269-272. Rao, S. S., and A. P. Grollman, 1967. Cycloheximide resistance in yeast: A property of the 60s ribosomal subunit. Biochem. Biophys. Res. Commun. 29:696-704. Romanoff, A. L., 1943. Differentiation in respiratory activity of isolated embryonic tissues. J. Exp. Zool. 93:1-26. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17p, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 73: 565-573. Snedecor, G. W., 1956. Two or more random samples of measurement data. Pages 237-290 in: Statistical Methods, 5th ed. G. W. Snedecor, ed. The Iowa State University Press, Ames, IA. Tang, L.K.L., 1977. Different feedback regulation and LHRH-responsiveness in adenohypophyseal cell cultures. Endocrinol. Res. Commun. 4:217-232. Wilson, S. C , and P. J. Sharp, 1975. Changes in plasma concentrations of luteinizing hormone after injection of progesterone at various times during the ovulatory cycle of the domestic hen (Gallus domesticus). J. Endocrinol. 67:59-70.

Downloaded from http://ps.oxfordjournals.org/ at Mount Royal College on June 8, 2015

mone. Gen. Comp. Endocrinol. 39:215-221. Kamel, F., and L. C. Krey, 1982. Gonadal steroid modulation of LHRH-stimulated LH secretion by pituitary cell cultures. Mol. Cell. Endocrinol. 26:151-164. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1978. A cytoplasmic progesterone receptor in hen pituitary and hypothalamic tissues. Endocrinology 102:1207-1213. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1979a. Nuclear progesterone receptor in the hen pituitary and hypothalamus. Endocrinol. Jpn. 26:501-506. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1979b. Cytoplasmic progesterone receptor concentrations in the hen hypothalamus and pituitary: Difference between laying and nonlaying hens and changes during the ovulatory cycle. Biol. Reprod. 20:581-585. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1979c. Effects of progesterone, estradiol and testosterone on cytoplasmic progesterone receptor concentrations in the hen hypothalamus and pituitary. Biol. Reprod. 21:639-646. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1981. Progesterone receptors in hypothalamus and pituitary of the hen during the ovulatory cycle. Pages 35-42 in: Recent Advances of Avian Endocrinology. G. Pethes, P. Peczely, and P. Rudas, ed. Pergamon Press, Budapest, Hungary. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1987. Presence of estrogen receptors in the hen hypothalamus and pituitary. Endocrinology 120: 582-588. Kawashima, M., M. Kamiyoshi, and K. Tanaka, 1989. Presence of androgen receptors in the hen hypothalamus and pituitary. Acta Endocrinol. 120:217-224. Kawashima, M., M. Kamiyoshi, K. Tanaka, M. Hattori, and K. Wakabayashi, 1982. Effect of progesterone on pituitary cells of the hen (Gallus domesticus) during the ovulatory cycle for pro-