Servomechanism of Prolactin and Progesterone in Regulating Uterine

Servomechanism of Prolactin and Progesterone in Regulating Uterine Gene Expression Beverly S. Chilton, Shailaja K. Mani, and D. W. Bullock Department of Cell Biology and Anatomy (B.S.C.) Texas Tech University Health Sciences Center Lubbock, Texas 79430 To investigate the interaction of PRL and progesterone in regulating uterine gene expression, we have quantitated the concentration of PRL receptor and of uteroglobin (UG) mRNA in the endometrium of rabbits of different ages and after treatment with different hormones. During uterine differentiation in 2- to 4-week old rabbits, a marked increase in unoccupied uterine PRL receptor number was observed, presumably increasing uterine sensitivity to PRL. Receptor values for 4-week old rabbits were comparable to values for sexually mature, estrous females, but were lower than in 5-day pseudopregnant (PSP) animals. When total PRL receptor was determined by Scatchard analysis after in vitro desaturation with MgCI2, PSP animals again expressed the highest receptor concentration with no changes in the dissociation constant (Kd) values. To determine whether progesterone regulates uterine PRL receptor, long term ovariectomized rabbits (>12 weeks) were treated with various combinations of hormones, and unoccupied and total uterine PRL receptors were determined. Progesterone treatment resulted in the highest concentration of both unoccupied and total PRL receptor after desaturation and removal of anti-ovine PRL antibodies with MgCI2. The value for total uterine PRL receptor was equivalent to the value for mammary gland, and the Kd values (2-4 x 10~10 M) were similar. Treatment of long term ovariectomized rabbits with progesterone, with or without estradiol, produced an increase (P < 0.05) in the UG mRNA content, which also occurred in PSP animals. PRL alone had no effect on UG mRNA but PRL plus progesterone increased (P < 0.05) UG mRNA in a dose-dependent manner. We propose a servomechanism by which PRL acts morphologically and biochemically to enhance the uterine response to progesterone. (Molecular Endocrinology 2: 1169-1175, 1988) INTRODUCTION PRL receptors have been identified in steroidogenic organs (testis, ovary, and adrenal glands) and target tissues for steroid hormones, especially uteri from sheep (1), pigs (2), rats (3), mink (4), and rabbits (5, 6). The rabbit uterus is a target organ for progesterone, which acts directly on uterine epithelial cells (7, 8) to produce a rise in the steady state level of uteroglobin (UG) mRNA (9) through an increase in the rate of transcription of the UG gene (10). Thus UG provides a useful marker for the molecular mechanism of action of progesterone (11,12). PRL acts on the rabbit uterus to cause endometrial hypertrophy and glandular differentiation (13,14). These changes are accompanied by an increase in the concentration of cytosol estrogen and progesterone receptor (15) when long term ovariectomized (LTOVX) rabbits are treated with PRL. The sequential treatment of LTOVX rabbits with PRL plus progesterone induces UG secretion 4-fold higher than in LTOVX rabbits treated with progesterone alone (15). Collectively, these data support the hypothesis that a dynamic relationship exists between PRL and progesterone in the regulation of uterine function (15). To define the mechanism whereby PRL augments the uterine response to progesterone, we quantitated changes in PRL receptor during uterine development and after hormonal treatment. To investigate whether PRL modulates the progesterone-induced accumulation of UG mRNA, LTOVX animals were treated with different hormone regimens, and UG mRNA levels were quantitated by slot-blot hybridization to a 32P-labeled genomic DNA probe. We report here that progesterone regulates the uterine PRL receptor in adult animals and suggest a mechanism for the mutual effects of these hormones on UG mRNA. RESULTS 0888-8809/88/1169-1175$02.00/0 Molecular Endocrinology Copyright © 1988 by The Endocrine Society PRL receptor assays were characterized with membranes from rabbit mammary glands after in vivo de1169 Department of Cell Biology (S.K.M., D.W.B.) Baylor College of Medicine Houston, Texas 77030 Vol2No. 12 MOL ENDO-1988 1170 100 ~ D • unoccupied total 80 | | eon jE oc = 40 Q. O E r. 20 D 2-WK B 1 4-WK ESTROUS Age or Hormonal Status 8 PSP Fig. 1. Uterine PRL Receptor Concentrations in 2-Week (2wk) and 4-Week-(4-wk) Old Juveniles, in Sexually Mature Estrous Rabbits, and in 5-Day PSP Rabbits Values are expressed as mean ± SEM, and mean values with the same letter designation are not significantly different (P > 0.05). ing sites rather than changes in the affinity of the receptor. Ovariectomized rabbits (Fig. 2) showed a significant reduction in the concentration of unoccupied uterine receptor (6.8 ± 0.4 fmol/mg protein) compared to estrous controls, suggesting that the concentration of PRL receptor may depend on ovarian hormones. As shown in Fig. 2, when LTOVX animals were injected with various combinations of steroid hormones, progesterone treatment resulted in a significant increase in the concentration of PRL receptor. This stimulation was not further enhanced by pretreatment with PRL, and the Kd values for all treatment groups were similar to those of the mammary gland and uterine tissue from estrous control animals. Pretreatment with PRL plus estradiol resulted in a significant decrease in the stimulation of PRL receptor by progesterone (Fig. 2). In the absence of PRL, estradiol alone decreased the stimulation of PRL receptor by progesterone, although the decrease was not significant. Animals injected with PRL produce anti-ovine PRL (oPRL) antibodies which may contaminate membrane preparations and cause an artificial increase in the determination of PRL receptor (18). There was no evidence of anti-oPRL antibodies in group 1 and 2 animals that were treated with PRL alone, or in group 5 and 7 animals that were treated with steroid hormones. Animals in groups 3,4, and 6, however, that were injected sequentially with PRL and steroid hormones, produced measurable quantities of anti-oPRL antibodies (mean ± SEM, 18,294 ± 4381 cpm or 36.2 ± 8.7% of the total counts, compared to estrous controls, 188 ± 12 cpm or 0.38 ± 0.02% of the total counts). As the antibodies are removed from membrane preparations by MgCI2 (17), the assay for total PRL receptor (18) achieves antibody-free conditions. As shown in Fig. 3, progesterone treatment of LTOVX rabbits resulted in the highest concentration of total uterine endometrial S£ T T 30- I a. a a. co 2 0 - LTOVX PR. 1PRL.P 2PRLP 2PRUE«P E»P P Hormonal Status Fig. 2. Unoccupied PRL Receptor Concentrations in Uterine Endometrium of LTOVX Rabbits Treated with PRL (1 or 2 mg); 1 mg PRL Followed by Progesterone (1 PRL + P); 2 mg PRL Followed by Progesterone (2PRL + P); 2 mg PRL, Followed by Estradiol, Followed by Progesterone (2PRL + E + P); Estradiol Followed by Progesterone (E + P); or Prog (...truncated)