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Differential Effects of Progestin and Relaxin on the Synthesis and Secretion of Immunoreactive Prolactin in Long Term Culture of Human Endometrial Stromal Cells^*

HUI HUI ZHU, JUN RONG HUANG, JAMES MAZELLA, MIRIAM ROSENBERG and LINDA TSENG

Department of Obstetrics and Gynecology, State University of New York Stony Brook, New York 11794

Address all correspondence and requests for reprints to: L. Tseng, Department of Obstetrics and Gynecology, School of Medicine, State University of New York, Stony Brook, New York 11794.

PRL secretion from human endometrium is a continuous process extending from the luteal phase of the menstrual cycle throughout the entire gestational stage. We have developed a long term primary cell culture system to elucidate the hormonal requirements for this sustained production of PRL. The effects of medroxyprogesterone acetate (MPA), progesterone, and relaxin (RLX) on the production of immunoreactive PRL were investigated. MPA stimulated cell growth and PRL production rate during days 5–20 of culture. Progesterone was 20–40% less effective in stimulating PRL than MPA. Stimulation of PRL was continued 1–2 weeks after MPA withdrawal. Relaxin did not promote cell growth. However, it induced the PRL production which fluctuated during the long term culture. The maximal response to RLX was 2- to 3-fold higher or similar to that of MPA. Only five of nine endometrial specimens examined responded to RLX alone. The effect of MPA plus RLX was significantly greater than that of MPA or RLX alone. The highest production rate was shown in cells treated with MPA and then RLX in sequence. After a month of culture, the production rates (micrograms of PRL per 0.1 mg cell DNA/day) under various culture conditions (A, control; B, MPA; C, MPA for 10–15 days and no hormone afterward; D, both MPA and RLX; and E, MPA and RLX in sequence) were: A, about 0–0.01 (n = 12); B, 2.5 ± 0.9 (n = 8); C, 4.8 ± 2.5 (n = 8); D, 5.7 ± 3.0 (n = 5); and E, 11 ± 3.7 (n = 7); mean ± SD; n, number of specimens).

Endometrial stromal cells were incubated with [³⁵S]methionine, and [³⁵S]immunoreactive PRL and other secretory proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to characterize the size and isoforms of immunoreactive PRL. PRL was one of the five major secretory proteins (23–25K, 32K, 42K, 78K, and 150K daltons, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition) induced by MPA and RLX in endometrial stromal cells. More than 90% of immunoreactive PRL was secreted into the medium. The apparent mol wt of immunoreactive PRL were 21K, 23K (the predominant size), and 25K daltons. Results obtained from the incorporation of [¹⁴C]glucosamine into immunoreactive PRL indicated that both 23K and 25K PRL contained glycosylated PRL. A 45K-dalton glycosylated immunoreactive PRL was also present in the culture medium. The isoforms of 23–25K PRL had pI values ranging from 5.2–5.8. The majority of the endometrial PRL was focused at pI 5.7, whereas pituitary PRL, run in parallel, was focused at pI 5.4 and 5.7.

These results show that multiple sizes of immunoreactive PRL are secreted from endometrial stromal cells under the influence of progestin and RLX. Stimulation of PRL is sustained for 1–2 weeks after progestin withdrawal. The production rate is maximal when stromal cells are treated with progestin and RLX in sequence. The stimulation of PRL by progestin and RLX is accompanied by four other major secretory proteins.

^* This work was supported by USPHS Grant HD-19247 from the NIH.

On sabbatical leave from December 1989 to November 1990 at OB/GYN, SUNY at Stony Brook. Permanent address: Agricultural Research Organization, Institute of Animal Sciences, Bet Degan, Israel.

Received March 26, 1990.

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