Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium.

Proenkephalin mRNA has previously been shown to be expressed in the rodent uterus with varying levels during the estrous cycle. To examine for the potential regulation of proenkephalin gene expression by steroid hormones in a primate displaying a menstrual cycle and to define the functional tissue within the uterus expressing this transcript, we have used Northern blot analysis of extracted RNA from isolated uterine tissue subtypes from normal adult rhesus macaques obtained during the menstrual cycle and from ovariectomized females under different physiological steroid hormone treatments. A strong band of proenkephalin mRNA of 1.3 kilobases was detected almost exclusively in the proliferative endometrium from monkeys in the follicular phase of the cycle. No proenkephalin mRNA was detected in secretory endometrium obtained from monkeys in the luteal phase. When ovariectomized macaques were implanted with silastic capsules of 17 beta-estradiol, proenkephalin mRNA was detected in the endometrium but not the myometrium of the estradiol-treated animals. No proenkephalin mRNA was detected in ovariectomized control animals. Under these conditions, we were unable to detect proenkephalin mRNA in ovariectomized macaques implanted with separate silastic capsules of 17 beta-estradiol and progesterone or in decidual tissue from early or late pregnancy. These results suggest that in the primate uterus 1) proenkephalin mRNA is expressed primarily in the endometrium of the uterus, 2) expression of the proenkephalin gene is regulated by 17 beta-estradiol in the endometrium, and 3) this effect of estradiol is antagonized by progesterone.

Effects of progesterone on decidual prolactin production by organ cultures of human endometrium.

The effects of progesterone on decidual PRL (dPRL) production by human endometrium were investigated by culturing explants of proliferative (n = 20) and secretory (n = 12) endometrium in Dulbecco's Modified Eagle's Medium supplemented with 10 mM HEPES buffer, 0.1% gelatin (wt/vol), and antibiotics for 6 days and in medium containing 50 ng/ml of progesterone for 6-28 days. The culture medium was replaced daily, and the spent medium was assayed for dPRL. Representative explants were fixed for histological examination after 1, 2, 7, 14, and 28 days in vitro. When explants of endometrium obtained throughout the cycle were cultured in the presence of progesterone, dPRL production was stimulated. Proliferative endometria required 2-5 days in culture before dPRL was measured in the medium. Thereafter, dPRL production increased gradually for the duration of the experiment. Progesterone also induced glandular secretion and stromal differentiation (decidualization) in these tissues. Cultures of proliferative endometrium that did not receive progesterone did not produce detectable amounts of dPRL. When explants of secretory endometrium were cultured in DMEM without exogenous progesterone, dPRL was released into the medium for 2-5 days; however, dPRL production by the cultures that did not receive progesterone declined to undetectable levels, while that by the progesterone-treated cultures increased steadily. Explants of both proliferative and secretory endometrium that were fixed for histological examination after 28 days of culture in the presence of progesterone were composed predominately of large stromal cells that resembled the decidual cells of pregnancy endometrium.

Steroid production by early pregnancy human placental villi in culture.

Organ cultures prepared from human placentae obtained at 7-12 weeks of gestation were maintained for 3-13 days in Dulbecco's modified Eagle medium (DMEM). The addition of pregnenolone to the medium resulted in a dose-related increase in progesterone production and the addition of androstenedione resulted in a dose related increase in oestrogen production. More oestrone than oestradiol was measured in medium collected at the end of the first day of culture, but, on all subsequent days, oestradiol was the predominant oestrogen produced when androstenedione was added to the culture medium. When villi were incubated with [3H]androstenedione immediately after dissection most of the radiolabelled oestrogen recovered from the tissue and medium was oestrone; however, more [3H]oestradiol was recovered when villi were tested after 3 days of culture in DMEM. The addition of oestrone to the culture medium resulted in a dose related increase in oestradiol production with oestradiol accounting for a larger proportion of the total oestrogen in the day 2 and 3 medium samples than in the day 1 samples. These data demonstrate that the enzymes required for biosynthesis of progesterone and oestrogen from exogenous substrate are maintained for at least 13 days when early pregnancy placental villi are cultured in serum-free DMEM. However, a temporal change in the pattern of oestrogen synthesis does occur in culture, such that oestradiol rather than oestrone becomes the major product of androstenedione metabolism.

Effect of short-duration progesterone treatment on decidual prolactin production by cultures of proliferative human endometrium.

The effect of short-term progesterone (P) treatment in vitro on decidual prolactin (dPRL) production by human endometrium was investigated. Cultures prepared from proliferative endometrium received medium containing P for 3 hours, 1 day, or 3 days. The culture medium was then changed daily for 7 to 14 days, and the amounts of dPRL in the spent medium were measured by radioimmunoassay. The results of these experiments indicated that even a short exposure to elevated concentrations of P is adequate to stimulate dPRL production and that the resulting pattern of dPRL production is determined by the duration of the P treatment.

Evidence for a nonpituitary source of amniotic fluid prolactin.

Prolactin levels were determined during a gonadotropin-induced pregnancy following hypophysectomy for a chromophobe adenoma. Maternal plasma prolactin concentrations did not vary significantly from prepregnancy values throughout gestation, remaining between 25 and 35 ng/ml. Fetal prolactin levels were 55 ng/ml and maternal levels were 29 ng/ml at delivery. Amniotic fluid prolactin concentration was approximately 100 ng/ml. Decidual tissue isolated from the maternal surface of the chorion released significant amounts of prolactin into the medium during a 24-hour incubation. Final concentrations of prolactin in the incubation medium were as high as 196 ng/ml. It is concluded that after hypophysectomy (1) prolactin is present in the maternal circulation during pregnancy, and the concentration does not change significantly throughout gestation; (2) fetal and amniotic fluid prolactin concentrations near term do not differ significantly from those reported for normal pregnancy; and (3) the capacity of the decidua to release prolactin in vitro is not diminished compared with normal term decidua, suggesting a nonpituitary source of amniotic fluid prolactin.

The significance of lymphocytic-leukocytic infiltrates in interpreting late luteal phase endometrial biopsies.

The effectiveness of the endometrial biopsy in diagnosing luteal phase defects as a cause of infertility depends upon the accurate determination of a histologic date based on the morphologic features of tissue. The criteria--edema, predecidual reaction, stromal mitosis, and lymphocytic-leukocytic infiltrate--used to interpret such biopsies were based on changes occurring in the normal ideal menstrual cycle. The present study examines the criteria used for dating endometrium as applied to endometrial biopsies for luteal phase deficiency. It was determined that one of these criteria, lymphocytic and leukocytic infiltration, correlated with subsequent onset of menses and not with the other indications of histologic maturity during the late secretory phase.

Prolactin production by human endometrium during the normal menstrual cycle.

Prolactin (PRL) synthesis by human decidua from term pregnancies has been reported. The present study examined the tissue content and in vitro production of prolactin by "decidualized" and "nondecidualized" endometrium unassociated with pregnancy. Tissue obtained throughout the menstrual cycle was dated histologically. When proliferative endometrium (N = 16) or secretory endometrium prior to day 22 (N = 6) was examined, no PRL was detected in the tissue or medium after a 24-hour incubation at 37 degrees C in Gey's buffer. Total PRL in tissue and medium measured by radioimmunoassay increased significantly from 1.2 +/- 0.3 ng/100 mg of tissue at cycle days 22 to 24 (N = 4) to 5.3 +/- 2.4 ng/100 mg of tissue at cycle days 25 to 26 (N = 7). The addition of 100 micrograms/ml of cycloheximide to the medium prevented the net increase in PRL content during incubation. It is concluded that PRL is synthesized by endometrium during the normal menstrual cycle and that the appearance and degree of synthesis and decidualization of the stroma correspond.

Prolactin production during in vitro decidualization of proliferative endometrium.

Decidua obtained in the late luteal phase of the human menstrual cycle has been shown to produce immunoreactive prolactin (PRL). The amount of PRL produced is a function of the extent of decidual differentiation. Further, the maintenance of decidualization and PRL production in specimens of late luteal phase in explant culture is dependent on the presence of progesterone (P). To further examine P's effect on decidualization, PRL production was monitored during P-induced decidualization of proliferative endometrium in vitro. Samples of proliferative endometrium obtained from six hysterectomy specimens were cultured in Dulbecco's modified Eagle medium in the presence of no hormones, 200 pg/ml estradiol (E2) 20 ng/ml P, and 20 ng/ml P with 200 pg/ml E2. It was found that P alone or with E2 caused PRL production and histologic decidualization. However, E2 slowed the histologic progression of P-induced decidualization on days 2 and 4 and decreased the rate of PRL production (p less than 0.01) on days 6, 8, and 10 of culture. These data indicate that P alone is capable of inducing decidualization and initiating PRL production. Further, E2 can modify the rate and/or extent of this P-mediated phenomenon.

Term decidua response to estradiol and progesterone.

Immunoreactive prolactin (PRL) is produced from decidualized endometrium from the late luteal phase until the time of delivery. The induction of decidualization and the initiation of PRL production in the proliferative endometrium is dependent on progesterone (P) in vitro. This induction process is slowed by estradiol (E2). To determine whether this hormone dependency extends to term, decidua from labor and nonlabor term pregnancies was cultured in explant for response to P (100 ng/ml) and E2 (10 ng/ml) as evidenced by PRL production. On the first (n = 7) and eighth to ninth (n = 9) days of explant culture in nonlabor patients, P exposure resulted in significantly (p less than 0.01) more PRL production than in nonhormonal controls, and this increase was not inhibited by E2 at either time interval. In labor-exposed decidua, no significant response was noted to either P or E2 over 24 hours of culture. In all groups, tissue variability in PRL production was extensive. In nonlabor decidua, a significant interaction between basal PRL production and response to progesterone was noted (p less than 0.01). The role of P as an initiator and stimulator of PRL production extends to term; however, no clear effect of E2 is demonstrated. In labor-exposed decidua, this P response is eliminated. Whether this is a result of, or occurs prior to, labor is undetermined.

Decidual prolactin production by organ cultures of human endometrium: effects of continuous and intermittent progesterone treatment.

Explants of human endometrium were cultured in serum-free nutrient medium and the effects of continuous and intermittent progesterone treatment on decidual prolactin (dPRL) production compared. The synthesis of dPRL was induced in cultures of proliferative and secretory endometrium when progesterone (50 ng/ml) was added to the medium. The amount of dPRL produced by these cultures increased gradually during 41 days of continuous progesterone treatment. When progesterone was provided for only the first 14 days of a 28-day cycle, dPRL production continued to increase during the first wk of culture in the absence of exogenous hormone and then began to decline. A similar pattern was elicited during a second 28-day cycle. Explants of endometrium fixed for histologic examination after either continuous or intermittent progesterone treatment contained large "decidualized" stromal cells. These findings indicate that progesterone can induce and maintain decidualization and dPRL synthesis in organ cultures of human endometrium, dPRL production increases immediately after progesterone is withdrawn, and long-term dPRL production is not maintained in the absence or progesterone.

Decidual production of prolactin in late gestation: further evidence for a decidual source of amniotic fluid prolactin.

The capacity of human decidual tissue to synthesize prolactin de novo throughout late gestation was investigated and correlated with the levels of prolactin (PRL) in amniotic fluid. Maximal concentrations of PRL in both amniotic fluid and samples of decidua were found prior to the thirtieth week of gestation and declined simultaneously until term. A high correlation (r = 0.90, p < 0.00005) was found when the levels of PRL in amniotic fluid and the initial (preincubation) content of PRL in decidua from the same patient were compared. A very high correlation (r = 0.96, p < 0.00005) was seen between the ability of the decidua to produce prolactin in vitro and the corresponding levels of prolactin in amniotic fluid. No significant difference in any parameter tested was noted with respect to either the sex of the fetus or the mode of delivery (p > 0.05). These data are interpreted as indicating (1) that decidual tissue varies throughout late gestation, in both its initial content of prolactin and its ability to synthesize prolactin de novo, in a manner which correlates to a high degree with variations in amniotic fluid prolactin levels and (2) that the decidual tissue is the major source of amniotic fluid prolactin.

Amniotic fluid and decidual prolactin during pregnancy in rhesus macaques.

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.

Thyroxine uptake and metabolism by fetal sheep after intra-amniotic thyroxine injection.

Thyroxine (T4) uptake from amniotic fluid was investigated in fetal sheep. Samples of fetal and maternal blood and of amniotic fluid were obtained from indwelling catheters at specific intervals after intra-amniotic injection of T4. T4 and reverse T3 (rT3) were measured by radioimmunoassay. Basal levels of T4 were 7.3 +/- 0.92, less than 2, and 6.28 +/- 0.49 microgram/dl in the fetus, amniotic fluid, and ewe, respectively. Basal levels of rT3 were 3,858 +/- 214, 189 +/- 62, and 385 +/- 20 pg/ml in the fetus, amniotic fluid, and ewe, respectively. T4 and rT3 rose progressively in the fetus with maximum concentrations of 25 to 30 microgram/dl T4 by 10 hours and 11,000 to 14,000 pg/ml rT3 by 20 hours after intra-amniotic injection of 500 microgram of T4. These concentrations returned toward baseline by 50 and 70 hours for T4 and rT3, respectively. The increase of fetal T4 was proportional to the amount of T4 injected in a range of 250 and 2,500 microgram. Esophageal ligation abolished the changes in fetal T4 but not rT3. Amniotic fluid rT3 increased with time after intra-amniotic injection of T4 and returned to baseline long after amniotic fluid T4 had reached basal levels. This pattern persisted despite esophageal ligation. T4 was converted to rT3 during incubation in amniotic fluid in vitro. It is concluded that (1) substantial amounts of T4 are taken up by fetal fetal sheep from the amniotic fluid by deglutition, (2) increases in fetal concentrations of T4 and rT3 are related to the amount of T4 added to the amniotic fluid, (3) amniotic fluid and fetal rT3 concentrations increase following intra-amniotic injection of T4 in the absence of significant uptake of T4 by the fetus, and (4) significant amounts of T4 may be converted to rT3 in vitro during incubation of T4 in amniotic fluid.

Prolactin production by luteal phase defect endometrium.

The production of prolactin (PRL) by explants of late secretory endometrium obtained during normal cycles (n = 61) and that by similar explants obtained during luteal phase deficient (n = 17) cycles was compared. The amount of prolactin produced in vitro correlated with the degree of histologic decidualization in both normal and luteal phase defect endometria. However, samples of luteal phase defect endometrium produced significantly less prolactin (p less than 0.01) than did control tissues of the same ideal menstrual dates. These data indicate that the amount of prolactin produced by late secretory endometrium in explant culture can be used as an additional criterion for the diagnosis of luteal phase defects and may also provide a method for evaluating the response of the endometrium to progesterone.

Stimulatory activity of serum on prolactin production by human decidua.

Fetal calf serum (FCS) enhances the synthesis and secretion of prolactin (PRL) by explants of human decidua incubated in Gey's balanced salt solution. In order to further characterize the factors involved in the prolactin-stimulating activity (PSA) of serum, decidua was incubated in Gey's buffer alone and in buffer containing either FCS, bovine serum albumin (BSA), or human serum (HS). When buffer was supplemented with varying concentrations of FCS, ad dose-related PSA was apparent between 0.1% and 10%. The PSA of FCS was not altered by eithr heating (85degrees C for 30 minutes), boiling, or dialysis. The protein fraction of FCS, precipitated with trichloroacetic acid, redissolved in dilute NaOH, and dialyzed against Dulbecco's phosphate buffer, demonstrated PSA comparable to that of whole serum. A similar concentration of BSA added to Gey's buffer did not stimulate the production of PRL by decidua. The PSA of human serum obtained from euprolactinemic men and women was not different from that of FCS. These data indicate that FCS and HS contain a substance(s) (1) which stimulates the secretion of PRL by human decidua, (2) which is very stable, being resistant to dialysis, boiling, and denaturation by trichloroacetic acid, (3) and which is most likely a large polypeptide with a molecular weight greater than 12,000 or a smaller moiety tightly bound to a protein.