Observation of production of immunoactive prolactin by normal human connective tissue in cell culture.

Data from our in vitro studies indicate a new source of prolactin (PRL)-like activity, normal human connective tissue. Fascial cells from primary culture and subsequent passages produced an extracellular antigen which specifically reacted in a radioimmunoassay RIA developed to detect human pituitary PRL. An initial peak or first surge of fascial PRL-like activity occurred between 4 and 15 d in primary culture. Ibuprofen, cytotoxic levels of 0.01% azide, or 7.5 mM EDTA and medium lacking serum [fetal bovine serum (FBS)] significantly (P less than or equal to 0.05) reduced PRL-like activity levels, whereas female steroids, 257 to 342 milliosmolarity, 1 to 3.6 mg/ml glucose, 2 to 20% FBS, and dialyzed FBS (MWCO approximately 1 kDa) were without effect. Optimum production of PRL-like activity occurred at pH 7.3. A second surge began after 18 d and continued until passage indicating that perhaps two populations of cells produced PRL-like activity in primary culture. Production of PRL-like activity by cells from early passages (1 and 2) became detectable at confluence, was serum-dependent, showed two patterns (tonic, rising to plateau), and averaged 3.2 fg.cell-1.3 d-1 feed interval. Cells from late passages showed morphologic damage from repetitive trypsinization, aging, and reduced production of PRL-like activity with aberrant production pattern. Production of PRL-like activity was maintained in an unusual long-term culture. These in vitro studies demonstrate the most recently recognized and ubiquitous source of human extrapituitary PRL or PRL-like activity, normal connective tissue (fascia).

Human uterine luminal fluid volumes and prolactin levels in normal menstrual cycles.

Human uterine luminal fluid has not been well characterized. Prolactin is produced in vitro by decidualized human endometrium and is secreted in vivo into the uterine luminal fluid of cynomolgus monkeys. Uterine luminal fluid prolactin has not been demonstrated in vivo in human beings. To study cyclic uterine luminal fluid volumes and prolactin levels, uterine luminal fluid was aspirated 8 to 13 times each during a single menstrual cycle in six ovulatory women. No anesthesia was used. Seventy of 75 (93%) attempts were successful; there were no complications. Serum estradiol, progesterone, luteinizing hormone, and prolactin levels were assayed every 1 to 3 days. Uterine luminal fluid volume and prolactin were normalized to the luteinizing hormone peak (day 0). Uterine luminal fluid volumes were relatively constant until the early luteal phase, when they then decreased. Uterine luminal fluid prolactin was detectable in all samples. Mean values were stable until day +3 or +4, after which they rose to a peak at day +9 or +10. This study establishes that (1) frequent uterine luminal fluid sampling is possible; (2) uterine luminal fluid volume decreases in the luteal phase; (3) uterine luminal fluid prolactin is detectable in vivo in women and its concentration increases in the luteal phase; and (4) cyclic human uterine luminal fluid prolactin levels differ from those in cynomolgus monkeys and from those anticipated from human in vitro studies.

Uterine fluid and prolactin secretion in the ovulating cynomolgus monkey.

Immunoreactive prolactin is produced by late secretory human endometrium in vitro. Human myometrium in explant culture also produces prolactin. A primate model with the use of the cynomolgus monkey is described that allowed repeated samplings of uterine secretions in vivo. The uterine secretory prolactin thus measured appears immunoreactively similar to human serum prolactin, and the pattern of secretions reflects the previously described pattern of endometrial prolactin production in vitro.

Prolactin production from proliferative phase leiomyoma.

In vivo and in vitro endometrial stromal synthesis of prolactin occurs after progesterone-induced decidualization. Synthesis of prolactin by myometrium in vitro suggests that cells whose embryologic origin is the loose mesenchyme surrounding the paramesonephric ducts may retain the capacity to synthesize prolactin. Since physiologic myometrial synthesis of prolactin has not been demonstrated in vivo, prolactin genome expression in pathologic conditions was considered. Follicular phase leiomyomas were diced to 8 mm3 and cultured in Dulbecco's modified Eagle's medium (DMEM) with either no hormones, estradiol 200 pg/ml, progesterone 20 ng/ml, or estradiol and progesterone. Media were sampled and changed every other day for 8 days, followed by culture in tritium-labeled leucine DMEM for 2 days. Portions of leiomyomas were homogenized for initial prolactin content, and all samples were assayed for prolactin by radioimmunoassay. Follicular phase leiomyomas contained prolactin (47 +/- 15 ng/gm) in excess of normal serum values. Synthesis was demonstrated during all time periods from leiomyomas not exposed to progesterone. Progesterone variably suppressed the synthesis of prolactin until after 144 hours of culture. Determination of molecular weight on a 60 by 1.5 cm Sephadex G-100 column revealed identical estimates for pituitary, decidual, and leiomyoma prolactin. Tritium-labeled leucine incorporation into prolactin was confirmed by immunoprecipitation of Sephadex G-100 column fractions. Similar antigenicity was confirmed by parallel dilution curves for pituitary, decidual, and leiomyoma prolactin. Preliminary bioactivity in lymphoma proliferation assays confirmed prolactin activity. The conclusion reached was that proliferative phase leiomyomas contained elevated prolactin presumably secondary to in vivo synthesis. This synthesis was confirmed in vitro.

Human myometrium: a new potential source of prolactin.

Human myometrium is shown for the first time to produce prolactin in vitro. This prolactin is similar to pituitary prolactin by criteria of immunologic identity, gel chromatography and bioassay. The de novo synthesis of myometrical prolactin is supported by no detectable prolactin in initial tissue homogenate, nondetectable prolactin production during the first 24 hours of culture, cycloheximide inhibition of prolactin production with recovery of production in control medium, and tritiated leucine incorporation into prolactin. Although human myometrium is capable of producing prolactin without the addition of exogenous hormones, the addition of estrogen and progesterone, respectively, enhances and suppresses prolactin production in contrast to decidualized human endometrium where opposite effects on prolactin production are found.