Gap junctions form between rat endometrial stromal cells as they undergo decidualization. We have examined the steady-state levels of the gap junction transcripts, connexins 26 and 43 (cx26 and cx43), during artificially induced decidualization in vivo and found that they have a temporal pattern similar to that observed in pregnancy. An in vitro model of decidualization was then used. Endometrial stromal cells from rat uteri sensitized for decidualization were cultured for 24, 48, or 72 h before total RNA was extracted and subjected to Northern blot analyses to determine the steady-state levels of cx26 and cx43 transcripts. The analyses revealed that cx26 transcript steady-state levels decreased, whereas those for cx43 increased, from 24 to 72 h. Using an anti-cx43 antibody, punctate immunofluorescent signals were observed around the periphery of the cells, suggesting that cx43 had been assembled into membrane plaques. The presence of functional gap junctions between the cells was determined in vitro by two dye-coupling methods: preloading and scrape-loading. Calcein (995 Da) and a membrane-bound dye, dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate (933 Da), were preloaded into 5% of the endometrial stromal cells before plating. The percentage of preloaded cells that transferred calcein to adjacent cells increased from 10% at 3 h after plating to 40% at 6 h. To determine whether or not cells maintain the ability to dye-couple throughout the culture period, carboxyfluorescein (CF; 376 Da) and rhodamine dextran (9.3 kDa) were introduced into cells by scraping the cells with a scalpel, and the distribution of dyes was determined 20 min later. In cells cultured for 24, 48, or 72 h, only CF was transferred to cells distal to the scrape line. The results from these experiments suggest that stromal cells can dye-couple throughout the culture period (3-72 h) and indicate that functional gap junctions form between endometrial stromal cells as they undergo decidualization in vitro.

Possible roles for neutrophils in the response of sensitized rat uteri to a deciduogenic stimulus were investigated. Ovariectomized rats were sensitized for the decidual cell reaction by the administration of estradiol and progesterone. Sesame oil was injected into one uterine horn as a deciduogenic stimulus on the equivalent of Day 5 of pseudopregnancy. Neutrophil numbers in the endometrium, as determined by microscopy after application of the stimulus, were dependent on time and stimulation. Very few neutrophils were found in the endometrium before stimulation. To determine whether neutrophils were involved in the uterine responses to a deciduogenic stimulus, rats were depleted of circulating neutrophils by prior treatment with either methotrexate (MET) or antineutrophil serum (ANS). The increase in endometrial vascular permeability, as quantified with [125I]-BSA 10 h after stimulation, was not significantly affected by MET or ANS treatment, nor was the extent of decidualization, as indicated by the weight of the uteri 5 days after stimulation. Endometrial neutrophil numbers were significantly lower in the MET- and ANS-treated animals compared to their respective controls. In the uterus, activity of myeloperoxidase, an enzyme found mainly in neutrophils, was significantly reduced in MET-treated animals. To investigate the role of neutrophils in blastocyst implantation, we treated rats with ANS early on Day 5 of pregnancy. On Day 6, neither the numbers of implantation sites nor their weights were affected by ANS treatment. ANS treatment on Day 5 of pregnancy did not attenuate the numbers and weights of placentae and fetuses on Days 19 and 20 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)