No abstract

The pattern of proteins synthesized and secreted in response to progesterone by guinea-pig endometrial epithelial cell cultured with estradiol-17 beta was investigated. Glandular epithelial cells were maintained in culture for 3 days on growth medium, then washed three times with a steroid-free medium. After this period, 2 x 10(-8) M estradiol-17 beta or 2 x 10(-8) M estradiol-17 beta plus 5 x 10(-7) M progesterone were added to the medium for 48 h. To study biochemical changes, the proteins in medium and in cells were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. The addition of progesterone to estradiol-17 beta in the culture medium caused a change in the patterns of cellular and secreted proteins: one-dimensional gel electrophoresis showed that variation of 8 cellular proteins and 12 secreted proteins was caused by progesterone. Induction of individual proteins by progesterone treatment was observed by two-dimensional gel electrophoresis: one cellular protein (Mr 49,000; pI 5.90) and one secreted protein (Mr 14,300; pI 4.80) were specifically induced and might serve as markers of progesterone action.

In the guinea pig, the estrous cycle is characterized by a constant measurable level of plasma progesterone with two peaks: the first one associated with the peak of plasma estradiol-17 beta occurring at proestrus and the second, during diestrus, more pronounced at the time at which the level of estradiol-17 beta is undetectable. The progesterone receptor content is the highest on day 1 and the lowest on day 10 of the estrous cycle, which lasts 16.3 +/- 1.5 days (n = 37; mean +/- SD). There is a positive correlation between the plasma level of estradiol-17 beta and the progesterone receptors detected immunocytochemically in both endometrial epithelial and stromal cells. The general morphology of the endometrium during proestrus and estrus is consistent with an estrogenic stimulation, i.e., a smooth and regular surface of the endometrium and the presence of numerous microvilli on the cell surface. However, a moderate secretory activity also occurs in proestrus and estrus. During postestrus, the glandular cells display an increase in characteristic secretory features which parallels the rise of progesterone in the plasma.

The effects of oestradiol-17 beta, progesterone and oestrone-3-sulphate were studied in primary cultures of guinea-pig endometrial glandular epithelial cells. Comparative ultrastructural studies were performed by means of transmission electron microscopy on cells grown either without hormones or with oestradiol-17 beta (2 nmol/l), oestradiol-17 beta (2 nmol/l) plus progesterone (50 nmol/l), or oestrone sulphate (0.1 mumol/l). In the control medium, without steroid hormones, the majority of epithelial cells were poorly differentiated, although numerous small mitochondria were present and abundant lipid droplets could be observed. Oestradiol-17 beta stimulated metabolic activity in the cells. Progesterone added to oestradiol-17 beta-primed cells stimulated the development of the endoplasmic reticulum-Golgi system. Oestrone sulphate induced a higher level of differentiation characterized by large clear mitochondria, well-developed Golgi complexes, and active nuclei, suggesting secretory activity. In all cases, the cultured cells displayed deep invaginations of the nuclear membrane associated with nuclear pores, known as nucleolar channels. After treatment with oestrone sulphate these channels were associated with a characteristic reticular nucleolus. We conclude that cultured endometrial epithelial cells display secretory activity in response to treatment with oestradiol-17 beta plus progesterone, or with oestrone sulphate.

Samples of human endometrium were maintained in organ culture for 6 days on growth medium. On Day 6, ultrastructural studies were performed on endometrial explants, demonstrating that human endometrium grown in organ culture preserved its normal structure. The effect of oestrone sulphate was studied on the endometrium explants. The endometrium was cultured on harvest medium for 4 days to ensure the complete removal of endogenous steroids, the tissues were then incubated with 10(-7) M oestrone sulphate for 24 h. The oestrone sulphatase known to interfere with oestrone sulphate metabolism was present in endometrial organ culture. By incubation with estrone sulphate for 24 h it was demonstrated that oestrone sulphate is hydrolysed to active oestrogens.