METHODS: Female rats, once rendered hypothyroid via oral administration of methimazole (% in drinking water) for twenty-one days were mated with fertile euthyroid male rats at 1:1 ratio. Pregnancy was confirmed by the presence of vaginal plug and this was designated as day-1. Thyroxine (20, 40 and 80 μg/kg/day) was then subcutaneously administered to pregnant, hypothyroid female rats for three days. A day after last injection (day four pregnancy), female rats were sacrificed and expression of thyroid hormone receptors (TR-α and β), retinoid X receptor (RXR) and extracellular signal-regulated kinase (ERK1/2) in uterus were quantified by Western blotting while their distribution in endometrium was visualized by immunofluorescence.
Concentrations of testosterone and androstenedione in the spent medium were measured at 24 h, 48 h, and 72 h using RIA and ELISA, respectively. When cocultured with GCs, TCs produced an increased amount of testosterone and androstenedione than TCs cultured alone ( , Figure 2(a) for testosterone and Figure 2(c) for androstenedione). Interestingly, when TCs were cultured alone, the production of testosterone and androstenedione showed a declining trend along time, which may reflect the adaptation change to in vitro conditions lacking the supports from other cell types. In the spent medium of GCs cultured alone, the levels of testosterone (Figure 2(b) ) and androstenedione (Figure 2(d) ) were both diminished in comparison with TCs. And no alterations were observed following different time of in vitro culture ( ). Taken together, these results indicated that GCs could promote TCs production of testosterone and androstenedione.