11beta-Hydroxysteroid dehydrogenases (11beta-HSDs) interconvert active corticosterone and inert 11-dehydrocorticosterone. In tissue homogenates, 11beta-HSD type 1 (11beta-HSD-1) exhibits both 11beta-dehydrogenase (corticosterone inactivating) and 11beta-reductase (corticosterone regenerating) activities, whereas 11beta-HSD type 2 (11beta-HSD-2) is an exclusive dehydrogenase. In the rat testis, 11beta-HSD has been proposed to reduce glucocorticoid inhibition of testosterone production, promoting puberty and fertility. This hypothesis presupposes dehydrogenation predominates. 11beta-HSD-1 immunoreactivity has been localised to Leydig cells. However, recent studies suggest that 11beta-HSD-1 is predominantly an 11beta-reductase in many intact cells. We therefore examined the expression and reaction direction of 11beta-HSD isozymes in cultures of intact rat Leydig cells. Reverse transcriptase PCR demonstrated expression of 11beta-HSD-1, but not 11beta-HSD-2 mRNA in rat testis. Primary cultures of intact rat Leydig cells showed predominant 11beta-reductase activity, activating 50-70% of 11-dehydrocorticosterone to corticosterone over 3 h, whereas 11beta-dehydrogenation was <5%. Although both dexamethasone (10 nM) and corticosterone (1 microM) modestly inhibited LH-stimulated testosterone production by Leydig cells, inert 11-dehydrocorticosterone (1 microM) had similar effects, suggesting 11beta-reductase is functionally important. Carbenoxolone (10(-5) M) inhibited 11beta-reduction in intact Leydig cells. However, although carbenoxolone reduced Leydig cell testosterone production, this also occurred in the absence of glucocorticoids, suggesting effects distinct from modulation of corticosteroid access to Leydig cells. In conclusion, rat Leydig cell 11beta-HSD-1 is unlikely to reduce glucocorticoid access to testicular receptors. More likely, 11beta-reductase amplifies glucocorticoid action, perhaps to maintain Leydig cell metabolic and endocrine functions.

The major protein secreted by Day-15 guinea-pig endometrium cultured for 24 h had a molecular weight of 68.3 kDa on SDS-PAGE. This protein had no effects on phospholipase (PL) A2 activity in vitro and prostaglandin (PG) production by Day-7 guinea-pig endometrium in culture. Following purification, proteins present in fractions F1, F2, F7, F7:2 and F7:3 (which comprised <15% of the total amount protein secreted) increased PLA2 activity in vitro. The major proteins in F7:2 had molecular weights of 13.9 and 15.6 kDa on SDS-PAGE, but they had no effect on endometrial PG synthesis in the concentration used (20 mg/ml). Unpurified endometrial secreted proteins had no effect on PLA2 activity, but stimulated endometrial PG synthesis. This stimulation was lost following purification of the proteins, and may have been due to a large amount of contaminating serum albumin. The mechanism by which oestradiol acting on a progesterone-primed guinea-pig uterus stimulates endometrial PGF2alpha synthesis by a process which is dependent upon increased endometrial protein synthesis still remains obscure.

The co-culture of Day-15 guinea-pig conceptures or Day-15 pregnant guinea-pig endometrium with Day-15 non-pregnant guinea-pig endometrium had no inhibitory effect on PGF2α output from the non-pregnant endometrium. Unpurified proteins secreted by the Day-15 guinea-pig conceptuses, or these proteins purified by Blue Sepharose CL-6B and ion-exchange column chromatography also had no inhibitory effect on PGF2α output from Day-15 non-pregnant guinea-pig endometrium cultured in vitro. However, following the further purification of guinea-pig conceptus secreted proteins on Sephadex G-75SF, the proteins present in fraction F3:4 inhibited PGF2α output from the Day-15 non-pregnant guinea-pig endometrium during the first 6 h of culture. The major protein present in F3:4 had a molecular weight of 38.2 kDa on SDS-PAGE. Proteins present in F3:4 formed only a minor proportion of the total proteins secreted. Nevertheless, the anti-luteolytic factor secreted by the guinea-pig conceptus may be this 38.2 kDa protein, but further study is required.

Sodium fluoride (10 mM) caused a slow increase in the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and, to a lesser extent, PGE-2 from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. This stimulatory action of sodium fluoride was not prevented by using calcium-free Krebs' solution. There was also a faster stimulation of 6-keto-PGF-1 alpha output from the Day-7 guinea-pig uterus produced by sodium fluoride, and this quicker response was abolished by using calcium-free Krebs' solution. TMB-8 (an intracellular calcium antagonist) inhibited the stimulatory action of sodium fluoride on the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus. W-7 and trifluoperazine (calmodulin antagonists) and neomycin (an inhibitor of phospholipase C) had no inhibitory effect on the increases in outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus produced by sodium fluoride. These results indicate that sodium fluoride slowly stimulates uterine PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha synthesis in the guinea-pig uterus by mobilizing intracellular calcium by a mechanism which apparently does not involve the activation of phospholipase C or the participation of calmodulin (or a related compound). The initial, faster stimulation of 6-keto-PGF-1 alpha synthesis in the Day-7 guinea-pig uterus by sodium fluoride is dependent upon extracellular calcium.

The outputs of prostaglandin (PG) F-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-7 and Day-15 guinea-pig endometrium were neither stimulated nor inhibited by cholera toxin and pertussis toxin. This indicates that PG synthesis by guinea-pig endometrium is not controlled by toxin-sensitive G-proteins. Short-term treatment of guinea-pig endometrium in culture with sodium fluoride stimulated PG output, suggesting that endometrial PG synthesis may be regulated by a fluoride-sensitive G-protein. Long-term treatment of guinea-pig endometrium in culture with sodium fluoride inhibited endometrial PG synthesis, and this was due to an inhibition of endometrial protein synthesis. Human alpha-interferon had no inhibitory effect on the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-15 guinea-pig endometrium in culture. It appears that the anti-luteolytic factor secreted by guinea-pig conceptus is not an alpha-interferon and is therefore probably different from ovine trophoblast protein-1.