Effect of testosterone on gonadotropin response to DBcAMP, cAMP binding, and cAMP production in pituitary cultures.

The role of testosterone (T) in the modulation of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) sensitivity to DBcAMP was examined in the pituitary monolayer cultures prepared from intact young female rats. Hormone contents in media and cell homogenates were determined by radioimmunoassays. Incubation with 8 and 4 mM DBcAMP for 4 h consistently induced a significant (P less than 0.05) increase in FSH and LH release, respectively. Pretreatment with 10 nM T for 4 days reduced the minimal dose of DBcAMP required to stimulate FSH release (2 vs. 8 mM) but had no effect on the DBcAMP-induced LH release. Data indicate that T treatment for 4 or 7 days stimulated total FSH contents (sum of hormone contents in medium and cells). Similarly, incubation with 10 mM DBcAMP for 4 h significantly increased total FSH content per dish in both the T-treated and non-T-treated cultures. Neither T nor DBcAMP had any effect on LH production under these conditions. Intracellular cAMP was significantly increased to three- to eightfold of control after T treatment for 3 or 6 h, respectively. Furthermore, cAMP-binding activities were significantly increased after T treatment for 1 or 4 days (174 or 422% of control). Our previous data indicate that estrogen increases LH production, cAMP binding, cAMP production, and LH sensitivity to DBcAMP, and these data indicate that T exerts stimulatory effects on FSH in a similar fashion. These results support the concept that the two gonadotropins are regulated independently via different gonadal steroids.

Luteinizing hormone response to N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate in aged, young and androgenized rats: estradiol effect in vitro.

The studies in this report were designed to investigate whether the loss of pituitary luteinizing hormone (LH) responses to N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) in aged noncycling rats was the result of age, endocrine status associated with diestrus, or noncyclic low estrogen status. Pituitary monolayer cultures were prepared from female Fischer 344 rats. Aged (18-month-old) persistent diestrous (PD) rats, young diestrous (D) rats, or noncycling neonatally androgenized-constant estrous (AN-CE) rats were used. Enzymatically dispersed cells were maintained in the same batch of medium supplemented with dextran-coated charcoal adsorbed serums. Total LH contents were 1.75 +/- 0.04, 1.15 +/- 0.03, and 1.71 +/- 0.02 micrograms LH/dish in Day 5 cultures prepared from aged PD, young D, and AN-CE rats, respectively. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated LH release in cultures prepared from young D and AN-CE rats but inhibited LH release in cultures prepared from aged PD rats even though a 4-h incubation with 10 nM LH releasing hormone (LHRH) stimulated LH release similarly in cultures of all three types of cells. The loss of DBcAMP-induced LH release in cultures prepared from aged PD rats was reversed by 17 beta-estradiol (E2). This treatment also reduced the basal LH release and increased the cellular LH content. These results indicate that the loss of DBcAMP-induced LH response in the aged rat is not an irreversible aging phenomenon but appears to be associated with the chronically low E2 status of aged PD rats but not young cycling D or noncycling AN-CE rats.

Estradiol stimulation of pituitary cAMP production and cAMP binding.

The role of 17 beta-estradiol (E2) in the modulation of N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced hormone release was examined in pituitary monolayer cultures prepared from intact and ovariectomized female rats. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated both luteinizing hormone (LH) and prolactin (PRL) release in pituitary cultures prepared from rats at diestrus and from cycling rats at random stages of the estrous cycle. However, DBcAMP failed to stimulate the LH or PRL release in cultures prepared from ovariectomized rats in which the basal LH and PRL release was approximately three-fold and one-tenth of that in cycling rats, respectively. Pretreatment with 1 nM E2 augmented or restored the DBcAMP-induced LH release but not the DBcAMP-induced PRL release in cultures prepared from cycling or ovariectomized rats, respectively. Furthermore, E2 treatment alone of cultures prepared from cycling rats significantly increased intracellular cAMP concentrations and cAMP-binding activities by at least twofold over that of the non-E2-treated controls. The E2-induced rise in cellular cAMP concentration preceded the E2-induced rise in cAMP binding. These results indicate that the priming effect of E2 on pituitary LH responsiveness to DBcAMP is associated with increased cAMP production and cAMP binding.

Estradiol stimulation of LH response to LHRH and LHRH binding in pituitary cultures.

The relationship between 17 beta-estradiol (E2) stimulation of luteinizing hormone (LH) response to LH-releasing hormone (LHRH) and E2 effect on LHRH binding was examined in pituitary monolayer cultures prepared from female rats. E2 pretreatment significantly (P less than 0.05) augmented the LHRH-induced LH release to 158-180% of the non-E2-treated controls. The maximal E2-priming effect could be observed after 1 day of treatment. E2 treatment for 3 days stimulated [D-Ala6]luteinizing hormone-releasing hormone (LHRHa) binding to about 1.5-fold that of the non-E2-treated controls without affecting the dissociation constant of LHRH receptor (Kd = 4 X 10(-10) M). The stimulatory effect of E2 on cell proliferation as determined by [3H]thymidine incorporation was also observed 3 days after treatment. However, E2 stimulation of LH accumulation in the cultured cells could be detected as early as 4 h after treatment. These results indicate that E2-priming effect on pituitary LH response to LHRH is initially associated with an increase in cellular LH content and later associated with increases in LHRH binding and in an index of cell proliferation that may include the LH-producing cells.