Morphine, naloxone and the gonadotrophin surge in ewes

Possible endogenous opioid peptide regulation of the preovulatory gonadotrophin surge was examined in ewes during the breeding season. Intact ewes (n = 54) were synchronized by treatment for 12 days with intravaginal sponges releasing medroxyprogesterone acetate. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion prior to and during the gonadotrophin surge were not affected by naloxone (0.33 mg/kg body wt per h) administered from the time of medroxyprogesterone acetate withdrawal until 30 h after the onset of oestrus (n = 6). Morphine was administered in 4 patterns: (i) 0.25 mg morphine/kg body wt per h from medroxy-progesterone acetate withdrawal until 30 h after the onset of oestrus (n = 6), (ii) 0.25 mg morphine/kg body wt per h from 24 to 48 h after medroxyprogesterone acetate withdrawal (n = 6), (iii) 0.50 mg morphine/kg body wt per h from 24 to 36 h after medroxyprogesterone acetate withdrawal (n = 6) and (iv) 0.50 mg morphine/kg body wt per h from 18 to 30 h after medroxyprogesterone acetate withdrawal (n = 6). Oestrus and the gonadotrophin surge were delayed, but not blocked, in all cases of morphine administration (P less than 0.05). Inconsistent effects of morphine on circulating oestradiol and gonadotrophin concentrations prior to the gonadotrophin surge suggest that the delays are not due to reduced gonadotrophic support of ovarian oestradiol output. Morphine may reduce responsiveness of central behavioural and gonadotrophin surge-generating centres to the oestradiol signal. The absence of effects of naloxone on gonadotrophin secretion suggest that suppression of LH secretion by opioid peptide activity is reduced after the end of the luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chorionic gonadotropin and uterine dialogue in the primate

Implantation is a complex spatio-temporal interaction between the growing embryo and the mother, where both players need to be highly synchronized to be able to establish an effective communication to ensure a successful pregnancy. Using our in vivo baboon model we have shown that Chorionic Gonadotropin (CG), as the major trophoblast derived signal, not only rescues the corpus luteum but also modulates the uterine environment in preparation for implantation. This response is characterized by an alteration in both the morphological and biochemical activity in the three major cell types: luminal and glandular epithelium and stromal fibroblasts. Furthermore, CG and factors from the ovary have a synergistic effect on the receptive endometrium. Novel local effects of CG which influence the immune system to permit the survival of the fetal allograft and prevent endometrial cell death are also discussed in this review. An alternate extracellular signal-regulated kinase (ERK) activation pathway observed in epithelial endometrial cells and the possibility of differential expression of the CG/LH-R isoforms during gestation, open many questions regarding the mechanism of action of CG and its signal transduction pathway within the primate endometrium.     

ACTH and growth hormone relationship in fetal rat adrenal steroidogenesis in vitro.

The effects of growth hormone and ACTH, alone or in combination, on fetal rat adrenal steroidogenesis in vitro were examined on the last day of intrauterine development. ACTH increased, while growth hormone did not affect fetal adrenal weight. ACTH increased fetal rat adrenal steroidogenesis, hydroxylation of 4-14C-progesterone to corticosterone, 18-hydroxy-11-deoxycorticosterone, 11-hydroxycorticosterone and aldosterone. Growth hormone alone had no effect on fetal adrenal steroidogenesis. ACTH and growth hormone administered together increased the conversion of progesterone to the above mentioned steroids to a greater extent than ACTH alone. The results indicate that growth hormone may participate in the fetal rat adrenal steroidogenesis potentiating the effects of fetal pituitary ACTH.     

Changes in the relationship between cerebrospinal fluid and blood composition produced by ACTH treatment in conscious sheep

It has been proposed that an increase in CSF osmolality could be involved in the genesis of hypertension by activation of central nervous system receptors involved in cardiovascular regulation. ACTH induced hypertension in the sheep is an adrenally dependent model of steroid induced hypertension. This study reports the effect of ACTH administration (20 g/kg/day) for 5 days on the composition of cerebrospinal fluid (CSF) and blood (plasma) in conscious sheep. ACTH increased CSF and plasma osmolality within 24 h associated with parallel increases in both blood and CSF glucose concentrations and plasma and CSF sodium concentration. Plasma potassium fell within 24 h, but CSF potassium did not change over the 5 days of ACTH treatment. Neither calcium nor magnesium changed in either plasma or CSF. CSF phosphate increased and plasma phosphate decreased. CSF and plasma bicarbonate were elevated with ACTH. Plasma chloride decreased after 5 days of ACTH treatment but was not associated with a change in CSF. The relevance of the measured changes in CSF osmolality and composition to the mechanisms involved in the production of ACTH-induced hypertension will be subject of further experimentation.     

ACTH receptor: Ectopic expression, activity and signaling

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH₃₉ in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH₃₉ with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH₃₉ in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and –07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH₃₉, implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH₃₉ and, in addition, ACTH₃₉ promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH₃₉ in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH₃₉, implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH₃₉. However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.