Steroidogenic refractoriness of bovine adrenocortical cells to dibutyryl cyclic AMP

The long-term effects of dibutyryl cyclic AMP [(Bu)₂-cAMP] on steroidogenesis in bovine adrenocortical cells maintained in primary culture were investigated. During the first 36 h, total steroid production by cells incubated with (Bu)₂-cAMP increased progressively. Thereafter, however, there was a marked fall in steroid output. During the first 36 h adrenocortical cells incubated in the presence of (Bu)₂-cAMP produced substantially more C₁₉-steroids and 17α-hydroxylated C₂₁ -steroids than did cells incubated in the absence of (Bu)₂-cAMP. By 48 h, however, such steroid secretion by cells incubated in the continued presence of (Bu)₂-cAMP declined toward control levels. By contrast, the secretion of corticosterone and 11-deoxycorti-costerone was consistently less by cells maintained in the presence of (Bu)₂-cAMP than by cells maintained in its absence. These results suggest that refractoriness results, at least in part, from events which occur distal to the formation of cAMP. The action of ACTH and (Bu)₂cAMP to promote the secretion of 17α-hydroxylated C₂₁-steroids and C₁₉-steroids, on the other hand, appears to reflect an increase in the rate of cholesterol side-chain cleavage, as well as an increase in 17α-hydroxylase and possibly also 17,20-lyase activities.     

The releasing action of calcium upon cyclic AMP-dependent meiotic arrest in hamster oocytes

The effect of increasing cytoplasmic calcium on cyclic adenosine monophosphate (cAMP)-dependent meiotic arrest (%GV where GV is germinal vesicle) in hamster oocytes was investigated. The hypotheses tested were that calcium is required for the spontaneous maturation of hamster oocytes, elevation of calcium in the oocyte-cumulus complex can antagonize cAMP-dependent meiotic arrest, and the intraoocyte level of cAMP remains unchanged, but heterologous metabolic coupling decreases, concomitant with calcium-stimulation of germinal vesicle breakdown (GVBD). Levels of cAMP were elevated by culturing cells in the presence of dibutyryl cAMP (dbcAMP), isobutylmethylxanthine (IBMX) or forskolin and intracellular levels of calcium were manipulated by altering the CaCl2 concentration in the medium and/or by utilizing EGTA or A23187. Intracellular cAMP was determined by RIA, functional metabolic coupling was assessed by determination of the fraction of radiolabeled uridine marker transferred from the cumulus mass to the oocyte, and meiotic stage was determined cytogenetically. Compared with the proportion of oocytes that underwent meiotic maturation in control medium containing 1.53 mM CaCl2, that of cumulus-free (denuded) oocytes was unaffected by culture in the absence of added CaCl2, while that of cumulus-enclosed (intact) oocytes was significantly decreased (%GV = 59.5 +/- 4.8 and 4.2 +/- 0.9 in 0 and 1.53 mM CaCl2, respectively, P less than 0.001, where GV is germinal vesicle). EGTA prevented, in a dose-dependent manner, the spontaneous maturation of denuded oocytes that occurred in 0 mM CaCl2 (ID50 = 0.05 mM, where ID50 is the dose of EGTA that inhibited GVBD in 50% cultured oocytes). In contrast, compared with the control, less than 1 mM EGTA failed to increase the %GV of intact oocytes, although 5 mM EGTA significantly increased meiotic arrest. The %GVBD of oocytes cultured in medium containing 0 mM CaCl2 was dose-dependent on A23187 for both intact oocytes (ID50 = 3.0 microM) and for denuded oocytes cultured in the presence of 0.5 mM EGTA (ID50 = 2.7 microM). Elevated extracellular calcium significantly antagonized dbcAMP-maintained meiotic arrest in both types of oocyte and the %GV was significantly correlated with the pH of the medium [(r) = -0.78 and -0.60 for intact and denuded oocytes, respectively, P less than 0.001 in both cases]. Both CaCl2 and A23187 induced dose-dependent antagonistic effects on forskolin-maintained meiotic arrest in intact oocytes but neither antagonism was accompanied by significant dose-dependent decreases in either the intraoocyte content of cAMP or the extent of heterologous metabolic coupling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the mode of action of gonadotrophin-inhibiting material (anti-LH) isolated from human urine.

Gonadotrophin-inhibiting material (GIM) was able to inhibit the binding of 125I-hCG to rat Leydig cells, suggesting that its inhibiting properties are due to its ability to complete for the hCG binding sites on Leydig cells. Binding of fluorescein isothiocyanate-tagged GIM to Leydig cells was also seen. The effect of GIM on hCG-induced adenylate cyclase activation and testosterone production was also studied. It was found that there was a dose-dependent inhibition of both these effects.     

Abdominal vagotomy decreased the number of ova shed and serum progesterone levels on estrus in the cyclic hamster.

The effects of abdominal vagotomy (AVGT) on ovarian function were studied in cyclic hamsters. AVGT significantly decreased the number of ova shed (AVGT: 10.5 +/- 1.5 ova/hamster, sham: 15.8 +/- 0.7 ova/hamster; P less than 0.05) and serum progesterone levels (AVGT: 2.1 +/- 0.3 ng/ml, sham: 3.9 +/- 0.7 ng/ml; P less than 0.05) on the morning of estrus. However, progesterone concentrations in the corpora lutea and non-luteal ovary on estrus in the AVGT and sham groups were similar. The serum estradiol levels in both groups on proestrus increased from 0900 h (AVGT: 75 +/- 10 pg/ml, sham: 72 +/- 6 pg/ml) to 1500 h (AVGT: 204 +/- 27 pg/ml, sham: 196 +/- 35 pg/ml) but there was no significant difference between the two groups. Partial degranulation of ovarian mast cells was not increased in the AVGT group. Also, vasoactive intestinal peptide (VIP) content in the ovary was not increased by AVGT at 0900 h on proestrus (AVGT: 60.1 +/- 16.8 pg/ovary, sham: 37.2 +/- 14.3 pg/ovary). These results indicated that AVGT interferes with normal ovulation and results in an increase in the number of atretic follicles, but that these effects by AVGT seemed not to be mediated through ovarian mast cells and VIP.     

Involvement of prostaglandins in LH-induced superovulation in the cyclic hamster

Osmotic minipumps containing 400 micrograms ovine LH were inserted subcutaneously (sc) on day 1 (estrus) at 09:00-10:00h of the cycle in the hamster. This treatment induced increased ovarian blood flow by day 3 and superovulation of 30.0 +/- 1.4 ova at the next estrus compared to controls (16.5 +/- 0.8 ova). The continuous infusion of LH throughout the cycle increased prostaglandin F (PGF) and decreased prostaglandin E (PGE) in the growing follicles destined to ovulate and suppressed a day 3 increase in PGF concentrations in the nonluteal ovarian remnant devoid of the larger follicles. Indomethacin, a cyclooxygenase inhibitor, given sc (2 or 4 mg regimens) at 12:00-14:00h on days 1 and 2, at 09:00h and 17:00h on day 3 and at 09:00h on day 4 of the cycle to LH-infused and saline treated animals suppressed ovarian prostaglandin levels, prevented the superovulation and prevented the increased ovarian blood flow. Exogenous PGF2 alpha or PGE2 restored the superovulatory effect of LH infusion in the presence of indomethacin. The results suggest that the superovulation in response to continuous LH infusion may be mediated in part by prostaglandins via altered ovarian blood flow.     

Development of persistent ovarian follicles during synchronization of estrus influences the superovulatory response to FSH treatment in cattle

The synchronization of estrus with synthetic progestins or progesterone (P(4)) results in the development of a large, persistent ovarian follicle. The objectives of the present study were to determine if development of a persistent ovarian follicle during synchronization of estrus suppresses recruitment of additional follicles during FSH treatment. On Day 5 of the estrous cycle (estrus = Day 0), beef cows were treated with 0.5 or 2.0 P(4) releasing intravaginal devices (PRIDs) for 8 d (Experiment 1, n = 20), 5 or 2 d (Experiment 2, n = 44) before initiation of FSH treatment. Prostaglandin F(2alpha) (25 mg) was administered on Days 5 and 6. Superovulation was induced with 24 mg of recombinant bovine FSH (rbFSH, Experiment 1) or 28 mg of FSH-P (Experiment 2) over a 3- or 4-d period, respectively. The PRIDs were removed concurrently with the 5th injection of rbFSH or FSH-P. There was a treatment-by-day interaction (P < 0.001) for the concentration of 17beta-estradiol in cows treated for 8, 5 or 2 d before FSH treatment. In Experiment 1, FSH treatment initiated 8 d after insertion of a 0.5 PRID did not affect the number of CL (6.9 +/- 1.4 vs 6.7 +/- 1.6), ova/embryos (3.7 +/-1.3 vs 3.0 +/- 1.3) and transferable embryos (2.4 +/- 0.9 vs 3.0 +/- 0.9) compared with that of the 2.0 PRIDs. In Experiment 2, FSH treatment initiated 5 d after insertion of a 0.5 PRID decreased the number of CL (4.0 +/- 0.5 vs 8.3 +/- 0.8; P < 0.001), ova/embryos (3.0 +/- 0.6 vs 5.9 +/- 1.2; P < 0.03) and transferable embryos (2.3 +/- 0.6 vs 5.1 +/- 1.0; P < 0.03) compared with that of a 2.0 PRID, respectively. Initiation of FSH treatment 2 d after insertion of a 0.5 PRID compared with a 2.0 PRID had no affect on the number of CL (8.0 +/- 2.1 vs 8.7 +/- 1.2), total ova (4.8 +/- 1.4 vs 6.9 +/- 1.4) and transferable embryos (2.9 +/- 1.2 vs 6.1 +/- 1.7). In conclusion, treatment with low doses of P(4) (0.5 PRID) for 5 d but not for 2 or 8 d before initiation of FSH treatment results in the development of a dominant ovarian follicle, which reduces recruitment of ovarian follicles, and the number of CL, total ova and transferable embryos.     

Antidyslipidemic action of fenofibrate in dyslipidemic-diabetic hamster model

Fenofibrate is the ligand for PPARalpha subtype that mediates the action of its agonists' in lipid metabolism. How fibrate exerts hypolipidemic effect? The mechanism is studied in a newly developed high-fat fructose enriched diet induced dyslipidemia-diabetic hamster model. Fenofibrate lowered the basal plasma lipids like TC, TG, PL, FFA, glycerol, VLDL, and LDL, but HDL was increased. The activity of lipoprotein lipase in liver, adipose tissue, and small intestine was upregulated. However, that of triglyceride lipase was downregulated in liver. It has also improved the insulin secretion and plasma glucose lowering, caused by impairment in insulin secretion due to high-fat load. The drug was found effective in reducing body weight and diet due to rise in leptin level. Fenofibrate also enhanced the fecal excretion of total lipids, cholic acid, and deoxycholic acid probably by the activation of 7alpha cholesterol hydroxylase enzyme. Thus, causing broad-spectrum lipid lowering along with inhibition of hepatic lipid biosynthesis and maintaining lipid-glucose homeostasis.     

Functional compartments in cyclic nucleotide action

Cyclic AMP-dependent protein kinase (cAMP-PK) is a ubiquitous enzyme that, when activated by cAMP, is capable of phosphorylating a variety of intracellular proteins. The central postulate of cAMP-mediated hormone action is that hormones regulate intracellular cAMP concentration and cAMP-PK mediates the effects of this second messenger. Although this postulate accurately describes cAMP action in certain systems, it does not adequately provide for recent observations of the accumulation of cAMP and the activation of protein kinase without the anticipated effects on protein kinase's substrates. Both biochemical and cytochemical technics provide evidence that hormonally-specific regulation of cAMP action occurs and is important. Our thesis is that hormonal regulation of metabolic events via cAMP is localized intracellular phenomenon. We propose that occupation of some cell-surface hormone receptors leads to cAMP accumulation and the activation of protein kinase in subcellular compartments, with the consequent phosphorylation of specific, rather than all, substrates of protein kinase. circumstances potentially contributing to this specificity include: (a) physical and kinetic compartmentation of hormone-receptor-adenylate cyclase complexes non-randomly within the cell membrane; and, (b) a fixed spatial relationship of hormonally activated adenylate cyclase and specific intracellular regions by the participation of cytoskeletal proteins.     

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.     

PGE1-mediated cyclic AMP refractoriness: effects of cycloheximide and indomethacin.

Human synoviocytes in culture respond to prostaglandin E1 (PGE1) by increasing their intracellular concentration of cyclic AMP. Readdition of PGE1 to cells previously treated with PGE1 elicits no change in the intracellular concentration of cyclic AMP. This refractory state is partially prevented by the inhibitors of protein synthesis, puromycin (PM) and cycloheximide (CH). Indomethacin (IM), which reduces angiotensin tachyphylaxis, does not prevent the occurrence of refractoriness to PGE1 with respect to accumulation of cyclic AMP. This agent does alter the release of cyclic AMP from human synovial cells. We postulate that other factors, independent of new protein synthesis, are necessary for the development of the complete PGE1 refractory state in these cells.     

Progesterone levels measured every two hours in the cyclic hamster.

Four groups of unanesthetized hamsters were bled by cardiac puncture three times a day at 4-hr intervals for each day of the 4-day estrous cycle. Serum progesterone (P) was determined by RIA. On day 1 of the cycle (day of ovulation) there was a trend for excursions in P at 4- to 6-hr intervals; this was followed on day 2 by a relatively steady level of P of 6-8 ng/ml. P dropped drastically after 0200 of day 3, indicating the onset of luteolysis. A significant increase in P occurred at 1600 of day 3 which was presumably nonluteal in origin. A series of cyclic fluctuations in P began at 1600 of day 4 (proestrus) comparable to the pattern observed on day 1 of the cycle.     

Acute effects of unilateral ovariectomy on follicular development in the cyclic hamster

Hamsters were hemispayed at 09:00 h on Day 3 of the cycle (Day 1 equal to ovulation) and were killed 1 h after injection of [3H]thymidine at 09:00, 12:00, 17:00 or 22:00 h. Unilateral ovariectomy had no effect on the number of Stage 1 or Stage 2 follicles, but there were significantly fewer Stage 3 follicles between 10:00 and 13:00 h. This decrease was not encountered in intact hamsters and was reflected in an increase in the number of Stage 6 (antral) follicles. At 13:00 h there was no difference in the number of atretic follicles between the experimental and control groups. It is concluded that preantral follicles with 6-7 layers of granulosa cells were recruited within 4 h after unilateral ovariectomy and transformed into antral follicles.