Organization of 3β-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358–1364] of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and cytochrome P450_scc (cyt. P450_scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3β-HSD and cyt. P450_scc is observed during the purification of 3β-HSD from mitochondria. The behaviour of 3β-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450_scc. Immunoprecipitations from mitochondria with either anti-cyt. P450_scc or anti 3β-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 μM between 3β-HSD and P450_scc was obtained. These observations suggest that P450_scc and 3β-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.     

Effects of human chorionic gonadotropin (hCG) and phenobarbital on the reproductive performance of fat-tailed Ghezel ewes

Increased ewe prolificacy and lambing rates, under certain conditions of sheep husbandry, may be economical and desirable. This paper reports on the effects of human chorionic gonadotropin (hCG) and phenobarbital (PB) on fertility and lambing rate in fat-tailed Ghezel sheep. One-hundred and five cyclic ewes (1.5 to 7.5 years of age) were randomly allotted to seven groups. Estrus was synchronized with two intramuscular injections of a prostaglandin F₂ analogue (PGF₂), eight days apart. Four groups were randomly assigned to the hCG and three groups to PB experiments. Twenty-four hours after the second injection of PGF₂, the ewes in four groups were injected (i.m.), either with 1 ml normal saline (control) or with 125, 250 or 500 IU hCG. Prolificacy of the ewes which conceived at first estrus increased in ewes receiving 500 IU of hCG, as compared with control group (an increase of 0.5 lamb/ewe) but fertility and lambing rate decreased in hCG-treated groups (P<0.05). Nine days after the second injection of PGF₂, ewes in the three remaining groups were orally fed empty gelatin capsules (control), 1.0 or 1.5 g of PB per day until the next estrus. Prolificacy and lambing rate increased (P<0.05) in ewes which received 1.0 g of PB (1.57 and 146.5%, respectively), as compared with the control groups (1.17 and 100.3%, respectively). It is concluded that daily feeding of 1.0 g of PB from day 7 of the estrous cycle until the next estrus period is an effective method of increasing lambing rate. hCG also increases the prolificacy, but it greatly decreases fertility and lambing rates. Since hCG is more convenient to administer on a flock basis, further experiments to improve its efficacy are warranted.     

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates

The side-chain cleavage of cholesterol by cytochrome P-450_scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3β-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450_scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450_scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20-hydroxycholesterol, 22R-hydroxycholesterol and 20,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450_scc occur at approximately the same rate.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.     

Inducing ovulation with hCG improves the fertility of dairy cows during the warm season

This study was designed to assess the effects of human chorionic gonadotrophin (hCG), given within a timed artificial insemination program, on plasma progesterone concentrations and subsequent fertility in lactating dairy cows during the warm and cold seasons of the year. Cows were treated intramuscularly with GnRH-agonist (Day 0) and PGF₂ (Day 7) followed by either GnRH-agonist (GPG treatment; 60 animals) or hCG (GPH treatment; 60 animals) on Day 9. All cows were fixed-time inseminated (TAI) 16–22 h after the end of treatment. To determine plasma progesterone levels, blood was withdrawn from all animals on Days 3, 6, 9, 12 and 15 after TAI. During the warm period, the pregnancy rate recorded at TAI was similar for the GPG and GPH groups (20% vs. 23%) while the cumulative pregnancy rate within 30 days of TAI was lower (P < 0.05) for the GPG than the GPH group (36% vs. 63%). No differences were observed during the cold period. During the warm period, embryo losses between Days 28 and 45 after TAI were greater (P < 0.05) for the GPG group compared to the GPH group (36% vs. 5%) while again no differences emerged during the cold period. Mean plasma progesterone levels were higher (P < 0.05) in the GPH group than GPG group on Days 3, 6 and 9 post-insemination. Our findings indicate that the use of hCG to induce ovulation in a timed artificial insemination protocol increases plasma progesterone levels and improves fertility in dairy cows during the warmer period of the year.     

A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 Aromatase)

In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the “I-helix model” by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (−)-R-vorozole, (−)-S-fadrozole, R-liarozole and (−)-R-aminoglutethimide. It is found that (+)-S-vorozole, (−)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu³⁰² and Asp³⁰⁹) and C-terminus (Ser⁴⁷⁸ and His⁴⁸⁰). The weak activity of (−)-R-aminoglutethimide might be attributed to a lack of interaction wit the C-terminus.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Examination of cyclic changes in bovine luteal echotexture using computer-assisted statistical pattern recognition techniques

B-mode sonography is a well-established diagnostic tool for determination of cycle stage in gynaecology. The aim of this study was to determine whether computer-assisted texture analysis of B- mode sonographic images of bovine luteal glands provides further information about the animal's plasma progesterone concentration and cycle stage. Four Simmenthal cows were examined during two consecutive estrous cycles with an ultrasound device equipped with a 7.5 MHz microconvex probe. During each examination three B-mode images of the corpus luteum (CL) were digitized and analyzed off-line using a computer-assisted texture analysis program. Size, echogeneity, and echotexture of the CL were characterized by the following texture parameters: area of cross-sectional planes of the CL (A), mean gray level (MGL), correlation (CORR), run percentage (RPERC), and long-run emphasis (LREM). Plasma progesterone levels (P4) were also determined. All parameters showed characteristic changes during the estrous cycle (P < 0.05). Variance component estimates for the effect of Day of estrous cycle on A, MGL, CORR, RPERC, and LREM were 56.6%, 64.6%, 77.6%, 89.9%, and 86.0%, respectively, and 20.6%, 24.5%, 7.2%, 0.0%, and 14.0% for the influence of the individual cow. The factor estrous cycle within cows was responsible for 22.8%, 10.9%, 15.2%, 10.1%, and 0.0% of the variability of A, MGL, CORR, RPERC and LREM values, respectively. Cyclic changes were similar in A and P4. In contrast to P4, which decreased already between Days –5 and –3 (Day 0 = ovulation), A stayed at constant high values until Day –3. Mean MGL values were higher (P < 0.05) on Days 7, 9, and 13 compared to Days 3 and –3. Mean CORR values were constantly high (P > 0.05) during the first days after ovulation and decreased continuously (P < 0.05) between Days 5 and 13. Thereafter, mean CORR values remained low (P < 0.05) until the next ovulation, except on Day –3 (P < 0.05). Mean RPERC rose between Days 1 and 9 from low to high values (P < 0.0001) remained at these high values (P > 0.05) between Days 9 and 15, and decreased (P < 0.05) afterwards to baseline values on Day –1. Mean LREM inclined steeply (P < 0.0001) from minimum to maximum between Days 1 and 5. From Days 7 to –3, mean LREM remained (P > 0.05) at a constant level close below the maximum value, and decreased to baseline values on Day –1. The results of this study show that statistical pattern recognition techniques provide new information about the luteal glands, thus facilitating a more accurate differentiation between different cycle stages in cows.     

Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy and at day 200 in cows. The hypophysis is not obligatory after day 50 nor the ovaries after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE(2) and PGF(2alpha) synthesis), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased (P< or =0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased (P< or =0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 (P> or =0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater (P< or =0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased (P< or =0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ (P> or =0.05). Concentrations of PGE were increased (P< or =0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased (P< or =0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF(2alpha) were increased in media (P<0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF(2alpha) in media declined (P< or =0.05) and did not differ (P> or =0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.     

Transcriptional regulation of aromatase in placenta and ovary

Our goal is to define the cellular and molecular mechanisms for tissue- and cell-specific, developmental and hormonal regulation of the human CYP19 (aromatase P450/P450arom) gene in estrogen-producing cells. In this article, we review studies using transgenic mice and transfected cells to identify genomic regions and response elements that mediate CYP19 expression in placenta and ovary, as well as to define the molecular mechanisms for O₂ regulation of differentiation and CYP19 gene expression in human trophoblast cells in culture. We also highlight recent findings regarding LRH-1 versus SF-1 mRNA expression and cellular localization in the mouse ovary during the estrous cycle and various stages of pregnancy. Spatial and temporal expression patterns of mRNAs encoding these orphan nuclear receptors in comparison to those of P450arom and 17-hydroxylase/17,20-lyase mRNAs, suggest an important role of LRH-1 together with SF-1 in ovarian steroidogenesis.     

Ability of deglycosylated human chorionic gonadotropin (dghCG) to block luteal function and establishment of pregnancy in bonnet monkeys (Macaca radiata).

The ability of deglycosylated hCG (dghCG) prepared by deglycosylation of a clinical hCG (3000 IU/mg) preparation, to block luteal function during regular cycles as well as luteal rescue in simulated and mated cycles of female bonnet monkeys (M. radiata) has been evaluated. The cycle length (C:28 vs E:24 days) and the total progesterone produced during the luteal phase was significantly reduced (by 45%, P < .05) by injecting 450 micrograms of dghCG/day (in split doses) on days 18, 19, and 20 of cycle. At the doses tested the dghCG used did not exhibit any agonistic activity in the female monkey. In a second experiment injection of 200 micrograms of dghCG/day on days 18-20 of cycle blocked the normal response of the luteal tissue to exogenous hCG (10 micrograms of a 12,000 IU/mg preparation) injected on day 23 of cycle. In a third experiment no pregnancies occurred when a group of 5 animals were injected dghCG (450 micrograms dghCG/day) on days 18-21 of their mated cycle. Animals chosen for this study were proven fertile regularly cycling monkeys and these were cohabited with males between days 9 and 14 of cycle. Each of the monkeys was exposed to 3 consecutive treatment cycles. During post-treatment phase 2 out of 3 monkeys exposed to males became pregnant. The study clearly demonstrates that it is possible to block normal luteal function as well as luteal rescue of the female monkey by using dghCG in the right dose and mode.     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Reproductive studies of Brahman cattle III. Comparison of weight, progesterone content, histological characteristics, and 3β-hydroxysteroid dehydrogenase activity in corpora lutea of Brahman, Hereford, and Brahman × Hereford heifers

Sixty corpora lutea (CL), 30 from day 8 and 30 from day 13 of the estrous cycle were collected from 10 Brahman, 10 Hereford and 10 Brahman × Hereford F-1 (B×H) heifers and compared for weight, progesterone concentration and progesterone content. 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and histological and morphological differences were evaluated in CL from 10 animals from each breed at each day.

The Brahman CL were significantly smaller than either Hereford or B×H Cl, 2.616, 3.836 and 4.211 g, respectively. No statistically significant differences were detected for luteal progesterone concentration or content, however, Brahman and B×H CL tended to have less progesterone per CL than did Hereford CL. The histology and morphology of the luteal tissue appeared similar for the three breeds, since there were no detectable differences in the organization, apparent population of cells per area, or the cell types present in the CL. Brahman CL had significantly higher 3β-HSD activity than Hereford or B×H. Day 13 corpora from all breeds had higher 3β-HSD enzyme than CL from day 8 of the cycle. It is evident from this study that major differences exist in CL from Brahman and B×H as compared to Hereford.     

The pharmacology of neurosteroidogenesis

In adrenal cortex and other steroidogenic tissues including glial cells, the conversion of cholesterol into pregnenolone is catalyzed by the cytochrome P450_scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450_scc limits the rate of pregnenolone biosynthesis. Participating in this mechanism are DBI (diazepam binding inhibitor), an endogenous peptide that is highly expressed in steroidogenic cells and some of the DBI processing products including DBI 17–50 (TTN). DBI and TTN activate steroidogenesis by binding to a specific receptor located in the outer mitochondrial membrane, termed mitochondrial DBI receptor complex (MDRC). MDRC is a hetero-oligomeric protein: only the subunit that includes the DBI and benzodiazepine (BZD) recognition sites has been cloned. Several 2-aryl-3-indoleacetamide derivatives (FGIN-1-X) with highly selective affinity (nM) for MDRC were synthesized which can stimulate steroidogenesis in mitochondrial preparations. These compounds stimulate adrenal cortex steroidogenesis in hypophysectomized rats but not in intact animals. Moreover, this steroidogenesis is inhibited by the isoquinoline carboxamide derivative PK 11195, a specific high affinity ligand for MDRC with a low intrinsic steroidogenic activity. Some of the FGIN-1-X derivatives stimulate brain pregnenolone accumulation in adrenalectomized-castrated rats. The FGIN-1-X derivatives that increase brain pregnenolone content, elicit antineophobic activity and antagonize punished behavior in the Vogel conflict test in rats. These actions of FGIN-1-X are resistant to inhibition by flumazenil, a specific inhibitor of BZD action in GABA_A receptors but are antagonized by PK 11195, a specific blocker of the steroidogenesis activation via MDRC stimulation. It is postulated that the pharmacological action of FGIN-1-X depends on a positive modulation of the GABA action on GABA_A receptors mediated by the stimulation of brain neurosteroid production.