Side-chain Cleavage Enzyme Activity Research Articles

Localization of Gonadotropin‐Releasing Hormone (GnRH), Gonadotropin‐Inhibitory Hormone (GnIH), Kisspeptin and GnRH Receptor and Their Possible Roles in Testicular Activities From Birth to Senescence in Mice

The changes in distribution and concentration of neuropeptides, gonadotropin-releasing hormone (GnRH), gonadotropin-inhibitory hormone (GnIH), kisspeptin, and gonadotropin-releasing hormone receptor (GnRH-R) were evaluated and compared with reproductive parameters, such as cytochrome P450 side-chain cleavage (P450 SCC) enzyme activity, androgen receptors (AR) in the testis and serum testosterone levels, from birth to senescence in mice. The results showed the localization of these molecules mainly in the interstitial and germ cells as well as showed significant variations in immunostatining from birth to senescence. It was found that increased staining of testicular GnRH-R coincided with increased steroidogenic activity during pubertal and adult stages, whereas decreased staining coincides with decreased steroidogenic activity during senescence. Similar changes in immunostaining were confirmed by Western/slot blot analysis. Thus, these results suggest a putative role of GnRH during testicular pubertal development and senescence. Treatment with a GnRH agonist ([DTrp(6), Pro(9)-NEt] GnRH) to mice from prepubertal to pubertal period showed a significant increase in steroidogenic activity of the mouse testis and provided further support to the role of GnRH in testicular pubertal maturation. The significant decline in GnRH-R during senescence may be due to a significant increase in GnIH synthesis during senescence causing the decrease in GnRH-R expression. It is considered that significant changes in the levels of GnRH-R may be responsible for changes in steroidogenesis that causes either pubertal activation or senescence in testis of mice. Furthermore, changes in the levels of GnRH-R may be modulated by interactions among GnRH, GnIH, and kisspeptin in the testis.

Regulatory Mechanisms ofCordyceps sinensison Steroidogenesis in MA-10 Mouse Leydig Tumor Cells

Cordyceps sinensis (CS) is an herbal medicine that increases steroidogenesis in Leydig cells and improves male reproductive dysfunction. We have found that CS stimulates Leydig cell steroidogenesis through the protein kinase A and protein kinase C signaling pathways. In the present study, we sought to determine the mechanisms of CS-stimulated steroidogenesis in MA-10 mouse Leydig tumor cells. Using pharmacological approaches, we found that de novo protein synthesis, protein transcription, a calcium signal, and a mitochondria electrochemical gradient were required for CS-stimulated steroidogenesis in MA-10 cells. mRNA expression of steroidogenic acute regulatory protein was activated by CS. However, CS had an adversary effect on P450 side-chain cleavage enzyme activity, but not in 3β-hydroxysteroid dehydrogenase enzyme, in regulating MA-10 cell steroidogenesis. In conclusion, de novo protein synthesis, increased steroidogenic acute regulatory protein mRNA expression, and the mitochondria electrochemical gradient were involved in CS-stimulated steroidogenesis in MA-10 cells.

PINEAL-ADRENAL INTERACTIONS IN DOMESTIC MALE PIGEON EXPOSED TO VARIABLE CIRCADIAN LIGHT REGIMES AND EXOGENOUS MELATONIN

This study was aimed to see the histophysiological changes of the pineal and adrenal glands under altered photoperiodic conditions due to the administration of melatonin in the male domestic pigeon, Columba livia Gmelin. Young adult male domestic pigeons were exposed to long photoperiod (LP; 20 hr light: 4 hr dark), short photoperiod (SP; 4 hr light: 20 hr dark) exogenous melatonin (MEL; 20 microg/100 g body weight/day) was administered for 60 days in the primary breeding and regressive phase. At the end of experiment, adrenal and pineal glands were quickly dissected and processed for histology, ultrastructure study, biochemical, histochemical and immunohistochemical analysis. In the primary breeding phase, the number of mitochondria and rough endoplasmic reticulum were increased in the adrenal gland in MEL-LP group, while lipid granules were also increased in the subcapsular zone. In MEL-SP treated group, however, the number of mitochondria decreased. Pinealocytes were increased in size and well-developed Golgi complexes were present near the cell nucleus after induction with MEL-LP treatment during the primary breeding phase. No remarkable changes were noticed in the number of mitochondria. In the regressive phase-I, the adrenocortical cytoplasm showed similar morphological features both in MEL-LP treatment and control groups. In pinealocytes, few rough endoplasmic reticulum and lipid droplets and moderate number of mitochondria were present. In MEL-SP treatment, increased number of mitochondria in the adrenocortical cells and decreased nuclear diameter of the pinealocytes were noticed. Few mitochondria were observed within the pinealocyte cytoplasm. Side chain cleavage enzyme (immunocytochemical) activity was increased in the subcapsular zone in MEL-LP treated group. The present data indicates that the changes in pineal and adrenocortical tissue histophysiology might be due to melatonin rhythm and light/dark regime which act as a modulator in the male domestic pigeon.

Hormetic-Like Biphasic Dose-Response Relationships Are Resumed in Endocrine Profile During Lead Poisoning in Male Reproduction

ISEE-130 Objective: The objective of this study was to predict the mode of inhibitory actions of lead on steroidogenesis acute regulatory (StAR) protein expression and steroidogenic enzymes on steroidogenesis in MA-10 mouse Leydig tumor cells. Material and Methods: We evaluated our data with linear and second-order polynomial models. The equation and correlation coefficient were calculated by linear regression analysis for the logarithm of Pb concentration versus percent (%) of control and by the second-order polynomial analysis for the logarithm of Pb concentration versus progesterone production or StAR protein expression given as integrated optical density. Results: The correlation coefficients R2 by second-order polynomial were 0.96 for human chorionic gonadotropin-stimulated progesterone production, 0.79 for dibutyryl cAMP-stimulated progesterone production, 0.99 for the expression of StAR protein, 0.92 for P450 side-chain cleavage enzyme activity, and 0.96 for 3b-hydroxysteroid dehydrogenase activity. Conclusions: The hormetic-like biphasic dose-response relationships might be resumed in adaptation of the endocrine profile during lead poisoning in male reproduction.

Cellular mechanisms and modulation of activin A- and transforming growth factor beta-mediated differentiation in cultured hen granulosa cells.

Undifferentiated granulosa cells from prehierarchal (6- to 8-mm-diameter) hen follicles express very low to undetectable levels of LH receptor (LH-R) mRNA, P450 cholesterol side chain cleavage (P450scc) enzyme activity, and steroidogenic acute regulatory (StAR) protein, and produce negligible progesterone, in vitro, following an acute (3-h) challenge with either FSH or LH. It has previously been established that culturing such cells with FSH for 18-20 h induces LH-R, P450scc, and StAR expression, which enables the initiation of progesterone production. The present studies were conducted to characterize the ability of activin and transforming growth factor (TGF) beta, both alone and in combination with FSH, to promote hen granulosa cell differentiation, in vitro. A 20-h culture of prehierarchal follicle granulosa cells with activin A or transforming growth factor beta (TGFbeta)1 increased LH-R mRNA levels compared with control cultured cells. Activin A and TGFbeta1 also promoted FSH-receptor (FSH-R) mRNA expression when combined with FSH treatment. Neither activin A nor TGFbeta1 alone stimulated progesterone production after 20 h culture. However, preculture with either factor for 20 h (to induce gonadotropin receptor mRNA expression) followed by a 3-h challenge with FSH or LH potentiated StAR expression and progesterone production compared with cells challenged with gonadotropin in the absence of activin A or TGFbeta1 preculture. Significantly, activation of the mitogen-activated protein (MAP) kinase pathway with transforming growth factor alpha (TGFalpha) (monitored by Erk phosphorylation) blocked TGFbeta1-induced LH-R expression, and this effect was associated with the inhibition of Smad2 phosphorylation. We conclude that a primary differentiation-inducing action of activin A and TGFbeta1 on hen granulosa cells from prehierarchal follicles is directed toward LH-R expression. Enhanced LH-R levels subsequently sensitize granulosa cells to LH, which in turn promotes StAR plus P450scc expression and subsequently an increase in P4 production. Significantly, the finding that TGFbeta signaling is negatively regulated by MAP kinase signaling is proposed to represent a mechanism that prevents premature differentiation of granulosa cells.

INHIBITORY ACTIONS OF LEAD ON STEROIDOGENESIS IN MA-10 MOUSE LEYDIG TUMOR CELLS

The inhibitory actions of Pb on StAR protein expression and steroidogenic enzymes on steroidogenesis were analyzed by both linear and 2nd order polynomial models in MA-10 mouse Leydig tumor cells. Lead acetate, ranging from 10(-8) M to 10(-5) M, caused inhibitory effects on StAR protein expression and steroidogenic enzymes. The correlation coefficients R2 (linear vs. 2nd order polynomial) were 0.93 vs. 0.96 for human chorionic gonadotropin-stimulated progesterone production, 0.38 vs. 0.79 for dibutyryl cAMP-stimulated progesterone production, 0.03 vs. 0.99 for the expression of StAR protein, 0.6 vs. 0.92 for P450 side-chain cleavage enzyme activity, and 0.52 vs. 0.96 for 3beta-hydroxysteroid dehydrogenase activity. Thus, 2nd order polynomial model showed higher correlation coefficients than the linear model for predicting inhibitory actions of Pb on StAR protein expression and the activities of steroidogenic enzymes after exposure of Pb on steroidogenesis in MA-10 cells.

In vivo implants of β-sitosterol cause reductions of reactive cholesterol pools in mitochondria isolated from gonads of male goldfish ( Carassius auratus)

β-Sitosterol, a phytosterol found in high concentrations in pulp mill effluents, has been proposed as one of the causative agents for steroid depressions observed in fish exposed to pulp mill effluents. Previous studies have suggested a cholesterol-mediated mechanism; however, it is unknown how β-sitosterol depresses gonadal steroidogenesis. In this study, adult male goldfish ( Carassius auratus) were exposed for 24–31 days to β-sitosterol (55% of a phytosterol mixture or 96% pure; 150 μg/g; Silastic implant) after which gonadal mitochondria were isolated. Pregnenolone production, an indicator of the size of the pool of reactive cholesterol, was then measured in the isolated mitochondria. Sterol exposure did not affect P450 side-chain cleavage enzyme (converts cholesterol to pregnenolone) activity but did decrease the size of the mitochondrial pool of reactive cholesterol, suggesting β-sitosterol is impeding cholesterol transfer across the mitochondrial membrane. This finding is supported by the observation that 25-hydroxycholesterol, which passes through mitochondrial membranes without need for a membrane transporter, restores β-sitosterol-induced reductions in pregnenolone production.

Inhibitory effect of digoxin on testosterone secretion through mechanisms involving decreases of cyclic AMP production and cytochrome P450scc activity in rat testicular interstitial cells.

1. In vivo and in vitro experiments were performed to examine inhibitory effects of digoxin on testosterone secretion and to determine possible underlying mechanisms. 2. A single intravenous injection of digoxin (1 microg kg(-1)) decreased the basal and human chorionic gonadotropin (hCG)-stimulated plasma testosterone concentrations in adult male rats. 3. Digoxin (10(-7) - 10(-4) M) decreased the basal and hCG-stimulated release of testosterone from rat testicular interstitial cells in vitro. 4. Digoxin (10(-7) - 10(-4) M) also diminished the basal and hCG-stimulated production of cyclic 3':5'-adenosine monophosphate (AMP) and attenuated the stimulatory effects of forskolin and 8-Br-cyclic AMP on testosterone production by rat testicular interstitial cells. 5. Digoxin (10(-4) M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450sec) activity (conversion of 25-hydroxy cholesterol to pregnenolone) in the testicular interstitial cells but did not influence the activity of other steroidogenic enzymes. 6. These results suggest that digoxin inhibits the production of testosterone in rat testicular interstitial cells, at least in part, via attenuation of the activities of adenylyl cyclase and cytochrome P450sec.

Inducible expression of protein kinase Calpha suppresses steroidogenesis in Y-1 adrenocortical cells.

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCalpha isoform mediates this suppression, we have developed Y-1 cell lines in which PKCalpha is expressed from a tetracycline-regulated promoter. Induction of PKCalpha expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCalpha expression is increased. However, expression of PKCalpha has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCalpha but not PKCepsilon or PKCzeta. These studies define for the first time the role of a specific PKC isoform (PKCalpha) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.

The relationship between testicular activity, accessory sex glands, and circulating steroid concentration during the reproductive cycle in a male Indian vespertilionid bat,Scotophilus heathi

The relationship between spermatogenesis, accessory sex glands, Leydig cell activity, and circulating concentrations of testosterone and androstenedione in male Scotophilus heathi, the greater yellow bat, was studied. Scotophilus heathi accumulated fat prior to winter dormancy at Varanasi, India. Spermatogenesis was seasonal and extended over the period November to March, with the testes becoming quiescent during winter. Monthly changes in testis and accessory sex gland masses showed two peaks, in November and January. Accessory gland secretory activity and fructose concentration showed only one peak, during January and February, which coincided with the mating period. The mass in the epididymides and their histological changes reflect the influence of testicular spermatogenesis. However, sperm were found in the cauda epididymidis during the spermatogenically quiescent period of winter dormancy. The Leydig cells showed intense side chain cleavage (SCC) enzyme activity from August to December. In late January and February, males had smaller Leydig cells and low SCC activity. Monthly changes in serum testosterone concentration included two peaks in November and January, coinciding with peak spermatogenic activity, whereas androstenedione showed only one peak, an unusually high concentration in November. The circulating serum androstenedione concentration may be responsible for the unique reproductive activity noted in this bat.

A novel activity for a group of sesquiterpene lactones: inhibition of aromatase

A group of eleven sesquiterpene lactones isolated from different Asteraceae species from north-western Argentina were investigated for their inhibitory action on the estrogen biosynthesis. Seven of them, of different skeleton types, were found to inhibit the aromatase enzyme activity in human placental microsomes, showing IC 50 values ranging from 7 to 110 μM. The most active were the guaianolides 10-epi-8-deoxycumambrin B (compound 1), dehydroleucodin (compound 2) and ludartin (compound 3). These compounds were competitive inhibitors with an apparent K i=4 μM, K i=21 μM and K i=23 μM, respectively. Compounds 1 and 2 acted as type II ligands to the heme iron present in the active site of aromatase cytochrome P450 (P450arom). Besides, all of them failed to affect the cholesterol side-chain cleavage enzyme activity on human placental mitochondrias. This is the first report on the aromatase inhibitory activity of this group of natural compounds.

Estrogen Regulation of Placental P-450 Cholesterol Side-Chain Cleavage Enzyme Messenger Ribonucleic Acid Levels and Activity During Baboon Pregnancy

Estrogen Regulation of Placental P-450 Cholesterol Side-Chain Cleavage Enzyme Messenger Ribonucleic Acid Levels and Activity During Baboon Pregnancy

Estrogen regulation of placental P-450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid levels and activity during baboon pregnancy.

The present study was conducted to determine whether estrogen regulates the P-450 cholesterol side-chain cleavage (P-450scc) enzyme component of the progesterone biosynthetic pathway in the placenta during the second half of baboon pregnancy. Placental estrogen formation was suppressed by removing the fetus, i.e. fetectomy, and thus fetal adrenal C19-steroid estrogen precursors, on day 100 of baboon gestation (term = 184 days). P-450scc activity and messenger ribonucleic acid (mRNA) levels were then determined in placentas obtained on day 160 after fetectomy alone and after fetectomy and s.c. administration of the estrogen precursor and rostenedione or estradiol benzoate. Placentas were maintained in situ after fetectomy, and placental villi were comprised of syncytiotrophoblasts that seemed morphologically normal, based on their histology and immunocytochemical expression of pregnancy-specific-beta 1-glycoprotein. In untreated baboons, peripheral serum estradiol increased with advancing gestation, and mean (+/-SE) concentrations were 1.22 +/- 0.05 ng/ml on days 101-160 of gestation. After fetectomy serum estradiol concentrations decreased to 24% (P < 0.01) of normal. Androstenedione or estradiol administration after fetectomy increased serum estradiol levels to values that were 57% (P < 0.01) of, or 90% (P < 0.001) greater than intact controls, respectively,. Placental P-450scc specific activity, determined on a mitochondrial-enriched fraction of villous tissue, was 281.1 +/- 15.0 pmol pregnenolone plus progesterone formed per mg mitochondrial protein in untreated control baboons. Fetectomy resulted in a 52% decrease (P < 0.001) in placental P-450scc activity. Administration of androstenedione or estradiol after fetectomy increased P-450scc activity to values that were not significantly different from control. P-450scc mRNA levels were quantified by competitive RT-PCR. P-450scc mRNA levels in placental villous tissue of fetectomized baboons was 38% lower (P < 0.01) than that in the intact controls (110.9 +/- 5.9 attomoles/microgram RNA). The administration of androstenedione after fetectomy restored P-450scc mRNA to a level that was not different from the untreated controls. The results of this study show that there was close association between the levels of estrogen and the specific activity of and the mRNA levels for placental P-450scc in the second half of baboon pregnancy. Therefore, we propose that the P-450scc enzyme that catalyzes the conversion of substrate cholesterol to pregnenolone is regulated, for the most part, by estrogen in the primate placenta.

Insulin-like growth factor I enhancement of steroidogenesis by bovine granulosa cells and thecal cells: dependence on de novo cholesterol synthesis.

Studies were conducted to determine the importance of de novo cholesterol synthesis and cholesterol side-chain cleavage enzyme in the action of IGF-I in bovine granulosa and thecal cells. Granulosa and thecal cells from bovine follicles were cultured for 2 days in 10% fetal calf serum and then treated with luteinizing hormone (100 ng/ml) and IGF-I (0 or 100 ng/ml) for an additional 2 days in serum-free medium. During the last 24 h of treatment, cells were concomitantly treated with simvastatin (0, 0.5 or 5 micrograms/ml) or 25-hydroxycholesterol (0 or 10 micrograms/ml). Simvastatin, a potent inhibitor of the key enzyme controlling de novo cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, completely inhibited (P < 0.05) progesterone production by granulosa cells and progesterone and androstenedione production by thecal cells. Simvastatin also inhibited (P < 0.05) granulosa cell and thecal cell proliferation. Concomitant treatment with mevalonate, an immediate product of HMG-CoA reductase, attenuated the inhibitory effect of simvastatin on progesterone and androstenedione production by thecal cells and blocked the inhibitory effect of simvastatin on cell proliferation. The addition of 25-hydroxycholesterol, a substrate for cholesterol side-chain cleavage enzyme, had no effect (P > 0.10) on IGF-I-stimulated progesterone or androstenedione production by thecal cells and actually inhibited (P < 0.05) IGF-I-stimulated progesterone production by granulosa cells. These results provide indirect evidence indicating that stimulation of HMG-CoA reductase is an important locus of IGF-I action in bovine granulosa and thecal cells, whereas IGF-I has little or no effect on side-chain cleavage enzyme activity in these same cell types under the culture conditions employed.

Exposure to bleached kraft pulp mill effluent reduces the steroid biosynthetic capacity of white sucker ovarian follicles

White sucker exposed to bleached kraft mill effluent (BKME) show a number of reproductive alterations correlated to reduced circulating reproductive steroid levels. The purpose of this experiment was to examine the steroid biosynthetic capacity of white sucker ovarian follicles undergoing vitellogenesis to determine if reductions in steroid production contribute to the reduced circulating steroid levels found during this stage of reproduction. We also examined the mechanisms responsible for steroid production within the ovary to determine where BKME exerts its impacts. Follicles collected from BKME-exposed fish during early and late vitellogenesis had reduced testosterone and 17β-extradiol production under basal and human chorionic gonadotropin stimulated conditions. Production of cyclic 3′,5′ adenosine monophosphate (cAMP) by ovarian follicles was similar between sites suggesting impacts occurred post-cAMP formation. Reduced conversion of exogenous testosterone to 17β-estradiol during August collections demonstrates reduced levels of the aromatase enzyme in BKME-exposed follicles during early vitellogenic stages. Measurement of pregnenolone production in October indicates similar P450 side chain cleavage enzyme activity between sites, suggesting disruptions downstream of pregnenolone formation were responsible for the reduced steroid productivity in ovarian follicles from BKME-exposed fish during this stage of the reproductive cycle.

Induction of rat granulosa cell steroidogenic enzyme activities and their messenger ribonucleic acids by a splenocyte-derived factor

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20α-dihydroprogesterone (20α-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20α-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22( R)-hydroxycholesterol. While both FSH-and PSF-stimulated progesterone and 20α-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3β-hydroxysteroid dehydrogenase (3β-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH. A similar pattern was also observed with 3β-HSD mRNA levels. In contrast, FSH stimulated only minimal increases in 20α-hydroxysteroid dehydrogenase (20α-HSD), whereas PSF stimulated larger and more rapid increases in 20α-HSD activity, which were maximal by 3 days. These studies indicate that the slower onset of PSF-stimulated progesterone production is due, at least in part, to slower induction of mRNAs for P450scc and 3β-HSD leading to slower increases in the activities of their respective enzymes. Additionally, the higher 20α-OH-P levels produced by PSF may be due to greater induction of 20α-HSD activity.

Development of cytochrome P-450 side chain cleavage mRNA levels in neonatal ovaries of normal and hypogonadal (hpg) mice

Cytochrome P-450 side chain cleavage (P-450scc) is vital for biosynthesis of gonadal steroids. In this study changes in P-450scc mRNA levels and cholesterol side-chain cleavage (CSCC) enzyme activity have been measured during development in the ovary of the normal mouse and the hypogonadal (hpg) mouse which lacks circulating gonadotrophins. Levels of P-450scc mRNA were measured using a semi-quantitative reverse transcription-polymerase chain reaction technique for which part of the 5' sequence of a mouse P-450scccDNA was sequenced. On the day of birth P-450scc mRNA was present at low levels in the mouse ovary. Thereafter there was no change in P-450scc mRNA levels for 5 days after which time levels increased significantly to reach a peak around day 10. Activity of CSCC showed a similar pattern of development although activity was not detectable on days 1 and 3. In the hpg mouse P-450scc mRNA levels were normal on day 1 but did not increase thereafter up to 15 days. These results show that there is gonadotrophin-independent expression of P-450scc mRNA in the mouse ovary at birth when only primordial follicles and stromal tissue are present. As the ovary develops after 5 days changes in P-450scc mRNA levels become gonadotrophin-dependent and coincide with maturation of secondary follicles.

Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine increases the ovarian progesterone synthesis of PMSG-treated rats by regulating 3 beta-HSD activity

Little is known about the dopamine system in the ovary. The present study has been undertaken to investigate the effect of dopamine (DA) on the ovarian steroidogenic enzymes of pregnant mare serum gonadotropin (PMSG)-treated immature rats. Ovarian cells from PMSG-treated rats were cultured for 1-5 hours with or without DA, D1 agonists or bulbocapnine (Bul)(D1 antagonist). Progesterone (P) and estradiol (E2) in the media were assayed by specific RIAs. The enzyme activities were assayed by adding radioactive substrates in the media before incubation. DA and D1 agonists increased P in the media which was caused by the increment of 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) activity because cholesterol side chain cleavage enzyme (CSCC) activity showed no significant change. The stimulating effects of DA and DA agonists on P and 3 beta -HSD activity were inhibited by Bul. DA showed no effect on 17 alpha-hydroxylase activity. DA decreased 17.20 lyase activity, but this decrement was probably a non specific effect. DA alone did not affect the E2 level in the media and aromatase activity. The present results suggest that DA mainly stimulated 3 beta -HSD activity of the PMSG-treated rat ovary which regulated P synthesis.

The effects of GnRH agonist on steroidogenesis in the rat ovary

The effects of GnRH agonist (buserelin) on in vitro ovarian steroidogenesis were studied using DES-treated immature rats and PMS-treated immature rats. The estradiol and progesterone secreted by the cultured ovarian cells and the activities of various enzymes of steroid-metabolism were examined with or without gonadotropins (FSH or hCG), and the effects of 10(-6)-10(-12) M of buserelin on those indices were observed for 3-72 hours. In addition, the kinetic study of ovarian GnRH receptor was performed using 125I-labelled buserelin and crude ovarian cell membrane fraction of PMS-treated rats. The Scatchard analysis revealed the specific high affinity and low capacity ovarian GnRH receptor (Kd = 0.92 nM and Bmax = 0.57 fmol/mg tissue). The FSH-stimulated cholesterol side chain cleavage enzyme (CSCC) activity of the DES-treated rats was suppressed in a dose-dependent manner by buserelin. Estradiol release and aromatase activity were increased by 10(-8) M buserelin within 48 hours from the beginning of the incubation of the PMS-treated rat ovarian cells, but were suppressed after 48 hours. Buserelin increased basal progesterone secretion and both activities of CSCC and of 3 beta-hydroxysteroid dehydrogenase of PMS-treated rat ovarian cells incubated without hCG, which were suppressed by buserelin co-incubated with 100 IU/ml of hCG. These results suggested that GnRH plays a physiological role in ovarian steroidogenesis binding the specific receptor and that GnRH promotes the development of the follicle through increased estrogen synthesis in the early stage of the folliculogenesis and the luteinization in the late stage of the follicular development through increased progesterone and decreased estradiol production and the luteolysis in the luteinized cells by hCG through decreased progesterone secretion.