Cholesterol Side-chain Cleavage Cytochrome Research Articles

VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis.VDR is widely expressed in testis cells such as Leydig cells, Sertoli cells, and sperm. The levels of steroids are critical for sexual development. In the early stage of steroidogenesis, cholesterol is converted to pregnenolone (precursor of most steroid hormones) by cholesterol side-chain lyase (CYP11A1), which eventually synthesizes the male hormone testosterone. This study aims to reveal how VDR regulates CYP11A1 expression and affects testosterone synthesis in murine Leydig cells. The levels of VDR, CYP11A1 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Targeted relationship between VDR and Cyp11a1 was evaluated by dual-luciferase reporter assay. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). Phylogenetic and motif analysis showed that the Cyp11a1 family had sequence loss, which may have special biological functions during evolution. The results of promoter prediction showed that vitamin D response element (VDRE) existed in the upstream promoter region of murine Cyp11a1. Dual-luciferase assay confirmed that VDR could bind candidate VDREs in upstream region of Cyp11a1, and enhance gene expression. Tissue distribution and localizatio analysis showed that Cyp11a1 was mainly expressed in testis, and dominantly existed in murine Leydig cells. Furthermore, over-expression VDR and CYP11A1 significantly increased testosterone synthesis in mice Leydig cells. Active vitamin D3 (VD3) and Vdr interference treatment showed that VD3/VDR had a positive regulatory effect on Cyp11a1 expression and testosterone secretion. VDR promotes testosterone synthesis in male mice by up-regulating Cyp11a1 expression, which played an important role for male reproduction.

1420P The pharmacokinetics and the pharmacodynamic effect of ODM-208, an inhibitor of cholesterol side-chain cleavage enzyme (CYP11A1)

ODM-208 is a first-in-class, oral, non-steroidal small molecule that selectively and completely inhibits the mitochondrial cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme, the first and rate limiting step in steroidogenesis, leading to deficiency of endogenous steroid hormones. The pharmacokinetics (PK) of ODM-208 and the pharmacodynamics (PD) of ODM-208 by measuring serum testosterone levels were studied in the Cypides phase 1 trial in males with progressive mCRPC, who had received at least one prior line of both 2nd generation AR pathway inhibitor (ARPI) and taxane chemotherapy.

HC diet inhibited testosterone synthesis by activating endoplasmic reticulum stress in testicular Leydig cells.

Emerging epidemiological studies indicate that hypercholesterolaemia is a risk factor for testosterone deficiency. However, the underlying mechanism is unclear. Testicular Leydig cells are the primary source of testosterone in males. To identify the effect and mechanism of cholesterol overload on Leydig cell function, rats were fed with a HC (HC) diet to induce hypercholesterolaemia. During the 16‐week feeding period, serum testosterone levels were reduced in a time‐dependent manner in rats fed the HC diet. Accordingly, these steroidogenic enzymes within the Leydig cells, including steroidogenic acute regulatory protein (StAR), cholesterol side‐chain cleavage cytochrome P450 (P450scc) and 3β‐hydroxysteroid dehydrogenase (3β‐HSD), were down‐regulated. Notably, the HC‐fed rats showed evident endoplasmic reticulum (ER) stress in the testis, including a dilated ER as an evident pathological change in the Leydig cell ultrastructure, up‐regulated ER stress biomarker (binding immunoglobulin protein) levels and activation of the activating transcription factor 6 (ATF6)‐related unfolded protein response pathway. Further analysis showed that when 4‐phenyl butyric acid (4‐PBA) was used to block ER stress in HC‐fed rats for 8 weeks, the testosterone deficiency was significantly alleviated. Our findings suggested that high dietary cholesterol intake affected serum testosterone levels by down‐regulating steroidogenic enzymes and that activated ER stress might serve as the underlying mechanism.

Morphological rearrangement of the cortical region, in aging ovaries.

The ovary is a structurally dynamic organ that alters with age. Modifications in the paracrine status influence the capacity of aging oocytes to develop normal embryos. Despite the importance of understanding the cellular and molecular mechanism involved in the process of ovarian aging, histological changes remain poorly understood. Correlating the process of folliculogenesis and somatic cell function during ovarian aging is essential to explain the reproductive decline of aged mammalian species, including humans. Here, we performed a morphological and immunohistological study on the ovaries of chinchilla rabbits that varied in age from one to 34-months. The spatiotemporal expression of the cholesterol side-chain cleavage cytochrome P450scc (CYP11A) and the smooth muscle actin (SMA) were analyzed. A significant histological rearrangement of immunodetected cells in theca interna, theca externa and the interstitial tissue around the follicles occurred. The expression of CYP11A1 decreased considerably in antral follicles of aging ovaries. Moreover, we found that the secondary interstitial gland developed extensively, and a remarkable rearrangement of the surface epithelium occurred in aging ovaries. In contrast to ovaries during the reproductive period, the immunohistological changes demonstrate that the interstitial gland became the most abundant tissue during the aging of ovaries. Thus, the current study provides new data for understanding the alteration of somatic cell function in elderly ovaries and how this affects their declined fertility.

Effect of weaning age on cortisol release in piglets

The effect of weaning age on the adrenal cortex, which plays a vital role in the stress response, is currently unknown. Therefore, plasma adrenocorticotropic hormone (ACTH) and cortisol levels, weights and relative weights of adrenal glands, and steroidogenesis-related protein and enzyme expression levels in piglets weaned on different days were determined. Piglets weaned at 35 days had significantly lower ACTH levels than those weaned at 14 or 21 days, and cortisol levels of piglets weaned at 21, 28, and 35 days were significantly lower than those of piglets weaned on day 14. Adrenal gland weights of piglets weaned at 28 and 35 days and relative adrenal gland weights of piglets weaned at 35 days were significantly lower than those of piglets weaned at 14 days. However, no significant difference was detected in the expression of melanocortin-type 2 receptor mRNA, which is associated with weaning age. Steroidogenic acute-regulatory (StAR) mRNA and cholesterol side-chain cleavage cytochrome P450 mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 or 21 days, and P450 11β mRNA expression levels in piglets weaned at 28 and 35 days were significantly lower than in those weaned at 14 days. Therefore, early-weaned piglets exhibited increased adrenal gland weights and StAR and steroidogenic enzyme expression, all of which contributed to high cortisol levels. The high plasma ACTH and cortisol levels in early-weaned piglets indicate that these animals would be greatly affected by stress.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Isolation and identification of ovarian theca-interstitial cells and granulose cells of immature female mice.

Theca-interstitial cells (TICs) and granulosa cells (GCs) are important components of follicles that support follicle development and hormone secretion, and are considered to be important cell models for basic research. However, no method currently exists for simultaneously isolating TICs and GCs from a single ovary of the immature mouse. Here, we sought to develop such a protocol using mechanical dissection combined with brief collagenase-DNase digestion. Morphological characteristics and molecular markers were detected to identify TICs and GCs. In isolated TICs, cholesterol side chain cleavage cytochrome P450 (P450scc) was expressed abundantly, but anti-Mullerian hormone (AMH) was expressed only at very low levels. This expression profile was reversed in GCs. In addition, TICs secreted large amounts of testosterone (T) and minimal amounts of estradiol (E2 ), while the converse was found in GCs. T concentrations rose gradually in TIC culture media as the concentration of added luteinizing hormone (LH) was increased. In GCs, E2 secretion increased as the follicle-stimulating hormone (FSH) concentration increased. Thus, mechanical dissection combined with collagenase-DNase digestion is a simple, effective and reproducible method for obtaining large numbers of highly purified and hormonally stimulated TICs and GCs from one ovary.

Initiation of active immunization against testosterone during early puberty alters negative feedback regulation of the hypothalamic-pituitary-testicular axis in rabbits

To investigate the effects of antitestosterone immunization, initiated during early puberty, on hypothalamic-pituitary-testicular feedback in rabbits, 16 early pubertal male rabbits were randomly allocated into 2 groups (n = 8), control or immunized against testosterone-3(O-carboxymethyl)oxime-BSA in Freund adjuvant at 4 mo of age (with a booster immunization 4 wk later). Blood samples (for antibody titers and hormone concentrations) were collected at 2- or 4-wk intervals after immunization. Compared with controls, antitestosterone immunization triggered: a substantial and sustained antibody response (P < 0.01); increases in serum concentrations of luteinizing hormone (LH) and testosterone and testis weight and volume (P < 0.05); hyperplasia of testicular interstitial tissue with clustered and hypertrophic Leydig cells; and greater (P < 0.05) enzyme protein and messenger RNA (mRNA) expression levels for testicular cholesterol side-chain cleavage cytochrome P-450, 17α-hydroxylase cytochrome P-450, and 3β-dydroxysteroid dehydrogenase. Furthermore, immunoneutralization of testosterone upregulated mRNA expressions for genes in sex steroid negative feedback loops, including androgen receptor (AR), estrogen receptor alpha (ER-α), kisspeptin encoded gene (kiss-1) and kisspeptin receptor (G-coupled receptor 54) and gonadotropin-releasing hormone (GnRH) in the hypothalamic arcuate nucleus, GnRH receptor and LH-β in pituitary, and AR, inhibin-α and βA subunits in testes (P < 0.05). However, immunization did not affect mRNA expressions for follicle-stimulating hormone β, AR, and ER-α in pituitary, or ER-α in testes. We concluded that antitestosterone immunization in male rabbits, initiated during early puberty, increased GnRH mRNA expression, and in turn LH synthesis by reducing testicular feedback signaling. Reduction of direct steroidal effects on the testis may also have increased testosterone secretion. Consequently, there was an accelerated testicular development during puberty and enhanced testicular function after puberty, which likely conferred prolonged reproductive advantages.

Active immunisation against pregnenolone reduces testicular steroidogenesis and GnRH synthesis in rabbits

To investigate the effects of active immunisation against pregnenolone on reproductive traits in rabbits, 16 early pubertal male rabbits (4mo old) were randomly and equally allocated into two groups, control or immunised against pregnenolone–hemisuccinate–BSA in Freund's adjuvant (with a booster 4wk later). Blood samples (for antibody titres and hormone concentrations) were collected at 2 or 4wk-intervals after immunisation until rabbits were killed, 24wk after the primary immunisation. Compared to controls, rabbits immunised against pregnenolone had increased serum antibody titres (P<0.01) and decreased serum concentrations of both testosterone and LH (P<0.01 for each). At 24wk after the primary immunisation, testes were severely atrophied, spermatogenesis was arrested and steroidogenesis was suppressed, as evidenced by lesser amounts of testicular cholesterol side-chain cleavage cytochrome P-450 and 17α-hydroxylase cytochrome P-450 mRNA (P<0.05). Furthermore, the amounts of mRNA for GnRH in the arcuate nucleus (Arc), of the hypothalamus and GnRH receptor and LH-β in the pituitary and genes in sex-hormone negative feedback loops (androgen receptor, oestrogen alpha receptor, kisspeptin encoded gene and kisspeptin receptor) in the Arc were decreased in pregnenolone-immunised rabbits compared to controls (P<0.05). It was concluded that immunisation against pregnenolone directly blocked testicular steroidogenesis, which reduced synthesis of hypothalamic GnRH and subsequently synthesis of pituitary LH by abolishing the permissive action of sex steroids on hypothalamic GnRH neurons, thereby disrupting spermatogenesis. This was apparently the first report that active immunisation against pregnenolone was a means of immunological castration

Selective inhibition of steroidogenic enzymes by ketoconazole in rat ovary cells.

OBJECTIVEKetoconazole (KCZ) is an anti-fungal agent extensively used for clinical applications related to its inhibitory effects on adrenal and testicular steroidogenesis. Much less information is available on the effects of KCZ on synthesis of steroid hormones in the ovary. The present study aimed to characterize the in situ effects of KCZ on steroidogenic enzymes in primary rat ovary cells.METHODSFollowing the induction of folliculogenesis in gonadotropin treated rats, freshly prepared ovarian cells were incubated in suspension for up to four hours while radiolabeled steroid substrates were added and time dependent generation of their metabolic products was analyzed by thin layer chromatography (TLC).RESULTSKCZ inhibits the P450 steroidogenic enzymes in a selective and dose dependent manner, including cholesterol side-chain cleavage cytochrome P450 (CYP11A1/P450scc), the 17α-hydroxylase activity of CYP17A1/P450c17, and CYP19A1/P450arom, with IC50 values of 0.3, 1.8, and 0.3 μg/mL (0.56, 3.36, and 0.56 μM), respectively. Unaffected by KCZ, at 10 μg/mL, were the 17,20 lyase activity of CYP17A1, as well as five non-cytochrome steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase-Δ5–4 isomerase type 1 (3βHSD1), 5α-reductase, 20α-hydroxysteroid dehydrogenase (20α-HSD), 3α-hydroxysteroid dehydrogenase (3α-HSD), and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1).CONCLUSIONThese findings map the effects of KCZ on the ovarian pathways of progestin, androgen, and estrogen synthesis. Hence, the drug may have a potential use as an acute and reversible modulator of ovarian steroidogenesis in pathological circumstances.

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.

Differentiation of Steroid-Producing Cells and Folliculogenesis in the Developing Ovary of the Nile TilapiaOreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.

Cloning and expression of medaka cholesterol side chain cleavage cytochrome P450 during gonadal development

Cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a) is responsible for the first step in steroidogenesis, catalyzing the conversion of cholesterol to prognenolone. To investigate the differentiation of steroid-producing cells and the function of sex steroids during gonadal differentiation in the teleost fish, medaka (Oryzias latipes), we isolated the full length cDNA of medaka P450scc and analyzed the expression pattern of P450scc mRNA during gonadal development using in situ hybridization. At hatching, and just after the initiation of morphological sex differentiation, we did not detect any P450scc expression in both sexes. In male gonads, expression of P450scc was detected in the interstitial somatic cells 15 days after hatching following the formation of the seminiferous tubule precursor, and was maintained in the interstitial somatic cells throughout testicular development. In the female gonad, expression of P450scc was initially detected in interstitial somatic cells 5 days after hatching. Subsequently, the expression of P450scc was continuously detected in the interstitial somatic cells of the developing ovary. This expression pattern of P450scc differed from that of female specific steroidogenic enzyme P450arom. Both P450scc and P450arom expressing cells, only P450scc expressing cells, and only P450arom expressing cells were observed. Our results suggest that expression of steroidogenic enzymes is regulated by various mechanisms during ovarian development.

Profiles of Circulating Steroid Hormones, Gonadotropins, Immunoreactive Inhibin and Prolactin During Pregnancy in Goats and Immunolocalization of Inhibin Subunits, Steroidogenic Enzymes and Prolactin in the Corpus Luteum and Placenta

The current study was performed to follow up the circulating hormonal changes and to correlate the findings with the physiological activity of the corpus luteum (CL) and placenta during pregnancy in goats. Blood samples were collected weekly from five goats during pregnancy for measuring steroid and protein hormones. A gradual increase was observed in immunoreactive (ir-) inhibin, with maximal levels at the 17th week. The plasma concentrations of estradiol and prolactin (PRL) showed nearly similar patterns during pregnancy, where they declined to basal levels during the first 4 weeks post-breeding and then increased significantly, with the maximal concentration during late pregnancy. The plasma FSH and LH concentrations were maintained at basal levels throughout the gestation period. The plasma progesterone concentration abruptly increased in the first week post-breeding and remained at high values throughout the pregnancy period. Immunohistochemical localization of inhibin alpha, beta(A), beta(B) and steroidogenic enzymes cytochrome P450 aromatase, 3alpha-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 and cholesterol side-chain cleavage cytochrome P450 in the cyclic and pregnant goat CL revealed positive immunoreactivity without affinity differences between the luteal and pregnancy stages. The placental syncytiotrophoblasts also showed positive staining, except for inhibin beta(A) and 3betaHSD. The giant binucleate cells of the placenta showed positive immunoreactions to PRL. These results suggest that the high concentrations of ir-inhibin, estradiol and PRL during late pregnancy are of placental origin and that the placenta may have a vital role in the maintenance of pregnancy, regulation of mammary growth and preparation for kidding and lactation in goats.

Expression of steroidogenic enzymes in porcine polycystic ovaries.

In the present study the expression pattern of the cholesterol side-chain cleavage cytochrome (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and aromatase (P450(arom)) was analyzed in the health and polycystic ovaries of gilts by means of the Western blot and immunohistochemistry. The polycystic status of ovaries was induced by i.m. dexamethasone (DXM) injections on days 7-21 of the estrous cycle. Macroscopic observation of ovaries of DXM-treated gilts revealed the presence of cysts (1-2 cm in diameter, with a mean number of 7.0+/-1.2 per ovary), a decrease (P<0.05) in number of small follicles (1-3 mm in diameter), as well as the lack of medium-sized follicles (4-6 mm in diameter) and corpora lutea, as compared to the control animals. The expression of P450(scc) (P<0.01), 3beta-HSD (P<0.05) and P450(arom) (P<0.001) proteins in the cysts was higher than in the medium-sized follicles of the control gilts. Moreover, DXM injections resulted also in an enhancement (P<0.05) in the level of P450(scc) protein in the walls of small follicles as compared to the control gilts. Following DXM administration the immunoreactivity (IR) of P450(scc) in the primordial follicles was lower than in the control group. Comparing to the control gilts, the reaction for this enzyme in DXM-treated animals was observed in secondary follicles, while for 3beta-HSD, in primordial, primary, as well as secondary follicles. The immunostaining for P450(scc) (theca cells) and P450(arom) (granulosa cells) in the small follicles of the DXM-treated gilts were more prominent than those found in the gonads of control animals. However, IR for P450(scc) was not found in the granulosa cells of small follicles in the gilts receiving DXM. The intensity of P450(scc) and P450(arom) labelling was distinctly enhanced in the cysts as compared to the medium follicles of the control animals. Furthermore, in contrary to the medium follicles of the control animals, faint IR for 3beta-HSD was found in the granulosa cell layer of cysts. Our data revealed that both the expression of P450(scc), 3beta-HSD and P450(arom) and localization of these enzymes in polycystic ovaries were different from those, found under physiological conditions. These results suggested that above-mentioned enzymes may, by influencing the ovarian steroid synthesis, play an essential role in the creation and/or course of cystic ovarian disease.

222 CHARACTERIZATION OF CO-CULTURES OF GRANULOSA CELLS AND THECA CELLS AS EXPLANTS OF BOVINE OVARY FOLLICULAR WALLS

Granulosa cells (GC) and theca cells (TC) luteinize in culture because of the presence of serum in the medium and, as a consequence, the production of progesterone (P4) increases and that of 17β-estradiol (E2) decreases. The follicular phase of the bovine estrous cycle is characterized by the presence of E2, as well as aromatase activity, that is turned off in the atresia or in the luteal phase of the bovine estrous cycle. We propose to characterize 2 co-culture systems utilizing slices of follicular walls (explants) containing both GC and TC and to cultivate them in nondefined medium (NDM; TCM199+serum) or defined medium (DM; α MEM+polyvinyl alcohol). To prepare the explants, 80 follicles measuring 4 to 6 mm in diameter were selected. Concentrations of P4 and E2 were evaluated by RIA after 24, 48, and 72 h of culture and values were corrected by tissue weight (10 mg). Morphological analysis of the explants was stained with hematoxylin and eosin. The total mRNA from GC-TC explants was purified and the expression of the following genes was estimated by RT-PCR: bovine 3 beta-hydroxysteroid dehydrogenase (HSD3B1), cholesterol side-chain cleavage cytochrome P450 (CYP11A1), 17|3-hydroxylase (CYP17), aromatase cytochrome P450 (CYP19), steroidogenic acute regulatory protein (StAR), follicle-stimulating hormone receptor (FSHr), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) as internal control. The results were analyzed by two-way ANOVA followedby Bonferroni least-significant tests. Microscopic analyses showed that cells cultured in NDM lost their polyhedral shape and acquired a fibroblast-like form. Defined medium maintained GC morphology with low cytoplasm:nucleus ratio and GC-GC contact, similar to in vivo cells. In NDM, the concentration of P4 increased as opposed to that of E2, which decreased after 48 h of culture. In DM, the concentrations of P4 and E2 were maintained in the first 48 h of culture. The level of P4 in NDM was higher than in DM during all periods tested. Our data show that CYP11A1 expression remains absent in NDM for follicles explants that initially did not express CYP11A1, whereas in DM the expression of this gene appears after 24, 48, or 72 h of culture, evidence that indicates rescue from atresia. It is important to notice that gene expression of steroidogenic enzymes in DM were higher than in NDM. Very low levels of CYP11A1 and CYP19 were observed in the NDM group. On the other hand, in the DM group there was a progressive increase of all the steroidogenic enzymes and FSHr, which reached a peak at 48 h of culture. In conclusion, our results suggest that DM showed a stimulating effect on steroidogenic activity of GC-TC co-culture similar to that observed in vivo for functional growing follicle, whereas in NDM the explants seemed to mimic luteinizing follicles. Grants from FAP-DF, Finatec, CNPq, and Capes.

Changes in expression of cytochrome cholesterol side chain cleavage enzyme in the brain of morphine-dependent rats

Objective To evaluate the changes in the expression of cytochrome cholesterol side chain cleavage enzyme (P450scc) in the brain of morphine-dependent rats. Methods Twenty-four male SD rats aged 4-8 months weighing 180-200 g were randomly divided into 3 groups (n = 8 each): group Ⅰ normal saline (group NS), group Ⅱ morphine dependence (group MD) and group Ⅲ morphine withdrawal (group MW). In group MD and MW, the rats were given intraperitoneally increasing doses of morphine starting from 5 mg/kg to 10, 15, 20, 30, 40 and 50 mg/kg twice a day for 7 days. In group NS, the rats were given equal volume of normal saline instead of morphine. The rats were decapitated 1 h after last injection in group NS and MD. In group MW, naloxone 2 mg/kg was given 1 h after last injection, and then the animals were decapitated 30 min after withdrawal symptoms were observed. The brains were immediately removed and the frontal cortex, hippocampus, striatum and thalamus were separated. The expression of P450see was determined by Western blot. Results The expression of P450scc in the frontal cortex, hippocampus and striatum was significantly decreased in group MD and MW compared with group NS (P<0.05). Conclusion The down-regulation of P450scc expression might be involved in the development of morphine dependence, but it is not involved in the morphine withdrawal. Key words: Morphine dependence; Brain; Cholesterol side-chain cleavage enzyme