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

2010 ◽  
Vol 22 (1) ◽  
pp. 269
Author(s):  
L. P. Salles ◽  
R. B. Vasconcelos ◽  
I. Oliveira e Silva ◽  
L. V. M. Gulart ◽  
F. A. G. Torres ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Estrous Cycle ◽  
Granulosa Cells ◽  
Internal Control ◽  
Regulatory Protein ◽  
Defined Medium ◽  
Aromatase Activity ◽  
Steroidogenic Enzymes ◽  
Theca Cells

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.

scholarly journals Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

Reproduction ◽  
2002 ◽  
pp. 651-661 ◽  
Author(s):  
HA Garverick ◽  
G Baxter ◽  
J Gong ◽  
DG Armstrong ◽  
BK Campbell ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Gnrh Agonist ◽  
Follicular Growth ◽  
Steroidogenic Enzymes ◽  
Lh Receptor ◽  
Theca Cells ◽  
Follicular Wave ◽  
Follicle Size ◽  
Bovine Somatotrophin

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

149 EFFECTS OF FSH ADDED IN DEFINED MEDIUM MATURATION ON THE PRE-IMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax, Bcl-2, AND CONNEXIN 43 GENES IN BOVINE IVP BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
L. V. M. Gulart ◽  
L. Gabriel ◽  
L. P. Salles ◽  
G. R. Gamas ◽  
D. K. Souza ◽  
...  
Keyword(s):  
Internal Control ◽  
Connexin 43 ◽  
Calf Serum ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Blastocyst Rate

FSH at low concentrations affect embryo production. In vitro culture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivo has led to the development of several systems of oocyte in vitro maturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1 and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n = 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1 BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1 of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries.

scholarly journals The Acute and Chronic Effects of Adrenocorticotropin on the Levels of Messenger Ribonucleic Acid and Protein of Steroidogenic Enzymes in Rat Adrenal in Vivo*

Endocrinology ◽  
1998 ◽  
Vol 139 (9) ◽  
pp. 3913-3922 ◽  
Author(s):  
Jean-Guy Lehoux ◽  
Alain Fleury ◽  
Lyne Ducharme
Keyword(s):  
Cytochrome P450 ◽  
Western Blotting ◽  
Steroidogenic Enzymes ◽  
Chronic Stimulation ◽  
Western Blotting Analysis ◽  
Star Protein ◽  
Protein Levels ◽  
Blotting Analysis ◽  
Acth Treatment

Abstract The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220–370% and 300–350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5–1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11β-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3β-hydroxysteroid dehydrogenase (3βHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210–270%) and a decrease in the levels of 3βHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3βHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570–870%, respectively). The levels of 3βHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3βHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3βHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3βHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. Under chronic stimulation by ACTH, adrenals were hypertrophied, and the expression of many steroidogenic enzymes was modified, particularly the level of StAR protein was increased in the ZG and ZFR, confirming the importance of this protein in the control of steroidogenesis in a situation similar to that of Cushing’s syndrome.

Glucocorticoid Action upon SIRPa and SHP-1 mRNA Expression in Autoimmune Hemolytic Anemia (AIHA).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3843-3843
Author(s):  
Ana Carolina Almeida ◽  
Sara T.O. Saad ◽  
Maria de Lourdes R.B. Castro ◽  
Antonio Condino Neto
Keyword(s):  
Mrna Expression ◽  
Internal Control ◽  
Regulatory Protein ◽  
Protein A ◽  
U937 Cells ◽  
Blood Monocytes ◽  
Healthy Donors ◽  
Positive Direct

Abstract AIHA is characterized by a high erythrocyte destruction rate associated to autoantibodies directed against blood cell antigens and is usually treated with glucocorticoid (GC). SIRP-a (Signal Regulatory Protein-a)) is an inhibitory receptor in phagocytes and a direct substrate for the phosphatase SHP-1, an important regulator of macrophage proliferation and activation. SIRP-a activation and consequent phosphorylation of immunoreceptor tyrosine-based inhibitory motifs, occur by the binding to CD47 on the erythrocyte membrane, and allow SHP-1, SHP-2 and SHIP recruitment, which in turn dephosphorylate specific protein substrates involved in the mediation of several physiologic effects. The aim of this study was to evaluate the “in vitro” and “in vivo” effects of GC on SIRPa and SHP-1 expression. Peripheral blood monocytes (PBM) were isolated from AIHA patients with and without GC therapy. For the “in vitro” studies PBM from healthy donors and U937 cells were cultured for 48 hours with Dexamethasone (Dexa-1mM) and/or IFNg (100U/ml) and TNFa (1000U/ml). SIRPa and SHP-1 mRNA expression was determined by Real Time PCR, using b-actin expression as an internal control and PBM from healthy donor as a calibrator. AIHA patients underwent clinical and immunohematological investigation. SIRPa and SHP-1 mRNA expression was significantly increased in U937 cells and normal PBM treated with IFN/TNF alone (Mean±SD, SIRPa U937: 4.0±3.2, p=0.009; PBM: 14.9±9.0, p=0.004)(SHP- 1 U937:8.1±6.9, p=0.0002; PBM 12.3±7.1, p=0.006) or associated with Dexa (Mean SIRPa U937:2.7±1.3, p=0.02; PBM 36.1±34.5, p=0.0001)(SHP-1 U937 :5.7±4.2, p=0.002; PBM: 18.9±16.0, p=0.001) compared to basal conditions (Mean SIRPa U937 :1.8±2.1; PBM 3.8±3.9)(SHP 1 U937 :1.7±2.2; PBM 3.9±4.0). Regarding treatment with Dexa alone, SIRPa and SHP-1 were significantly increased in normal PBM but not in U937 (SIRPa 24.8±12, p=0.0004, SHP-1 19.2±6.2, p=0.0001). SIRP-a and SHP-1 expression was significantly higher in PBM from AIHA patients (SIRPa 5.6±1.9, SHP-1 6.1±1.8 n=6) compared to normal (SIRPa 1.8±1.40; p=0.008, SHP-1 2.4±2.1, n=10; p=0.01). After GC therapy, SIRP-a and SHP-1 expression was similar in PBM of AIHA patients (SIRPa 0.6±0.3, SHP-1 0.9±0.18, n=5) compared to healthy donors. AIHA patients studied before GC therapy showed positive direct antiglobulin test (DAT) with anti-IgG and C3d, low hemoglobin (7.7±3.3g/dl) and hematocrit (23.6±11.6%), and reticulocytosis (258.1±194.5). AIHA patients studied after GC therapy showed DAT with only anti-IgG, normal level of hemoglobin (12.1±1.6), hematocrit (35.1±4.8) and reticulocytes (71.8±42.3). In the present study, SIRPa and SHP1 mRNA were higher in mature monocytes compared to U937 and upregulated by dexametasone and IFN /TNF. This result could suggest that this pathway is involved in the reduction of phagocytosis by GC. However, patients with AIHA showed upregulation of these proteins in basal conditions and after GC treatment and hemolysis reduction, SIRPa and SHP1 mRNA expression decreased to normal levels. A balance between inhibition and activation signals determined the macrophage phagocytic activity. Increased SIRP-a and SHP-1 expression in AIHA patients before GC therapy could be a consequence of a homeostatic mechanism, activated by massive phagocytosis, with the purpose of inhibiting the predominant activating signals.

Effects of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extracts on the Secretion of Progesterone and Estradiol In Vivo and In Vitro

2007 ◽  
Vol 232 (9) ◽  
pp. 1181-1194 ◽  
Author(s):  
Shih-Min Hsia ◽  
Chih-Lan Yeh ◽  
Yueh-Hsiung Kuo ◽  
Paulus S. Wang ◽  
Wenchang Chiang
Keyword(s):  
Ethyl Acetate ◽  
Enzyme Activities ◽  
Regulatory Protein ◽  
Endocrine System ◽  
Female Rats ◽  
Aromatase Activity ◽  
Water Fraction ◽  
Protein Expressions

Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for dysfunction of the endocrine system. However, there have been few studies on the effects of adlay seed on the endocrine system. In the present study, both the in vivo and in vitro effects of methanolic extracts of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with four different solvents: water, 1-butanol, ethyl acetate, and n-hexane. Four fractions, namely, AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction), and AHM-Hex ( n-hexane fraction), were respectively obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and were challenged with different reagents, including human chorionic gonadotropin (hCG; 0.5 IU/ml), 8-bromo-adenosine-3′,5′-cyclic monophosphate (8-Br-cAMP; 0.1 m M), forskolin (10 μ M), 25-OH-cholesterol (10 μ M), and pregnenolone (10 μ M), in the presence or absence of AHM (100 μg/ml). The functions of steroidogenic enzymes, including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc), protein kinase A (PKA), and aromatase activity, were investigated. The expression of StAR mRNA was also explored by using real-time reverse transcription–polymerase chain reaction. In the in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via inhibition of (i) the cAMP-PKA signal transduction pathway, (ii) cAMP accumulation, (iii) P450scc and 3β-HSD enzyme activities, (iv) PKA, P450scc and StAR protein expressions and StAR mRNA expression, and (v) aromatase activity in rat GCs. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of the cAMP pathway, enzyme activities, and the protein expressions of P450scc and StAR in rat GCs.

Immunohistochemical localization of steroidogenic enzymes and comparison with hormone production during follicle development in the pig

1999 ◽  
Vol 11 (6) ◽  
pp. 337 ◽  
Author(s):  
Ellen M. Shores ◽  
Morag G. Hunter
Keyword(s):  
Granulosa Cells ◽  
Follicular Development ◽  
Staining Intensity ◽  
Immunohistochemical Localization ◽  
Vital Role ◽  
Steroidogenic Enzymes ◽  
Hormone Production ◽  
Theca Cells ◽  
Enzyme Distribution ◽  
Follicle Size

The steroidogenic enzymes, P450 aromatase (P450 arom ) and P450 17a-hydroxylase (P450 17a ), were precisely located within the healthy porcine follicle by immunohistochemistry. Enzyme distribution was examined throughout follicular development during natural oestrous cycles (n = 14 gilts) and was compared with steroid production by healthy whole and theca-only follicles. All follicles 2 mm or more in diameter were either fixed for immunohistochemistry (n = 380 of which 197 were assessed as healthy) or incubated as whole (n = 110) or theca-only (n = 110) follicles to measure steroidogenesis. P450 17a was confined to the theca layer. The number of positive cells and staining intensity increased with follicle size. P450 arom was consistently detected in the granulosa layer of follicles measuring 6 mm or more in diameter and those cells furthest from the antrum were most strongly stained. P450arom was also detected in the theca layer of these large follicles. Whole and theca-only follicles produced oestradiol and androstenedione, and the levels of both hormones increased with follicle size (P<0.001). Whole follicles produced more oestradiol (P<0.001), but less androstenedione (P = 0.01) than theca-only follicles of the same size. Although granulosa cells contained P450 arom and synthesized oestradiol, only theca cells contained P450 17a. Theca cells therefore provided granulosa cells with androgen substrate. In addition, theca cells possessed P450 arom , making them capable of independent oestradiol production, which may be required to trigger the LH surge. This study confirms the vital role of theca cells in follicular steroidogenesis in the pig.

Effect of melatonin on bovine theca cells in vitro

2018 ◽  
Vol 30 (4) ◽  
pp. 643 ◽  
Author(s):  
T. Feng ◽  
L. F. Schutz ◽  
B. C. Morrell ◽  
M. C. Perego ◽  
L. J. Spicer
Keyword(s):  
Cell Proliferation ◽  
Cytochrome P450 ◽  
Ovarian Function ◽  
Cell Function ◽  
Regulatory Protein ◽  
Theca Cell ◽  
Theca Cells ◽  
Dependent Inhibition ◽  
Steroidogenic Acute Regulatory

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8–22 mm) follicles and treated with various hormones in serum-free medium for 24 h or 48 h. Melatonin caused a dose-dependent inhibition (P < 0.05) of LH+insulin-like growth factor 1 (IGF1)-induced androstenedione and progesterone production. Also, melatonin inhibited (P < 0.05) LH+IGF1-induced expression of steroidogenic acute regulatory protein (StAR) mRNA (via real-time polymerase chain reaction) in theca cells, but it had no effect (P > 0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P < 0.05) the number of bovine theca cells in both LH- and LH+IGF1-treated cultures. In conclusion, melatonin may act as an endocrine regulator of ovarian function in cattle by stimulating theca cell proliferation and inhibiting differentiation via inhibition of hormone-induced steroidogenesis.

scholarly journals Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles

Reproduction ◽  
2003 ◽  
pp. 709-716 ◽  
Author(s):  
G Maillet ◽  
A Benhaim ◽  
H Mittre ◽  
C Feral
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Cell Apoptosis ◽  
Annexin V ◽  
Rapid Loss ◽  
Theca Cells ◽  
Preovulatory Follicles ◽  
Apoptotic Effect

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.

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