Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis†

2019 ◽  
Author(s):  
P Asiabi ◽  
E C R Leonel ◽  
E Marbaix ◽  
M M Dolmans ◽  
C A Amorim
Keyword(s):  
Cytochrome P450 ◽  
Luteinizing Hormone ◽  
Granulosa Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Expression Intensity ◽  
Theca Cells ◽  
Perilipin 2 ◽  
Cytochrome P450c17

Abstract The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2, play a role in the cascade after luteinizing hormone–choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 μm), and large (>3000 μm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.

scholarly journals Regulation of Angiotensin Type 2 Receptor in Bovine Granulosa Cells

Endocrinology ◽  
2008 ◽  
Vol 149 (10) ◽  
pp. 5004-5011 ◽  
Author(s):  
Valério M. Portela ◽  
Paulo B. D. Gonçalves ◽  
Angela M. Veiga ◽  
Edmir Nicola ◽  
José Buratini ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Theca Cells ◽  
Protein Levels ◽  
Estradiol Secretion ◽  
Morphogenetic Protein ◽  
Angiotensin Type 2 Receptor ◽  
Igf I ◽  
Follicle Atresia

Angiotensin II (AngII) is best known for its role in blood pressure regulation, but it also has documented actions in the reproductive system. There are two AngII receptors, type 1 (AGTR1) and type 2 (AGTR2). AGTR2 mediates the noncardiovascular effects of AngII and is expressed in the granulosa cell layer in rodents and is associated with follicle atresia. In contrast, expression of AGTR2 is reported to occur only in theca cells in cattle. The objective of the present study was to determine whether AngII also plays a role in follicle atresia in cattle. RT-PCR demonstrated AGTR2 mRNA in both granulosa and theca cells of bovine follicles. The presence of AGTR2 protein was confirmed by immunofluorescence. Abundance of AGTR2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas in theca cells, it did not change. Granulosa cells were cultured in serum-free medium, and treatment with hormones that increase estradiol secretion (FSH, IGF-I, and bone morphogenetic protein-7) increased AGTR2 mRNA and protein levels, whereas fibroblast growth factors inhibited estradiol secretion and AGTR2 protein levels. The addition of AngII or an AGTR2-specific agonist to granulosa cells in culture did not affect estradiol secretion or cell proliferation but inhibited abundance of mRNA encoding serine protease inhibitor E2, a protein involved in tissue remodeling. Because estradiol secretion is a major marker of nonatretic granulosa cells, these data suggest that AngII is not associated with follicle atresia in cattle but may have other specific roles during follicle growth.

Expression of 17β-hydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production

1994 ◽  
Vol 143 (1) ◽  
pp. 139-150 ◽  
Author(s):  
S A Ghersevich ◽  
M H Poutanen ◽  
H K Martikainen ◽  
R K Vihko
Keyword(s):  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Protein Concentration ◽  
Hydroxysteroid Dehydrogenase ◽  
Enzyme Protein ◽  
P450 Aromatase ◽  
Human Granulosa Cells ◽  
Cytochrome P450 Aromatase ◽  
In Cells

Abstract The aim of this study was to examine the expression and regulation of type 1 17β-hydroxysteroid dehydrogenase (type 1 17-HSD) enzyme protein and mRNA, and 17-HSD activity in human granulosa cells. The cells were obtained from patients taking part in an in vitro fertilization programme. The cells from each patient were divided into two groups: cells obtained from preovulatory follicles (LGC=granulosa cells from large follicles ≥ 18 mm in diameter), and cells from other visible follicles (SGC=granulosa cells from small follicles, less than 15 mm in diameter). The identity of 17-HSD enzyme protein expressed in human granulosa cells with placental cytosolic 17-HSD (type 1 17-HSD) was assessed by immunoblot analysis using polyclonal antibodies, and the enzyme was immunolocalized in the cytoplasm of granulosa cells. Type 1 17-HSD protein concentration, 17-HSD and cytochrome P450 aromatase (P450arom) activities and oestradiol (OE2) production in cells from LGC were significantly lower than the corresponding values obtained in SGC in the same patient (paired t-test). The type 1 17-HSD protein concentration, 17-HSD activity and P450arom activity were 140±16% (mean±s.e.m.), 121±22% and 113±26% higher in cells from SGC, which was also reflected in a 70±12% higher OE2 production in these cells. In freshly isolated cells from LGC or SGC, a high correlation between 17-HSD and P450arom activities was observed (r=0·93, P<0·001). In long-term cultured cells, type 1 17-HSD was stably expressed at least until day 9, while P450arom expression decreased. In addition, treatments with gonadotrophins did not affect type 1 17-HSD protein concentration and 17-HSD activity. In contrast to this, both P450arom activity and OE2 production were significantly increased (P<0·05). The data, therefore, suggest that type 1 17-HSD and P450arom are expressed in parallel during the latest stages of follicular maturation but, in cultured granulosa-luteal cells, the enzymes are regulated by distinct mechanisms. Journal of Endocrinology (1994) 143, 139–150

scholarly journals 11?-Hydroxysteroid dehydrogenase Type 1 in obesity and Type 2 diabetes

Diabetologia ◽  
2004 ◽  
Vol 47 (1) ◽  
pp. 1-11 ◽  
Author(s):  
T. M. Stulnig ◽  
W. Waldh�usl
Keyword(s):  
Type 2 Diabetes ◽  
Hydroxysteroid Dehydrogenase

Activin-A, but not inhibin, regulates 17β-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells

2000 ◽  
Vol 73 (5) ◽  
pp. 203-210 ◽  
Author(s):  
Sergio Ghersevich ◽  
Lateef Akinola ◽  
Tadeusz Kaminski ◽  
Matti Poutanen ◽  
Veli Isomaa ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Activin A

Steroidogenesis by avian ovarian cells: effects of luteinizing hormone and substrate availability

1983 ◽  
Vol 244 (5) ◽  
pp. E487-E493 ◽  
Author(s):  
B. L. Marrone ◽  
F. Hertelendy
Keyword(s):  
Luteinizing Hormone ◽  
Granulosa Cells ◽  
Synergistic Effects ◽  
Theca Cells ◽  
Ovarian Cells ◽  
P Accumulation ◽  
Preovulatory Follicles ◽  
Extensive Metabolism ◽  
Increased Sensitivity ◽  
Stimulation Of

The production of progesterone (P) and estrogen (E) by enzymatically dispersed granulosa and theca cells from chicken preovulatory follicles was examined in 3-h incubations. Accumulation of the P produced by granulosa cells was significantly reduced by the addition of theca cells, whereas E production was increased. The decrease in P accumulation was shown to be due to extensive metabolism of P by theca cells. There were no synergistic effects of luteinizing hormone (LH) and any substrate tested on E production by theca cells. Maturation of granulosa cells was characterized by an increased sensitivity to LH stimulation of P production, but there was no change in pregnenolone conversion to P. Conversely, maturation of theca cells was accompanied by decreased in both sensitivity to LH and the ability to convert substrates to E. The results are discussed in terms of the contribution of each cell type in the production of steroids by chicken follicles during maturation.

scholarly journals Expression of Key Androgen-Activating Enzymes in Ovarian Steroid Cell Tumor, Not Otherwise Specified

2020 ◽  
Vol 8 ◽  
pp. 232470962093341
Author(s):  
Evana Valenzuela Scheker ◽  
Amita Kathuria ◽  
Ashwini Esnakula ◽  
Hironobu Sasano ◽  
Yuto Yamazaki ◽  
...  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Steroidogenic Enzymes ◽  
Androgen Excess ◽  
Ovarian Steroid ◽  
Variable Expression ◽  
Not Otherwise Specified ◽  
Chain Cleavage ◽  
Cleavage Enzyme

To characterize the expression of steroidogenic enzymes implicated in the development of ovarian steroid cell tumors, not otherwise specified (SCT-NOS). We present 4 ovarian SCT-NOS evaluated by immunohistochemical staining of steroidogenic enzymes as an approach to define this entity pathologically. All 4 ovarian SCT-NOS showed increased expression for cholesterol side-chain cleavage enzyme (CYP11A1), 17α-hydroxylase (CYP17A1), 17β-hydroxysteroid dehydrogenase 1 (HSD17B1), aldo-ketoreductase type 1 C3 (AKR1C3), 3β-hydroxysteroid dehydrogenase 2 (HSD3B2), 5α-reductase type 2 (SRD5A2), steroid sulfatase (SULT2A1), estrogen sulfotransferase (EST), and aromatase (CYP19A1). Expression was negative for 21-hydroxylase (CYP21A2) and 17β-hydroxysteroid dehydrogenase 2 (HSD17B2). 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) and 5α-reductase type 1 (SRD5A1) showed variable expression. Our analysis reveals a novel finding of increased expression of AKR1C3, HSD17B1, SRD5A2, SULT2A1, and EST in ovarian SCT-NOS, which is clinically associated with androgen excess and virilization. Further studies are needed to validate these enzymes as new markers in the evaluation of hyperandrogenic ovarian conditions.

scholarly journals Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase

1998 ◽  
Vol 20 (1) ◽  
pp. 99-110 ◽  
Author(s):  
FM Rogerson ◽  
J Courtemanche ◽  
A Fleury ◽  
JG LeHoux ◽  
JI Mason ◽  
...  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Cdna Libraries ◽  
Sexually Dimorphic ◽  
High Affinity ◽  
Liver And Kidney ◽  
Low Levels ◽  
Type 3

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

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