Endometriosis does not Alter Aromatase Gene Expression (CYP19A1) in                     Mural Lutein-granulosa Cells of Women Undergoing Assisted Reproduction                     Techniques – a Pilot Study

Purpose The aim of this study was to quantify aromatase gene expression in mural lutein-granulosa cells of women with endometriosis undergoing assisted reproduction techniques (ART, IVF or ICSI). Methods: a case-control study was performed on 11 women with endometriosis (all stages, ASRM criteria) and 11 women with male or tubal causes of infertility undergoing ART. There was no difference between the groups regarding age, amount of gonadotrophins used, days of induction, follicles, and eggs collected. Mural lutein-granulosa cells were harvested from pre-ovulatory follicles during oocyte retrieval and correctly isolated. After cells lyses and storage into Trizol LS Reagent®, RNA extraction and cDNA synthesis were performed. Quantification of relative gene expression for CYP19A1 (aromatase) was performed by real time PCR, using SYBR Green reagents. All experiments were performed in duplicates. Results there was no difference between the groups in the quantitative gene expression of CYP19A1 (aromatase) gene on mural lutein-granulosa cells (P>.05, Mann Whitney). Conclusions These results suggest that this enzyme, aromatase, may have a complex and refined control of its gene expression on this population of granulosa cells, and in spite of previous evidence showing its reduced activity on these cells in endometriosis, gene expression per se may not be affected by the disease.

Download Full-text

The Orphan Nuclear Receptors NURR1 and NGFI-B Modulate Aromatase Gene Expression in Ovarian Granulosa Cells: A Possible Mechanism for Repression of Aromatase Expression upon Luteinizing Hormone Surge

Endocrinology ◽

10.1210/en.2004-0889 ◽

2005 ◽

Vol 146 (1) ◽

pp. 237-246 ◽

Cited By ~ 32

Author(s):

Yimin Wu ◽

Sagar Ghosh ◽

Yoshihiro Nishi ◽

Toshihiko Yanase ◽

Hajime Nawata ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Receptor ◽

Granulosa Cells ◽

Intracellular Camp ◽

Orphan Nuclear Receptors ◽

Aromatase Gene ◽

Ovarian Granulosa Cells ◽

A Genome ◽

Responsive Gene ◽

Aromatase Gene Expression

Ovarian granulosa cells play pivotal roles in many aspects of ovary functions including folliculogenesis and steroidogenesis. In response to FSH and LH, the elevation of intracellular cAMP level in granulosa cells leads to activation of multiple ovarian genes. Here, we report findings from a genome-wide study of the cAMP-responsive gene expression profiles in a human granulosa-like tumor cell line, KGN. The study identified 140 genes that are either activated or repressed by 2-fold or greater after stimulation by the adenylyl cyclase activator forskolin. The induction patterns of some cAMP-responsive genes were further analyzed by quantitative real-time PCR. Consistent with previous observations, the LH-responsive genes, such as the nuclear receptor 4A subfamily (NURR1, NGFI-B, and NOR-1), were rapidly but transiently induced, whereas the FSH-responsive gene CYP19 encoding aromatase was induced in a delayed fashion. Interestingly, ectopic expression of NURR1 or NGFI-B severely attenuated the cAMP-responsive activation of the ovary-specific aromatase promoter. Reduction of the endogenous NURR1 or NGFI-B by small interfering RNA significantly elevated aromatase gene expression. The cis-elements responsible for NURR1/NGFI-B-mediated repression were mapped to the minimal aromatase promoter sequence that confers camp responsiveness. Furthermore, the DNA-binding domain of NURR1 was required for the repression. Taken together, these results strongly suggest a causal relationship between the rapid decline of aromatase mRNA and induction of nuclear receptor subfamily 4A expression, which concomitantly occur upon LH surge at the later stages of ovarian follicular development.

Download Full-text

172 P450 AROMATASE GENE EXPRESSION, ESTRADIOL PRODUCTION, AND DOMINANT FOLLICLE DEVIATION IN BOS TAURUS AND BOS INDICUS DAIRY HEIFERS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab172 ◽

2013 ◽

Vol 25 (1) ◽

pp. 234

Author(s):

E. K. N. Arashiro ◽

S. Wohlres-Viana ◽

M. P. Palhao ◽

L. S. A. Camargo ◽

M. Henry ◽

...

Keyword(s):

Gene Expression ◽

Bos Taurus ◽

Rna Extraction ◽

Bos Indicus ◽

Dominant Follicle ◽

Aromatase Gene ◽

P450 Aromatase ◽

Dairy Heifers ◽

Follicular Waves ◽

Aromatase Gene Expression

It is well documented that the size of the dominant follicle at deviation is smaller in Bos indicus compared with in Bos taurus breeds. The physiological mechanisms underlying this difference, however, are unknown. The aim of the present study was to evaluate the dynamic of oestradiol production during follicle development close to the expected moment of deviation in Bos taurus and Bos indicus dairy heifers. Intrafollicular concentration of oestradiol (E2) and P450 aromatase gene expression in granulosa cells (GC) were evaluated in Gir (n = 10) and Holstein (n = 10) heifers. Follicular waves were synchronized with an intravaginal progesterone device (1 g, Sincrogest, Ourofino Agropecuária, São Paulo, Brazil) and benzoate oestradiol (2 mg im, Sincrodiol, Ourofino Agropecuária). Ultrasonography evaluations (MyLab30 Vet Gold, Esaote, Genova, Italy, with a 7.5-MHz transducer) were performed every 24 h to detect the emergence of the new follicular waves. The largest follicle of each wave was individually aspirated by ovum pickup before, at the expected diameter, or after deviation in both Gir (4.6 ± 0.2, 6.3 ± 0.2, and 8.5 ± 0.6 mm, respectively) and Holstein heifers (6.0 ± 0.5, 8.6 ± 0.4, and 10.2 ± 0.2 mm, respectively), as previously described (Arashiro et al. 2012 Reprod. Fertil. Dev. 24, 175). Follicular fluid (FF) samples were centrifuged and the supernatant stored at –20°C until E2 and progesterone (P4) determination by RIA. The pellet of GCs was washed twice with PBS, kept in RNAlater, and frozen at –20°C until RNA extraction and reverse transcription. Relative transcript quantification was performed by real-time PCR. The β-actin gene was used as control. Samples of FF with E2:P4 ratio <1 or presenting contamination by theca cells (detected by the expression of 17α-hydroxylase) were not used for statistical analyses. Concentration of E2 in FF was evaluated between breeds and among follicle size classes by ANOVA and differences among means compared by Student t-test or Tukey’s test, respectively. Within breeds, relative gene expression was accessed by pair-wise fixed reallocation randomization test (software REST®). Results are shown as mean ± SEM. In both breeds, concentration of E2 in FF progressively increased with follicular diameter (P < 0.05). Intrafollicular concentration of E2 (ng mL–1) was greater (P < 0.05) in Holstein than in Gir before (58.5 ± 11.7 v. 8.8 ± 2.0), at expected (226.0 ± 49.9 v. 78.9 ± 21.0), and after follicle deviation (579.1 ± 45.0 v. 185.0 ± 34.9). Interestingly, however, follicles with similar diameters (~6 or 8 mm) showed similar (P > 0.05) E2 concentrations between Holstein and Gir. Moreover, in both breeds, the relative expression of P450 aromatase gene in GC first increased (3.9 ± 2.4 and 67.5 ± 52.8 for Holstein and Gir, respectively; P < 0.05) at the same stage of follicular development (8 mm). The present results suggest that the smaller size of follicles at deviation in Bos indicus is not related to an earlier increase in intrafollicular E2 production. CNPq, CAPES, and Fapemig (CVZ APQ 02863/09).

Download Full-text

Effect of aromatase inhibitor (fadrozole) on proliferation, estradiol production and telomerase activity in pig granulosa cells in vitro

Czech Journal of Animal Science ◽

10.17221/136/2009-cjas ◽

2009 ◽

Vol 54 (No. 12) ◽

pp. 566-574 ◽

Cited By ~ 2

Author(s):

E. Chronowska ◽

M. Tománek ◽

T. Kott

Keyword(s):

Gene Expression ◽

Aromatase Inhibitor ◽

Granulosa Cell ◽

Granulosa Cells ◽

Telomerase Activity ◽

Culture Conditions ◽

Large Follicle ◽

Aromatase Gene ◽

Small Follicle ◽

Aromatase Gene Expression

The objective of the present work was to study the effect of a nonsteroidal aromatase inhibitor (fadrozole) on proliferation, estradiol production, aromatase expression and telomerase activity (TA) in pig granulosa cells (GC) from small (1–2 mm) and large (5–7 mm) follicles. The cells were treated with fadrozole for 48 h and 72 h in basal and FSH-stimulated conditions. Fadrozole caused a decrease (P < 0.05) of 3H-thymidine incorporation in granulosa cells derived from small (1–2 mm) and large follicles (5–7 mm). The proliferative potential of small-follicle GC was significantly higher (P < 0.01) under all culture conditions. Estradiol production was suppressed (P < 0.01) in both granulosa cell populations cultured in the presence of fadrozole for 48 and 72 h. Fadrozole caused a decrease (P < 0.05) of aromatase gene expression in small-follicle granulosa cell incubated for 72 h and in large-follicle GC after 48 h of culture. Large-follicle GC were characterized by a higher (P < 0.01) level of estradiol production and aromatase gene expression. Telomerase activity decreased (P < 0.05) in large-follicle granulosa cells incubated in the presence of an aromatase inhibitor for 72 h. The TA level in large-follicle granulosa cells was higher (P < 0.01) in comparison to small-follicle GC in all culture conditions after 72 h of incubation. The results of the present study suggest the important role of telomerase in the process of follicular growth and development.

Download Full-text