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Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

Summers AF, Pohlmeier WE, Sargent KM, Cole BD, Vinton RJ, Kurz SG, McFee RM, Cushman RA, Cupp AS, Wood JR - PLoS ONE (2014)

Bottom Line: We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance.There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired.These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
Aspiration of bovine follicles 12-36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

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High A4 Cows Have Increased Steroidogenic Gene Expression in Theca Cells.Quantitative RT-PCR results for StAR (A), LHCGR (B), CYP11A1 (C), CYP17A1 (D), and GATA6 (E) in theca cells collected from dominant follicles of High A4 (black bars, n≥12) and Low A4 (white bars, n≥19) cows. The geometric mean of GAPDH and RPL-15 was used as an endogenous control. Data for CYP11A1, CYP17A1, LHCRG, and GATA6 were log transformed to meet normal distribution assumptions. Graphs represent a fold change in mRNA abundance with Low A4 set as control (1). The mean ± SEM normalized values are presented. *, P≤0.05.
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pone-0110683-g004: High A4 Cows Have Increased Steroidogenic Gene Expression in Theca Cells.Quantitative RT-PCR results for StAR (A), LHCGR (B), CYP11A1 (C), CYP17A1 (D), and GATA6 (E) in theca cells collected from dominant follicles of High A4 (black bars, n≥12) and Low A4 (white bars, n≥19) cows. The geometric mean of GAPDH and RPL-15 was used as an endogenous control. Data for CYP11A1, CYP17A1, LHCRG, and GATA6 were log transformed to meet normal distribution assumptions. Graphs represent a fold change in mRNA abundance with Low A4 set as control (1). The mean ± SEM normalized values are presented. *, P≤0.05.

Mentions: Theca and granulosa cells are important regulators of steroid synthesis in the ovary. Therefore, the mRNA abundance of steroidogenic enzymes was analyzed. In theca cells, the mRNA abundance of StAR, which traffics cholesterol from the outer to inner mitochondrial membrane was not different (P = 0.46, Fig. 4A) between High A4 and Low A4 cows. However, the abundance of CYP11A1 mRNA, which is responsible for the conversion of cholesterol to pregnenolone, was 6.5-fold greater (P = 0.05, Fig. 4C) in High A4 compared with Low A4 theca cells. Furthermore, CYP17A1 which converts pregnenolone to 17-OH pregnenolone and ultimately dehydroepiandrosterone (DHEA), exhibited an 18.4-fold increase (P = 0.01, Fig. 4D) in mRNA abundance in High vs. Low A4 theca cells. Luteinizing hormone directly increases expression of steroidogenic enzymes and the transcription factor, GATA-binding factor 6 (GATA6) has previously been reported to increase promoter activities of CYP11A and CYP17A[38]. In the theca cells of High A4 cows, the expression of LHCGR (P = 0.01, Fig. 4B) mRNA was increased 13.1-fold and GATA6 (P = 0.01, Fig. 3E) mRNA abundance was increased 30-fold. Although we report increased expression of steroidogenic enzyme gene expression in theca from High A4 cows, there was no difference (P≥0.19) in HSD3B or CYP19A1 steady-state transcript levels in granulosa cells collected from High A4 and Low A4 dominant follicles (data not shown).


Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

Summers AF, Pohlmeier WE, Sargent KM, Cole BD, Vinton RJ, Kurz SG, McFee RM, Cushman RA, Cupp AS, Wood JR - PLoS ONE (2014)

High A4 Cows Have Increased Steroidogenic Gene Expression in Theca Cells.Quantitative RT-PCR results for StAR (A), LHCGR (B), CYP11A1 (C), CYP17A1 (D), and GATA6 (E) in theca cells collected from dominant follicles of High A4 (black bars, n≥12) and Low A4 (white bars, n≥19) cows. The geometric mean of GAPDH and RPL-15 was used as an endogenous control. Data for CYP11A1, CYP17A1, LHCRG, and GATA6 were log transformed to meet normal distribution assumptions. Graphs represent a fold change in mRNA abundance with Low A4 set as control (1). The mean ± SEM normalized values are presented. *, P≤0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4199720&req=5

pone-0110683-g004: High A4 Cows Have Increased Steroidogenic Gene Expression in Theca Cells.Quantitative RT-PCR results for StAR (A), LHCGR (B), CYP11A1 (C), CYP17A1 (D), and GATA6 (E) in theca cells collected from dominant follicles of High A4 (black bars, n≥12) and Low A4 (white bars, n≥19) cows. The geometric mean of GAPDH and RPL-15 was used as an endogenous control. Data for CYP11A1, CYP17A1, LHCRG, and GATA6 were log transformed to meet normal distribution assumptions. Graphs represent a fold change in mRNA abundance with Low A4 set as control (1). The mean ± SEM normalized values are presented. *, P≤0.05.
Mentions: Theca and granulosa cells are important regulators of steroid synthesis in the ovary. Therefore, the mRNA abundance of steroidogenic enzymes was analyzed. In theca cells, the mRNA abundance of StAR, which traffics cholesterol from the outer to inner mitochondrial membrane was not different (P = 0.46, Fig. 4A) between High A4 and Low A4 cows. However, the abundance of CYP11A1 mRNA, which is responsible for the conversion of cholesterol to pregnenolone, was 6.5-fold greater (P = 0.05, Fig. 4C) in High A4 compared with Low A4 theca cells. Furthermore, CYP17A1 which converts pregnenolone to 17-OH pregnenolone and ultimately dehydroepiandrosterone (DHEA), exhibited an 18.4-fold increase (P = 0.01, Fig. 4D) in mRNA abundance in High vs. Low A4 theca cells. Luteinizing hormone directly increases expression of steroidogenic enzymes and the transcription factor, GATA-binding factor 6 (GATA6) has previously been reported to increase promoter activities of CYP11A and CYP17A[38]. In the theca cells of High A4 cows, the expression of LHCGR (P = 0.01, Fig. 4B) mRNA was increased 13.1-fold and GATA6 (P = 0.01, Fig. 3E) mRNA abundance was increased 30-fold. Although we report increased expression of steroidogenic enzyme gene expression in theca from High A4 cows, there was no difference (P≥0.19) in HSD3B or CYP19A1 steady-state transcript levels in granulosa cells collected from High A4 and Low A4 dominant follicles (data not shown).

Bottom Line: We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance.There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired.These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
Aspiration of bovine follicles 12-36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.

Show MeSH
Related in: MedlinePlus