Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory Follicles

Granulosa cells express vascular endothelial growth factor A (VEGFA), and VEGFA mRNA levels increase as bovine follicles reach preovulatory status. To further evaluate the role of VEGFA isoforms in follicular development, cows were either synchronized with a modified Co- Synch protocol (CIDR) or treated with melengestrol acetate (MGA) with subsequent aspiration of the dominant follicles. Higher mRNA levels for the antiangiogenic isoform, VEGFA_164B, along with AMH and CARTPT in E2-inactive follicles suggest that these factors are markers for unhealthy, atretic follicles. In contrast, higher mRNA levels for the proangiogenic isoform, VEGFA_164, in E2-active follicles indicate that this isoform may help predict healthy ovulatory follicles

Follicular Vascular Endothelial Growth Factor A Expression Before and After the LH Surge

Granulosa cell expression of VEGFAisoforms in dominant bovine follicles was evaluated. Collection of granulosa cells via follicle aspiration revealed altered expression of the proangiogenic VEGFA_164 isoform but not the antiangiogenic VEGFA_164B isoform prior to and after the LH surge. Expression of VEGFA_164 declines as both the LH surge and ovulation approaches. In addition, VEGFA_164 and VEGFA_164B expression prior to the LH surge was positively correlated with FSHR and CYP19A1 expression, suggesting that VEGFA expression may be regulated by FSH. These data indicate differential expression of VEGFA isoforms may be an important feature of bovine dominant follicle development

Oocyte mRNA and Follicle Androgen Levels Associated with Fertility

The environment that the oocyte develops in (follicle) and the mRNA that is produced (mRNA abundance) during development were examined. Androgen levels within the follicle were higher in heifers (≤ 2 years) that never established a pregnancy compared to cows that stayed in the herd at least 3 years and had at least one successful pregnancy. These high androgen levels were associated with increased abundance of several candidate mRNAs in the cumulus-oocyte complex (COC), which includes the oocyte and somatic cells immediately surrounding the oocyte, isolated from the dominant follicle. The data suggest that androgen levels represent a marker for oocyte quality which could be used to select for females to retain in the herd

Androgen Excess in Beef Cows Results in Altered Theca Cell Gene Expression and Fertilityand

Within the University of Nebraska–Lincoln physiology herd, two sub-populations of cows with different concentrations of androstenedione have been identified. Androstenedione is a precursor for estradiol production, and androstenedione concentration is increased 24.5-fold in the high androstenedione cows. Our objective was to determine the cause of increased androstenedione production in high androstenedione cows and the effects on theca cell and oocyte gene expression. High androstenedione cows had increased steroidogenic enzyme abundance in theca cells and altered oocyte mRNA abundance. Increased androgen production in high androstenedione cows is associated with altered gene expression and/or mRNA stability during oocyte growth and maturation, which may reducefertility

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype

Altered Theca and Cumulus Oocyte Complex Gene Expression, Follicular Arrest and Reduced Fertility in Cows with Dominant Follicle Follicular Fluid Androgen Excess

Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; \u3e40 ng/ml; mean = 102) and another with Low A4 (/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 (

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype

Granulosa Cell Exposure to Excess Androgens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess

Within the UNL physiology herd, a group of cows have been identified with excess androgen (androstenedione, A4) in their dominant follicle (30 fold higher than controls) and a 17% reduction in calving rate, suggesting subfertility. Th e objective was to identify altered granulosa cell gene expression that could be preventing these cells from converting excess androgen into estrogen. Microarray analysis suggests these granulosa cells experience inhibited proliferation resulting in a reduced total population of cells. Improved understanding of the causes of this phenotype may provide beef producers with tools to identify potentially subfertile cattle and improve reproductive efficiency

Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity

Heifers and cows that were culled from the herd due to failure to become pregnant were categorized into groups with low (\u3c 2 year), moderate (\u3e2 and \u3c 6 year) or high (≥ 6 year) fertility. Antral follicle counts were numerically lower in the low group and increased in the moderate- and high-fertility group. Granulosa cells from dominant follicles in moderate- and high-fertility cows had a greater ratio of Vascular Endothelial Growth Factor 164 (VEGF164) to VEGF164B compared to the low-fertility cows. Furthermore, there was more CARTPT in granulosa cells from subordinate follicles in moderate- and high-fertility cows than low. Gene expression is altered in granulosa cells from cows differing in fertility, suggesting these are candidate genes that may be used as markers to assist in determining reproductive longevity in beef cows

Ovarian and follicle phenotypic measurements for Low A4 and High A4 cows.

1<p>Dominant follicle follicular fluid A4 concentration of <20 ng/mL.</p>2<p>Dominant follicle follicular fluid A4 concentration of >40 ng/mL.</p>3<p>Follicle aspirations were taken 12–48 hours after two injections of Prostaglandin F2alpha (PG).</p>4<p>Ovariectomies were conducted after modified cosynch protocol 36 hours after PG.</p>5<p>Where AFC = antral follicle count.</p><p>Ovarian and follicle phenotypic measurements for Low A4 and High A4 cows.</p