Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article “A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity.

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these four cell types. Analysis of the RNA present in each bovine cell type using Affymetrix microarrays yielded new cell-specific genetic markers, functional insight into the behavior of each cell type via Gene Ontology Annotations and Ingenuity Pathway Analysis, and evidence of small and large luteal cell lineages using Principle Component Analysis. Enriched expression of select genes for each cell type was validated by qPCR. This expression analysis offers insight into cell-specific behaviors and the differentiation process that transforms somatic follicular cells into luteal cells

Reproductive Aging Influences Ovarian Function in Beef Cows

Anti-Müllerian Hormone (AMH) has been associated with follicle number and age of the ovary. Therefore, our hypothesiswas that AMH was a biomarker for both follicle number and ovarian function in the beef cow. Ovaries were collected by flank laparotomy. The number of follicles increased as cows aged from 1.5 to 6 years and began to decrease thereafter; however, the size of the ovary continued to increase with advanced age. Expression of the AMH gene increased with increasing follicle number in 2-year-old beef cows. These results suggest that heifers with larger ovaries will have greater numbers of follicles and greater productivity, allowing them to stay in the production herd longer. AMH could be used to identify heifers of high reproductive potential at a very young age

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

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

VEGFA splicing: divergent isoforms regulate spermatogonial stem cell maintenance

Despite being well-known for regulating angiogenesis in both normal and tumorigenic environments, vascular endothelial growth factor A (VEGFA) has been recently implicated in male fertility, namely in the maintenance of spermatogonial stem cells (SSC). The VEGFA gene can be spliced into multiple distinct isoforms that are either angiogenic or antiangiogenic in nature. Although studies have demonstrated the alternative splicing of VEGFA, including the divergent roles of the two isoform family types, many investigations do not differentiate between them. Data concerning VEGFA in the mammalian testis are limited, but the various angiogenic isoforms appear to promote seminiferous cord formation and to form a gradient across which cells may migrate. Treatment with either antiangiogenic isoforms of VEGFA or with inhibitors to angiogenic signaling impair these processes. Serendipitously, expression of KDR, the primary receptor for both types of VEGFA isoforms, was observed on male germ cells. These findings led to further investigation of the way that VEGFA elicits avascular functions within testes. Following treatment of donor perinatal male mice with either antiangiogenic VEGFA165b or angiogenic VEGFA164 isoforms, seminiferous tubules were less colonized following transplantation with cells from VEGFA165b-treated donors. Thus, VEGFA165b and possibly other antiangiogenic isoforms of VEGFA reduce SSC number either by promoting premature differentiation, inducing cell death, or by preventing SSC formation. Thus, angiogenic isoforms of VEGFA are hypothesized to promote SSC self-renewal, and the divergent isoforms are thought to balance one another to maintain SSC homeostasis in vivo

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in Granulosa Cells Using \u3ci\u3epDmrt-1-Cre\u3c/i\u3e or \u3ci\u3eAmhr2-Cre\u3c/i\u3e Reduces Fertility by Arresting Follicular Development and by Reducing Litter Size in Female Mice

Because VEGFA has been implicated in follicle development, the objective of this study was to determine the effects of granulosa- and germ cell-specific VEGFA loss on ovarian morphogenesis, function, and female fertility. pDmrt1-Cre mice were mated to floxed VEGFA mice to develop granulosa-/germ cell-specific knockouts (pDmrt1-Cre;Vegfa-/-). The time from mating to first parturition was increased when pDmrt1-Cre;Vegfa-/- females were mated to control males (P = 0.0008) and tended to be longer for heterozygous females (P \u3c 0.07). Litter size was reduced for pDmrt1-Cre;Vegfa-/- females (P \u3c 0.007). The time between the first and second parturitions was also increased for heterozygous females (P \u3c 0.04) and tended to be increased for pDmrt1-Cre;Vegfa-/- females (P \u3c 0.07). pDmrt1-Cre;Vegfa-/- females had smaller ovaries (P \u3c 0.04), reduced plasma estradiol (P \u3c 0.007), fewer developing follicles (P \u3c 0.008) and tended to have fewer corpora lutea (P \u3c 0.08). Expression of Igf1r was reduced (P \u3c 0.05); expression of Foxo3a tended to be increased (P \u3c 0.06); and both Fshr (P \u3c 0.1) and Sirt6 tended to be reduced (P \u3c 0.06) in pDmrt1-Cre;Vegfa-/- ovaries. To compare VEGFA knockouts, we generated Amhr2-Cre;Vegfa-/- mice that required more time from mating to first parturition (P \u3c 0.003) with variable ovarian size. Both lines had more apoptotic granulosa cells, and vascular staining did not appear different. Taken together these data indicate that the loss of all VEGFA isoforms in granulosa/germ cells (proangiogenic and antiangiogenic) causes subfertility by arresting follicular development, resulting in reduced ovulation rate and fewer pups per litter. Includes 2 supplementary files

Transcriptomes of bovine ovarian follicular and luteal cells

Affymetrix Bovine GeneChipR 1.0 ST Array RNA expression analysis was performed on four somatic ovarian cell types: the granulosa cell (GCs) and theca cells (TCs) of the dominant follicle and the large luteal cells (LLCs) and small luteal cells (SLCs) of the corpus luteum. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE83524). Subsequent ANOVA determined genes that were enriched genes that were enriched (\u3e/= 2 fold more) or decreased

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

Loss of Vascular Endothelial Growth Factor A (VEGFA) Isoforms in the Testes of Male Mice Causes Subfertility, Reduces Sperm Numbers, and Alters Expression of Genes That Regulate Undifferentiated Spermatogonia

Vascular endothelial growth factor A (VEGFA) isoform treatment has been demonstrated to alter spermatogonial stem cell homeostasis. Therefore, we generated pDmrt1-Cre;Vegfa−/− (knockout, KO) mice by crossing pDmrt1-Cre mice to floxed Vegfa mice to test whether loss of all VEGFA isoforms in Sertoli and germ cells would impair spermatogenesis. When first mated, KO males took 14 days longer to get control females pregnant (P \u3c .02) and tended to take longer for all subsequent parturition intervals (9 days; P \u3c .07). Heterozygous males sired fewer pups per litter (P \u3c .03) and after the first litter took 10 days longer (P \u3c .05) to impregnate females, suggesting a more progressive loss of fertility. Reproductive organs were collected from 6-month-old male mice. There were fewer sperm per tubule in the corpus epididymides (P \u3c .001) and fewer ZBTB16-stained undifferentiated spermatogonia (P \u3c .003) in the testes of KO males. Testicular mRNA abundance for Bcl2 (P \u3c .02), Bcl2:Bax (P \u3c .02), Neurog3 (P \u3c .007), and Ret was greater (P = .0005), tended to be greater for Sin3a and tended to be reduced for total Foxo1 (P \u3c .07) in KO males. Immunofluorescence for CD31 and VE-Cadherin showed no differences in testis vasculature; however, CD31-positive staining was evident in undifferentiated spermatogonia only in KO testes. Therefore, loss of VEGFA isoforms in Sertoli and germ cells alters genes necessary for long-term maintenance of undifferentiated spermatogonia, ultimately reducing sperm numbers and resulting in subfertility