Brief Summary of Minutes of Annual Meeting

Objective 1: Identify intracellular signaling pathways and gene expression regulatory mechanisms within the ovary, embryo, or female reproductive tract that promote oocyte growth and maturation, fertilization, early embryonic development, and establishment and maintenance of pregnancy.

MicroRNA play a key regulatory role in luteal rescue during early pregnancy, and likely target proteins involved in exptracellular matrix events.

The phenotype of macrophages within the CL changes with luteal status. Later stages of luteal regression and luteal rescue during early pregnancy are associated with macrophage protein expression that supports antiinflammatory and tissue remodeling events.

Acute zinc depletion promotes progesterone accumulation in media from cumulus cells or CL tissue cultured in vitro.

Conceptus secretory proteins alter lympocyte numbers and functions to promote tolerance at the fetal-maternal interface during early pregnancy in dairy heifers.

miRNAs regulate E2/P4 biosynthesis and follicle formation by targeting mRNAs involved in steroidogenesis and steroid bovine fetal ovaries.

Identification of the impact that thermal stress has on ovarian signaling is resulting in a mechanistic map being developed, upon which strategies to ameliorate seasonal infertility can be based.

Molecular signaling protein alterations from lipopolysaccharide or thermal stress treated gilts have been identified.

Determined influence of a physiological stressor, obesity, on the ovarian capacity to respond to a toxic environmental stress.

Identified molecular proteins within the oocyte that contribute to viability of the oocyte.

Determined that the increase in early luteal phase concentrations of progesterone attained by providing supplemental selenium in a 1:1 blend of organic and inorganic forms may advance development of the uterine endometrium, facilitating early embryonic growth and the establishment of a viable pregnancy.

Determined that CCN1 expression in KGN and HGrC1 granulosa cells is regulated by PGF2α, involving the protein kinase C pathway. Further, the diversity of PKC isoforms (conventional, novel and atypical) in cells may, in part, explain the differential effects of Calphostin C and the Pseudo Substrate on CCN1 expression in KGN cells.

Early plane of nutrition impacts the progression of uterine gland development in heifers, and that this outcome may be mediated by changes in the expression of several local controllers of gland development.

Prepubertal exposure to the SERM tamoxifen alters the morphology of the bovine reproductive tract as well as signaling pathways associated with tract development.

Maternal obesity modifies a subset of important mediators of embryonic and extraembryonic development in ovine and porcine conceptuses.

Maternal obesity modifies uterine gene expression during early pregnancy.

Established that uterine factors act cooperatively to promote embryonic and placental events in ruminants.

New regulators of interferon-tau expression identified.

Interleukin-6 improves development of the inner cell mass (ICM) in bovine embryos.

Described the relationship between periconceptional heat stress and subsequent productivity of both primiparous and multiparous Holstein dairy cows.

Determined that early weaning of beef heifers can have long-term effects on physiology. Some of these changes have implications for lifetime productivity.

Obese female mice exhibit evidence of ovarian inflammation which in turn alters the transcription of oocyte mRNAs. The increased inflammation, and in particular increased Tnfa expression, was correlated with increases in the family of gut microbes, Lachnospiraceae.

Induction of oxidative stress using hydrogen peroxide decreased the degradation of Pou5f1 and Dppa3 transcripts during oocyte maturation and resulted in increased expression of the POU5F1 and DPPA3 proteins in 1- and 2-cell embryos, respectively.

Identified obesity-dependent changes in the protein expression of RNA binding proteins that recognize AU-rich sequences in the 3’UTR of mRNAs. Changes in these proteins will have a significant impact on the stability of oocyte mRNAs.

Short-term, acute treatment of primary follicles with TNFa increased phosphorylation of NFkB p65 but not STAT3 in the oocyte.

Objective 2: Identify intercellular interactions between somatic cells of the ovary, somatic cells and germ cells, or somatic cells and the embryo that promote follicular growth, oocyte maturation, early embryonic development, and establishment and maintenance of pregnancy.

The presence of the growth hormone receptor (GHR) AluI(-) allele in Holstein cows is associated with increased serum IGF-I concentrations and a shorter calving to conception interval during lactation ie. higher fertility.

Determined that a reproductive management strategy designed to increase insemination of cows in estrus after NPD resulted in similar reproductive performance (time to pregnancy) than a typical resynchronization of ovulation protocol used in dairy farms.

Characterized ovarian connexin proteins 37 and 43 to understand communication between somatic cells and the female gamete.

Validated a cell fate mapping approach that has allowed us to determine the identity and fate of cells within the mesenchymal tissue surrounding growing follicles that are direct targets of hedgehog (HH) signaling and that contribute to the formation of the theca cell layer.

Determined that uterine size has an effect on fertility in lactating dairy cows with cows with a larger uterus having lower fertility.

Determined that expression of estrus was associated with increased fertility and reduced pregnancy loss in fixed time AI programs.

Determined that Bos indicus and Bos taurus cattle differ in their ability to produce viable offspring following nutrient restriction during early pregnancy.

Found that there is a second period of CL regression that results in loss of pregnancy and is maintained by a local mechanism (as evidenced by contralateral CL regression in pregnant animals). Maintaining progesterone during this period (Day 30-60 of pregnancy) can maintain pregnancies indicating the inappropriate CL regression may be responsible for some pregnancy loss in this time period.

Found that Trio carriers have increased ovulation rate, independent of AMH or antral follicle count due to increased SMAD6 leading to decreased granulosa cell proliferation and a reduced follicle growth rate with follicle deviation happening at the same time but at a reduced follicle size.

Determined that the lack of Ca2+ oscillations is not the not the main reason of ICSI failure in the bovine.

Determined that bovine sperm heads are highly resistant sperm head decondensation.

Sperm epigenome (chromatin dynamics) as well as functional genome (macromolecules) are associated with bull fertility.

In collaboration with Andrea S. Cupp, we have identified cows with evidence of chronic and sporadic anovulation which likely contributes to the subfertility of these cows. The primary abnormality is the presence of excess androgens in follicular fluid, decreased circulating Sex Hormone Binding Globulin concentrations, and altered circulating NEFAs.

Hypothesized that animals that appear to enter puberty at a young age and then become acyclic are more likely to fall into the high intrafollicular A4 group as cows.

Showed that ovarian cortex from cows classified as High A4 secrete more A4 into the culture medium. Furthermore, treatment of cortex explants with VEGFA increases follicle progression independent of classification based on intrafollicular A4 concentrations.

Plans for next reporting period:

Investigate differences in uterine immune cell function between heifers and mature, lactating dairy cows.

Studies to characterize expression of oocyte-specific lncRNAs during early embryogenesis and identify ZNFO target genes will be carried out.

The work on prostaglandin F receptors in bovine corpora lutea will be continued and if translocation is shown to occur from Day 4 to Day 10, further studies during maternal recognition of pregnancy may be initiated.

Further studies of interference with early pregnancy in the sheep model of bovine mastitis and analyses of changes in white blood cell types in postpartum, lactating dairy cows in contrast to heifers will be carried out.

Examine the effects of INT and PAG on uterine immune cell function.

Continue efforts to commercialize viral and early pregnancy diagnostics for cattle.

Examine effects of omega-3 FA on immune function in transition dairy cows.

TNFα SNP genotyping will be completed on the DNA sample set collected from 470 dairy cows as an ongoing collaborative project.

Complete the GHR, IGF-I and TNFα genotyping of additional 110 dairy cows for comparison and analysis of phenotypic data for DMI, energy balance, insulin, IGF-I and fertility during early lactation.

Compare plasma IGF-I levels during lactation in cows with GHR and IGF-I SNP’s.

Investigate different treatments to improve sperm head decondensation following sperm head injection in bovine oocytes.

Examine the outcome of changing the concentration of certain ions in the extracellular media to stimulate oscillations, better egg activation and initiation of embryo development following ICSI in the bovine.

Continue work on PI3K and microRNA regulation of chemical metabolism.

Investigating impacts of central metabolism alterations on reproductive function.

Investigate the impact of glyphosate, a widely used herbicide, on ovarian signaling and the potential for glyphosate to be considered an ovotoxicant.

Determine the regulation of CCN1 expression in bovine granulosa cells by estradiol, gonadotropins and PGF2α.

Determine the regulation of CCN1 expression in KGN and HGrC1 cells by growth factors.

Determine the signaling pathways (e.g. MEK/ERK and PI3K/Akt) associated with the regulation of CCN1 expression in KGN and HGrC1 cells.

We will increase the number of replicates for Gli1-CreER^T2 – tdTomato mice treated with TM at different ages and stages of ovarian development (prior to formation of follicles in addition to labeling follicles in the process of growth) as well as replicates for Gli1LacZ mice.

Investigate molecular, cellular and physiological mechanisms regulating embryo and fetal development.

Complete studies to determine the impact of heat stress on follicular fluid composition and oocyte competency in dairy cattle.

Continue studies to determine if Bos indicus mitochondrial transfer can be used to improve thermal resistance of Bos taurus oocytes and embryos.

Complete studies to describe changes in conceptus and uterine transcriptomes in obese ewes.

Continue studies to examine the impact of fescue endophyte toxicity on follicle development, oocyte maturation and early embryonic development.

Continue studies to understand how embryonic lineage commitment events are controlled in bovine embryos, and specifically with fate decisions for primitive endoderm formation.

Decipher the roles of interleukin-6 and its downstream signaling factor, STAT3 in controlling the development, maintenance and proliferation of inner cell mass cells and epiblast cells in bovine embryos.

Initiate studies to better understand how conceptus elongation, trophoblast cell proliferation, and germinal disk development are controlled during early pregnancy in cattle.

Continue studies examining how bovine subspecies genotype and nutritional status impact fetal outcomes.

Implement granulosa cell study which examines the MAPK/ERK signaling pathway in granulosa cells of small follicles (3-5mm) as potential mechanism of resistance to immune-mediated apoptosis and follicular atresia.

Determine the effect of supplementation of rumen-protected methionine on reproduction.

Determine the changes in gene expression in the CL during normal pregnancy or in cows that have not been inseminated.

Determine the timing of regression of an accessory CL in pregnant lactating cows and heifers.

Determine the physiology of anovulation in lactating dairy cows.

Continue to work on the high ovulation rate genotype of beef cattle with Dr. Brian Kirkpatrick. Carriers of this allele have an average ovulation rate of 4, whereas non-carrier halfsiblings have an ovulation rate of ~1.

Determine the accuracy of pregnancy diagnosis on Day 21 using various methods in ET recipients.