SAES-422 Multistate Research Activity Accomplishments Report

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Basic Information
Additional Info

Status: Approved

Basic Information

Project No. and Title: NE1027 : Ovarian Influences on Embryonic Survival in Ruminants
Period Covered: 10/01/2008 to 09/01/2009
Date of Report: 06/19/2009
Annual Meeting Dates: 06/18/2009 to 06/19/2009

Keth Inskeep, West Virginia University; Milo Wiltbank, University of Wisconsin; Ron Butler, Cornell University (NY); Dave Townson, University of New Hampshire; Paul Tsang, University of New Hampshire; Joanne Fortune, Cornell University, (NY); Joy Pate, Penn State University; Francisco Diaz, Penn State University; Robert Milvae, University of Connecticut

Brief Summary of Minutes of Annual Meeting

The meeting was called to order at 3:15 pm by Joy Pate, Chair. No administrative advisors were present at the meeting, but Terry Etherton, Chair of Dairy and Animal Science, PSU, welcomed the group on Friday morning and led a brief discussion on multistate projects and the importance and future of reproductive biology programs.

Station reports began with presentations for Objective 1: .

Objective 1: Identify genetic, morphological and physiological attributes of the ovary considered to improve fertility in ruminants

WV (Inskeep): Fetal ovary-expressed microRNAs have been cloned from bovine fetal oocytes and expression profiling completed. One novel miRNA was identified. Future analyses of predicted mRNA targets for these miRNAs should improve our understanding of their roles in regulating gene expression during embryogenesis and folliculogenesis.

NY (Fortune): It was discovered that insulin in culture medium is what has caused spontaneous activation of primordial follicles. In insulin-deplete medium, an effect of kit-ligand on activation of bovine primordial follicles was observed.

NY(Butler): Dry matter intake is positively correlated with volume of the CL and negatively correlated with plasma P4. Liver catabolic enzymes were not affected by DMI. An unanswered question is, how does DMI increase P4 metabolism? (catabolism).

WI (Wiltbank): A high concentration of P4 before AI seems to be more important to increasing conception rates than high P4 after AI. In vitro, PUFAs decrease P4 catabolism in liver slices, However, this effect could not be replicated in vivo, perhaps because they could not achieve high enough PUFA concentrations in vivo.

PA (Diaz): New investigator on the project. His contributions to the project will be to address the following two questions:
1) In the cow, are cumulus cells more mural-like or more distinct?
2) Do the factors that affect oocyte quality in the mouse also do so in the cow?

PA (Pate): A means for separation of luteal endothelial cells that yields relatively pure, highly viable cells was developed. This method employs a series of filtration steps based on cell size, rather than magnetic or flow cytometric sorting. The isolated endothelial cells were placed in culture and could be used for functional studies.

NH (Tsang): Active MMP-2 secreted by endothelial cells is decreased by P4. TIMP-2 is increased by P4, but TIMP-1 is decreased by P4.

CT (Milvae): Oxytocin receptors are on small luteal cells.

Legacy attachment file:

[Minutes]

Accomplishments

Thirty percent of beef cows and 50% of dairy cow pregnancies are lost due to embryonic mortality resulting in a loss of 1.4-1.2 billion dollars in the US. Problems associated with embryonic mortality may be due in part to oocytes that are not competent to be fertilized. It is our hypothesis that the microenvironment of the oocyte and granulosal cells during follicular growth and oocyte maturation contributes to the developmental potential of the oocyte. Our data indicates that altered metabolic hormone and sex steroid levels results in changes in gene expression. Our future experiments will determine how these changes in gene expression affect markers of oocyte quality including chromatin condensation and meiotic resumption. There are very few markers that distinguish a good oocyte from one that is not fertile. Thus, identifying good markers or indicators of competent oocytes we may be able to understand more and diagnose factors that contribute to embryonic mortality. Furthermore, defining genes that are important for establishing an oocyte of high quality may identify new targets for reversing the deleterious effects of a persistent follicle and increase the fertility of dairy and beef cattle.

Impacts

Primordial follicles comprise the "follicular reserve" which will supply the female with follicles and gametes throughout her reproductive life. Thus a greater understanding of how the pool of resting follicles is formed and of the signals that allow or inhibit activation and subsequent growth is of potential practical importance.
Higher DMI and liver catabolic enzyme activity in lactating dairy cows were related to decreased plasma progesterone concentration. DMI negatively affects plasma progesterone and P4/CLV probably via increased liver blood flow, but clearly not by increasing liver catabolic enzyme activities ie. Higher blood flow carrying progesterone past ongoing level of enzyme activity in individual cows. In liver, total enzyme activity negatively impacts plasma progesterone with CYP2C being more important for progesterone catabolic activity than CP3A.
Conditioned medium and cell extracts need to be concentrated (10-fold) before analysis by zymography and immunoblotting for MMPs and TIMPs, respectively. So far, the variability between our experiments may be explained by different culture conditions (i.e. type of medium used EGM versus DMEM) that may favor one endothelial cell subtype over another in mixed cultures of microvascular endothelial cells.
Alternation of the expression of CD8/18 intermediate filaments in luteal cells failed to influence Fas ligand-induced apoptosis, suggesting these filaments do not regulate Fas expression of Fas-mediated signaling as seen in other cell types. However, given the fact that acrylamide-induced disruption was transient and that genetically-induced overexpression caused filament aggregation, we are exploring alternative approaches and mechanisms by which CD8/18 filaments could impair apoptosis of ovarian steroidogenic cells.
Identification of microRNAs in bovine oocytes and early embryos, including a novel microRNA, opens a door for further studies of the regulatory effects on gene expression during folliculogenesis and early embryogenesis. Basic studies in sheep have increased our information on regulation of luteal function during the estrous cycle, specifically in regard to nuances of actions of endothelin and oxytocin.
Luteal endothelial cells were isolated using magnetic separation or a sequential filtration method. Both procedures yield endothelial cell populations of similar purity, but the filtration method allows for a much greater yield of cells. With this newly developed procedure, it should be possible to provide endothelial cells for a greater variety of experimental procedures.
These are the first data collected in any species to demonstrate a functional shift from anti-inflammatory to pro-inflammatory T lymphocyte subsets isolated from the CL and suggests local regulation of lymphocyte function within the CL. Differences in T lymphocyte subsets within the CL compared to those in the peripheral circulation implies tissue-specific regulation of lymphocyte trafficking and/or proliferation.
Temporal changes in adiponectin receptors expression in the CL and in T cells throughout the estrous cycle were observed. The putative role of adiponectin as a local or systemic regulator of luteal function remains to be determined.
Following maternal recognition of pregnancy PGRMC1and PGR are downregulated in the CL, whereas <i>mPRB</i> is upregulated. It may be inferred from these data that progesterone signalling is altered within the CL to facilitate luteal maintenance during early pregnancy.
Temporal changes in Derlin-1 were observed during the lifespan of the CL. Association of Derlin-1 with class I MHC in th CL suggests a possible role in controlling presentation to T lymphocytes to prevent unwanted activation.

Publications

Stevenson, JS, Tifany SM, Inskeep EK. Maintenance of pregnancy in dairy cattle after treatment with human chorionic gonadotropin or gonadotropin-releasing hormone. J. Dairy Sci 2008. 91:3092-3101.

Rhinehart JD, Starbuck-CLemmer MJ, Flores JA, Milvae RA, Yao J, Poole DH, Inskeep EK. Low peripheral progesterone and late embryonic / early fetal loss in sucked beef and lactating dairy cows. Theriogenology 2008; 71:480-490.

Devonish, EH, Knights, M, Inskeep, EK. Effect of expected peripheral concentration of progesterone on ovulation rate and litter size in Barbados Black berry ewes. Sheep and Goat Research Journal 2009; 24:17-25.

Jordan, KM, Inskeep EK, Knights M. Use of gonadotropin releasing hormone to inprove reproductive responses of ewes introduced to rams during seasonal anestrus. Anim Reprodu Sci 2009;

Goravanahally, MP, Salem M, Yao J, Inskeep EK, Flores JA. Differential gene expression in the bovine corpus luteum during transition from early phase to midphase and its potential role in acquisition of luteolytic sensitivity to prostaglandin F2 alpha. Biol Reprod 2009; 80:980-988.

Odhiambo, JF, Poole DH, Hughes L, DeJarnette Jm, Inskeep EK, Dailey RA. Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1.
http://dx.doi.org/10.1016/j.theriogenology.2009.04.013.

Jyothsma T, Lee KB, Tripurani SW, Smith GW, Yao J. Role of importin alpha 8, a new member of the importinalpha family of nuclear transport proteins, in early embryonic development in cattle. 2009 DOI: 10.1095/biolreprod.109.077396.

Keator CS, Hoagland TA, McCracken Ja, Milvae RA. Intrauterine infusion of BA-610, an endothelin type A receptor antagonist, delays luteolysis in dairy heifers. Dom Anim Endocr 2008; 34:411-418.

Rhinehart JD, Starbuck-Clemmer MJ, Flores JA, Milvae RA, Poole DH, Inskeep EK. Low peripheral progesterone and late embryonic / early fetal loss in sucked beef cows and lactaging dairy cows. Theriogenology 2009; 71:480-490.

Casale P. Luteolytic effects of endothelin-1 in the ewe. MS Thesis, U of Connecticut, 2009.

Camp M. The role of nitric oxide in ovine luteolysis. MS Thesis, University of Connecticut 2009.

Cherry JA, Xiaoying H, Rueda BR, Davis JS, Townson Dh. Microvascular endothelial cells of the bovine corpus luteum: A comparative examination of the estrous cycle and pregnancy. Journal of Reproduction and Development 2008. 54: 183-191.

Ferguson DE. Temporal expression of progesterone and proteins associated with prostaglandin F2-alpha-induced luteolysis in the sheep. MS Thesis, University of New Hampshire, 2009.

Sa Filho oG, Lemley CO, Wilson ME, Hillegass J, Vasconcelos JLM, Butler WR,. Relationships between dry matter intake (DMI), plasma progesterone (P4), and liver catabolic enzymes in lactating dairy cows. Abstract accepted for presentation at Joint Annual Meeting ADSA/ASAS, Montreal, Canada, July 2009.

Castaneda-Gutierrez E, Pelton SH, Giolbert RO, Buter WR. Effect of peripartum dietary energy supplementation of dairy cows on metabolites, liver function and reproductive variables. Anim Reprod Sci 2009; 112: 301-315.

Yang MY, Muruvi W, Fortune JE. Effects of steroids and kit ligand / kit on follicle formation in fetal bovine ovaries in vitro. Biology of Reproduction 2009; Special issue, proc of the 42nd annual meetin of the society for the study of reproduction.

Muruvi, W, Fortune JE. Kit ligand-KIT interaction is required for follicle activation and mediates insulin-stimulated activation of bovine primordial follicles in vitro. Biology of Reproduction 2009; Special issue, proceedings of the 42nd annual meeting of the society for the study of reproduction.

Souza Ah, Gumen A, Silva EP, Cunha AP, Guenther JN, Peto CM, Caraviello DZ, Wiltbank MC. Supplementation with estradiol-17-b before the last gonadotropin-releasing hormone injection of the ovsynch protocol in lactating diary cows. Journal of Dairy Science 2007; 90:4623-34.

Silva E, STerry RA, Kilb D, Wiltbank MC< Fricke PM. Effect of pretreatment with prostaglandin F2Alpha before resynchronization of ovulation on fertility of lactating dairy cows. Journal of Dairy Science 2007; 90: 5509-5517.

Souza Ah, Sartori R, Guenther JN, Caraviello D, Monson R, Wilbank MC. Effect of semen source and dose of FSH on superovulatory response and embryo production in Holstein heifers. Animal Reproduction 2008; 4:70-76.

Brusveen DJ, Cunha AP, Silva CD, Cunha PM, STerry RA, Silva EPB, Guenther JN, Wiltbank MC. Altering the time of the second gonadotropin-releasing hormone injection and artificial insemination during ovsynch affects pregnancies per AI in lactating dairy cows. Journal of Dairy Sciences 2008 91:1044-1052.

Souza AH, Ayres H, Ferreira RM, Wiltbank MC. A new presynchronization system (double-ovsynch( increases fertility at first postpartumtime AI in lactating diary cows. Theriogenology 80:208-15.

Stevenson JS< Tenouse DE, Krisher RL, Lamb GC, Dahlen CR, Pursley JR, Bello NM, Fricke PM, Wiltbank MC, Brusveen DJ, Burkhart M, Yougqist RS, Garverick HA. Detection of anovulation by heatmount detectors and transrectal ultrasonography before treatment with progesterone in a timed insemination protocol. Jounral of Dairy Science 2008; 91:2901-2915.

Silva C*, Wood JR*, Salvador L, Kostetskii I, Williams CJ, and Strauss JF 3rd (2009) Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone, Mol Reprod Dev, 76(1) 11-21, *authors contributed equally to the manuscript

JR, Slattery R, Clopton DT, Smith J, Beavers KA, Pohlmeier W, Bergman JW, Moline KV, Cupp AS (2009) Reproductive Ageing Influences Ovarian Function in Beef Cows, University of Nebraska Beef Report, in press

Wood JR, Yang Z, Mack EM, Cockerill KA, Hofman LA, and Smith JE (2008) Expression profile of MTOC-associated genes and growth factors in the ovary, Nebraska Physiological Society 11th Annual Meeting, Omaha, NE

Slattery R, Clopton D, Wood JR, Cushman R, and Cupp A (2009) Vascular endothelial growth factor (VEGF) mRNA isoforms are altered in bovine granulosa cells (GC) by circulating progestin concentrations (P4) and may indicate follicle status and ooctye competence Midwest Animal Science Annual Meeting (J. Animal Science) 87 (Supp 1) 85

Cushman RA, Wood JR, Echternkamp SE, and Cupp AS (2009) Influence of exogenous progestin on ovarian function in beef cows. Midwest Animal Science Annual Meeting (J. Animal Science) 87 (Supp 1) 239.

Smith JE, Yang Z, Yaw T, and Wood JR (2009) IGF-1 Dependent Changes in Histone H3 Modifications are Associated with Akt Signaling and the Expression of Cell Survival Genes, Society for the Study of Reproduction 42nd Annual Meeting, Pittsburgh, PA

Davis, TL and Pate, JL. 2007. Proliferation of major histocompatibility nonrestricted gamma delta T cells is stimulated by bovine luteal cells. Biol. Reprod. 77:914-922

Hou, X., Arvisais, E.W., Jiang, C., Chen, D, Roy, S.K., Pate, J.L., Hansen, T.R., Rueda, B.R. and Davis, J.S. 2008. Prostaglandin F2a (PGF2a) stimulates the expression and secretion of TGFB1via induction of the early growth response 1 gene (EGR1) in the bovine corpus luteum. Mol. Endocrinol. 22:403-414

Ndiaye, K, Poole, DH and Pate, JL. 2008. Expression and regulation of functional oxytocin receptors in bovine T lymphocytes. Biol. Reprod. 78:786-793

Winkelman, LA, Elsasser, TH, Lucy, MC, Pate, JL and Reynolds, CK. 2008. Suppressor of cytokine signaling-2 mRNA increases after parturition in the liver of dairy cows. J. Dairy Sci. 91:1080-1086

Ndiaye, K., Lussier, J. and Pate, J.L. 2008. Characterization of DER1-Like Domain Family Member 1 (DERL1) and its Expression in Bovine Corpus Luteum. World Congress on Reproductive Biology, abstract, no. 19.

Poole, D.H., Ndiaye, K., Pate, J.L., 2008. Prostaglandin F2a induces proinflammatory T lymphocyte phenotypes in the bovine corpus luteum. Biol Reprod. Abstracts, no. 609

Poole, D.H., Ndiaye, K., Pate, J.L., 2008. Potential role of secreted phosphoprotein 1 in the bovine corpus luteum. World Congress on Reproductive Biology, abstract no. 34

Ndiaye, K., Poole, D.H., Hansen, T.R., Pate, J.L., 2008. Expression profiles of cytokine and progesterone receptor messenger ribonucleic acids (mRNA) in bovine corpus luteum during pregnancy. Biol. Reprod. Abstract no. 400

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