NE1027: Ovarian Influences on Embryonic Survival in Ruminants

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[06/10/2008] [06/19/2009] [06/11/2010] [06/11/2011] [07/05/2012]

Date of Annual Report: 06/10/2008

Report Information

Annual Meeting Dates: 06/09/2008 - 06/09/2008
Period the Report Covers: 10/01/2007 - 09/01/2008

Participants

Patty Cromack, Assistant Director-Ma Agricultural Station;
Keth Inskeep, West Virginia Univeristy;
Jennifer Wood, University of Nebraska;
Milo Wiltbank, University of Wisconsin;
Ron Butler, Cornell University (NY);
Dave Townson, University of New Hampshire;
Paul Tsang, University of New Hampshire;
Ming Yang, Cornell University (NY)

Brief Summary of Minutes

Keith Inskeep was the Chair of the meeting since Dr. Milvae could not attend the meeting for medical reasons. Ron Butler nominated Keith Inskeep to act as chair. The meeting was called to order at 9:15 am. Prior to the presentation of reports, Patty Cromack was introduced. She provided background for the MA Experiment Station. The situation of budgets for traveling was discussed. It is clear that there is less funding available to investigators via Hatch and regional projects.

Station reports began with presentations for Objective 1.

NE (Wood): Oocytes were collected from PCOS women with high insulin to test the hypothesis that abnormal oocytes are produced in these patients and this might underlie high abortion in the first trimester. Most of the genes considered to be abnormally expressed were either 1.8- to 10-fold greater than normal. Many of the genes that appear to be abnormal were associated with spindle organization, centrosome, cell-cycle checkpoint and homologous recombination. To study the identified genes, Dr. Wood is trying to develop culture of follicles in vitro from ovaries of 10d-old mice. Gene expression was affected by insulin concentration in the cultures, particularly in the expression of StAR, aromatase, Nek2 and TACC1. Gene expression was also dependent on stage of the estrous cycle. Protein expression also changes during the cell cycle. In another experiment, cows were fed MGA to produce persistent follicles. The cumulus cells were isolated from persistent follicles (16 mm). These will be used to compare gene expression in persistent vs.normal oocytes.

NY (Fortune): The overall question being pursued in this lab is, how does the follicle form? Insulin seems to be responsible for the activation of the follicle, which is the transition from primordial to primary follicle. Culture of follicles with Kit ligand did not produce the same effect as culture with insulin. Insulin caused hypertrophy in the follicles, which was not observed with Kit ligand. Kit ligand also did not support the transition from primary to secondary follicle. The ability to become activated (follicles) appears to be associated with the property to be arrested at the diplotene stage. In large animals, newborns have primary, primordial and antral follicles at birth. Estradiol inhibits activation of the follicles, possibly by delaying the progression to the diplotene stage.

NY (Butler): In this lab, there has been a long-time interest in the relationship of follicle diameter to fertility success in superovulated animals. Generally, moderately larger follicles produce more estradiol and pregnancy is slightly higher. In high producing cows, high metabolic activity and luteal function is observed, but progesterone may be lower because it is rapidly metabolized. This results in less biological effect of progesterone in high producing cows. This lead to the hypothesis that progesterone would be higher and activity of metabolic enzymes lower in pregnant compared to nonpregnant cows. Following timed AI and OVsynch, pregnancy rate did not differ between cows that ovulated a single follicle vs. cows with double ovulations (producing two CL). Cows had greater plasma progesterone than heifers. Nonpregnant and pregnant cows had similar plasma concentrations of progesterone. There was no relationship of pregnancy status with CYP450 RNA or metabolic activity of CYP450 enzyme, which degrades progesterone. Considerable variation was observed among samples in the activity and mRNA of these enzymes, which may make the association with function very difficult.

WI (Wiltbank): Follicles developed after Ovsynch are smaller and produce less estrogen than those developed during a normal cycle. Therefore, the goal of this experiment was to supplement with estradiol. Administration of 1 mg of estradiol closely corresponded to the in vivo concentrations observed in a normal estrous cycle. Manifestation of estrus was improved, but pregnancy rates did not change. However, this seemed to depend on the size of the follicles; estrogen only improves fertility when the follicles are intermediate in size. With ovsynch, almost 50% of the animals did not ovulate or had higher than normal progesterone.

Data in the porcine CL, which does not regress in response to PGF2alpha, suggests that there is a transcription factor that cannot respond to the injection of PGF2alpha. Jun may not be turned on in the pig and this delays the response. This is different than data published using the cow

WV (Yao/Inskeep): Investigations are underway to determine the role of miRNAs in the compromised fertility associated with persistent follicles. miRNA 181a may be associated with the silencing of nucleoplasmin. Nucleoplasmin 2 is important for decondensation of the sperm head, but it needs to be downregulated afterwards. The expression of 181miRNAs matches the degradation pattern of NPM2.

NH (Tsang): Mixed endothelial cells were isolated from a midcycle bovine corpus luteum. MMP-2 activity was identified in this population of cells. TIMP2 is present in the CL, which could interact with the MMPs, forming dimers. In western blots, the molecular weight of TIMP2 was different than expected, which could be due to dimerization with itself or with MMPs.

NH (Townson): The question being addressed is if functional differences exist in endothelial cell subpopulations during different stages of the cycle. No differences in lectin binding to the CL were observed when cells isolated from CL of the cycle vs pregnancy were compared. In addition, expression of several additional proteins was examined, but did not differ between groups.

WV (Flores): Differential gene expression between early and late CL was analyzed using microarrays. Many genes were differentially expressed. Some of the identified geneshave been confirmed with PCR. The same genes also appear to change following administration of PGF2alpha.

Endothelin receptor A may be important to induce luteolysis. Studies using the inhibitor, BQ-610, have produced confounding results, sometimes inhibiting luteolysis while other times not. Other factors may be involved and need to be elucidated.

Objective 2 and related experiments:

WI (Wiltbank): The problem is how to detect animals that are not cycling? Ultrasonography and progesterone analyses are useful to determine if there is a corpus luteum on the ovary. The incidence of anovulation in lactating dairy cows is approximately 25%. Many cows in Ovsynch do not ovulate in the appropriate time, which greatly decreases the rates of conception. Double Ovsynch seems to be more reliable and appears to synchronize and improve conception rates (approximately 50%), which is 15% greater than regular Ovsynch.

MA (Fissore): PLCzeta has been localized to the equatorial region in bull sperm, and its functional characterization continues. Injection activates oocytes to develop to the blastocyst stage.

WV (Inskeep for Bob Dailey): Following proteomics of bull sperm samples, spermadhesin was found to be present in most bulls. However, the isoform 13 only appeared in bulls of low fertility.

Business:

Dr. Joy Pate became new Chair. Jennifer Woods become Director. Rafael Fissore became new secretary. Next meeting will be in Penn State, and Dr. Pate will host the meeting. The time of the meeting remains underdetermined, but it might remain in the first weeks in June. The group appreciates the efforts of Dr. Milvae as past Chair.

At 5:20 pm the meeting was adjourned.

Minutes Attachment:

Attachment

Accomplishments

Publications

C. O. Lemley, S. T. Butler, W. R. Butler, and M. E. Wilson. Short communication: insulin alters hepatic progesterone catabolic enzymes cytochrome P450 2C and 3A in dairy cows. J.Dairy Sci 91 (2):641-645, 2008. Ferguson DE, Tsang PCW, Schreiber DT, Keator CS, Milvae RA, McCracken JA. 2007. In vivo changes in protein expression of the gelatinases, matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the ovine corpus luteum (CL) after multiple pulses of prostaglandin F2 alpha (PGF2±). The annual meeting of the Society for the Study of Reproduction in San Antonio, Texas; Biol Reprod (special issue): abstract #103, pp.99-100. Cherry JA, Hou Xiaoying, Rueda BR, Davis JS, Townson DH 2008 Microvascular endothelial cells of the bovine corpus luteum: A comparative examination of the estrous cycle and pregnancy. Journal of Reproduction and Development 54(3): 183-191. Yoon SY, Fissore RA. Release of phospholipase C zetaand [Ca2+]i oscillation-inducing activity during mammalian fertilization. Reproduction. 2007. 134:695-704. Ross PJ, Beyhan Z, Iager AE, Yoon SY, Malcuit C, Schellander K, Fissore RA, Cibelli JB. Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta. BMC Dev Biol. 2008. 8:16. Wurst A. K., A. B. Dixon, and E. K. Inskeep. 2007. Lack of effect of transrectal ultrasonography with restraint on lambing rate and prolificacy in ewes, Theriogenology 68:1012-1016. Goravanahally, M. P., A. Sen, E. K. Inskeep, and J.A. Flores. 2007. PKCe and an increase in intracellular calcium concentration are necessary for PGF2± to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells. Reprod. Biol. Endocrinol. 5:37-43. Lingenfelter, B.M., Dailey, R.A., Inskeep, E.K., Vernon, M.W., Poole, D.H., Rhinehart, J.D. and Yao, J. 2007. Microarray analysis of gene expression in granulosal cells from persistent follicles in cattle. Anim. Reprod. Sci. 104:405-413. Doerr, M. D, M. P. Goravanahally, J. D. Rhinehart, E. K. Inskeep and J. A. Flores. 2008. Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2&-induced luteolysis of the sheep corpus luteum. Biol. Reprod. 78:688-696. Wurst, Aimee Kathryn 2007. Effects of Ovulation of Aged Follicles, Pregnancy Diagnosis By Ultrasonography, and Treatments During Lactation On Reproduction in Ewes, Dissertation, West Virginia University, [On-line Abstract]. Available: https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5347 Lingenfelter, Brandon Michael (2008). The Effect of Follicular Aging on Gene Expression in oocytes and Granulosal Cells, Dissertation, West Virginia University, [On-line Abstract]. Available: https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5783 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 F2± (PGF2±) 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

Impact Statements

Elucidation of the molecule(s) responsible for the initiation of Ca2+ oscillations in the cow should facilitate the use of its cRNA or protein as a parthenogenetic agent in Assisted Reproductive Technologies, such as cloning and Intracytoplasmic Sperm Injection (ICSI). In addition, given the conserved nature of this molecule among mammals and poultry, it is possible that reagents could be developed to test for certain cases of infertility/subfertility in sires prior to their progeny test and/or wide use in the field. Moreover, given that PLCz can faithfully reproduce oscillations by the sperm in bovine eggs, and the ability to mount oscillations is acquired progressively during maturation, it is possible that the ability of eggs to mount oscillations can be used to assess the quality of different maturation media, and possible developmental competence.
Basic studies have increased our information on regulation of luteal function during the estrous cycle, specifically in regard to expression of genes in corpora lutea that are insensitive (Day 4) or sensitive (Day 10) to regression by prostaglandin F2±.
The finding that microRNA 181a somehow promotes silencing of the key regulatory gene, Nucleoplasmin 2, in early bovine embryos may be very important to understanding embryonic loss. This gene involved in chromatin remodeling is essential for early development, but must be degraded during embryonic genome activation in order for embryonic development to continue.
The identification of a marker of low fertility in bulls may be useful in increasing conception rates of artificially inseminated cows, especially dairy cattle, in which low fertility is a major limiting factor.
Understanding the unique structural and physiological attributes of endothelial cells of the bovine corpus luteum can lead to novel strategies to control and possibly enhance ovarian function, and thus fertility, in cattle.
The microenvironment of the oocyte and granulosa 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, which are hallmark characteristics of persistent follicles of the beef and dairy cow, results in changes in ovarian gene expression. Defining genes that are important for establishing an oocyte of high quality will identify new targets for reversing the deleterious effects of a persistent follicle and increase the fertility of dairy and beef cattle. These data should also prove to be an important model that can be extrapolated to oocyte quality in humans.
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 since it may suggest methods for increasing the follicular reserve in cattle and hence, enhancing fertility.
Plasma progesterone concentrations in proportion to size of corpus luteum and liver steroid catabolic enzyme activities were compared in lactating dairy cows and non-lactating heifers. High feed intake in cows increased abundance of mRNA for catabolic enzymes in liver and resulting in greater metabolic clearance rates of progesterone. Plasma progesterone levels were greater in cows than heifers, similar for single versus double ovulation, and not different between pregnant and non-pregnant cows until > 15 days after insemination. The results of this study contribute to understanding relationships of luteal tissue mass and functionality in response to the high metabolic rates during peak lactation and necessary for pregnancy.

Date of Annual Report: 06/19/2009

Report Information

Annual Meeting Dates: 06/18/2009 - 06/19/2009
Period the Report Covers: 10/01/2008 - 09/01/2009

Participants

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

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.

Minutes Attachment:

Attachment

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.

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

Impact Statements

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 mPRB 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.

Date of Annual Report: 06/11/2010

Report Information

Annual Meeting Dates: 06/10/2010 - 06/12/2010
Period the Report Covers: 10/01/2009 - 09/01/2010

Participants

Brief Summary of Minutes

Opening Remarks:

David Towson. Welcome and room is reserved until 5:00 pm. Lunch will be here and there will be welcoming words from Jon Wraith, Associate Director, NH Agricultural Station.

Joy: requests for 2008 publications to all members of the group. What that USDA want regarding publications? Papers, abstracts, thesis, Station reports. Adele, indicated more is better. Then, it was decided that Papers, thesis and Station reports were all useful. Do not include abstracts.

Joy: Rafael is presently secretary and will move to Chair; Jennifer will move from Director to Secretary and a new director will need to be selected.

Station Reports:

MA-Station-Rafael Fissore-Umass Amherst.

Bovine PLCzeta can be a very useful tool to test developmental competence, as it initiates sperm-like oscillations in bovine eggs.
Definition of repeat breeders. A question that needs to be addressed is that oocytes in lactating cows the follicular size may be larger than in heifers, but more importantly they may have much longer exposures to hormones, which may seriously compromise the developmental competence of oocytes.
This will be very interesting to test whether the oocytes markers decrease, as aging happens in the follicle.

Nebraska Station- Jennifer Wood.

Interested in examining the effect of metabolism on oocyte development. Using a -/- agouti mutant that overeats as a model. Significant amounts of fat in the carcass.
These animals become infertile by about 6 months. The notion is that there is not a normal signal for ovulation. The thought is that gonadal fat pad may very important in limiting the reproductive capacity of these animals.
Leptin levels are much higher in these animals than in the controls, even as early as 12 wks.
Energy balance may be more critical than the absolute weight. Looking at genes in granulosa cells in these animals, StAR expression was up in the mutant mice while Egr-1 was decreased.
BXD animals crosses obtained from the progeny of C57Bl and DBAs. They have several 30 lines and will subject them to different diets to see how they respond in terms of oocyte quality and fertility.
In a cow project, testing the number of antral follicles in culled cows showed that culled cows at later times seem to have more antral follicles.

Erdogan Memili - Mississippi State U

Effect of nutrition/metabolism, lactation, and immunology on reproduction
Focus = embryo development/programming; epigenetic changes during development
Molecular mechanisms of oocyte quality and embryonic developmental competence
Role of sperm in fertility - differentially expressed proteins/SNPs associated with bull fertility
- DNA damage associated with low fertility
Maternal influence - Bovine germinal vesicle and cumulus cell proteomics study - Reproduction
- DNA methylation/chromatin remodeling of oocyte/cumulus cells
Embryonic genome activation - chromatin remodeling, DNA methylation, and LAG (leukocyte antigen G; immune function) from 1-cell to 8-cell

Ned Place - Cornell

Photoperiod and fertility; primordial follicle activation
Model = Siberian hamsters (long-day breeders); more AMH in SD breeders - suppresses folliculogenesis
Responsiveness of cows to ovarian stimulation - initial studies in hamsters = model for responsiveness; during short days - poor response/low ovulation rate while during long days - good response/high ovulation
Some genes were up regulated in the long day animals vs. short when expression was analyzed with microarrays. They got 50% coverage of the gene expression profiles in oocytes. There were 98 genes that were differentially expressed among these genes, one of which was aromatase. Animals exposed to LDays will eventually lose all the ovarian mass prematurely compared to animals that have been raised with the normal amount of light and photoperiod.

Jon Wraith - Associate Director-NH Station.

Comments. Welcoming remarks. Arrived from Montana State. Happy to be here. Trying to re-invigorate agriculture in NH.

Joy Pate - Penn State

Smad4 KO in oocyte (ZP3 CRE) or granulosa only - phenotype (Francisco Diaz).
- These mice have more follicles and oocytes than controls. The same results were obtained with pharmacological inhibitors of this pathway. It seems that Smad4 mutant oocytes are larger, but have fewer granulosa cells. These results suggest that oocytes control granulosa cells proliferation, which is well known finding.
AMH - prevents follicular maturation due to reduced granulosa proliferation; as follicles grow lose AMHR resulting in AMH insensitivity (AL Johnson)
Interaction between luteal cells and T cell lymphocytes; Contact-mediated and contact-independent effects
- Brefeldin A (BFA) blocks conventional secretion of proteins and increases unconventional secretion of proteins.
- Luteal cell conditioned medium induces luteal cell dependent proliferation of T cells; if you add BFA too, T cell proliferation is enhanced suggesting an unconventional secreted product that causes the proliferation
- Gamma-delta T cell proliferate in absence of contact; if you add CD4 or CD8 (alpha-beta) cells with luteal cells, there is poor proliferation of T cells (this is during midcycle)
- At CL regression, contact between luteal cells and gamma-delta is required for T cell proliferation
miRNA in luteal cells (day 4 vs day 10 vs d18 (pregnant and non-pregnant))
- microarrays for miRNA - shows differences
- knock-down Dicer and Drosha - identifying functional end-points

Paul Tsang - U New Hampshire

Bovine Corpus Luteum Capillary Endothelial Cell Cultures

4 subtypes of endothelial cells- what are proportions of these types during corpus luteum development . What is functional importance
Migration of endothelial cells dependent on MMP production; regulation of MMP-2 by progesterone - high concentration of progesterone inhibits MMP-2 activity; endogenous inhibitor (TIMP1) also decreased upon progesterone treatment. However, TIMP2 levels increased upon progesterone treatment
MMP2 and MMP9 detected in Penn State CL lysates
Developed protocol to isolate and culture endothelial cells from CL
Problems with cryopreservation; primary cultures decided to investigate culture and cryopreservation properties
How many passages? - 2 passages, cells look good (appropriate morphology)
Cryopreservation - DMSO concentration (10% instead of 2.5%) cells recovered much better.
Future studies:
- endothelial cell verification (factor VIII, acetylated LDL, progesterone in conditioned medium) - interestingly, endothelial cell conditioned medium contains detectable progesterone. Probably contaminating luteal cells - how to get rid of these contaminating cells?
- Comparison studies using bovine adrenal capillary endothelial cells; clonal cultures

Milo Wiltbank - U Wisconsin

Role of progesterone in fertility
Pre-AI progesterone
- In first lactation animals - Double Ovsynch increases fertility dramatically (but not in older animals); produces high progesterone due to production of a second CL - High P4 =higher pregnancy per AI (50% versus 37%); also less pregnancy loss (P = 0.054); less double ovulation (therefore increased twinning) - double ovulations still relatively high
- Double Ovsynch with extra PGF2a more complete luteolysis appx 5% increase in PP/AI-first service (not statistically significant) but not necessarily important for improving fertility
Post-AI progesterone
- Change - 5 days after AI add hCG or supplement with CIDR
- CIDR + hCG in lactating cows raises P4 to match heifers
- However, there is no effect of this treatment on conception rate suggesting that P4 prior to AI is more important than post-AI (smaller group of animals)
- In ~3000 lactating cows, hCG did have an effect (37% v 42%) (second study with larger group of numbers)
Endometrial thickness near timed AI (ultrasound)
- When P4 is high, endometrial thickness is low (~6.9 mm); as P4 drops precipitously with PGF2a injection, endometrial thickness increases (9.2 9.4 mm)
- P4 concentration seems to determine endometrial thickness (inversely correlated)
- Compared groups (endometrial thickness < 8mm versus >8 mm); endometrial thickness was an important indicator for fertility (e.g improved pregnancy /AI) predicts about ½ of the fertility
- Mechanism is not known??

Ron Butler - Cornell

Embryo survival in lactating versus non-lactating dairy cows (oocyte quality); embryo transfer to lactating cows vs. virgin Holstein heifers
Effects of conjugated linoleic acid (CLA) on reproduction
- CLA-10,12 isoform improves dairy cow fertility (meta-analysis)
- Possible mechanisms = earlier first ovulation, increased plasma IGF-1, improved estradiol production from the ovarian follicle, no effect on uterine PGF2a production
Trans (18:1) fatty acids which may increase CLA improved reproduction due to increased fertilization rate and embryo quality
Developed IVM/IVF protocol
- Parthenogenesis studies first
- CLA during IVM only - no statistical differences in MII maturation, cleavage rate, or blastocyst development
- CLA during whole culture (IVM ) - start to see an effect however they seem to be inhibitory especially in blastocyst rate
- Next step - repeat above doing IVM/IVF. Also, include in samples from lactating and non-lactating animals and embryo transfers

Brad Hillman

Appx. 1 year out for re-write; start planning organization of writing; want to begin review process at beginning of 2012.
Size of group - small; not necessarily a problem unless loose critical mass
NIFA - 25% of Hatch must go to multi-state - make sure they are true collaborative endeavors-grant writing/manuscripts
Submit meeting minutes within 60 days (SAE 422??) - Rubie Mize = assistant for Brad
Emphasize collaborations and importance of these collaborations for re-write and during development of new application

Adele Turzillo

Overview and Update of NIFA programs - see handouts

New Director: Milo Wiltbank - Joy nominates, several individuals second the nomination - passed unanimously

Plan for Re-write

Groups:

Oocyte/Embryo
CL
Metabolism/Lactation/Reproductive Efficiency

Groups communicate over the course of this year and submit plan at next year's meeting

Block out 2 days for meeting in order to spend time on re-write

Location for 2011: Washington DC - meet at USDA building - Adele would host

Minutes Attachment:

Attachment

Accomplishments

Publications

1. Piccinato CA, Sartori R, Sangsritavong S, Souza AH, Grummer RR, Luchini D, Wiltbank MC. 2010. In vitro and in vivo analysis of fatty acid effects on metabolisms of 17 beta-estradiol and progesterone in dairy cows. Journal of Dairy Science 93:1934-1943. 2. Araujo RR, Ginther OJ, Ferreira JC, Palhao MM, Beg MA, Wiltbank MC, 2009. Role of follicular estradiol-17² in determining the time of luteolysis in heifers. Biology of Reproduction PMID 19264702. Mar 4. 3. Souza AH, Cunha AP, Silva EPB, Gumen A, Ayres H, Guenther JN, Wiltbank MC, 2009. Comparison of gonadorelin products in lactating dairy cows: efficacy based on induction of ovulation of an accessory follicle and circulating LH profiles. Theriogenology 72:271-279. 4. Brusveen DJ, Souza AH, Wiltbank MC, 2009. Effects of additional prostaglandin F2± and estradiol-17² during Ovsynch in lactating dairy cows. Journal of Dairy Science 92: 1412-1422. 5. Souza AH, Viechnieski S, Lima FA, Silva FF, Araujo R, Bo GA, Wiltbank MC, Baruselli PS, 2009. Effects of equine chorionic gonadotropin and type of ovulatory stimulus in a time-AI protocol on reproductive responses in dairy cows. Theriogenology 72:10-21. 6. Bamber RL, Shook GE, Wiltbank MC, Santos JE, Fricke PM, 2009. Genetic parameters for anovulation and pregnancy loss in dairy cattle. Journal of Dairy Science 92:5739-5753. 7. Watters RD, Wiltbank MC, Guenther JN, Brickner AE, Rastani RR, Fricke PM, Grummer RR, 2009. Effect of dry period length on reproduction during the subsequent lactation. Journal of Dairy Science 92:3081-3090. 8. Checua CM, Beg MA, Gastal MO, Wiltbank MC, Parrish JJ, Ginther OJ, 2009. Effect of suppression of FSH with a GnRH antagonist (acyline) before and during follicle deviation in the mare. Reproduction in Domestic Animal 44:504-511. 9. Watters, RD, Wiltbank, MC, Guenther JN, Brickner AE, Rastani RR, Fricke PM, Grummer RR, 2009. Effects of dry period length on reproduction during the subsequent lactation. Journal of Dairy Science 92: 3081-3090. 10. Kneebone J, Tsang PCW, Townson DH. 2010 IDEXX SNAP® tests detect antibiotic residues in powered milk products. Journal of Dairy Science, Accepted 11. Townson DH, Putnam AN, Sullivan BT, Guo L, Irving-Rodgers HF, 2010. Expression and distribution of cytokeratin 8/19 intermediate filaments in bovine antral follicles and corpus luteum: An intrinsic mechanism of resistance to apoptosis? Histology and Histopathology 25:889-900 12. Duncan AR, 2009. Disruption of cytokeratin 18-containing intermediate filaments in bovine luteal cells: Effects on Fas expression, progesterone secretion, and FasL-induced apoptosis. MS Thesis, University of New Hampshire. 13. Forcina JJ, 2009. Genetic over-expression of cytokeratin 18 in bovine luteal cells: Effects on intermediate filament formation and Fas ligand-induced apoptosis. MS Thesis, University of New Hampshire. 14. Cushman RA, Wood JR, Slattery R, Clopton DT, Smith J, Beavers KA, Pohlmeier W, Bergman JW, Moline KV, Cupp AS (2010) Reproductive Ageing Influences Ovarian Function in Beef Cows, University of Nebraska Beef Report, p16 15. Cooney MA, Malcuit C, Banyoon C, Holland MK, Fissore RA, DCurz NT (2010) Species-Specific Differences In Activity and Nuclear Localization of Murine and Bovine Phospholipase C, Zeta 1. Biol Reprod 83(1):92-101. 16. Tripurani, S.K., Xiao, C., Salem, M. and Yao, J. 2010. Cloning and analysis of fetal ovary microRNAs in cattle. Animal Reproduction Science. 120:16-22. 17. Odhiambo JF, Poole DH, Hughes L, DeJarnette JM, Inskeep EK, Dailey RA. 2009. Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1. Theriogenology. 72: 566-571. 18. Inskeep K. 2009. Induction of estrus as a reproductive management tool in anestrous ewes. Proceedings Great Lakes Dairy Sheep Symposium. Ed by DL Thomas. Albany NY Nov 12-14, 2009. pp 94-110. 19. Perea-Ganchou F, Inskeep EK. 2010. Infertilidad asociada con la duracion de la fase luteal en vacas postparto. In Manejo Reproductivo de la Vaca Posparto. Ed by German E. Portillo Martinez. Cuadernos Cientificos Girarz 7:205-221. Maracaibo, Venezuala. 20. Inskeep EK. 2010. Time-dependent embryotoxicity of the endogenous luteolysin prostaglandin F2± in ruminants. Chapter 10, pp 240-255 in Endocrine Toxicology. C. Eldridge and J.T. Stevens, Eds. Informa Healthcare, London 21. Inskeep EK, Dailey RA. 2010. Maximizing Embryonic and Early Fetal Survival in Dairy Cattle. In WCDS Advances inDairy Technology 22:51-69. Lorraine Doepel, Editor. 22. Relling, AE, Pate, JL, Reynolds, CK and Loerch, SC. 2010. Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide mRNA concentrations in growing wethers. J. Anim. Sci. 88: 737-748 23. Ndiaye, K, Lussier, JG, and Pate, JL. 2010. Molecular characterization and expression of DERL1 in bovine ovarian follicles and corpora lutea. Reprod. Biol. Endocrinol. 8:94 (http://www.rbej.com/content/8/1/94) 24. Pate, JL, Toyokawa, K, Walusimbi, S, and Brzezicka, E. 2010. The Interface of the Immune and Reproductive Systems in the Ovary: Lessons Learned from the Corpus Luteum of Domestic Animal Models. Amer. J. Reprod. Immunol. 64:275-286. 25. Smitz, J., R.L. Stouffer, E.E. Telfer, T.K. Woodruff, M.B. Zelinski, M.M. Dolmans, J. Donnez, J.E. Fortune, O. Hovatta, K. Jewgenow, H.M. Picton, C. Plancha, L.D. Shea, 2010. Current achievements and future research directions in ovarian tissue culture, in-vitro follicle development and transplantation: Implications for fertility preservation. Human Reproduction Update 16:395-414. 26. Fortune, J.E., M.Y. Yang, and W. Muruvi, 2010. The earliest stages of follicular development: Follicle formation and activation. Reproduction in Domestic Ruminants VII, M.C. Lucy, J.L. Pate, M.F, Smith, and T.E. Spencer (eds). Nottingham Univ. Press, Nottingham, UK, p. 203-216.

Impact Statements

Date of Annual Report: 06/11/2011

Report Information

Annual Meeting Dates: 06/09/2011 - 06/10/2011
Period the Report Covers: 10/01/2010 - 09/01/2011

Participants

Brief Summary of Minutes

June 9, 2011 - Meeting called to order by Rafael Fissore at 9:00A. Members present include Rafael Fissore, Joy Pate, Ron Butler, Joanne Fortune, Milo Wiltbank, Keith Inskeep, Jianbo Yao, Dave Townson, Paul Tsang, Jorge Flores, and Jennifer Wood. In addition, Dan Poole (Post-doc, Alan Johnson) and Adele Turzillo and Mark Mirando (NIFA administrators) were in attendance

Opening remarks:

Joy Pate asked about the proper mechanism for reporting minutes - members discussed emailing reports and a list of publications to the secretary (Jennifer Wood - 2011); Adele Turzillo indicated that Brad Hillman should receive the minutes and how we distribute them is up to the group

The group decided to order in lunch and work through in order to get station reports done today; Joanne Fortune moved that Friday's meeting would be held in the hotel instead of at the Waterfront due to the convenience, it was seconded and unanimously agreed upon.

Station Reports

Objective A

1. Follicle/Oocyte

a. Persistent versus growing follicle - no updates on this part of the project by the WVU (K. Inskeep) - this portion of the project has been completed

b. Jianbo Yao - Oocyte data FIGLA cloning and expression in oocyte and early embryos - collaborating with George Smith (Michigan State) to study oocyte-specific and maternal effect genes in bovine model; FIGLA drives zona pellucida expression and genes essential for folliculogenesis and early development but expression during embryogenesis has not been defined. Cloning (previously just predicted but 3'UTR and 5'UTR was not correct so they cloned using RACE) and expression in tissues demonstrated only expressed in gonads (male and female). QPCR shows highest expression in GV, decreases during maturation and continues to decrease during pre-implantation development (similar to known maternal effect genes) - Western blots agree with mRNA data (although GV v MII is not particularly different at the protein level). Then investigated how FIGLA mRNA is downregulation is carried out (miRNA). miR-430 degrades maternal effect genes - miR-212 site in FIGLA 3'UTR. miR-212 expressed during cleavage stage and peaks at 4C-8C stage. Demonstrated specificity of miR-212 for FIGLA and exogenous miR-212 mimics regulation of FIGLA mRNA during embryonic development.

c. Jen Wood - obesity factors and oocyte mRNAs - showed changes in oocyte mRNA abundance in obese compared to normal-weight mice. Based on the hormone profiles of the mice, it is the current hypothesis is that adipocytokines including leptin, TNFa, and/or insulin alter transcriptional or post-transcriptional regulation of mRNAs during oocyte growth and maturation, respectively. Bob Dailey suggested that thyroid function may be altered in these mice; it is also important to track the ovulatory ability in LY v HFD v ND; future experiments will dissect the effect of insulin resistance versus other hormones on ovulation and oocyte quality. In a second project, the effect of granulosa cell steroidogeneic efficiency on cumulus-oocyte gene expression was shown using a beef cow model. Specifically, when androstenedione levels are elevated with respect to estrogen, there is global increases in COC mRNA abundance.

d. Rafael Fissore - oocyte quality and conception failure in repeat breeders and uterine environment in repeat breeders. Focus = Ca2+ homeostatic machinery in mammalian oocytes and in particular role of PLC zeta. SERCA and PMCA regulation is not very well understood although importance is documented. What causes differences in sensitivity to PLC zeta between different species? IVF causes calcium oscillations but ICSI will cause one calcium pulse but not sustained. If ICSI is combined with bPLC zeta then calcium oscillations initiated so why cannot bovine sperm cause oscillations. In mouse oocytes at PN - oscillations stop due to sequestration of PLC zeta. IP3 receptors are insensitive - low calcium in stores in bovine/human oocytes. What happens to oocyte Ca2+ homeostasis during maturation? Calcium stores increase from GV to MII in mouse oocytes while influx decreases from GV to MII. What regulates increase in Ca2+ stores? During growth there is a leak of Ca2+ from store. Stim1 is in ER and interacts with Orai1 s in the plasma membrane which opens that channel and allows Ca2+ influx. When Ca2+ is high Stim1 is in ER and when Ca2+ is low it translocates to plasma membrane and interacts with Orai1. High Ca+ influx or store prevents meiotic maturation. Using FRET to measure Ca2+ in ER and compare to Ca+ levels in cytoplasm - noted that during first 4 oscillations Ca2+ comes essentially from the stores.

e. Ron- preovulatory follicle, CL size - no update this year - study completed and data reported in previous years. Report from Ned Place (absent from the meeting) handed out by Ron.

f. Joanne Fortune - activation of primordial follicles - use fetal bovine ovaries - isolate cortex and culture to track activation of primordial follicles and over time there is good activation. Has moved to defining how follicles form and when the attain ability to undergo activation. Around day 90 first primordial follicles form, 140 days first primary follicles. What causes gap? Is it intrinsic or environmental inhibitor? Looked at effect of steroid hormones on bovine follicle formation -both E2 and P4 (1 uM) inhibit follicle formation; no effect of DHT (or testosterone) - have not looked at ER, PR expression in fetal ovaries. In vivo E2 and P4 levels may decline during progression of fetal development that allows for follicle development. Looked at E2 and P4 in cortical culture medium - E2 decreases while P4 increases - so what is happening with P4 - is it the receptor. Question- is the effect intrinsic to oocytes or dependent on ovarian environment? Effects of FGF-18 on secretion of estradiol by ovarian pieces - increasing dose of FGF-18 but not FGF-10 inhibits estradiol and P4 (not as fast as E2) secretion.

g. Milo Wiltbank - regulation of follicle selection - high ovulation rate heifers (4 per cycle) working with Brian Kirkpatrick to determine genetic determinant of phenotype . Physiologically FSH levels after aspiration peaks one day after aspiration and declines while in multiple ovulators FSH remains elevated through 3.5 days after aspiration. No E2 yet but have collected follicular fluid. Similar phenotype to sheep (multi-ovulators) but region of genome in heifers does not contain those same genes (i.e. BMP15, Gdf9/Smads). Also not necessarily intrafollicular given the FSH characteristic. - USDA projection of daughter pregnancy rate versus milk yield (inversely proportional). However last 5 years, pregnancy rate is dramatically increasing due to improved breeding techniques (i.e. timed-AI). Used pre-Synch, ov-Synch - what if animals are bred following pre-Synch; also compared estrus detection and AI. Estrus detection shows much reduced pregnancy detection - probably due to development of persistent follicle. Timed AI removes milk yield issue and enables ovulation of an oocyte from a smaller follicle so improve conception rates. Low P4 double Ovsynch - reduce luteal function so lower P4 during final oocyte growth stage - low P4 negatively impacts embryo quality.

h. Dan Poole (Post-doc for Alan Johnson) - AMHRII regulation by oocyte factors - recruitment of dominant follicle. Hypothesis is that unselected follicles block FSH responsiveness while in selected follicles there is a block to AMH signaling. Characterized AMHR2 in different follicle sizes (decreases with follicle size). BMP2, BMP6, and BMP15 all increase AMH while BMP15 (and high BMP6) increase AMHR2. Exogenous AMH inhibits Cyp19 expression in mural granulose. So propose that oocyte factors in a small follicle can affect granulosa cell AMH and therefore E2. As oocyte grows factors cannot impact granulosa due to distance.

2. Corpus Luteum

a. NH - Paul Tsang and Dave Townson - Endothelial cells - problem with contamination during culture and purity of cells; working with Joy to overcome problems. - remaining items in the written report

b. WV - Jorge/Keith - calcium homeostasis in bCL - PGF2a increases cytoplasmic calcium in ovine LLC (receptor on large cells) - due to mobilization of internal stores; however, later studies demonstrated that both mobilization and influx are important; using single cell prep to compare calcium responsiveness between SLC, LLC and endothelial cells; regulation of calcium levels mediates PGF2a dependent decreases in P4 levels. In current year determining developmental differences in expression of genes that regulate calcium homeostasis in bCL. Selected candidate genes (e.g. ER channels/pumps; plasma membrane channels/pumps) and differentially expressed mRNAs validated at protein level. Model = during developing CL, the RyR is reduced keeping Ca2+ sequestered in ER but in mature CL RyR is increased plus SERCA activity is low so more calcium in cytoplasm and this is mechanism for reduced P4 production by mature CL. - 2 shots of PGF2; is it timing or is it a true effect of multiple doses - important to look at calcium homeostasis factors. What is role of oxytocin produced by LLC (or delivered via bloodstream) on SLC; treated ewes with atosiban (mimics oxytocin) and P4 levels monitored.

c. PA - Joy - PRAME in the bCL (novel and preliminary) - low in most normal cells except testis; also expressed in cancer cells. Also expressed in mid to late CL (not day 4); propose that PRAME inhibits RARB signaling. T lymphocytes and luteal cells - what types of physical interactions occur and what chemical signals mediate this? - T cell receptors on luteal cells (MHC + Tcell receptor. Do interactions with luteal cells impact T cell phenotype (function)? When T-cells are exposed to luteal cells causes anti-inflammatory program. Luteal cell secretions and T cell proliferation -incubated T cells with mid-cycle luteal cell conditioned medium and demonstrated T cell proliferation - characterizing protein secreted in medium using size exclusion and gel electrophoresis - next year will run 2D gels for sequencing. miRNA in the regulation of luteal development - Dicer and Drosha knockdown in CL affects apoptosis. Microarray - day 4 versus day 10. Picked 10 miRNAs that are important and validated (confirmed 3 of 10)-focus on miRA 126 and miRNA 34a. Used Ingenuity Pathway Analysis and identified candidate mRNA targets including PPARA, PTEN, NRG1, etc&

Objective B

1. Embryo

a. Luteal sensitivity - project completed

b. Ron - Embryo transfer - embryo survival in lactating versus non-lactating cows - still working on identifying embryos. CLA supplementation and reproduction in lactating cows; CLA supplement shortens open period by appx. 40 days due to increased IGF-1, earlier ovulation postpartum, and increased follicular estrogen production. Took preliminary data to commercial dairy and analyzed pregnancy survival. Also carried out an intensive study in a smaller group of animals and also looked at gene expression in tissues and collected follicular fluid (E2, IGF1). CLA supplementation in intensive study - milk fat production was lower and energy balance improved with no differences in milk production - higher plasma E2, and plasma IGF1 but IGF1 mRNA in tissues was not changed. In field study CLA cows - milk fat was lower as well as milk yield - body condition was not different between groups and plasma IGF1 was not different until d60 (although very modest) - pregnancy rates were not different (pregnancy survival).

USDA Update

Adele - Documents in NIFA folder - Acting director (Dr. Roger Beechy left for family reasons); RFAs not released due to budget issues (challenge areas should be released soon??); NIFA Fellowship Grants - individual grants (not University) for graduate students (PhD) or post-docs; research must be related to challenge areas - take time to read through Climate change RFA and work on collaborations; New farm bill in 2012; Stakeholder workshop - NIFA and ARS - 18 breakout sessions (research area, commodity, emerging issues) - included producers, scientists, etc& - workshop summary will be posted on ARS and NIFA website. Discussion about the stakeholder meeting and PD meetings.

Dr. Debby Sheely, Assistant Director, NIFA Institute of Food Production and Sustainability - welcome and able to answer questions. Discrepancy between need for increased product for increased population and funding availability - audit of programs during farm bill rewrite; Training of young scientists at risk due to shifting of funds to large programs not affiliated with agriculture - priorities straight to President and they are important problems; ARS eligibility and competitive grant opportunities - unfair advantage; acknowledge agency when speaking - CRIS reports- all will help with auditing etc.

Adjourn for Dinner at 5:30P

Reconvened meeting at 8:30A June 10 at the River Inn

Discussed Site for Next Year

Cornell folks (Joanne Fortune and Ron Butler) will work on site for next year - perhaps Poconos or around Ithaca. Meet tentatively June 7, 2012 with travel on Wed and Fri - Perhaps following week if June 7 does not work out.

Rewrite

Debating ovarian function only or continue to include embryo

Title: Ovarian Influences on Reproductive Success in Ruminants

One Aim = Follicle/Oocyte - Rafael and Joanne will organize 2-3 topics - Oocyte (Rafael), Follicle Recruitment and Selection (Joanne) - discussions on design of experiment - persistent follicle model

One Aim = CL - Identify cellular signaling and inter-cellular interactions during CL development, function, and regression - Joy, Milo, Paul, Dave, Keith/Jorge, Bob

Leaders for each aim will put together outlines by end of July and groups will meet at SSR (Milo Wiltbank organizes this)

Dates - Adele indicated that suggested reviewers and submission should occur January 2012; Brad Hillman should be contacted for specific deadlines and organization of the proposal.

Meeting officially adjourned at 10AM

Minutes Attachment:

Attachment

Accomplishments

Publications

1. Mack EM, Smith JE, and Wood JR. IGF-1 has an Additive Effect on cAMP-Dependent Regulation of Granulosa Cell Gene Expression through Activation of Akt- and Erk1/2 Signaling, (submitted to Molecular Cellular Endocrinology) 2. Slattery RG, WE Pohlmeier, RM McFee, JG Kerl, VM Brauer, JR Wood, RA Cushman, AS Cupp. Females with low granulosa cell efficiency have excess follicular fluid androstenedione, reduced IGF-1 mRNA, greater expression of mRNA for VEGFA164b and reduced follicle health and fertility. (submitted to Reproduction) 3. Artec RA, Pohlmeier WE, McFee RM, Brauer V, Wood JR, Cushman R, and Cupp AS. Anti-angiogenic VEGFA164B isoform mRNA is more abundant in E2-inactive, atretic follicles while expression of angiogenic VEGFA isoforms is greater in granulosa cells from developing bovine follicles prior to the LH surge. (Submitted to Reproduction). 4. *Zhang B, *Tsang PCW, Pate JL, Moses MA 2011. A role for cysteine rich 61 in the angiogenic switch during the estrous cycle in cows: Regulation by prostaglandin F 2alpha. Biology of Reproduction. In Press. *These authors contributed equally to this work. 5. Sullivan BT, Cherry JA, Sakamoto H, Henkes LE, Townson DH, Rueda BR 2010 Cytokeratin-18 (CK18) expression inhibits cytokine-induced death of cervical cancer cells. International Journal of Gynecologic Cancer 20:1474-1481. 6. Galvao, K. N., M. Frajblat, W. R. Butler, S. B. Brittin, C. L. Guard, and R. O. Gilbert. Effect of early postpartum ovulation on fertility in dairy cows. Reprod. Dom. Anim., doi: 10.1111/j.1439-0531.2009.01517. 2009. 7. Yang JH, Menshenina J, Cunha GR, and Place N, Baskin LS. (2010). Morphology of mouse external genitalia: implications for a role of estrogen in sexual dimorphism of the mouse genital tubercle. J Urol. 184:1604-1609. 8. Piekarski DJ, Place NJ, and Zucker I. (2010). Facilitation of male sexual behavior in Syrian hamsters by the combined action of dihydrotestosterone and testosterone. PLoS ONE. 5(9):e12749. 9. Place NJ, Coscia EM, Dahl NJ, Drea CM, Holekamp KE, Roser JF Sisk CL, Weldele ML, and Glickman SE. (2011). The anti-androgen combination, flutamide plus finasteride paradoxically suppressed LH and androgen concentrations in pregnant spotted hyenas, but not in males. Gen Comp Endocrinol. 170:455-459. 10. Place NJ, Hansen BS, Cheraskin J-L, Cudney SE, Flanders JA, Newmark AD, Barry B, and Scarlett JM. (2011). Measurement of serum anti-Müllerian hormone concentrations in female dogs and cats before and after ovarihysterectomy. J Vet Diag Invest 23:524-527. 11. Schwacke LH, Zolman ES, Balmer BC, DeGuise S, George RC, Hoguet J, Hohn AA, Kucklick JR, Lamb S, Levin M, Litz JA, McFee WE, Place NJ, Townsend FI, Welss RS, and Rowles TK. (2011). Anemia, hypothyroidism, and immune suppression associated with polychlorinated biphenyl exposure in bottlenose dolphins (Tursiops truncatus) Proc Roy Soc B (in press). 12. Almeida J, Ball BA, Conley AJ, Place NJ, Liu IKM, Scholtz EL, Mathewson L, Stanley SD, and Moeller BC. (2011). Biological and clinical significance of anti-Müllerian hormone determination in blood serum of the mare. Theriogenology (in press). 13. Rutkowska J, Place NJ, Vincent SE and Adkins-Regan E. (2011). Adrenocortical response to mating, social interaction and restraint in the Japanese quail. Physiol Behav (in press). 14. Smitz, J., R.L. Stouffer, E.E. Telfer, T.K. Woodruff, M.B. Zelinski, M.M. Dolmans, J. Donnez, J.E. Fortune, O. Hovatta, K. Jewgenow, H.M. Picton, C. Plancha, L.D. Shea, 2010. Current achievements and future research directions in ovarian tissue culture, in-vitro follicle development and transplantation : Implications for fertility preservation. Human Reproduction Update 16:395-414. 15. Fortune, J.E., M.Y. Yang, and W. Muruvi, 2010. The earliest stages of follicular development: Follicle formation and activation. Reproduction in Domestic Ruminants VII, M.C. Lucy, J.L. Pate, M.F, Smith, and T.E. Spencer (eds). Nottingham Univ. Press, Nottingham, UK, p. 203-216. 16. Fortune, J.E., M.Y. Yang, and W. Muruvi, 2011. In vitro and in vivo regulation of follicular formation and activation in cattle. Reproduction, Fertility and Development 23:15-22. 17. Scaramuzzi, R.J., D.T. Baird, B.K. Campbell, M.-A. Driancourt, J. Dupont, J.E. Fortune, R.B. Gilchrist, G.B. Martin, K.P. McNatty, A.S. McNeilly, P. Monget, D. Monniaux, C. Vinoles Gil, R. Webb, 2011. Regulation of folliculogenesis and the determination of ovulation rate in ruminant. Reproduction, Fertility, and Development 23: 444-467. 18. Relling, AE, Pate, JL, Reynolds, CK and Loerch, SC. 2010. Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide mRNA concentrations in growing wethers. J. Anim. Sci. 88: 737-748 19. Ndiaye, K, Lussier, JG, and Pate, JL. 2010. Molecular characterization and expression of DERL1 in bovine ovarian follicles and corpora lutea. Reprod. Biol. Endocrinol. 8:94 (http://www.rbej.com/content/8/1/94) 20. Pate, JL, Toyokawa, K, Walusimbi, S, and Brzezicka, E. 2010. The Interface of the Immune and Reproductive Systems in the Ovary: Lessons Learned from the Corpus Luteum of Domestic Animal Models. Amer. J. Reprod. Immunol. 64:275-286. 21. Pickworth, CL, Poole, DH, Velleman, SG, Pate, JL, Loerch, SC, Fluharty, F L 2010. Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity. J Anim Sci. 2010 Oct 15. [Epub ahead of print] 22. Lucy, MC, Pate, JL Smith, MF and Spencer, TE, eds. 2010. Reproduction in Domestic Ruminants VII. Society of Reproduction and Fertility Volume 67. Nottingham University Press. 23. Tripurani, S.K., Lee, K.B., Wee, G., Smith, G.W., and Yao, J. 2011. MicroRNA-196a regulates bovine newborn ovary homeobox gene (NOBOX) expression during early embryogenesis. BMC Developmental Biology 11:25. doi:10.1186/1471-213X-11-25. 24. Tripurani, S.K., Lee, K.B., Wang, L.,Wee, G., Smith, G.W., Lee, Y.S., Latham, K.E. and Yao, J. 2011. A Novel Functional Role for the Oocyte-Specific Transcription Factor Newborn Ovary Homeobox (NOBOX) during Early Embryonic Development in Cattle. Endocrinology. 152:1013-1023. 25. Inskeep, E.K. 2011. Ruminant reproduction: recent findings and future challenges, a summary. In: Reproduction in Domestic Ruminants VII. M.F. Smith, M. Lucy and T. Spencer, Eds. 12 pp.

Impact Statements

Date of Annual Report: 07/05/2012

Report Information

Annual Meeting Dates: 06/06/2012 - 06/08/2012
Period the Report Covers: 10/01/2011 - 09/01/2012

Participants

Butler, Ron (wrb2@cornell.edu) - Cornell University;
Flores, Jorge (jflores@wvu.edu) - West Virginia University;
Fortune, Joanne (jf11@cornell.edu) - Cornell University;
Inskeep, Keith (einskeep@wvu.edu) - West Virginia University;
Keating, Aileen (akeating@iastate.edu) - Iowa State;
Milvae, Robert (Robert.milvae@uconn.edu) - University of Connecticut;
Ott, Troy (tlo12@psu.edu) - Penn State University;
Pate, Joy (jlp36@psu.edu) - Penn State University;
Thompson, Gary (gat10@psu.edu) - Penn State University;
Townson, Dave (dave.townson@unh.edu) - University of New Hampshire;
Tsang, Paul (paul.tsang@unh.edu) - University of New Hampshire;
Turzillo, Adele (aturzillo@nifa.usda.gov) - USDA NIFA;
Wood, Jennifer (jwood5@unl.edu) - University of Nebraska;
Yao, Jianbo (jianbo.yao@mail.wvu.edu) - West Virginia University;

Julio Giordano, Guest - new faculty member at Cornell

Brief Summary of Minutes

Minutes Attachment:

Attachment

Accomplishments

*Demonstrated a role for estrogen during primordial follicle establishment and how endocrine disrupting chemicals can have a negative impact on this process. *Identified a novel gene in the bovine fetal ovary that is a zinc finger protein. *Demonstrated that VEGFA isoform expression in granulosa cells, the ratio of E2/A4 in the follicular fluid, and oocyte mRNA abundance are altered in cows with reduced fertility. *Characterized the cellular expression of FGF2, VEGFA, VEGFR1, VEGFR2, and CYR61 in ovarian granulosa and theca during the ovulatory response *Determined a role for Fas ligand-induced apoptosis and identified a relationship between CK8/18 filaments, cFLIP, and ERK1/2 in granulosa cells *Identified a SNP in the growth hormone receptor associated with dairy cow fertility *Demonstrated that AMPK is involved in the response to activation of the prostaglandin F receptor in corpora lutea *Demonstrated a novel mechanism for protein secretion by luteal cells which are responsible for stimulation of T cells. One of the differentially secreted proteins in PGF-exposed luteal cells is alpha 2-macroglobulin which may mediate luteal cell-T cell interactions. *Showed that steroidogenic luteal cells express ALCAM, which may interact with receptors on T cells to activate T cell signaling pathways. *Demonstrated that luteal cells from regressing CL stimulate multiple types of T cells, whereas luteal cells from fully functional CL preferentially stimulate anti-inflammatory gamma-delta positive T cells. *Demonstrated that miRNA 126 is upregulated in Day 10 compared to Day 4 CL and is localized in luteal endothelial cells. VCAM-1 appears to be a target of miR126 in luteal endothelial cells, suggesting that this microRNA may be involved in regulating angiogenesis during luteal development. *Showed that the combined use of CIDR and PG600 does not increase pregnancy rates in ewes.

Publications

Abstracts Tripurani, S.K., Wang, L., Lee, K.B., Gabbine, W., Smith, G.W. and Yao, J. The SUMO-conjugating enzyme Ubc9 is developmentally regulated and interacts with activating transcription factor 7 interacting protein 2 (ATF7IP2) in cattle. 44th Annual Meeting Society for the Study of Reproduction, Portland, Oregon. July 31 - August 4, 2011. Tripurani, S.K., Lee, K.B., Smith, G.W. and Yao, J. Cloning and expression of bovine factor in the germline alpha (FIGLA) in oocytes and early embryos: a potential target of microRNA-212. 37th Annual Conference of the International Embryo Transfer Society, Orlando, Florida. January 8-11, 2011. Walusimbi, SS and Pate, JL. 2011. Luteal cells establish contact with T lymphocytes via the T cell receptor and induce deletion of gamma-delta T cells with a proinflammatory phenotype. SSR Abstract #76 Brzezicka, E. and Pate, J.L. 2011. Luteal cells induce gamma delta T cell activation through secretory factors. SSR Abstract #222 Maalouf, SW, Smith, CL and Pate, JL. 2011. MicroRNA in the development of the corpus luteum. SSR Abstract #228 Toyokawa, K, Liu, W and Pate, JL. 2011. Expression and regulation of PRAME in the bovine corpus luteum. SSR Abstract #221 Summers A, Cushman R, Smith JE, Lammers B, McFee R, Pohlmeier W, Brauer V, Sargent K, Lu N, Cupp AS, Wood JR (2011) Females with reduced fertility have excess androstenedione in follicular fluid, altered theca gene expression and increased VEGFA164b, maternal effect, and microRNA processing mRNA levels in cumulus-oocyte complexes, Society for the Study of Reproduction 43rd Annual Meeting, Portland, OR Wood JR, Yang Z, and Smith JE (2011) Genes involved in the immediate early response and epithelial mesenchymal transition are regulated by adipocytokines in the female reproductive tract, Society for the Study of Reproduction 43rd Annual Meeting, Portland, OR Schneider, A., L. F. M. Pfeifer, M. N. Corrêa, and W. R. Butler. Difference in the expression of components of the GHR/IGF-I axis in follicular granulosa cells and corpus luteum in cows. J.Dairy Sci. 94(E-Suppl.1):595, 2011. (Abstract) Esposito, G. and W. R. Butler. Effect of dietary conjugated linoleic acid on reproduction and tissue responses in dairy cows. J Dairy Sci. 94(E-Suppl.1):341, 2011. (Abstract) Peer-Reviewed Tripurani, S.K., Lee, K.B., Wee, G., Smith, G.W.. and Yao, J. 2011. MicroRNA-196a regulates bovine newborn ovary homeobox gene (NOBOX) expression during early embryogenesis. BMC Developmental Biology 11:25. doi:10.1186/1471-213X-11-25. Tripurani, S.K., Lee, K.B., Wang, L.,Wee, G., Smith, G.W., Lee, Y.S., Latham, K.E. and Yao, J. 2011. A Novel Functional Role for the Oocyte-Specific Transcription Factor Newborn Ovary Homeobox (NOBOX) during Early Embryonic Development in Cattle. Endocrinology. 152:1013-1023. Inskeep, E.K. 2011. Ruminant reproduction: recent findings and future challenges, a summary. In: Reproduction in Domestic Ruminants VII. M.F. Smith, M. Lucy and T. Spencer, Eds. 12 pp. Tian X and Diaz FJ. Zinc depletion causes multiple defects in ovarian function during the periovulatory period. Endocrinology: (2012) 153:873-886. Pickworth, CL, Poole, DH, Velleman, SG, Pate, JL, Loerch, SC, Fluharty, F L 2011. Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity. J Anim Sci. 89:355-366. Poole, DH and Pate, JL. 2012. Luteal microenvironment directs resident T lymphocyte function. Biol. Reprod. 86(2)29:1-10. Pate, JL. 2012. Commentary: It takes two to tango but four for the finale. Biol. Reprod. Doi:10.1095/biolreprod 112.099150. Pate, JL. Johnson-Larson, C.J., Ottobre, J.S. Life or Death decisions in the corpus luteum. Reprod. Domest. Anim. (in press) *Zhang B, *Tsang PCW, Pate JL, Moses MA 2011. A role for cysteine-rich 61 in the angiogenic switch during the estrous cycle in cows: Regulation by prostaglandin F 2alpha. Biology of Reproduction 85:261-268 *These authors contributed equally to this work. Townson DH, Combelles CMH 2012 Ovarian Follicular Atresia. In: Basic Gynecology Some Related Issues, In Tech Publishing, Atef Darwish (Ed), Rijeka, Croatia. pp. 43-76. ISBN 978-953-51-0166-6. McCracken JA, Custer EE, Schreiber DT, Tsang PCW, Keator CS, Arosh JA 2012. A new in vivo model for luteolysis using systemic pulsatile infusions of PGF2±. Prostaglandins and Other Lipid Mediators 97:90-96. Yang Z, Norwood KA, Kerl JG, and Wood JR (2012) Genes Involved in the Immediate Early Response and Epithelial-Mesenchymal Transition are Regulated by Adipocytokines in the Female Reproductive Tract, Mol Reprod Dev 79(2): 128-137 PMID: 22128093 Mack EM, Smith JE, and Wood JR (2012) IGF1 has an Additive Effect on cAMP-Dependent Regulation of Granulosa Cell Ovulatory Response Genes through Activation of Akt- and Erk1/2 Signaling, Reproduction (submitted 6/2012) Extension Bulletins Lu N*, Smith JE*, Brauer V, Summers A, Pohlmeier W, Beavers KA, McFee R, Sargent K, Kerl JG, Cushman RA, Cupp AS, Wood JR (2012) Oocyte mRNA and Follicle Androgen Levels are Associated with Fertility, Nebraska 2012 Beef Cattle Report, p.28 *authors contributed equally to the manuscript Theses/Dissertations Current Funding USDA Proposal number: 2009-01853 - Jorge Flores Proposal Title: Regulation of Corpus Luteum Lifespan: Developmental Differences in Signal Transduction Mechanisms Associated with the Luteal PGF2alpha Receptor, Total Direct and indirect costs: $338,416. USDA Proposal number: 2009-01853 - Jianbo Yao Proposal Title: Functional Characterization of Bovine Importin ±8, A New Member of the Importin ± Family Specifically Expressed in Oocytes and Early Embryos, Total Direct and indirect costs: $347,000. WV -140 Out-of-Season Breeding in Sheep, West Virginia State Legislature - E. K. Inskeep, P. E. Lewis, Marlon Knights, Direct Costs: $86,000 per year Increasing Adoption of Out-of-Season Breeding to Enhance Profitability of Sheep Producers in West Virginia GNE11-015, USDA/SARE GRAD/ EXT/COM Award Knights, M & D'Souza, K, 8/1/2011- 7/30/2012 $14,999.00 In-Kind Donation (2012) - 1250 CIDR-G Inserts (Retail Value $5.00 each) - Pfizer Animal Health - E. K. Inskeep and M. Knights $6,250.00 National Research Initiative Competitive Grant no. 2008-35203-04617 from the USDA National Institute of Food and Agriculture to JL Pate NRICGP/USDA Grant #2007-35203-18074,"Role of Cytokeratin 8/18 Filaments in Fas Ligand-Induced Apoptosis of Ovarian Steroidogenic Cells". D. Townson USDA (NRICGP, #2008-35203-05989); 9/30/09-8/31/012; PI - 15% effort, "Effects of Fetal Steroids on the Formation of Ovarian Follicles and on their Competence to Initiate Growth", JE Fortune NIH (R21 HD0602322); 07/01/09- 06/30/13; PI 20% effort, "Effects of Fetal Steroids on the Formation of Bovine Ovarian Follicles and on their Competence to Initiate Growth", JE Fortune

Impact Statements

Defining factors that regulate primordial follicle formation has implications on female reproductive longevity.
Understanding how the follicular environment impacts cell-cell communication and oocyte gene expression could improve oocyte quality.
Understanding intracellular death signaling may lead to improvements in follicular growth.
Understanding mechanisms regulating luteal cell function and regression may improve estrous synchronization protocols and dairy cow pregnancy rates.