Brief Summary of Minutes of Annual Meeting

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

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.