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NE1027: Ovarian Influences on Embryonic Survival in Ruminants

Annual/Termination Reports (SAES-422): [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: 06/09/08 to 06/09/08
  • Period the Report Covers: 10/2007 to 09/2008

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    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.


    URL: Copy of minutes

    Accomplishments:

    Impact Statements:
    1. 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.
    2. 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±.
    3. 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.
    4. 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.
    5. 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.
    6. 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.
    7. 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.
    8. 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.
    Last Modified: 05-Jan-2012

    Date of Annual Report: 06/19/2009

    Report Information:
  • Annual Meeting Dates: 06/18/09 to 06/19/09
  • Period the Report Covers: 10/2008 to 09/2009

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

    Station reports began with presentations for Objective 1: .

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

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

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

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

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

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

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

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

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


    URL: Copy of minutes

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

    Impact Statements:
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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.
    6. 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.
    7. 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.
    8. 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.
    9. 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.
    10. 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.
    Last Modified: 05-Jan-2012

    Date of Annual Report: 06/11/2010

    Report Information:
  • Annual Meeting Dates: 06/10/10 to 06/12/10
  • Period the Report Covers: 10/2009 to 09/2010

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    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.

    Nebraska Station- Jennifer Wood.

    Erdogan Memili - Mississippi State U

    Ned Place - Cornell

    Jon Wraith - Associate Director-NH Station.

    Joy Pate - Penn State

    Paul Tsang - U New Hampshire
    Bovine Corpus Luteum Capillary Endothelial Cell Cultures

    Milo Wiltbank - U Wisconsin

    Ron Butler - Cornell

    Brad Hillman

    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:


    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


    URL: Copy of minutes

    Accomplishments:

    Impact Statements:
    Last Modified: 06-Jan-2012

    Date of Annual Report: 06/11/2011

    Report Information:
  • Annual Meeting Dates: 06/09/11 to 06/10/11
  • Period the Report Covers: 10/2010 to 09/2011

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    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


    URL: Copy of minutes

    Accomplishments:

    Impact Statements:
    Last Modified: 05-Jan-2012

    Date of Annual Report: 07/05/2012

    Report Information:
  • Annual Meeting Dates: 06/06/12 to 06/08/12
  • Period the Report Covers: 10/2011 to 09/2012

    • Participants:
    Brief Summary of Minutes of Annual Meeting:

    URL: Copy of minutes
    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.

    Impact Statements:
    1. Defining factors that regulate primordial follicle formation has implications on female reproductive longevity.
    2. Understanding how the follicular environment impacts cell-cell communication and oocyte gene expression could improve oocyte quality.
    3. Understanding intracellular death signaling may lead to improvements in follicular growth.
    4. Understanding mechanisms regulating luteal cell function and regression may improve estrous synchronization protocols and dairy cow pregnancy rates.
    Last Modified: 19-Jul-2012
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