Duration: 10/01/2012 to 09/30/2017

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by stakeholders:

Impaired reproductive performance is a major cause of reduced productivity for ruminants and of reduced profitability for dairy and meat animal producers. The focus of the NE-1027 Regional Project (and its predecessor, the NE-1007, NE-161 Regional Project) has been to address nutritional, management, and environmental factors that impact ovarian activity and subsequent pregnancy rates in domestic ruminants. Our current goal is to continue this important investigative work, focusing on inter- and intra-cellular communication mechanisms that regulate oocyte growth and maturation, corpus luteum (CL) development, maintenance and regression, and early embryonic development. Alterations in inter- and intra-cellular communication due to metabolic and/or environmental stress will also be studied.

The objectives of this proposal are consistent with the second strategic goal identified in the NIFA Strategic Plan (2007-2012), which is to "Enhance the Competitiveness and Sustainability of Rural and Farm Economies". Under objective 2.2 [Provide research, education, and extension to increase the efficiency of agricultural production and marketing systems], we will evaluate our progress using performance criteria 2.2.7 [Increase and improve the reproductive performance of animals; CSREES knowledge area 301], 2.2.8 [Enhance understanding and improve application of animal nutrition; CSREES problem area 302], and 2.2.11 [Improve understanding of fundamental animal physiological processes; CSREES problem area 305]. Moreover, at a recent workshop, a group of more than 75 stakeholder scientists from federal, public, and private institutions across the United States recommended the following high impact areas as funding priorities for USDA/CSREES: development of the oocyte, follicle recruitment and development, identifying genes involved in gamete quality and embryo development, uterine-conceptus interactions with emphasis on embryonic and fetal survival, reproductive immunology, and gonadal development (Mirando and Hamernik, 2006). Our stakeholders include animal producers, the scientific community, and citizens of the region and the nation.

Importance of work and consequences if it is not done:
Improving fertility in ruminants requires fundamental knowledge about the influence of oocyte quality, follicular development, corpus luteum function, and oviduct and uterine environment on embryonic survival. The determination of ovarian and embryonic attributes of fertility in ruminants is critical to identify the underlying causes of anovulation, fertilization failure, luteal insufficiency, and early embryonic loss. Further experimental manipulation of these identified attributes will lead to a prioritization of future studies and management strategies. By enhancing basic knowledge of the underlying biology surrounding ovarian function and embryonic survival, new strategies can be developed for application by producers and veterinarians. Application of management strategies that are not based upon drug-development or use of new drugs is economical, user and consumer friendly, and preserves food quality and safety.

The technical feasibility of the work:

NE-1027 has historically been one of the most productive and cohesive multistate research groups nationwide. This is exemplified by large-scale collaborative projects that have been conducted and published by the group as a whole and thereby serves as a model for animal multistate projects [2, 3]. The technical members of NE-1027 (molecular biologists, cell physiologists, and animal scientists) are a diverse group of scientists with broad and complementary expertise in reproductive physiology of domestic ruminants. Furthermore, they have an established record of using the multistate, collaborative approach to accomplish project objectives. In the four previous years of this project, the twelve technical members of NE-1027 published over 80 refereed research papers, abstracts, theses, book chapters, and technical/extension publications. Accomplishments include: demonstrated interactions between immune and luteal cells which are important for luteal function; identified a novel shift in association of anti-inflammatory to pro-inflammatory cytokines with luteal cells during the luteal life span; established the existence of 4 endothelial cell subtypes within the CL and a method for purifying these cells in order to define functional importance of each subtype; determined a role for the endothelin receptor during luteolysis; defined the transcriptional profile of luteal cells; elucidated molecule(s) responsible for the initiation of Ca2+ oscillations during fertilization; demonstrated that moderately larger, not small preovulatory follicles and greater plasma estradiol concentrations on the day of AI increased the likelihood of subsequent pregnancy in cattle; determined that circulating progesterone concentrations are predictive of AI success; established a relationship between (1) metabolic rate and (2) luteal function, oocyte mRNA abundance, follicular steroidogenesis, and/or fertility; identified bovine miRNAs essential for successful folliculogenesis, luteal function, and early embryonic development; and demonstrated cellular and molecular actions of AMH signaling on bovine granulosa cell differentiation and predicted its role in regulating ovulation rate in a monovular species.

The advantages for doing the work as a multistate effort:

Advantages of performing this work as a multistate effort include overlapping approaches with collaborative efforts and technologies that can be directed toward several objectives simultaneously, and shared experience and data analyses that make interactions more beneficial. For example, members at different stations have contributed to unified animal and cell culture protocols and exchange samples to take advantage of unique validated procedures and will continue to do so in this project. Moreover, the combination of basic biological research and innovative applied research more effectively supports outreach programs and engagement, the goal of which is to improve reproductive performance in livestock more rapidly.

Likely impacts from successfully completing the work:

Fulfilling the objectives of this project will provide important new information concerning declining fertility among ruminants in the face of continuous improvement in production capability. Impacts include improved understanding of how oocyte quality, follicular/luteal function, and metabolic demands of production may alter key hormonal or cellular signals necessary for oocyte competence, fertilization, and embryo survival in early pregnancy. Preventive or therapeutic approaches to treat infertility and embryonic loss will benefit consultants (Nutritionists, Veterinarians, Educators, etc) and most importantly animal producers. Improved animal reproductive performance benefits the on-farm economy and sustains an agricultural production system that is highly competitive in the global economy.

Related, Current and Previous Work

ACCOMPLISHMENTS UNDER PREVIOUS OBJECTIVES:

A. Identify genetic, morphological and physiological attributes of the ovary considered to improve fertility in ruminants.

Follicle/Oocyte: Persistent versus growing follicle [4]- A number of important genes (mRNA binding, modification, polyadenylation, cap binding and transcription) were differentially expressed in oocytes from growing vs. persistent follicles in cows. Using microarray analysis, we showed down-regulation of many genes involved in protein metabolism, amino acid transport, and mitosis, but an up-regulation of apoptotic genes in granulosa cells of persistent follicles. Oocyte-specific genes [5-7]- We characterized several key oocyte-specific genes in cattle including NPM2, NOBOX and FIGLA and demonstrated that miRNAs play a significant role in the regulation of expression of these maternal effect genes during the maternal to zygotic transition in bovine embryos. Intrafollicular estrogens in growing follicles [8-10]- In beef cows, a subset of females that have increased intrafollicular androgen concentrations were identified. These same animals exhibited a decreased E2/A4 ratio which was correlated to altered granulosa cell VEGFA isoform expression and increased theca cell CYP17 and CYP11A expression. Finally, the abundance of miRNA processing and maternal effect genes in the cumulus-oocyte complex was increased in dominant follicles with high androgens. Oocyte quality in high-producing lactating dairy cows versus non-lactating dairy heifers [11-13]- PLCzeta which is localized to the equatorial region of bovine sperm initiated calcium oscillations in the bovine oocyte upon fertilization. The frequency and duration of these oscillations was correlated to the developmental competence of the oocyte. Recent studies indicate that the source of calcium is oocyte intracellular stores and its release is likely regulated by ER-associated proteins. Preovulatory follicle and corpus luteum size and progesterone clearance [14, 15]- There were no differences in circulating P4 or steroidogenic enzyme expression between pregnant vs. non-pregnant cows. However, heifers had lower P4 concentrations than cows. Furthermore, dry matter intake (DMI) was positively correlated with volume of the CL and negatively correlated with plasma P4; although, the effect of DMI on P4 metabolism is not known. Double Ovsynch resulted in higher P4 concentrations due to the production of a second CL and higher pregnancy rates. Conversely, supplementation of cows after AI with a CIDR or hCG raised P4 concentrations but did not improve conception rates. Thus, high P4 concentrations prior to but not after AI were associated with higher conception rates. Activation of primordial follicles [16, 17]- Insulin stimulated follicle activation in a bovine cortex in vitro culture system. Conversely, estradiol inhibited follicle activation. Preliminary studies suggest that estradiol may delay the progression of meiosis. Experiments using fetal bovine ovaries indicated that estradiol also regulates the formation of the primordial follicle pool.
Corpus luteum (CL): Endothelial cells (data in preparation for publication; collaborative effort between Pate, Tseng, and Townson) - In initial studies, mixed populations of endothelial cells secreted active MMP-2, TIMP-1, and TIMP-2. The expression of these MMPs, which stimulate endothelial cell migration, was regulated by P4. Furthermore, a characteristic expression profile was detected in mid-cycle CL. However, no differences in lectin binding or CL protein expression were detected in CL from cyclic versus pregnant cows. To determine functional differences in endothelial cell subpopulations, a method to isolate pure endothelial cell populations was developed in a collaborative effort between the PA and NH experiment stations. Using these isolated cells, differential expression of miRNAs in day 4 versus day 10 CL was identified. The miRNAs targeted several important signaling and transcription factors including PPARA, PTEN, and NRG1. In addition, studies showed that isolated endothelial cells interact with T lymphocytes. This interaction was dependent on the stage of the cycle and indicated important functional communications between luteal and immune cells. Prostaglandin F2alpha and endothelin-1 on luteolysis [18]- Microarray analyses identified differential gene expression between early and late CL. Many of these genes were regulated by PGF2alpha. Inhibitor studies using BQ610 which blocks endothelin-1 signaling via the endothelin receptor A indicated a role for this receptor during luteolysis.


B. Identify genetic and metabolic/nutritional attributes associated with embryo survival in ruminants.

Embryo: Luteal sensitivity to prostaglandin F2alpha [19]- PGF2a increased cytoplasmic calcium in ovine large luteal cells. Furthermore, PGF2a-dependent decreases in P4 concentrations were mediated by intracellular calcium concentrations. The PGF2a-dependent mechanisms regulating calcium influx and mobilization from intracellular stores included regulation of Ryanodine receptor (RyR) and smooth endoplasmic reticulum calcium ATPase (SERCA) gene expression. The ability of PGF2a to regulate the expression of RyR and SERCA was dependent on the stage of the CL (i.e. early versus late). Embryo transfer [20, 21]- The effect of nutritional supplements on embryo survival was determined. Specifically, lactating cows were fed a conjugated linoleic acid (CLA-10,12) and the effect on female fertility was assessed. CLA shortened the open period approximately 40 days and this effect was attributed to increased circulating IGF-1 concentrations, earlier postpartum ovulation, and increased follicular estrogen. Studies to determine the effect of CLA during IVM and IVF on embryo survival were initiated; however preliminary data indicated no statistical differences in MII maturation or blastocyst development. In addition to these studies, collaborative efforts between the West Virginia and Pennsylvania experiment stations determined factors that affect embryonic and fetal mortality in sheep [22, 23].

BACKGROUND OF CURRENT OBJECTIVES:

Follicle formation and the initiation of follicular growth: In cattle, primordial follicles form during fetal life and comprise the "follicular reserve" which is an essential source of new growing follicles and hence, make a significant contribution to reproductive longevity. However, little is known about the intracellular pathways involved in the formation and activation of primordial follicles and in early follicular growth. Yang and Fortune [17] have shown that fetal estradiol is a negative regulator of follicle formation and inhibits the initiation of follicular growth. The temporal pattern of estradiol production by the bovine fetal ovary is consistent with an in vivo role for this steroid. Furthermore, fetal bovine ovaries express nuclear estrogen receptors [24, 25]. However, nothing is known about the signaling pathways by which estradiol regulates follicle formation and activation.
Kit ligand (KITL) also regulates early follicular development including activation. In primordial and small primary follicles, the phosphatidylinositol-3 kinase (PI3K) pathway is activated when granulosa cell-expressed KITL binds to its receptor c-KIT on the oocyte [26]. c-KIT undergoes subsequent autophosphorylation ultimately leading to activation of PI3K and the key downstream molecule, AKT [27-33]. Phosphorylated AKT subsequently regulates a number of genes involved in promoting follicular survival [32] and controlling when the follicle becomes activated out of the primordial follicle pool [27]. Thus, understanding how this pathway is stimulated and inhibited is important for maintaining fertility in mammals.

Effect of follicular environment and intercellular communication on oocyte maturation and pregnancy rates: As the follicle transitions from pre-antral to antral stages, the follicular environment which includes hormones and growth factors deposited in the follicular fluid and bi-directional interactions between the oocyte and granulosa cells makes a significant contribution to follicular growth and oocyte maturation [34-36]. Abnormalities in the follicular environment have been associated with anovulation and reduced oocyte quality. For example, exposure of the oocyte to excess estrogens or androgens results in reduced ovarian function, oocyte quality and increased embryonic loss [37-39]. Metabolic disturbances due to undernutrition, a high metabolic rate, or obesity are also reflected in the follicular environment and have a detrimental effect on oocyte maturation and ultimately the developmental competence of the oocyte [40-42]. Likewise, the inflammatory response of the cow upon parturition and calving, the resumption of ovarian cycles, and pregnancy rates have been correlated [43-45] suggesting a potential role for pro-inflammatory cytokines on ovarian function. Finally increases or decreases in intercellular communication between the oocyte and cumulus granulosa cells affects the developmental potential of the oocyte [46-48]. While these studies emphasize the importance of the follicular environment on oocyte quality, mechanisms which link the status of the follicular environment to oocyte molecular phenotypes are not known.

Angiogenesis during the folliculo-luteal transition: After ovulation the somatic cells of the follicle undergo an important transformation into luteal cells which comprise the CL. Angiogenesis is a hallmark event that occurs during this transition and includes endothelial cell invasion from the vascular theca interna into the previously avascular granulosa cell compartment [49]. Currently, several known angiogenic regulators, such as vascular endothelial growth factor (VEGFa), basic fibroblast growth factor (FGF2), and matrix metalloproteinases (MMPs) are present in the follicular fluid and expressed by granulosa and theca cells [50-56]. However, it is unclear how these and other angiogenic regulators from the granulosa and theca cells interact to switch on angiogenesis following ovulation, when the collapsed follicular envelope transforms to become a functional CL. Collaborative studies between Pate and Tseng have identified cysteine-rich 61 as a potential novel regulator of angiogenesis at this transitional stage [57].

Calcium-dependent regulation of luteolysis: The CL must undergo luteolysis in order for the animal to return to cyclicity. Manipulation of luteolysis is also an important component of successful timed artificial insemination. Our previous studies indicated that reduced luteolysis of an early CL by PGF2a is due to temporal differences in signal transduction [18, 58]. It is generally accepted that PGF2a increases intracellular calcium concentration ([Ca2+]i) which subsequently inhibits P4 biosynthesis in target luteal cells [58]. Furthermore, the magnitude of [Ca2+]i increase is greater in the mature CL. Our data indicates that differences in expression of genes associated with calcium homeostasis may contribute to differential sensitivity of early and mature CL to PGF2a-dependent increases in [Ca2+]i [19, 58, 59]. Conversely, others indicate that the rise in [Ca2+]i may not always be inhibitory, but rather biphasic (i.e. initially stimulatory and then inhibitory). Distinguishing between these two hypotheses is important for understanding mechanisms of PGF2a-dependent luteolysis.

Factors required for successful early embryonic development: Embryonic development prior to its implantation in the uterus is dependent on several factors which are acquired during oocyte maturation and activated upon fertilization. For example, maternally-derived mRNAs play important roles during initial stages of embryonic development, before activation of the embryonic genome [60]. Some of the maternal transcripts are oocyte-specific and known as maternal effect genes. Examples of maternal effect genes that have been identified in mice include maternal antigen that embryos require (Mater), zygote arrest 1 (Zar1) and nucleoplasmin 2 (Npm2) [61-63]. In domestic animals, major activation of the embryonic genome takes place later as compared to rodents (e.g. 8-16-cell stage in cattle vs. 2-cell stage in mouse) suggesting potential species differences in mechanisms and mediators of the maternal-to-zygotic transition.

The ability of oocytes to initiate calcium oscillations after fertilization represents another important indicator of embryonic developmental competence. Following fertilization of in vitro matured eggs ~half of the activated zygotes reach the blastocyst stage [64, 65], which suggests that nearly 50% of the oocytes that undergo in vitro maturation are unable to support pre-implantation embryo development. Our previous studies showed that ~50% of in vitro fertilized oocytes were unable to mount Ca2+ oscillations which is a signal induced by the sperm and required for the initiation of development [66]. This inability to mount oscillations not only reflects abnormal Ca2+ homeostasis mechanisms in these oocytes but also may point to more general cellular and molecular defects.

In addition to oocyte-derived factors, certain sperm proteins also affect early embryonic development. For example, in the absence of Phospholipase C zeta, a sperm specific protein, sperm cannot induce Ca2+ oscillations in the oocyte resulting in a lack of oocyte activation and therefore male infertility [67]. Moreover, the low success of ICSI in large domestic species may be due to poor oocyte activation by the injected sperm [65]. Despite evidence that embryo developmental competence is influenced by sperm cytoplasmic factors, the repertoire of factors and their functional significance during embryonic development is poorly understood and represents an area of reproductive biology that may be targeted to improve rates of pregnancy.

The female reproductive tract supports embryonic and fetal growth. The oviduct provides the necessary micro-environment for gamete survival, maturation, fertilization and then development of the very early embryo (reviewed in [68]). Therefore the function of the oviduct is another essential factor contributing to successful embryonic development. P4 [69, 70], E2 [71, 72], prostaglandins [69, 73], and gonadotropins [74, 75] all regulate oviductal dynamics, yet the effect of endogenous and synchronization-induced changes in the endocrine milieu on oviductal function and early embryonic development is often overlooked and therefore remains poorly understood.

Role of the immune system on luteal function and maintenance of pregnancy: The maintenance of pregnancy is dependent on appropriate implantation and inhibition of luteolysis. Recent data suggests a potential role for immune cells on this process. In the ruminant the expression of signals like interferon-tau (IFNT) by the conceptus rescues luteal function and has an impact on systemic immune response through alteration of gene expression within the CL and endometrium and influencing peripheral blood immune cell populations. There is now evidence that IFNT also increases the expression of interferon-responsive genes not only in the CL but also peripheral blood cells [76]. Another potential role of IFNT is to modify immune response mechanisms in both the uterus and the CL. However, tissue specific changes in immune cell phenotype and function have not been clearly defined for later stages of gestation in ruminants. Immune cells are present in the endometrial epithelium and stroma as well as in the CL, and the functions of these cells must be modified to accommodate the semi-allogeneic fetus [76-81]. In summary, there is relatively little known regarding the phenotypes and functions of immune cells present in the endometrium during the key window of pregnancy recognition signaling in cattle, days 15-19 [82]. Furthermore, the identity of these cells at the fetal/maternal interface during maternal recognition of pregnancy is not known. Joy Pate, Troy Ott (PA) and David Townson (NH) have initiated a collaboration to address this topic, and submitted a grant proposal to NIFA. Furthermore, PA and NH have collaborated on the development of a new protocol for isolation and culture of luteal endothelial cells which is essential for addressing this gap.

It is clear from our previous accomplishments and these collective studies that endocrine hormone and intraovarian paracrine factors regulate important transitional events in the ovary and female reproductive tract including the initiation of follicular growth, nuclear and cytoplasmic maturation of the oocyte, fertilization, early embryonic development, and the function of the corpus luteum (CL). Likewise, communication between somatic and germ cells as well as ovarian or uterine cells and immune cells plays an essential role during follicular growth, luteal function, and implantation. However, a fundamental gap still exists regarding the signaling pathways and proteins which mediate these intra- and inter-cellular communications particularly in domestic livestock species. Furthermore, the impact of environmental factors including metabolic stress, inflammatory response, or external stimulation (e.g. estrus synchronization) on this communication has not been established. Achieving this goal has important implications for improving significant problems in dairy and beef cattle reproduction including anovulatory infertility and early embryonic loss. Thus, the overall objective of the current proposal is to delineate key mechanisms of intra- and inter-cellular communication among ovarian and embryonic tissues regulating transitional events of reproduction in ruminants.


STANDARD CRIS SEARCH

A search of the CRIS system was conducted using the following key words: follicle activation, follicular growth, oocyte maturation, oocyte quality, early embryonic death, corpus luteum function, and implantation. There are 3 current multi-state projects studying reproductive performance in domestic livestock. W1171 - Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock focuses on the manipulation of gametes and culture systems to develop transgenic animals that can be used for food and fiber production. The main impetus of NC1038 - Methods to Increase Reproductive Efficiency in Cattle is to develop breeding programs and estrous synchronization protocols to increase AI service rates. Finally, W2112 - Reproductive Performance in Domestic Ruminants studies the regulation of puberty onset, postpartum anestrus, seasonality of breeding, and factors causing early embryonic mortality with an emphasis on beef cattle and sheep. It is our view that our proposal complements these other projects with our long-term goal of improving female fertility of ruminants. However, our project will examine novel pathways and mechanisms regulating ovarian function and the establishment and maintenance of pregnancy in beef and dairy cattle.

Objectives

1. <b>Identify intracellular signaling pathways and gene expression regulatory mechanisms within the ovary, embryo, or female reproductive tract that promote oocyte growth and maturation, fertilization, early embryonic development, and establishment and maintenance of pregnancy.</b> Our working hypothesis is that regulation of signaling pathways and gene expression in somatic and germ cells by paracrine factors and steroid hormones ensures ovarian cyclicity, the production of developmentally competent oocytes, and the regulation of early embryogenesis.
   
2. <b>Identify inter-cellular interactions between somatic cells of the ovary, somatic cells and germ cells, or somatic cells and the embryo that promote follicular growth, oocyte maturation, early embryonic development, and establishment and maintenance of pregnancy.</b> Our working hypothesis is that bi-directional communication between somatic and germ cells of the ovary as well as between immune cells and components of the female reproductive tract is essential for the production of developmentally competent oocytes, maintenance of the CL, and implantation.
   

Methods

Objective 1: Identify intracellular signaling pathways and gene expression regulatory mechanisms within the ovary, embryo, or female reproductive tract that promote oocyte growth and maturation, fertilization, early embryonic development, and establishment and maintenance of pregnancy. What factors regulate establishment of the primordial pool during fetal development and the subsequent activation of follicular growth in adult animals (NY, IA)? In this study, our in vitro models of fetal bovine folliculogenesis will be used to elucidate estradiol-dependent mechanisms which contribute to the temporal regulation of follicle formation and activation [17]. We have evidence that 17b-estradiol (E2) inhibits follicle formation and competence to activate. Furthermore, it is known that the fetal ovary has the ERa and ERb receptors, but whether they are involved in E2's inhibition of follicle formation and capacity to activate is unknown. Thus, we will take advantage of the available agonists/antagonists of ERa and ERb to determine if one or both of these receptors is involved in transducing the effects of E2 on follicle formation and activation in the bovine fetal ovary using our in vitro fetal bovine ovary model [17, 83]. Specifically, cortical pieces will be obtained from 90-120 day-old fetal ovaries (when E2 has been shown to inhibit follicle formation and activation) and cultured for 10 days with E2, PPT (ERa selective), DPN (ERb selective), E2 + ICI 182,780 (a general antagonist), E2 + MPP dihydrochloride (ERa selective), and E2 + PHTPP (ERb selective). Since even "selective" agonists/antagonists can exert non-selective effects at high doses and binding affinities can vary by cell type, preliminary dose-response studies will be conducted to determine the lowest dose of each compound that is maximally stimulatory or inhibitory. These compounds have been selected and the experiments designed in consultation with Dr. Dennis Lubhan (U. MO-Columbia), who is an expert in the area of estrogen action. After 10 days of culture, ovarian cortical pieces will be fixed and processed for serial plastic sectioning and the sections analyzed for numbers, types, health and diameters of follicles. Based on previous literature, the phosphatidylinositol-3 kinase (PI3K) pathway has also been identified to maintain primordial follicle viability [84, 85], and to regulate the rate at which follicles are activated into the growing follicular pool in the adult animal [27, 29-31, 85, 86]. The latter function appears to depend on the ratio of nuclear to cytoplasmic Foxo3a which is regulated, in part, by PI3K signaling [27, 31]. However, gaps remain in understanding how PI3K and its downstream mediators are regulated and the implications of altered regulation on the reproductive longevity of the ruminant. Thus, immunoflourescent staining will be used to determine in which follicle types total AKT and pAKT, which are a downstream target of PI3K, are located in the bovine ovary [87]. Ovaries from heifers and high lactating cows will be compared to determine differences in this pathway. Additionally, potential activators and regulators of AKT (e.g. E2 or insulin) as well as downstream targets of AKT (e.g. FOXO3a) will be identified. Results will be shared with NY to facilitate studies of mechanisms by which follicles gain the competence to activate during fetal life. How is ovarian function including oocyte maturation altered by intrinsic or environmental factors (NY, NE, WV)? Our preliminary studies indicate that TNFa which is induced around calving due to inflammation of surrounding tissue (e.g. uterus, liver, adipose) negatively impacts fertility. To determine the impact of TNFa on folliculogenesis and oocyte maturation, an in vivo model of early versus delayed ovulation will be used. In this model, the early ovulators have reduced tissue metabolic and inflammatory signals which improve embryo survival. In complementary studies, early ovulation will be induced through dietary strategies. In each model, dairy cows (N=20) will be studied from 3 weeks pre-calving through 3 weeks of lactation and early or delayed ovulation detected via daily blood analysis. Blood and follicular fluid will be aspirated ~day 17 of lacatation for measurement of TNFa. In addition, haptoglobin, a reference for liver inflammation, and NEFA, an indicator of metabolic rate, will be measured. In a complementary trial, dairy cows (N=40) will be fed either a traditional moderate energy diet (1.60 Mcal/kg) or a low energy diet (1.34 Mcal/kg) containing straw beginning 30 days prepartum. Following calving cows will receive the same lactation diet (1.70 Mcal/kg) until week 4. Blood samples will be collected pre- and postpartum to monitor TNFa, NEFA and other metabolites. Ovarian activity will be monitored by ultrasound allowing comparisons between cows with early or delayed ovulation. In the final trial, the benefits of early ovulation after calving on uterine condition and fertility during the breeding period will be assessed. Ovarian activity will be recorded from calving to breeding in multiparous cows (N=100). During breeding, embryo transfer will be performed using in vitro produced female embryos. Pregnancy rates will be compared between cows that had early vs. delayed ovulation and the results correlated to P4 exposure in early lactation. Virgin heifers will provide a reference for performance of embryo transfer. Our previous studies have also demonstrated that the fertility of cows is dependent on intrafollicular androgen concentration. Furthermore, we have previously shown that the expression of Vascular Endothelial Growth Factor A (VEGFA) isoforms are correlated to increased intrafollicular androgen levels. To test our hypothesis that VEGFA signaling directly or indirectly regulates CYP17 transcription and thereby increases androgen levels, primary bovine theca cell cultures will be treated with recombinant VEGFA 164 (50 ng/mL). VEGFA dependent angiogenesis also increases oxidative stress. Thus, cells will also be treated with H2O2 (100 uM). After treatment, QPCR and quantitative Western blot analysis of CYP17 will be carried out. To establish how VEGFA 164 or H2O2 regulate CYP17 transcription, ChIP assays will be carried out using antibodies for FOXO1 or NFkB transcription factors which are the downstream targets of VEGFA and H2O2 signaling. It is our expectation that altered TNFa, androgen excess, increased VEGFA, and/or oxidative stress will negatively impact the molecular phenotype of the oocyte. Thus, to determine the consequence of increased TNFa, androstenedione (A4), VEGFA164, or H2O2 on oocyte maturation, cumulus oocyte complexes (COCs) will be matured in defined medium in the presence of recombinant TNFa (10 ng/mL), A4 (10-8 M), recombinant VEGFA164 (50 ng/ml), or H2O2 (100 uM) [88-92]. A subset of oocytes will be fertilized with bull sperm and embryo development assessed after 72 hr (embryonic cleavage) and 7 days (blastocyst development) of culture. Germinal vesicle stage oocytes prior to maturation, as well as MII-arrested oocytes from each treatment group with a visible polar body and no evidence of fragmentation or degradation will be collected and the expression of maternal effect genes and cumulus expansion factors which are a marker of oocyte quality will be assessed in the oocyte and cumulus cells using QPCR. We have identified novel transcripts in the bovine oocyte using deep sequencing. Thus, the functional significance of novel oocyte-specific genes on oocyte maturation and early embryonic development will be delineated. Specifically, we will clone the full-length cDNAs for previously identified novel genes, analyze their temporal expression during oocyte maturation and early embryogenesis, and elucidate the functional significance of these transcripts during early embryonic development [6, 93, 94]. The impact of TNFa, A4, VEGF164, and/or H2O2 on the expression profile of these novel transcripts will also be delineated. How is calcium concentrations regulated in the bovine oocyte and CL (WV, MA)? We previously showed that in vitro matured bovine oocytes have very low Ca2+ ER store ([Ca2+]ER) content and infer that this may compromise developmental competence. Thus, in the bovine oocyte, [Ca2+]ER will be correlated to the expression of Stromal Interaction Molecule-1 (STIM1)and Orai1, which are part of the Store Operate Ca2+ Entry (SOCE) mechanism of Ca2+ homeostasis. First, to determine if the [Ca2+]ER content changes in bovine oocytes during maturation, oocytes will be loaded with a fluorescent Ca2+ dye at different stages of maturation. The oocytes will subsequently be exposed to a Sarcoplasmic-Endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, thapsigargin (TG), which will empty [Ca2+]ER and promote a [Ca2+]i rise from the [Ca2+]ER content which can be calculated. Second, we will determine if bovine oocytes express STIM-1 and Orai1, the molecules thought to mediate SOCE. These experiments will be performed using oocytes at the GV, GVBD MI and MII stages and Western blots using specific antibodies against Human STIM1 and Orai1 proteins. Based on our results in mouse oocytes, we estimate that 50 oocytes will be needed per lane to detect expression of the endogenous proteins. The sequences of the peptides used to raise these antibodies are highly conserved among mammals, so we are confident that these antibodies will recognize the protein in bovine oocytes. Finally, we will determine if there are differences in STIM-1 and Orai1 expression between oocytes of heifers and high lactating cows and is this difference correlated to differences in [Ca2+]ER content in the oocyte. For these studies, after establishing the normal expression pattern and [Ca2+]ER content, we will obtain oocytes from heifers and high-producing lactating dairy cows from other members of the group and plan to compare the above-mentioned parameters between oocytes from these two different groups of animals. In addition to its role during fertilization, our recent studies indicate that the temporal regulation of intracellular Ca2+ concentrations makes an important contribution to luteolysis. Thus, the expression and phosphorylation status of the ER-calcium release channel (RYR2) and the ER-calcium-sequestering pump (ATPA2) during CL maturation will be determined and correlated to [Ca2+]i. Specifically, the expression of the ER-calcium release channel, RYR2, and the ER-calcium-sequestering pump (ATPA2) increases during CL maturation and appears to explain the temporal differences in PGF2a -induced increases in [Ca2+]i. Thus, we propose to use commercially available activators or inhibitors of the RYR2 channel and ATPA2 pump to determine if [Ca2+]i mediates the ability of PGF2a to regulate luteal progesterone biosynthesis. Depending on the results of these initial experiments, we would extend these observations at the whole animal level to examine the effects of these pharmacological agents on the ability of PGF2a to induce functional and structural luteolysis in the mature bovine CL. The temporal expression of genes associated with calcium binding and sequestration as well as the relationship between changes in [Ca2+]i and luteal progesterone biosynthesis in the developing and mature CL will also be determined. Our previous studies have indicated that there are no developmental differences in the expression of genes that participate in calcium homeostasis including PLC. Thus, in the current study, we will examine if changes in the activity of PLC contributes to the ability of PGF2a to induce a greater increase in [Ca2+]i in the mature CL. What role do sperm proteins play during early embryonic development (MS)? Even when spermatozoa appear normal in morphology and motility, and succeed during initial fertilization steps, they can still fail to complete fertilization or initiate normal embryonic development. Although recent studies indicate that spermatozoal proteins undergo posttranslational modifications such as phosphorylation [95, 96], there is no conventional system that exists for adequately predicting fertility of an ejaculate. Furthermore, very little is known about sperm proteins or their phosphorylation state during early embryonic development. Thus, the objective of this aim is to determine the sperm phenotype required for completion of fertilization and initiation of development. To achieve this goal, we will first perform IVF using sperm from three high and three low fertility bulls, and embryo culture to classify the infertile samples into those that fail to complete fertilization and those that produce defects in early embryonic development. We will then analyze total cell numbers of the resultant blastocysts. The rationale for this specific aim is that identifying the specific fertilization and developmental defects will allow us to understand the nature of non-compensable subfertility and infertility. Subsequently, we will identify differentially expressed proteins in spermatozoa of bulls with varying fertility, and the maternal proteins they interact in the oocyte. Specifically, we will determine differentially-expressed proteins using two-dimensional differential in-gel electrophoresis (2D-DIGE) in spermatozoa from high and low fertility bulls, and analyze differentially expressed proteins through microsequencing and mass spectrometry. Next, we will perform yeast two hybrid system to identify maternal proteins that bind to the key sperm proteins [97-99]. How does the ovary regulate oviductal function and early embryonic development (KY)? Ovarian-produced steroids are known mediators of oviductal function, and the manipulation of ovarian physiology by synchronization of estrus may be affecting the ability of the oviduct to promote early embryonic development. Thus, we will first determine oviductal gene expression which is regulated by ovarian steroids. Isolated sections of the oviducts ampulla and isthmus will be collected from normally cycling heifers during the mid-luteal phase (when circulating P4 is high) or the late follicular phase (when circulating E2 is high). Total RNA extracted from all tissues will be subjected to microarray hybridization, with the resultant data set analyzed to determine profiles of E2- and P4-dependent genes affecting oviductal secretion, remodeling and inflammation. In subsequent experiments the effect of estrous cycle manipulation on oviductal gene expression will be determined. In this study, isolated sections of the oviducts ampulla and isthmus will be collected from cycling heifers during the follicular phase of a non-manipulated estrous cycle, or during the follicular phase after estrus is synchronized with the use of prostaglandins, gonadotropins and/or exogenous steroids. The expression of genes affecting processes required for the maintenance of oviductal function and fertility (genes affecting secretion, remodeling and inflammation) will be examined by real-time PCR. When applicable, protein will be evaluated by Western blotting and/or immunohistochemistry. Objective 2: Identify inter-cellular interactions between somatic cells of the ovary, germ cells, embryonic cells, and/or immune cells that promote follicular growth, oocyte maturation, early embryonic development, and establishment and maintenance of pregnancy How does oocyte-cumulus interaction regulate oocyte cytoplasmic maturation (NE)? Bi-directional communication between the granulosa cells and oocyte is essential for optimal oocyte growth and maturation. This communication is mediated by transzonal projections (TZPs) from the granulosa cells. Previous studies have demonstrated hormonal regulation of TZP dynamics and gap junction function [36, 100-102]. However, the effect of the sex steroids and/or metabolic stress on this communication has not been examined. To this end, we will determine the expression (RNA and protein) of paracrine factors (GDF9, BMP15, KITL, and AMH) and connexin proteins (CX37, CX43) after treatment of cortical and IVM cultures with A4 (10-8M), recombinant VEGFA (50 ng/mL), H2O2 (100 uM), or TNFa (10 ng/mL). These genes exhibit a cell-type specific pattern and therefore we do not need to purify cells from the cortical samples. To address hormone-dependent changes in the establishment and maintenance of transzonal projections (TZPs), treated and untreated cortex samples will be digested with collagenase to release individual follicles. Isolated follicles or COCs from IVM will be fixed and TZPs visualized by immunoflourescence as described by Messinger and Albertini [103] and Combelles et al. [36]. Differences in the density of TZPs will be determined by confocal microscopy. Differences in TZP density will be correlated to changes in oocyte mRNA abundance of maternal effect genes described in objective A. What role does cysteine rich 61-connective tissue growth factor- nephroblastoma overexpressed (CCN1) play during the folliculo-luteal transition and in the development and maintenance of the bovine CL(NH, WI)? We previously found that the angiogenic inducer, CCN1, is highly expressed in the day 4 bovine CL [57], which led us to hypothesize that CCN1 may work in concert with angiogenic factors such as VEGFa and FGF2 to promote the folliculo-luteal transition. While granulosa, theca and luteal cells express VEGFa and FGF2, the interplay between them and CCN1 in the bovine follicle and CL remains to be elucidated. Therefore, in the present study, the temporal expression of CCN1, angiogenic factors and their receptors by granulosa and theca cells of periovulatory and preovulatory follicles, and by luteal cells, will be determined. Specifically, midcycle cows will be ovariectomized at 0 hr (control) or 12, 24 and 48 hrs after a single injection of Lutalyse (25 mg). The granulosa and theca cells isolated from follicles that are >10 mm in diameter will be analyzed by real time PCR and immunoblotting for CYR61, VEGFa, VEGFR1, VEGFR1, FGF2, MMP2, MMP9 and alpha and beta integrin subunits. Follicular fluid steroid hormone concentrations, angiogenic factors, and MMPs will be analyzed by radioimmunoassay, immunoblotting or ELISA, and zymography, respectively. In collaboration with Wisconsin, bovine luteal tissues collected over the estrous cycle (days 2, 4, 6, 8, 10, 12, 14, 16, and 18) will be analyzed for CCN1, its receptors (alpha and beta integrin subunits), angiogenic factors and their receptors (e.g.VEGFa, VEGFR1, VEGFR1, VEGFR2, FGF2), and proteolytic enzymes (MMP2, MMP9) by real time PCR, immunoblotting, ELISA or zymography. Further, plasma gonadotropin (e.g. LH) and P4 concentrations will be determined by radioimmunoassay. How do immune cells regulate luteal function and embryo implantation (PA, NH)? In order for immune cells to migrate into the CL, they must first interact with specific molecules expressed on endothelial cells. After extravasation, the immune cells can come into direct contact with steroidogenic cells, and it is likely that two-way cell-cell communication occurs, resulting in modulation of function of both steroidogenic and immune cells. Thus in the current study, two hypotheses will be tested : (1) leukocyte adhesion molecules expressed on luteal endothelial cells vary with functional state of the CL, facilitating infiltration of immune cells only after the CL is fully functional and (2) luteal steroidogenic cells interact with T lymphocytes by binding to the T cell receptor. Corpora lutea will be collected during the early stages of development (days 5-7 of the estrous cycle), which is before immune cells infiltrate into the tissue, and during luteal maintenance (days 12-15 of the estrous cycle), which is when immune cells actively migrate into the CL. Corpora lutea will be enzymatically dissociated and endothelial cells will be isolated by size-exclusion filtration. Expression of adhesion molecules will be evaluated by qPCR (RNA), Western blot (proteins) in isolated endothelial cells and by immunohistochemistry in frozen sections of luteal tissue. Identification of the adhesion molecules that are upregulated during luteal maintenance will allow for subsequent functional studies. Endothelial cells will be cultured with autologous T lymphocytes in a transwell system for analysis of adhesion and migration of lymphocytes. The role of specific adhesion molecules will be assessed using blocking antibodies. It is expected that these experiments will define which molecules are upregulated after rapid development of the CL and allow for adhesion and extravasation of immune cells through the capillary endothelium. These experiments will utilize cocultures of luteal steroidogenic cells with autologous T lymphocytes, and cell-cell interactions will be visualized using immunofluorescence confocal microscopy. The T cell receptor can be directly labeled with antibody, and antibodies to molecules known to bind the T cell receptor will be used to label steroidogenic cells. Luteal cells elicit distinct responses in T lymphocytes, and these responses will be tested in the presence of blocking antibodies, to determine the specificity of the interaction between steroidogenic cells and immune cells. Lymphocytes, macrophages and dendritic cells are also scattered throughout the endometrium, especially in the luminal epithelium. These cells are exposed to embryonic hormones and may facilitate or regulate adhesion and implantation. Therefore, we will test the hypothesis that the phenotypic profile of uterine-resident immune cells change during early pregnancy to allow for implantation and embryonic survival. To identify immune cells in the endometrium that regulate implantation, estrus will be synchronized in dairy heifers and one-half of the heifers will be inseminated at estrus. Animals will be sacrificed on Day 17 of the estrous cycle or early pregnancy and the uteri collected. The endometrium will be trimmed from the uterus, enzymatically digested, and immune cells will be isolated by magnetic separation. The phenotype of immune cells will be evaluated using flow cytometry. Uterine sections will also be collected for immunohistochemical analysis of immune cells. Cocultures of uterine epithelial cells and lymphocytes will be used to assess cell-cell interactions and the role of embryonic proteins, such as interferon tau, in modulation of those interactions will be evaluated. Initially, we will determine which cell types predominate in the bovine uterine endometrium (all T cell types, NK cells, macrophages and dendritic cells). More intensive experiments will focus on those cells that are present, and particularly those that change relative to pregnancy status.

Measurement of Progress and Results

Outputs

• Research data on factors regulating follicular activation and growth and the identification of novel genes affecting oocyte quality and early embryonic development.
• Information on mechanisms that link environmental and metabolic effects on oocyte quality and embryonic/fetal survival.
• Information on maternal and paternal factors affecting fertilization and the role of the oviductal environment on successful fertilization and the early embryo development.
• Information that will help producers make informed management decisions to enhance embryonic/fetal survival in ruminants.
• Present research results at professional meetings for animal scientists, veterinarians and other agricultural related professionals.
• Establish and characterize models that can be used for further research.

Outcomes or Projected Impacts

• Greater understanding of mechanisms involved in reproductive physiology of livestock species.
• New approaches to management strategies (e.g. estrous synchronization) that will enhance embryo survival and pregnancy rates.
• Correlations between molecular phenotypes and measurable markers which can be used to develop herd management strategies that minimize the effects of nutritional/metabolic stress on herd fertility.

Milestones

(2013): Established new genetic components and improved understanding of environmental factors that influence quality of oocytes and early embryos.

(2014): Established genetic, morphologic, and physiologic characteristics associated with immunologic activity and calcium flux within the CL.

(2015): Established roles of the oviduct and of sperm: oocyte interactions during fertilization and early embryonic development.

(2016): Established methods for isolation and conditions for growth of secondary follicles in vitro

(2017): Identified new markers based on understanding of ovarian function, oocyte quality and interactions in the reproductive tract that can help to maximize early embryo survival and longevity of animals in the herd.

Projected Participation

View Appendix E: Participation

Outreach Plan

The research described herein is largely basic. Application of findings from the basic research will be conducted at experiment stations and/or cooperating farms. Results will be communicated in annual reports, in refereed publications, in presentations at national meetings and on the websites of the experiment stations involved. Data will be presented to experiment station advisory boards, and to extension personnel and commodity groups in short courses and at field days. In addition, some extension specialists and agents will aid in collecting applied data and selecting cooperating farms. Research workers will prepare occasional news releases or participate in interviews. Through the latter venues, stakeholder input for further work and reactions to work in progress will be obtained. Thus an interactive system for dissemination of results and guidance in applications will be maintained throughout the project.

Organization/Governance

A regional Technical Committee will be organized in accordance with the Manual for Cooperative Regional Research (CSREES-OD-1082, 1977). The voting membership of the Regional Technical Committee shall include at least one representative from each cooperating Agricultural Experiment Station as appointed by the respective Director, a technical representative of each cooperation USDA-ARS research division and other participating organizations. Non-voting members shall consist of the Administrative Advisor and a consulting member representing CSREES.

All voting members of the Technical Committee are eligible for office. A chairperson, a secretary, and a third member of the committee will be elected for 2-year terms to compose an Executive Committee. The Technical Committee will meet at least annually. The chairperson, in consultation with the administrative advisor, will notify members of the time and place of meetings. The chairperson is responsible for the preparation of the annual report of the regional project. The secretary records the minutes and other duties as assigned by the Technical Committee. The Executive Committee may be delegated to conduct the business of the Technical Committee between meetings. Other subcommittees may be named by the chairperson as required.

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