Progress 01/15/12 to 01/14/18
Outputs
Target Audience:Researchers, farmers, veterinarians, graduate students, undergraduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate students have developed their skills in development and testing of hypotheses, experimental design and analysis of data. They were taught all of the procedures and assays necessary to perform these experiments. Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. Further professional development included the opportunity to present their results at annual meetings of the Society for the Study of Reproduction and the American Society of Reproductive Immunology. How have the results been disseminated to communities of interest?Publication in scientific journals and presentations at scientific and extension/stakeholder meetings What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Infertility in dairy cattle causes major financial losses for producers each year. A significant proportion of infertility in dairy cattle is due to early embryonic loss. The corpus luteum (CL) is a transitory endocrine gland that produces progesterone, which is essential for the establishment and maintenance of pregnancy. In the cow, regression of the CL at the end of an estrous cycle is initiated by uterine release of prostaglandin F2?, but if an embryo is present, embryonic signals prevent luteal regression. Thus, progesterone is maintained to allow for establishment of pregnancy. Although the exact mechanisms by which embryonic signals rescue the CL are unknown, it is clear that changes occur within the CL that facilitate survival of the tissue. We hypothesized that that development and survival of the CL is regulated by changes in expression of microRNA in the CL. MicroRNA are small, noncoding RNA, that can repress the translation of mRNA into proteins, thus serving as powerful regulators of cellular functions. Corpora lutea were collected from cows on day 4 and day 10 of the estrous cycle, or on day 17 of the estrous cycle and pregnancy. MicroRNA was profiled by microarray (Day 4/10 tissues) or by deep sequencing (Day 17 tissues). Day 17 tissues were also processed for sequencing of mRNA and proteomic analysis. Analyses of miR34a and miR126 targets were assessed by overexpression of the miRNA in primary cultures of luteal steroidogenic or endothelial cells. Gene ontology and pathway analysis were used to predict modulated functions and pathways. miRNA and protein datasets were integrated using mirPath 3.0. Cessation of luteal development was associated with upregulation of miRNA associated with cell cycle, cellular development and cell death. Validated targets of miR34a in luteal steroidogenic cells were Notch1 and YY1. PI3KR2 was directly targeted by miR126 in luteal endothelial cells. During luteal rescue, differentially expressed (DE) mRNA and predicted targets of DE miRNA were components of immune response pathways. Integration of DE miRNA and proteins indicated that miRNA regulate functions associated with steroid biosynthesis, removal of products of oxidative stress, and regulation of extracellular matrix. These data provide evidence that transitional states in the CL involve changes in miRNA expression. Processes that are directly regulated by miRNA include steroidogenic pathways, cellular proliferation, extracellular matrix remodeling, and immune cell signaling. This project has identified novel microRNAs that are expressed in the CL and provided entirely new information about the role of specific microRNAs in the function of the CL. Understanding how miRNAs regulate the CL may lead to new methods to enhance reproductive efficiency of dairy cattle and lower the costs of food production.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Pate JL, Maalaouf S, Hughes CH, Liu W. 2016. Micromanaging the CL: Regulation of transitional states in the corpus luteum by microRNA, Society for the Study of Reproduction.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Hughes CK, Maalouf S, Liu W-S, Pate J. 2016. Pathway analysis of differentially expressed mRNA and miRNA in the bovine corpus luteum during maternal recognition of pregnancy reveals functions involved in luteal rescue. Society for the Study of Reproduction.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Hughes CK and Pate JL. 2017. Differentially expressed proteins, transcripts, and miRNA in the corpus luteum during maternal recognition of pregnancy indicate matrix remodeling and miRNA regulation during luteal rescue. Proceedings of the 50th annual meeting of the Society for the Study of Reproduction, Washington, DC.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Pate JL and Hughes CK. 2017. Cell and networks the facilitate luteal survival for pregnancy success. Proceedings of the 4th World Congress on Reproductive Biology, Okinawa, Japan.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Pate JL, Maalouf SA and Hughes CK. 2017. MicroRNA as regulators of luteal function. Proceedings of the 37th annual meeting of the American Society of Reproductive Immunology, Chicago
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Progress 01/15/16 to 01/14/17
Outputs
Target Audience:Scientists, reproductive biologists, graduate students, technicians, and postdoctoral associates Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project How have the results been disseminated to communities of interest?Results have been presented at the annual meeting of the Society for the Study of Reproduction as an invited lecture and as a poster. The poster was presented by a graduate student, who was selected as a finalist in the poster competition. What do you plan to do during the next reporting period to accomplish the goals?Experiments will be completed in the upcoming year and two more manuscripts will be submitted
Impacts
What was accomplished under these goals?
The process of maternal recognition of pregnancy (MRP) requires rescue of the corpus luteum (CL), the transient endocrine gland that forms on the ovary and produces progesterone. This process is essential to the maintenance of pregnancy, yet little is known about pathways necessary for luteal rescue. Transcriptomic profiling was performed to compare the CL of day 17 of the estrous cycle to the CL of day 17 of pregnancy and to determine which functions may be modulated during MRP. Eight heifers were synchronized and, upon observation of estrus, four were bred and four left open. On day 17 after estrus, the heifers were slaughtered, pregnancy confirmed in bred heifers, and the CL were collected and snap frozen. RNA was isolated, reverse-transcribed, and strand-specific next-generation sequencing was performed with an Illumina HighSeq 2000. Clean reads were aligned using the reference genome Bos taurus UMD 3.1. Statistical analysis of differential expression was performed using DEseq in R. Transcriptomic profiling revealed 521 differentially expressed (DE) mRNA (p>0.05), 261 of which were more abundant in pregnancy and 260 of which were more abundant in the estrous cycle. Among these, 94 mRNA increased or decreased >2 fold. Sequencing results for seven mRNA were validated by qPCR and the selected mRNA showed the same pattern of expression in qPCR as in sequencing. Among the 521 DE mRNA, 335 mRNA were predicted by TargetScan to be targets of at least one DE micro(mi)RNA from the same tissues (Maalouf et al., Mol Cell Endocrinol, 2014). The expression of DE miRNA were not necessarily inversely correlated with the expression of their predicted target mRNA; the number of mRNA that changed in the same direction as their targeting miRNA and the number of mRNA that changed in the opposite direction as their targeting miRNA were approximately equal. mirPATH was used to predict pathways in which both DE miRNA and mRNA may be involved. The resulting pathways were related to lipid metabolism, matrix remodeling, immune system, and steroidogenesis. The mRNA DE in the CL during MRP were also compared to mRNA that changed during luteal regression (Mondal et al., Physiol Genomics, 2011). Twenty-three mRNA were DE both during MRP and during luteal regression, among which 20 changed in opposite directions. These genes may be key regulators of luteal regression and survival. Four pathway analysis programs, IPA, Serologix, PANTHER, and DAVID, were used to predict functions that may be differentially regulated during MRP based on the 521 DE mRNA. Three pathways, T cell receptor signaling, JAK STAT signaling, and TGF-beta signaling, were common to all four analyses. Pathways common to at least three analyses included cytokine receptor signaling, apoptosis signaling, oxidative stress signaling, ECM/integrin receptor signaling, and unfolded protein response. Overall, these data indicate modulation of immune response, matrix remodeling, and stress response during MRP and an interplay between transcriptomic changes and changes in miRNA expression
Publications
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Progress 01/15/15 to 01/14/16
Outputs
Target Audience:Animal scientists, reproductive biologists, graduate students, postdoctoral associates, and medical scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students, undergraduate students andpostdoctoral associates performed the research and were involved in all aspects of the project How have the results been disseminated to communities of interest?Results have been presented at the annual meeting of the Society for the Study of Reproduction, and have been published in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?We will be integrating the analyses of microRNA and mRNA sequencing and will begin work on predicted target pathways that may regulate function of the corpus luteum.
Impacts
What was accomplished under these goals?
The corpus luteum (CL) develops from the remnants of the ovulatory follicle and produces progesterone, required for maintenance of pregnancy in mammals. The differentiation of granulosal and thecal cells into luteal cells is accompanied by hypertrophy and hyperplasia of cells. As the CL matures, growth ceases and in ruminants, the tissue acquires the ability to undergo regression in response to prostaglandin F2alpha. The regulators of this transition are poorly understood. MicroRNA, which are posttranscriptional regulators of tissue development and function, are expressed in the CL. However, the pattern of their expression and their function during the transition from developing to functional CL is not known. The objectives of this study were to profile the expression of miRNA in developing vs. mature bovine CL and determine effects of miRNA on bovine luteal cell survival and function. Knockdown of drosha in midcycle (MC) luteal cells decreased progesterone and increased luteal cell apoptosis in the presence or absence of proinflammatory cytokines. Microarray analysis demonstrated that a greater number of miRNA were expressed in MC compared to D4 CL. Ingenuity pathway analysis (IPA) predicted that D4-specific miRNA regulate pathways related to carbohydrate metabolism, while MC-specific miRNA regulate pathways related to cell cycle and apoptosis signaling. Both predictions are consistent with a switch in the CL from a growing phase to a maintenance phase. One of the MC specific miRNA, miR-34a, was selected for further analysis. Overexpression of miR-34a in MC luteal cells resulted in decreased luteal cell proliferation, increased progesterone production, and inhibition of Notch1 and YY1 expression, but had no effect on luteal cell apoptosis. Overall, it was demonstrated that the transition from development to maximal function of the CL is accompanied by an increase in the expression of a number of miRNA, suggesting that these miRNA are involved in regulation of the proteins involved in cellular proliferation, angiogenesis and the capacity to undergo apoptosis. MiR-34a was expressed in fully functional (midcycle) CL, but expression was low (detected by qPCR) or absent (microarray) in developing CL. Overexpression of miR-34a in cultured luteal cells resulted in changes in progesterone production and proliferation of steroidogenic cells. Notch1 and YY1 were confirmed as targets of miR-34a in luteal cells. Overall, these data provide evidence that miRNA within the CL regulate the transition from development to function and that miR-34a regulates luteal cell proliferation and function via the regulation of NOTCH1 and YY1.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Maalouf SW, Liu W-S, Pate JL. 2015. MicroRNA in ovarian function. Cell Tissue Res. In press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Maalouf SW, Smith CL, Pate JL. Changes in microRNA expression during maturation of the bovine corpus luteum: Regulation of luteal cell proliferation and function by microRNA-34a. (submitted for publication)
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Progress 01/15/14 to 01/14/15
Outputs
Target Audience: Animal scientists, Reproductive biologists, graduate students, postdoctoral associates, andmedical scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project How have the results been disseminated to communities of interest? Results have been presented at the annual meeting of the Society for the Study of Reproduction, and have been published in Molecular and Cellular Endocrinology. What do you plan to do during the next reporting period to accomplish the goals? Continue to address the stated objectives and submit full-lenght manuscripts to peer-reviewed journals
Impacts
What was accomplished under these goals?
In the cow, regression of the corpus luteum (CL) is initiated by uterine release of prostaglandin F2 alpha, and rescue of the CL during maternal recognition of pregnancy is dependent on release of interferon-tau by the developing conceptus. The exact mechanisms that are responsible for determining the fate of the CL are unknown. Few studies have focused on post-transcriptional regulators, in particular the role of microRNA (miRNA), in luteal rescue or regression. In this study, CL were collected from heifers on day 17 of the estrous cycle (cyclic; n = 4) and day 17 of pregnancy (pregnant; n = 4). Total RNA (200 ng/ml) underwent Solexa 50 nt single-end sequencing using Illumina HiSeq 2000 sequencer. Full-length small RNA tags were then mapped to the bovine genome (UMD3.1) using the short oligonucleotide alignment program (SOAP) to analyze their expression and distribution. Only reads that mapped to the genome were aligned in miRBase (Release 19) to miRNA precursors with no mismatch, and to the mature miRNA. Unannotated small RNAs were used to predict novel miRNAs using Mireap prediction software (http://sourceforge.net/projects/mireap/). As a result, 85% of the annotated small RNA were identified as miRNA, 2% mapped to introns, exons, repeats or other types of small RNA, and remaining 13% were unannotated, and therefore potential novel miRNAs. The total number of annotated miRNA expressed in the CL was 551, of which ~40% had low reads (<5 reads), ~50% had reads between 5 and 4500, and ~10% had reads above 4500. Differential expression analysis using DEseq resulted in 11 miRNA that were significantly different (Padj < 0.05). Nine miRNA (miR-2443, miR-345, miR-1307, miR-181b-2, miR-2389, miR-378-1, miR-222, miR-6517 and miR-541) were downregulated and two miRNA (miR1-1 and miR-302b) were upregulated in the CL of pregnancy. Using TargetScan, we determined 172 predicted genes to be targeted by at least 3 or more of the differentially expressed miRNAs. Further analysis of these common targets for Gene Ontology (GO) demonstrated all these genes to be enriched for apoptosis and immune response signaling pathways. Moreover, 38 unannotated small RNAs were identified as novel miRNA that have not been reported for any species. Additional 188 miRNAs have been reported for other species but not for cattle. In conclusion, our data confirm the differential expression of miRNA in the CL at maternal recognition of pregnancy, and highlight a potential role for miRNA in modulating the immune response to rescue the CL at maternal recognition of pregnancy.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Maalouf SW, Liu W-S, Albert I, Pate JL. 2014. Regulating life or death: Potential role of microRNA in rescue of the corpus luteum. Mol Cell Endocrinol 398:78-88.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Maalouf, S., W-S. Liu, I. Albert, J. Pate. 2014. MicroRNA of the corpus luteum during maternal recognition of pregnancy. Society for the Study of Reproduction. Abstract 552.
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Progress 01/15/13 to 01/14/14
Outputs
Target Audience: Animal Scientists, Reproductive Biologists, and Graduate Students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students, undergraduate students and postdoctoral associates performed the research and were involved in all aspects of the project. How have the results been disseminated to communities of interest? Results have been presented at the annual meeting of the Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals? Continue to address the stated objectives and submit full-length manuscripts to peer-reviewed journals.
Impacts
What was accomplished under these goals?
The corpus luteum (CL) acquires luteolytic capacity around day 5 or 6 of the estrous cycle, followed by a cessation in growth and proliferation of cells around midcycle. The changes that regulate acquisition of luteolytic capacity, as well as the switch from the growth phase to a maintenance phase, are poorly understood. Notch signaling is an important regulator of several cellular processes such as proliferation, apoptosis, migration, and invasion. In tumors, Notch may have antiproliferative as well as oncogenic activities. In the corpora lutea of pregnant rats, Notch signaling was found to promote luteal cell survival and steroidogenesis. MicroRNAs play an important regulatory role during tissue development and function. These short nucleotide sequences bind to the 3’ end of mRNA and downregulate gene expression either through mRNA degradation or inhibition of translation. Previously, we have shown the upregulation of microRNA 34a (miR34a) in day 10 compared to day 4 bovine CL. This upregulation correlated with an increase in Notch1 mRNA but no change in Notch1 protein expression in D10 compared to D4 CL. Thus we asked the question, could miR34a regulate luteal cell survival and function by regulating Notch signaling? To answer this question, we transfected midcycle luteal cells with miR34a inhibitors and mimics and assayed for Notch 1 expression at different times after transfection. As suggested from our in vivo data, overexpression of miR34a in cultured luteal cells resulted in inhibition of Notch1 protein expression as early as 24h after transfection. To test whether the inhibition of Notch1 protein had any effect on luteal cell apoptosis, we assayed for caspase-3 activity, which plays a dominant role in both intrinsic and extrinsic apoptotic pathways. Transfection of luteal cells by miR34a was not enough to induce caspase activity; however, when cells were treated with a combination of tumor necrosis factor (TNF) alpha and interferon gamma (IFNG), overexpression of miR34a seemed to decrease caspase-3 expression. Furthermore, overexpression of miR34a increased progesterone secretion by transfected luteal cells. The increase in progesterone secretion in addition to the decrease in cytokine-induced caspase-3 expression are consistent with the suggestions from our in vivo studies that showed an increase of miR34a expression in D10, when cells are producing progesterone at optimum capacity. Our data are the first to show an effect of miR34a on Notch signaling and progesterone production in the corpus luteum and highlight a role for miRNA34a in function and survival of luteal cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Maalouf, SW and Pate, JL. 2013. MicroRNA34a regulates survival and function of luteal steroidogenic cells. Abstract. Biol. Reprod. Supplement, p. 135
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Progress 01/15/12 to 01/14/13
Outputs
OUTPUTS: During this initial funding phase of this grant, experiments were undertaken to address objective 1 and part of objective 3. All of the work has been conducted by a postdoctoral associate, with assistance from graduate students, so it directly impacts the education of the future generation of agricultural researchers. In addition, the project was discussed with a group of undergraduate students in the department, giving them an appreciation for the potential role of miRNAs in function of the corpus luteum. The results of the experiments were presented at the 2012 annual meeting of the Society for the Study of Reproduction. The first manuscript from these experiments is currently in preparation. PARTICIPANTS: Joy L. Pate served as the P.D. She directed the research and provided training opportunities for the postdoctoral associates and graduate students. Samar Maalouf and Koji Toyokawa are postdoctoral associates who performed the experiments. TARGET AUDIENCES: Knowledge gained from this project has been presented at the annual meeting of the society for the Study of Reproduction. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Luteal endothelial cells (LEC) are important in the development and function of the corpus luteum (CL). The rapid growth necessitates increased angiogenesis and a stable vasculature to support the growing tissue. Furthermore, at the end of the infertile cycle, endothelial cells are thought to be the first to undergo apoptosis and initiate the structural luteolysis of the CL. Therefore, understanding the regulation of LEC survival and function is important in order to understand the regulation of luteal development and regression. MicroRNA 126 (miR126) is known to regulate angiogenic signals and maintain vascular integrity. Moreover, miR126 is also involved in various cellular processes such as proliferation, migration, invasion, differentiation, and inflammation. We hypothesized that miR126 plays a role in the survival and function of luteal endothelial cells. Using microarray Analysis coupled with quantitative PCR and northern blot analysis, we showed that miR126 was significantly upregulated in midcycle CL compared to early (day4) CL. In situ hybridization assays demonstrated that miR126 was strictly expressed in LEC and not in steroidogenic cells. The upregulation of miR126 in midcycle CL correlated with activation of the AKT survival pathway depicted by an increase in phosphorylation of AKT and a decrease in expression of PI3K regulatory subunit 2 (PI3KR2), which negatively regulates the AKT pathway. Furthermore, a luteolytic dose of prostaglandin F2 alpha (PGF2alpha) downregulated miR126 (by 4 h) in midcycle CL. However, inhibition of miR126 expression in luteal endothelial cells isolated from midcycle CL did not affect the expression of the predicted target of miR126, PI3KR2. On the other hand, inhibition of miR126 expression in LEC induced the expression of another known target of miR126, vascular cell adhesion molecule 1 (VCAM-1), which is involved in leukocyte-endothelial adhesion and signal transduction. In conclusion, miR126 may play a role in endothelial cell function and survival in the CL.
Publications
- Maalouf, S. , K. Toyokawa, and J.L. Pate. 2012. MicroRNA-126 May Regulate Survival of Endothelial Cells in the Corpus Luteum. Biol. Reprod. (Abstract). Supplement p. 50
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