Jasmine Randhawa
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Teshome Wondie Alemu
Ongoing project: Effect of ketosis on ovarian functions of Holstein dairy cows
Brief description of the project:
High producing dairy cows during transition period from late gestation to early lactation undergo metabolic stress and negative energy balance, which results in the production of ketone bodies leads to ketosis. Depending on time and cause of increase in elevated circulating concentration of beta-hydroxy butyrate (BHB), ketosis is classified into type I and type II ketosis. Ketosis has been associated with declining reproductive performance of dairy cows. We hypothesize that metabolic stress associated with Type I and II ketosis have unique effects on ovarian follicular gene expression and compromises reproductive performances of dairy cows.
The following are the specific objectives to examine our hypothesis:
Objective 1
To evaluate the association of type I and type II ketosis on reproductive performance of early postpartum dairy cows.
Objective 2
To determine the status of ovarian functions and the transcriptome profile of the dominant-follicle granulosa cells in healthy, type I and type II SCK cows.
Objective 3
To examine the effect of beta-hydroxybutyrate (BHB) on cultured ovarian granulosa cell proliferation and differentiation.
Overall, the data from these three studies will generate novel information on the effect of ketosis on ovarian functions and fertility of dairy cows and therefore identify possible strategies for reducing the economic impact of SCK in Canadian dairy herds.
Yasmin's Project-> The Transition Cow: May the Odds be Ever in her FavourBrief Description:
The most common reason a dairy cow would be culled from a Canadian herd is reduced reproductive performance. At the onset of lactation, cows experience negative energy balance leading to metabolic stress, which may have adverse effects on ovarian functions. Metabolic stress in cows is characterized by generalized inflammation, oxidative stress, increased β-hydroxybutyric acid and decreased glucose. But, there may be additional metabolic parameters that may influence ovarian function. Several studies have reported mechanistic pathways of follicular development and ovulation using the murine model. However, such in vivo studies are scarce in monovulatory species. Most importantly, interaction between metabolic alterations during early lactation and molecular phenotypes of ovarian follicles remains poorly defined in cattle. By gaining a greater understanding of the fundamental causes of ovarian dysfunction we can better develop feeding strategies to improve animal performance. |
Project Aims:
Part A: Examining the role of mitogen activated protein kinase (MAPK) pathway on downstream gene-expression patterns following LH signaling in granulosa and theca cells of bovine ovulating follicles. Part B: Characterizing the metabolic stress and hepatic gene expression patterns and the molecular phenotype of the ovarian follicular environment of the lactating dairy cow. Collaborators: Thank you :) BioRep and ReproPel (Labs from Brazil), Macdonald Campus Farm, Ferme Norline, Abbatoir LeFaivre, Belisle Feed Solutions, Bordignon lab, Vasseur lab |
Medo's project -> Chromatin modifications associated with luteinizing hormone regulated genes in granulosa cells
Brief Description:
The preovulatory luteinizing hormone (LH) surge brings about remarkable tissue remodeling specifically within the ovulating follicle(s). These changes are brought about by a unique and sequential gene expression program, which is initiated by the activation of the LH receptors on granulosa cells of ovulating follicles. Extracellular regulated kinases 1 and 2 (ERK1/2) are activated by LH and have been implicated as key regulators of LH signaling. The gene expression program involves transient induction of genes such as progesterone receptor (Pgr), steroidogenic acute regulatory protein (Star) and prostaglandin synthase 2 (Ptgs2). Chromatin can be found in two forms, heterochromatin that is associated with the repression of genes and euchromatin that is associated with gene activation. Histone modifications in the promoter regions such as trimethylation of histone H3 at lysine 4 and acetylation of histones H3 and H4 at lysines 9 and 5, respectively are associated with activation of gene expression. On the other hand, trimethylation of H3 at lysines 9 and 27 have been shown to be associated with gene repression. How such changes in chromatin accessibility and histone modifications contribute during LH-induced gene expression in granulosa cells of ovulating follicles have not been thoroughly studied. |
Project Aims:
HYPOTHESIS:
Luteinizing hormone regulates the expression of genes during ovulation by altering chromatin accessibility and histone modifications.
OBJECTIVES:
OBJECTIVE 1: To ascertain which histone modifications are associated with the activation or repression of these genes by employing chromatin immunoprecipitation assays (ChIP) followed by qPCR. I will carry out ChIP assays using antibodies specific to modified histones associated with activation H3K9ac and H3K27ac, and histones associated with repression, H3K9me3 and H3K27me3. I expect to find that the histone modifications associated with activation are present when chromatin is accessible whereas those associated with repression are present when chromatin is inaccessible. We will associate these changes with active gene expression, by performing ChIP with polymerase II antibody.
OBJECTIVE 2: To determine the role of histone modifying enzymes such as histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT) and histone demethylases (HDM) in regulating the acetylation/methylation of histones and identify the transcription factors that are involved. I will use inhibitors such as DZNep (a HMT inhibitor) and TSA (a HDAC inhibitor) to investigate these procesess.
OBJECTIVE 3: To decipher the roles of ERK1/2 in LH regulated changes in chromatin accessibility and histone modifications. I will inhibit ERK1/2 using the inhibitor PD0325901 and then test for chromatin accessibility and histone modifications as described in objectives 1 and 2. I expect that this will outline the roles of MAPK3/1 in these processes.
HYPOTHESIS:
Luteinizing hormone regulates the expression of genes during ovulation by altering chromatin accessibility and histone modifications.
OBJECTIVES:
OBJECTIVE 1: To ascertain which histone modifications are associated with the activation or repression of these genes by employing chromatin immunoprecipitation assays (ChIP) followed by qPCR. I will carry out ChIP assays using antibodies specific to modified histones associated with activation H3K9ac and H3K27ac, and histones associated with repression, H3K9me3 and H3K27me3. I expect to find that the histone modifications associated with activation are present when chromatin is accessible whereas those associated with repression are present when chromatin is inaccessible. We will associate these changes with active gene expression, by performing ChIP with polymerase II antibody.
OBJECTIVE 2: To determine the role of histone modifying enzymes such as histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT) and histone demethylases (HDM) in regulating the acetylation/methylation of histones and identify the transcription factors that are involved. I will use inhibitors such as DZNep (a HMT inhibitor) and TSA (a HDAC inhibitor) to investigate these procesess.
OBJECTIVE 3: To decipher the roles of ERK1/2 in LH regulated changes in chromatin accessibility and histone modifications. I will inhibit ERK1/2 using the inhibitor PD0325901 and then test for chromatin accessibility and histone modifications as described in objectives 1 and 2. I expect that this will outline the roles of MAPK3/1 in these processes.
Milena's Project-> Investigating the Role of ERK1/2 Pathway in the Localization and Action of Nr5a2 in Murine Granulosa Cells during Ovulation
Brief Description:
Understanding the molecular mechanisms regulating ovulation is an enormous feat for reproductive biologists today. The complexity with which the female reproductive system manages, supports and maintains the many critical processes involved in the preparations of ovulation through to successful pregnancies is astonishing.Luteinizing Hormone (LH) surge regulates ovulation through complex signaling pathways and a unique gene expression program, which if disrupted lead to infertility. Recent studies using granulosa cell specific knockout mice show that the nuclear receptor 5 a2 (Nr5a2), which is expressed throughout follicular development, is critical for LH-induced gene expression. However, how cytoplasmic signaling pathways impinge upon the functions of Nr5a2 in LH stimulated granulosa cells has not been addressed before.
There is no doubt that Nr5a2 plays an important role as a nuclear receptor in various tissues including the ovary. Its activity as a transcription factor responsible for the regulation of important genes to support various function has been well demonstrated. In an attempt to contribute further knowledge and understanding to at least a small portion of these complex events, we have investigated the cellular localization and potential regulation of Nr5a2 by the ERK1/2 pathway. vulation is a complex process critical to female fertility.
Objectives:
The specific aim of this study was to investigate if ERK1/2 signaling regulates Nr5a2 function. In a follow-up study, we plan to demonstrate if Nr5a2 is regulated at the functional level by the ERK1/2 pathway using chromatin immunoprecipitation.
Understanding the molecular mechanisms regulating ovulation is an enormous feat for reproductive biologists today. The complexity with which the female reproductive system manages, supports and maintains the many critical processes involved in the preparations of ovulation through to successful pregnancies is astonishing.Luteinizing Hormone (LH) surge regulates ovulation through complex signaling pathways and a unique gene expression program, which if disrupted lead to infertility. Recent studies using granulosa cell specific knockout mice show that the nuclear receptor 5 a2 (Nr5a2), which is expressed throughout follicular development, is critical for LH-induced gene expression. However, how cytoplasmic signaling pathways impinge upon the functions of Nr5a2 in LH stimulated granulosa cells has not been addressed before.
There is no doubt that Nr5a2 plays an important role as a nuclear receptor in various tissues including the ovary. Its activity as a transcription factor responsible for the regulation of important genes to support various function has been well demonstrated. In an attempt to contribute further knowledge and understanding to at least a small portion of these complex events, we have investigated the cellular localization and potential regulation of Nr5a2 by the ERK1/2 pathway. vulation is a complex process critical to female fertility.
Objectives:
The specific aim of this study was to investigate if ERK1/2 signaling regulates Nr5a2 function. In a follow-up study, we plan to demonstrate if Nr5a2 is regulated at the functional level by the ERK1/2 pathway using chromatin immunoprecipitation.
Audrey's Project -> The Relationship between a Reproductive biomarker in milk and the Welfare Status of Dairy cows
Brief Description:
Canada boasts some of the most prized dairy genetics in the world. Despite the status of Canadian dairy genetics it is clear that current dairy cow longevity and lifetime production are not sustainable and profitable. Interestingly, reports have suggested that there may be a relationship between increased incidences of feet and leg injuries and reduced conception rates at the herd leve. Therefore, the effects of the comfort status of dairy cows on their reproductive performance require further study within the herd. For my MSc. research project, I hypothesize that dairy cows experiencing higher levels of comfort will have higher levels of Anti-Müllerian hormone (AMH) than dairy cows with a reduced comfort status. The goal of this project is to measure AMH levels in both plasma and skim milk, in order to determine if a non-invasive method can be used to assess the reproductive potential of a cow in relation to her welfare status. Project Aims: To validate a reproductive biomarker that can be used as a non- invasive and accessible assay to evaluate the reproductive performance of a dairy cow. This assay would then also have the potential of being used in future welfare projects to assess the reproductive consequences of the project design. |
Side Project -> An Ounce of Prevention: The effect Yeast Protein Supplement on Gluconeogenesis in Transition Cows
We hypothesized that supplementing gluconeogenic intermediates to transitioning dairy cows, via yeast microbial protein (YMP), would increase hepatic glucose production and promote fertility.
Collaborators: Belisle Feed Solutions, Macdonald Campus Farm, Ferme Norline, Abbatoir LeFaivre, Bordignon lab, Vasseur lab
We hypothesized that supplementing gluconeogenic intermediates to transitioning dairy cows, via yeast microbial protein (YMP), would increase hepatic glucose production and promote fertility.
Collaborators: Belisle Feed Solutions, Macdonald Campus Farm, Ferme Norline, Abbatoir LeFaivre, Bordignon lab, Vasseur lab