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Yuan Y.,University of Missouri | Krisher R.L.,University of Illinois at Urbana - Champaign | Krisher R.L.,National Foundation for Fertility Research
Methods in Molecular Biology | Year: 2012

Oocyte maturation is a critical component of in vitro embryo production. If not carried out in a precise manner under optimal conditions, subsequent fertilization and embryo development will be compromised. Here, we describe collection and in vitro maturation procedures in swine that maintain oocyte competence, resulting in successful embryo development following fertilization. These procedures can be used both for basic research purposes and large-scale production of mature oocytes for use in subsequent assisted reproductive technologies. © 2012 Springer Science+Business Media, LLC.


Paczkowski M.,National Foundation for Fertility Research | Schoolcraft W.B.,Colorado Center for Reproductive Medicine | Krisher R.L.,National Foundation for Fertility Research
Reproduction | Year: 2014

Fatty acid β-oxidation (FAO) is essential for oocyte maturation in mice. The objective of this study was to determine the effect of etomoxir (a FAO inhibitor; 100 mM), carnitine (1 mM), and palmitic acid (1 or 100 mM) during maturation on metabolism and gene expression of the oocyte and cumulus cells, and subsequent embryo development in the mouse. Carnitine significantly increased embryo development, while there was a decrease in development following maturation with 100 mM palmitic acid or etomoxir (P!0.05) treatment. Glucose consumption per cumulus-oocyte complex (COC) was decreased after treatment with carnitine and increased following etomoxir treatment (P!0.05). Intracellular oocyte lipid content was decreased after carnitine or etomoxir exposure (P!0.05). Abundance of Slc2a1 (Glut1) was increased after etomoxir treatment in the oocyte and cumulus cells (P!0.05), suggesting stimulation of glucose transport and potentially the glycolytic pathway for energy production when FAO is inhibited. Abundance of carnitine palmitoyltransferase 2 (Cpt2) tended to increase in oocytes (PZ0.1) after treatment with 100 mMpalmitic acid and in cumulus cells after exposure to 1 mM palmitic acid (PZ0.07). Combined with carnitine, 1 mMpalmitic acid increased the abundance of Acsl3 (P!0.05) and Cpt2 tended to increase (PZ0.07) in cumulus cells, suggesting FAO was increased during maturation in response to stimulators and fatty acids. In conclusion, fatty acid and glucose metabolism are related to the mouse COC, as inhibition of FAO increases glucose consumption. Stimulation of FAO decreases glucose consumption and lipid stores, positively affecting subsequent embryo development, while an overabundance of fatty acid or reduced FAO negatively affects oocyte quality. © 2014 Society for Reproduction and Fertility.


Katz-Jaffe M.G.,National Foundation for Fertility Research | Katz-Jaffe M.G.,Colorado Center for Reproductive Medicine | McReynolds S.,National Foundation for Fertility Research
Fertility and Sterility | Year: 2013

Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowledge of embryonic cellular processes. Copyright © 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.


Krisher R.L.,National Foundation for Fertility Research | Schoolcraft W.B.,Colorado Center for Reproductive Medicine | Katz-Jaffe M.G.,National Foundation for Fertility Research
Fertility and Sterility | Year: 2015

The advent of advanced omics technologies and the application of these techniques to the analysis of extremely limited material have opened the door to the investigation of embryo physiology in a focused, in-depth approach never before possible. The application of noninvasive metabolomic and proteomic platforms to understanding embryo viability permits the characterization of individual embryos in culture. Initial clinical data have highlighted the promise of these technologies for the development of noninvasive embryo selection criteria. In this way, a complex view of embryo function can be compiled and related to embryo development, quality, and outcome. Application of knowledge gained from omics will transform both our understanding of embryo physiology as well as our ability to select viable embryos for transfer in assisted reproductive technology. © 2015 American Society for Reproductive Medicine.


Gardner D.K.,University of Melbourne | Hamilton R.,National Foundation for Fertility Research | McCallie B.,National Foundation for Fertility Research | Schoolcraft W.B.,Colorado Center for Reproductive Medicine | Katz-Jaffe M.G.,National Foundation for Fertility Research
Reproduction | Year: 2013

Ammonium is generated in culture media by the spontaneous deamination of amino acids at 37 °C and through the metabolism of amino acids by human embryos. The appearance of ammonium is a time-dependent phenomenon and can compromise embryo physiology, development and viability. In this study, the effects of a gradient of ammonium on the development, metabolism and transcriptome of human and mouse embryos were investigated. Pronucleate oocytes were cultured in the presence of an ammonium gradient that mimicked the spontaneous deamination of Eagle's amino acids together with 1 mM glutamine. All embryos were cultured in sequential media G1/G2 at 5% O2, 6% CO 2 and 89% N2. Human embryo metabolism was assessed through a non-invasive fluorometric analysis of pyruvate consumption. Transcriptome analysis was performed on the resultant blastocysts from both species using a microarray technology. Embryo development prior to compaction was negatively affected by the presence of low levels of ammonium in both species. Human embryo metabolism was significantly inhibited after just 24 and 48 h of culture. Transcriptome analysis of blastocysts from both species revealed significantly altered gene expression profiles, both decreased and increased. Functional annotation of the altered genes revealed the following over represented biological processes: metabolism, cell growth and/or maintenance, transcription, cell communication, transport, development and transcription regulation. These data emphasize the enhanced sensitivity of the cleavage-stage embryo to its environment and highlight the requirement to renew culture media at frequent intervals in order to alleviate the in vitro induced effects of ammonium build-up in the environment surrounding the embryo. © 2013 Society for Reproduction and Fertility.


Paczkowski K.,National Foundation for Fertility Research | Silva E.,Urbana University | Schoolcraft W.B.,National Foundation for Fertility Research | Schoolcraft W.B.,Colorado Center for Reproductive Medicine | Krisher R.L.,National Foundation for Fertility Research
Biology of Reproduction | Year: 2013

The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 μM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 lM etomoxir in mouse oocytes, 100 μM etomoxir in bovine oocytes, and as little as 10 lM etomoxir in porcine oocytes (P < 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P < 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P < 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway. © 2013 by the Society for the Study of Reproduction, Inc.


Krisher R.L.,National Foundation for Fertility Research | Prather R.S.,University of Missouri
Molecular Reproduction and Development | Year: 2012

In this essay, we propose that embryos express a metabolic phenotype necessarily different from that of differentiated somatic cells and more like that of rapidly proliferating cancer cells. This metabolic adaptation, known as the Warburg effect, supports rapid cell proliferation. One of the hallmarks of the Warburg effect is that pyruvate is directed away from the tri-carboxylic acid cycle and metabolized to lactate, resulting in a buildup of glycolytic intermediates. Although this is a comparatively inefficient way to generate ATP, this adaptation allows the cell to meet other critical metabolic requirements, including biomass production and redox regulation. Thus, utilization of WE gives proliferating cells a selective growth advantage. This model represents a completely new understanding of embryo metabolism in the context of a broad, interconnected network of metabolic mechanisms that influence viability, versus the current dogma of carbohydrate metabolism via oxidative phosphorylation. A more complete understanding of embryo metabolism is critical to better support embryo viability in vitro, and to avoid forcing embryos to adapt to suboptimal culture conditions at a significant cost to future growth and development. © 2012 Wiley Periodicals, Inc.


Katz-Jaffe M.G.,National Foundation for Fertility Research | Parks J.,National Foundation for Fertility Research | McCallie B.,National Foundation for Fertility Research | Schoolcraft W.B.,National Foundation for Fertility Research
Fertility and Sterility | Year: 2013

Objective: To investigate the impact of paternal aging on reproductive success. Design: Animal study. Setting: Research facility. Animal(s): Outbred CF1 mice. Intervention(s): Ten young male mice with proven fertility were mated routinely over 15 months with superovulated young females to assess in vivo and in vitro reproductive outcome. Main Outcome Measure(s): In vivo fertilization, in vivo fetal development, in vitro embryo morphology, and developmental outcome were assessed. Result(s): There were no differences observed for any reproductive end point until the paternal age of 12 months. At 12-15 months, in vivo fertilization was significantly decreased (35% vs. 78% at <12 months). Natural matings with males ≥12 months revealed significantly smaller fetuses (11.36 mm vs. 14.73 mm <12 months) and placental weight (0.10 g vs. 0.13 g at <12 months). In vitro blastocyst development showed a significant decline at ≥12 months, and in vitro blastocyst transfer resulted in a significant increase in pregnancy loss with males ≥12 months (61.5% vs. 0% at <12 months). In addition, the expression levels of Ace-1, Prm1, Prm2, and Smcp were observed to be decreased in sperm from males ≥12 months compared with young male control subjects. Conclusion(s): Results from this study indicate an abrupt reproductive deterioration at paternal midlife, with an adverse effect observed on natural conception, in vitro blastocyst development, implantation potential, and fetal viability. Copyright © 2013 Published by Elsevier Inc.


Yuan Y.,Urbana University | Wheeler M.B.,Urbana University | Krisher R.L.,Urbana University | Krisher R.L.,National Foundation for Fertility Research
Biology of Reproduction | Year: 2012

The objective of this study was to identify specific redoxrelated genes whose function contributes to oocyte quality and to characterize the role of redox homeostasis in oocyte development. We determined the redox genes glutaredoxin 2(GLRX2), protein disulfide isomerase family A, members 4 and 6(PDIA4, PDIA6), and thioredoxin reductase 1 (TXNRD1) were differentially expressed between adult (more competent) and prepubertal (less competent) porcine in vitro-matured (IVM) oocytes. The association between these genes and oocyte quality was further validated by comparing transcript abundance in IVM with that in in vivo-matured (VVM) prepubertal and adult oocytes. By maturing oocytes in variable redox environments, we demonstrated that a balanced redox environment is important for oocyte quality, and over-reduction of the environment is as detrimental as excess oxidation. Critical levels of reactive oxygen species (ROS) and glutathione (GSH) are required for oocyte competence. Elevated GSH and lower ROS in prepubertal oocytes suggest disrupted redox homeostasis exists in these cells. By further comparing GLRX2, PDIA4, PDIA6, and TXNRD1 expression levels in oocytes matured under thesedifferent redox environments, we found aberrant expression patterns in prepubertal oocytes but not in adult oocytes whenthe maturation medium contained high concentrations of antioxidants. These results suggest that prepubertal oocytes are less competent in regulating redox balance than adult oocytes,contributing to lower oocyte quality. In conclusion, aberrant redox gene expression patterns and disrupted redox homeostasis contribute to decreaseddevelopmental competence in prepubertal and IVM porcine oocytes. The balance between ROS and GSH plays an important role in oocyte quality. ©2012 by the Societyfor the Study of Reproduction, Inc.


Krisher R.L.,National Foundation for Fertility Research
Annual Review of Animal Biosciences | Year: 2013

The oocyte is at the center of the equation that results in female fertility. Many factors influence oocyte quality, including external factors such as maternal nutrition, stress, and environmental exposures, as well as ovarian factors such as steroids, intercellular communication, antral follicle count, and follicular fluid composition. These influences are interconnected; changes in the external environment of the female translate into ovarian changes that affect the oocyte. The lengthy period during which the oocyte remains arrested in the ovary provides ample time and opportunity for environmental factors to take their toll. An appropriate environment for growth and maturation of the oocyte, in vivo and in vitro, is critical to ensure optimal oocyte quality, which determines the success of fertilization and preimplantation embryo development, and has long-term implications for implantation, fetal growth, and offspring health. © 2013 by Annual Reviews.

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