Progress 12/15/01 to 12/31/06
Outputs
The long-term goal of this project was to study the role of gap junctions in the regulation of ovarian function. In study 1 entitled EXPRESSION OF GAP JUNCTIONAL CONNEXINS 26, 32 AND 43 mRNA IN OVARIAN PREOVULTORY FOLLICLES AND CORPORA LUTEA IN SHEEP we have demonstrated that 1) expression of Cx43 mRNA in granulosa and theca cells decreased after hCG-treatment; 2) expression of Cx26 mRNA in the CL tended to be greater on day 10 than on days 5 or 15; 3) expression of Cx43 mRNA was greater on day 5 than on days 10 and 15 of the estrous cycle; 4) expression of Cx26, but not Cx32 or Cx43 mRNA decreased during prostaglandin F2alpha (PGF)-induced luteal regression; and 5) expression of Cx32 mRNA was less than Cx26 and Cx43 mRNA. Thus, we have shown that the mRNA expression pattern of Cx26 and Cx43 changes during peri-ovulatory period and during luteal development. This suggests that Cx26 and Cx43 play a role in ovarian tissue remodeling during the critical time around
ovulation and throughout luteal tissue growth, differentiation and regression in sheep. In Study 2 entitled CONNEXIN 37 (Cx37) EXPRESSION IN SHEEP OVARIES we have used Cx37 a species specific probe developed in our laboratory, and determined that: 1) Cx37 protein was expressed in granulosa and cumulus oocyte complex compartments, ovarian blood vessels, and on the luteal cell borders, 2) expression of Cx37 mRNA was greater in granulosa than in theca cells of preovulatory follicles, 3) Cx37 mRNA expression in granulosa but not theca cells was affected by hCG-treatment, 4) Cx37 protein and mRNA expression were dependent on the stage of luteal development, and 5) Cx37 expression changed during PGF-induced luteal regression. Thus, Cx37 may play a role in follicular development and ovulation as well as in luteal tissue growth, differentiation and regression. In Study 3 entitled ROLE OF GAP JUNCTIONS IN REGULATION OF PROGESTERONE SECRETION BY OVINE LUTEAL CELLS IN VITRO we have demonstrated
that 1) progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional intercellular communication (GJIC) were affected by the day of the estrous cycle, cell density, and treatments (luteinizing hormone or dbcAMP); 2) the changes in progesterone secretion were positively correlated with changes in Cx43 mRNA expression and the rates of GJIC; 3) Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 3β-hydroxysteroid dehydrogenase, and 4) two Cx43 gene targeted sequences decreased Cx43 mRNA expression and progesterone production by luteal cells. Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by 1) the positive correlations between progesterone secretion and Cx43 mRNA expression and GJIC of luteal cells, and 2) the inhibition of Cx43 mRNA expression by siRNA that resulted in decreased production of progesterone by luteal cells. Overall, these studies demonstrated that gap
junctional connexin proteins are expressed in the corpora lutea, and that gap junctional communication and connexins play a role in regulation of steroidogenesis and likely other ovarian tissue function.
Impacts
Knowledge of the role of gap junctions in the regulation of ovarian function will help us to better understand how cell-cell interactions contribute to growth, differentiation and regression of the ovarian follicles and the corpus luteum (CL), and to hormonal regulation of ovarian function. Results of these studies will provide a solid foundation for future studies designed to further define a role of gap junctions in reproductive function. This information will impact future methods to improve fertility and reproduction in mammals, including humans and animals of economic importance. Additionally, understanding how cell-to-cell communication contributes to controlling growth, differentiation and regression of the ovarian follicle and the CL may ultimately help to understand regulation of cell growth in other normal tissues or uncontrolled cell growth such as occurs in tumors.
Publications
- Wendy J. Arndt, Anna T. Grazul-Bilska, Joel S. Caton, Ewa Borowczyk, Pawel P. Borowicz, Marcy Ward, Dale A. Redmer, Lawrence P. Reynolds and Kimberly A. Vonnahme. Cellular proliferation in fetal ovarian follicles from late pregnant sheep fed maintenance or restricted diets with normal or enhanced selenium concentrations. International Embryo Transfer Society meeting, Orlando, FL, 2006.
- Borowczyk Ewa, Mary Lynn Johnson, Jerzy J. Bilski, Magda A. Bilska, Pawel P. Borowicz, Lawrence P. Reynolds, Dale A. Redmer and Anna T. Grazul-Bilska. Role of cell-to-cell communication in regulation of progesterone secretion by luteal cells in vitro. Biol. Reprod, Special Issue, p. 167, abstr. no. 430, 39th Annual SSR Meeting, July , 2006.
- Borowczyk E. Role of gap junctions in regulation of luteal function. Ph.D. Thesis, North Dakota State University, 2006.
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Progress 10/01/05 to 09/30/06
Outputs
The long-term goal of this project was to study the role of gap junctions in the regulation of ovarian function. In study 1 entitled EXPRESSION OF GAP JUNCTIONAL PROTEIN CONNEXIN 37 (Cx37) IN THE CORPORA LUTEA DURING THE ESTROUS CYCLE AND PROSTAGLANDIN F2alpha (PGF) INDUCED LUTEAL REGRESSION IN SHEEP, we have used Cx37 antibody and a species specific probe developed in our laboratory, and determined that: 1) Cx37 protein was expressed in ovarian blood vessels and on the luteal cell borders, 2) Cx37 protein and mRNA expression were dependent on the stage of the estrous cycle, and 3) Cx37 expression changed during PGF-induced luteal regression. Thus, Cx37 may play a role in regulation of luteal tissue growth, differentiation and regression. In Study 2 entitled ROLE OF CELL-TO-CELL COMMUNICATION IN REGULATION OF PROGESTERONE SECRETION BY OVINE LUTEAL CELLS IN VITRO we have demonstrated that 1) progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional
intercellular communication (GJIC) were affected by the day of the estrous cycle, cell density, and treatments (luteinizing hormone or dbcAMP); 2) the changes in progesterone secretion were positively correlated with changes in Cx43 mRNA expression and the rates of GJIC; and 3) Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 3β-hydroxysteroid dehydrogenase (a marker of steroidogneic cells). Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by the positive correlations between progesterone secretion and Cx43 expression and GJIC of luteal cells. Overall, these studies demonstrated that gap junctional connexin proteins are expressed in the corpora lutea, and that gap junctional communication and gap junctional protein connexins play a role in regulation of steroidogenesis and likely other function of the corpora lutea.
Impacts
Knowledge of the role of gap junctions in the regulation of ovarian function will help us to better understand how cell-cell interactions contribute to growth, differentiation and regression of the ovarian follicles and the corpus luteum (CL), and to hormonal regulation of ovarian function. Results of these studies will provide a solid foundation for future studies designed to further define a role of gap junctions in reproductive function. This information will impact future methods to improve fertility and reproduction in mammals, including humans and animals of economic importance. Additionally, understanding how cell-to-cell communication contributes to controlling growth, differentiation and regression of the ovarian follicle and the CL may ultimately help to understand regulation of cell growth in other normal tissues or uncontrolled cell growth such as occurs in tumors.
Publications
- Borowczyk, E., Johnson, M.L., J.J. Bilski, P.P. Borowicz, Redmer, D.A., Reynolds, and Grazul-Bilska, A.T. Expression of mRNA for gap junctional connexins 26, 32 and 43 in ovarian preovulatory follicles and corpora lutea in sheep. Canadian Journal of Physiology and Pharmacology, 2006; 84: 1011-1020.
- Borowczyk, E., Johnson, M.L., Redmer, D.A., Reynolds, L.P., J.J. Bilski, and Grazul-Bilska, A.T. Gap junctional connexins 37 is expressed in sheep ovaries. Endocrine, 2006; 30: 223-230.
- Borowczyk Ewa, Mary Lynn Johnson, Jerzy J. Bilski, Magda A. Bilska, Pawel P. Borowicz, Lawrence P. Reynolds, Dale A. Redmer and Anna T. Grazul-Bilska. Role of cell-to-cell communication in regulation of progesterone secretion by luteal cells in vitro. Biol. Reprod, Special Issue, p. 167, abstr. no. 430, 39th Annual SSR Meeting, July , 2006.
- Borowczyk E. Role of gap junctions in regulation of luteal function. Ph.D. Thesis, North Dakota State University, 2006.
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Progress 10/01/04 to 09/30/05
Outputs
The long-term goal of this project is to study the role of gap junctions in the regulation of ovarian function. In Experiment 1 entitled INHIBITION OF CONNEXIN (Cx) 43 EXPRESSION BY siRNA DECREASES PROGESTERONE PRODUCTION BY LUTEAL CELLS we have evaluated the role of Cx43 in regulation of luteal function by using RNA interference (RNAi) technology. Luteal cells were incubated with or without control medium, transfection factor or siRNA. We have used four different targeted sequences (TS) of siRNA to suppress Cx43 expression. Then, the morphology of cells was evaluated, cells were collected and frozen for extraction of total cellular (tc)RNA, and medium was collected for evaluation of progesterone concentration by using radioimmunoassay. As a control of transfection efficiency, 3T3 cells and luteal cells were transfected with plasmid containing green fluorescent protein (GFP) gene or GFP plasmid plus siRNA for GFP. About 50-60% of the 3T3 cells and about 20-40% of the
luteal cells were transfected with GFP. siRNA suppressed GFP expression by 99-100% in both cell types. TS8 and 24, but not TS2 and 12, decreased (P<0.02) by about 40% Cx43 mRNA expression and progesterone production by luteal cells. The changes in Cx43 mRNA expression were highly correlated with changes in progesterone concentration in media (r = 0.543; P<0.01). These data demonstrated that inhibition of Cx43 mRNA expression by siRNA decreased production of progesterone by luteal cells, suggesting that gap junctions are involved in the regulation of progesterone production by luteal cells. In Experiment 2 entitled CONNEXIN 37 (Cx37) IS EXPRESSED IN THE OVARIAN FOLLICLES DURING PERI-OVULATORY PERIOD IN SHEEP we have cloned ovine Cx37, designed a species specific probe and determined the expression of mRNA for Cx37 in the ovarian follicles during peri-ovulatory period. The granulosa and theca layers were collected at 0, 2, 8, 12, 24 and 48 h after hCG injection on day 15 of the estrous
cycle. The quality and quantity of tcRNA were evaluated using Agilent 2100 bioanalyzer (Agilent Biotechnologies; Wilmington, DE). Expression of Cx37 mRNA was quantified by real-time RT-PCR using the ABI Prism 7000 sequence detection system and the protocols and solutions supplied by Applied Biosystem (Foster City, CA). Ovine-specific oligonucleotide probe and primer sets were developed and used (Primer Express, Applied Biosystems). Cx37 expression in granulosa tended to be greater (P<0.08) than in theca cells, and changed (P<0.05) after hCG-treatment in granulosa but not in theca cells. These data demonstrate that expression of Cx37 in the follicle changes during peri-ovulatory, indicating the role of gap junctions in the regulation of ovarian function.
Impacts
Knowledge of the role of gap junctions in the regulation of ovarian function will help us to better understand how cell-cell interactions contribute to growth, differentiation and regression of the ovarian follicles and the corpus luteum (CL), and to hormonal regulation of ovarian function. Results of these studies will provide a solid foundation for future studies designed to further define a role of gap junctions in reproductive function. This information will impact future methods to improve fertility and reproduction in mammals, including humans and animals of economic importance. Additionally, understanding how cell-to-cell communication contributes to controlling growth, differentiation and regression of the ovarian follicle and the CL may ultimately help to understand regulation of cell growth in other normal tissues or uncontrolled cell growth such as occurs in tumors.
Publications
- E. Borowczyk, M. L. Johnson, L. P. Reynolds, D. A. Redmer, J. J. Bilski, A. T. Grazul-Bilska. Expression of connexin (Cx) 37 in sheep corpora lutea during the estrous cycle and prostaglandin F2alpha (PGF)-induced luteal regression. American Society of Cell Biology meeting, San Francisco, 2005.
- Grazul-Bilska, A.T., Borowczyk, E., and Johnson, M.L. Inhibition of Cx43 expression by siRNA decreases progesterone production by luteal cells: preliminary study. Frontiers in Reproduction Symposium, Marine Biological Laboratory, Woods Hole, MS, 2005; Dakota Reproductive Biology Symposia, Fargo, 2005.
- E. Borowczyk, M. L. Johnson, L. P. Reynolds, D. A. Redmer, J. J. Bilski, A. T. Grazul-Bilska. Connexin 37 is expressed in sheep ovaries during the estrous cycle. Dakota Reproductive Biology Symposia, Fargo, 2005.
- Borowczyk, E., Johnson, M.L., Redmer, D.A., Reynolds, L.P., Navanukraw, C., and Grazul-Bilska A.T. Gap junctional proteins connexins (Cx) 26, 32, 37 and 43 mRNA expression in the ovine ovarian follicles during the periovulatory period. 97th Annual Meeting of the North Dakota Academy of Sciences, Grand Forks, ND, vol. 59, p. 33, 2005.
- Borowczyk, E., Johnson, M.L., Redmer, D.A., Reynolds, L.P., Navanukraw, C., and Grazul-Bilska, A.T. . Expression of gap junctional connexins (Cx) 26, 32, 37, and 43 in the ovine ovarian follicles during the periovulatory period in sheep. Biol. Reprod., Special Issue: p. 183, abstr. no. 462, 38th Annual SSR Meeting, July 24-27, 2005, Quebec City, QC, Canada, 2005.
- Borowczyk, E., Johnson, M.L. and Grazul-Bilska, A.T. Role of gap junctions in luteal function: Expression of connexins mRNA in corpora lutea during the estrous cycle. Frontiers in Reproduction Symposium, Marine Biological Laboratory, Woods Hole, MS, 2005.
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Progress 10/01/03 to 09/30/04
Outputs
The long-term goal of this project is to study the role of gap junctions in the regulation of luteal function. In Experiment 1 entitled CHANGES IN VASCULAR COMPOSITION AND APOPTOSIS IN THE CORPUS LUTEUM DURING PROSTAGLANDIN F2alpha (PGF)-INDUCED LUTEAL REGRESSION IN SHEEP we have demonstrated that after an initial increase, the endothelial component of the vascular bed decreases (P<0.001) during PGF-induced luteal regression. However, smooth muscle cell actin expression remained high during luteal regression, indicating a role of pericytes and vascular smooth muscle in luteolysis. In addition, the rate of cell death increased (P<0.001) dramatically by 12 h after PGF-treatment. We suggest that the high rate of cell death during early luteolysis is primarily due to loss of endothelial cells. The presence of pericytes and smooth muscle cells during early luteal regression may serve to regulate tissue remodeling and to maintain the integrity of larger blood vessels. In
Experiment 2 entitled EXPRESSION OF GAP JUNCTIONAL CONNEXIN 37 (Cx37) IN THE CORPORA LUTEA (CL) DURING THE ESTROUS CYCLE IN EWES we have cloned ovine Cx37, designed a species specific probe and determined the expression of mRNA for Cx37 in the CL throughout the estrous cycle. CL were collected on days 5, 10 and 15 after estrus cycle. Total cellular (tc) RNA was extracted by using Tri-reagent (MRL, Cincinnati, OH). The quality and quantity of tcRNA were evaluated using Agilent 2100 bioanalyzer (Agilent Biotechnologies; Wilmington, DE). Expression of Cx37 mRNA was quantified by real-time RT-PCR using the ABI Prism 7000 sequence detection system and the protocols and solutions supplied by Applied Biosystem (Foster City, CA). Ovine-specific oligonucleotide probe and primer sets were developed and used (Primer Express, Applied Biosystems). Cx37 expression in luteal tissues was the greatest (P<0.05) on day 5 of the estrous cycle, and then decreased (P<0.05) by about 40% on day 15 of the
estrous cycle. These data demonstrate that expression of Cx37 in the CL changes during the estrous cycle, indicating the role of gap junctions in the regulation of luteal function.
Impacts
Knowledge of the role of gap junctions in the regulation of luteal function throughout the corpus luteum life-span will help us to better understand how cell-cell interactions contribute to growth, differentiation and regression of the corpus luteum, and to hormonal regulation of luteal function. Results of these studies will provide a solid foundation for future studies designed to further define a role of gap junctions in reproductive function. This information will impact future methods to improve fertility and reproduction in mammals, including humans and animals of economic importance. Additionally, understanding how cell-to-cell communication contributes to controlling growth, differentiation and regression of the corpus luteum may ultimately help to understand regulation of cell growth in other normal tissues or uncontrolled cell growth such as occurs in tumors.
Publications
- Borowczyk, E., M.L. Johnson, D.A. Redmer, L.P. Reynolds, P. Borowicz, J.S. Luther, D. Pant, R.M. Weigl, J.J. Bilski,and A.T. Grazul-Bilska. 2004. Expression of connexins 26, 32 and 43 in the corpora lutea during the estrous cycle and prostaglandin F2alpha (PGF)-induced luteal regression in sheep. Biol. Reprod., Special Issue: p. 269, abstr. no. 768.
- Grazul-Bilska, A.T., J.J. Bilski, K.A. Vonnahme, A. Tepfer, E. Borowczyk, P. Borowicz, D. Pant, M.L. Johnson, R.M. Weigl, L.P. Reynolds, and D.A. Redmer. 2004. Changes in vascular composition and apoptosis in the corpus luteum (CL) during prostaglandin F2alpha (PGF)-induced luteal regression in sheep. Biol. Reprod., Special Issue: p. 275, abstr. no. 792.
- Borowczyk E., M.L. Johnson, D.A. Redmer, L.P. Reynolds, P.P. Borowicz, J.S. Luther, D. Pant, R.M. Weigl, J.J. Bilski and A.T. Grazul-Bilska. 2004. Gap junctional proteins connexin (Cx) Cx26, Cx32 and Cx43 mRNA expression in the corpora lutea during the estrous cycle and prostaglandin F2alpha (PGF)-induced regression in sheep. Annual Meeting of the North Dakota Academy of Sciences, Fargo, ND, vol. 58, p. 49.
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Progress 10/01/02 to 09/30/03
Outputs
The long-term goal of this project is to study the role of gap junctions in the regulation of luteal function. In the experiment entitled "Expression of genes for gap junctional proteins during the estrous cycle and during luteal regression induced by prostaglandin F2alpha (PGF) in ewes" we have determined the expression of genes for Cx26, Cx32, Cx43 in the corpora lutea (CL) throughout the estrous cycle and during luteal regression. CL were collected on days 5, 10 and 15 after estrus and 0, 4, 8, 12 and 24 h after injection of Estrumate (analog of PGF, 2 ml/injection; 125 mg/ml) on day 10 of the estrous cycle. Total cellular (tc) RNA was extracted by using Tri-reagent (MRL, Cincinnati, OH). The quality and quantity of tcRNA were evaluated using Agilent 2100 bioanalyzer (Agilent Biotechnologies; Wilmington, DE). Expression of specific genes was quantified by real-time RT-PCR using the ABI Prism 7000 sequence detection system and the protocols and solutions supplied by
Applied Biosystem (Foster City, CA). Ovine-specific oligonucleotide probe and primer sets were developed and used for each gene (Primer Express, Applied Biosystems). All genes were expressed in luteal tissues, however, the expression of Cx32 was very low. Expression of Cx26 tended (P<0.1) to be higher on day 10 than on days 5 and 15 of the estrous cycle, but expression of Cx43 was similar throughout the estrous cycle. Four hours after PGF administration the expression of Cx26 decreased (P<0.05) and remained at low level during regression. Expression of Cx43 increased (P<0.05) 8 h after PGF administration and then decreased to the initial level. Expression of Cx32 was not affected by the day of the estrous cycle or PGF treatment. These data demonstrate that expression of genes for gap junctional proteins changes during the estrous cycle and regression of the CL, indicating the role of gap junctions in the regulation of luteal function.
Impacts
Knowledge of the role of gap junctions in the regulation of luteal function throughout the corpus luteum life-span will help us to better understand how cell-cell interactions contribute to growth, differentiation and regression of the corpus luteum, and to hormonal regulation of luteal function. Results of these studies will provide a solid foundation for future studies designed to further define a role of gap junctions in reproductive function. This information will impact future methods to improve fertility and reproduction in mammals, including humans and animals of economic importance. Additionally, understanding how cell-to-cell communication contributes to controlling growth, differentiation and regression of the corpus luteum may ultimately help to understand regulation of cell growth in other normal tissues or uncontrolled cell growth such as occurs in tumors.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
The long-term goal of this project is to study the role of gap junctions in the regulation of luteal function. In the experiment entitled 'Cloning and characterization of gap junctional protein connexins present in ovine ovaries' we tested the hypothesis that the pattern of expression of gap junctional protein connexins (Cx) 26, 32 and 43 is affected by developmental stage of the corpora lutea (CL). Sheep-specific cDNA clones of portions of Cx43, 32, and 26 were prepared by Rt-PCR and sequenced for use as probes in ribonuclease protection assays (RPA). The complete coding sequences (CDS) for Cx43 and Cx32 have not been reported for sheep, whereas sheep Cx26 has been reported (Accession# U17592; Dong et al., 1994). For each connexin, a cDNA clone was selected from a unique region of the gene that would not cross-hybridize with other connexins. Primers for each connexin were taken from consensus regions of the gene with bovine sequences for Cx26. Primers were designed
using the internet Primer3 software (Rozen and Skaletsky, 1988). For Cx43, the forward primer was 5'-AGGGAAGGTGTGGCTGTC-3', starting at base 63 of the CDS, and the reverse primer was 5'-AGCATGAAGATGATGAAGATGG-3', starting at base 644; therefore the cloned PCR product was 582bp. For Cx32, the forward primer was 5'-GCCATTGGCCGAGTATG-3', starting at base 55 of the CDS, and the reverse primer was 5'-GAAGCAGTCCACTGTGTTGG-3', starting at base 540; the cloned PCR product was 486bp. For Cx26, the forward primer was 5'-CGCAAGTTCATCAGAGGAGAGA-3', starting at base 310 of the CDS, and the reverse primer was 5'-CCAGAACAAAATCGAATCAGCA-3', starting at base 659; the cloned PCR insert was 350bp. The homology between the cloned sheep and reported cow connexin insert was 97.6% (Cx43) and 98.9% (Cx32); for Cx26 homology with the insert reported for sheep was 98.7%. An RPA experiment revealed protected bands of the expected sizes in sheep heart or liver. For Cx32 and Cx26, protected bands (480 and 350bp,
respectively) were present in liver, but not in heart. Each Cx was seen as a separate band in a uniformly mixed sample of sheep heart and liver. In addition, Cx43 mRNA was detected in 10 microgram samples of corpus luteum from day 5, 10, and 15 of the estrous cycle (n=4/day), whereas Cx32 and Cx26 mRNA were not detected. The greatest expression of Cx43 was observed on day 15, less on day 10, and least on day 5 of the estrous cycle. These sheep-specific probes will allow us to evaluate and compare the expression of connexin mRNA with connexin protein expression in reproductive tissues. This will help to establish the role of gap junction-mediated intercellular communication in ovine ovarian and uterine tissues.
Impacts
(N/A)
Publications
- No publications reported this period
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