John E. Gadsby, Ph.D.
Professor of Physiology
Department of Molecular Biomedical Sciences
College of Veterinary Medicine
North Carolina State University
Ph.D.: University of Cambridge, U.K., 1979.
(Dr. Brian Heap - Advisor)
Postdoctoral Research Fellow:
Reproductive Endocrinology Program, Department of Pathology, University of Michigan 1979-1981.
Research Investigator/Mellon Young Scientist, Reproductive Endocrinology Program, Department of Physiology, University of Michigan. 1981-1985.
The general research focus of our laboratory is to improve our understanding of the control of the development, maintenance and regression of the corpus luteum (CL).
Our major animal model is the pig, and was selected for our studies because it displays some unique features and because it is also an economically important species. A significant problem in the swine industry is the inability to regulate estrous cycles or synchronize estrus in this species, and since this problem is associated with the peculiarity of the porcine CL, we hope to find ways to solve this problem through our research.
In addition, the pig may represent a valuable animal model for studies of the regulation of human ovarian function, for which rodent animal models are inadequate. For example our studies designed to improve our overall knowledge about the process of CL development (luteinization) and luteolysis may lead to a greater understanding of the causes of luteal phase defects, that account for 30-40% of the causes of infertility in the human female (Dal et al., Fertility and Sterility, 2005).
Our recent projects involve studies of the:
- Growth factors in luteinization (= CL development)
- Control of CL sensitivity to prostaglandin F-2α (luteolytic sensitivity = LS)
- Role of Toll-like receptors (TLR) in the corpus luteum
A. Growth factors in luteinization
This project addresses the control of the early stages of corpus luteum development/differentiation from follicle tissues (= luteinization), following ovulation. The luteinizing hormone (LH)-surge is generally regarded as the trigger of follicle ovulation and the resultant process of luteinization, but little is known of the control pathways involved. Although this project is designed to gain a basic understanding of the process of CL development in pigs, it has the potential to help us understand luteinization or luteal phase defects which account for 30-40% of the causes of infertility in the human female (Dal et al., Fertility and Sterility, 2005).
We have described the expression (at mRNA and protein levels) of IGF-I, the type IGF-I receptor and IGF-binding proteins (BP) 2-5 within the porcine corpus luteum. In particular we have demonstrated that the expression of IGF-I receptor, IGF-I and IGFBP-3 is greatest early in the estrous cycle, suggesting that the IGF-system plays an important (autocrine/paracrine) role in regulating early corpus luteum development and function. Confirming this idea, our in vitro cell culture studies indicate that ONLY luteal cells taken during the early luteal phase (days 4 and 7) display a steroidogenic response to exogenous IGF-I. We have also shown that the signal transduction pathway mediating the steroidogenic actions of IGF-I involves the PI-3-kinase pathway. We have also explored the physiological role of IGF-I using a novel in vivo (ovarian) infusion model. Finally, in collaboration with Dr. Jim Hammond (Hershey Medical Center, Penn State University), we have demonstrated the novel expression of IGF-system components (i.e. acid-labile subunit, mac25 and connective tissue growth factor) within the porcine ovary, which further extend our understanding of how the insulin-like growth factor system may act to control both follicular and luteal development/function in female swine.
We have begun to explore the regulation of gene expression during luteinization, using gene expression microarray analysis and have identified several genes whose expression is up-regulated or down-regulated. Form these preliminary studies, we chose in particular to examine the expression of Transforming Growth Factor (TGF)-β , the TGF-β receptors and the signal transduction pathway (SMAD proteins) at mRNA and protein levels in both an in vivo model (prepubertal gilts treated with PG-600 and hCG), and an in vitro model (granulosa cells in culture), of luteinization. Our results have shown that hCG in vivo increases the expression of TGF-β3 as well as TGF-β receptors type and II. Overall our data suggest that LH and TGF-β may act together to promote the physiological process of luteinization during the estrous cycle.
Finally, we have explored the control of neo-vascularization that occurs concomitantly with luteinization, and by examining the expression of vascular endothelial cell growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α in luteinizing porcine follicles and corpora lutea, it has become clear that these factors also play critical roles in luteinization in the pig.
B. Control of CL sensitivity to prostaglandin F-2α
This project seeks to understand the mechanism underlying the long recognized phenomenon that the corpus luteum of the pig lacks sensitivity to the luteolytic actions of prostaglandin F-2a for 12 days of the 18-day estrous cycle. Unlike the other domestic species (e.g. sheep, cow, mare), prostaglandin analog drugs cannot be used to regulate or synchronize estrous cycles in the pig, and thus currently there are few practical or cost effective approaches for controlling estrous cycles in swine.
We have shown that PGF-2α receptor (protein) concentrations on luteal cells increase at a time during the estrous cycle (day 13) when the CL becomes sensitive to the luteolytic actions of PGF-2α. At the mRNA level, PGF receptor expression increases as early as day 10, several days earlier than expression at the protein level. No increase occurs in corpora lutea of pregnant / pseudopregnant animals (which have prolonged luteal life-spans) on 13, suggesting that regulation of luteal receptors to PGF-2α may be a novel component of the maternal recognition of pregnancy.
In other studies, we have been able to induce premature luteolysis and shorten the estrous cycle in gilts by about 5-7 days, with repeated administration of PGF-2α analogs. The mechanism underlying this action appears not to be associated with increasing PGF-2α receptor levels, but may be via an inhibition of the luteal insulin-like growth factor system (i.e. via increased IGFBP-3 secretion).
In recent studies, we have examined the effects of Tumor Necrosis Factor (TNF)-α on porcine luteal cells in culture, and have shown that TNF-α, 1) blocks the stimulatory actions of IGF-I, and 2) sensitizes these cells to the luteolytic actions of PGF-2α. Also we have data indicating that TNF-α increases the expression of several protein kinase C isoforms, Endothelin (ET)-1 and ET-receptors, all of which are known to mediate the luteolytic actions of PGF-2α . In addition, we have observed that TNF-a promotes a shift in luteal PG synthesis favoring PGF-2a rather than PGE-2, which appears to be an important component in the control of luteolytic sensitivity in vitro. From these studies we developed the hypothesis that TNF-a, secreted by macrophages which invade the porcine corpora lutea in increasing numbers between day 4 and 12 of the cycle, plays a role in the development of luteolytic sensitivity in vivo.
In an effort to explore this hypothesis further, we examined the expression of endothelin (ET)-1 system components and PKC isoforms in porcine corpora lutea during the estrous cycle. We observed increased expression of enzyme Endothelin Converting Enzyme, ECE-1 (synthesizes ET-1 from precursor form) on day 10, and the novel protein kinase isoform epsilon (PKC-e) on day 13, showing a temporal pattern consistent with a role for ET-1 and PKC-e in mediating TNF-a’s role in controlling the onset of luteolytic sensitivity in the pig.
Finally we are exploring the potential role that apoptosis-associated genes play in the control of luteolytic sensitivity. Preliminary studies suggest that TNF-a stimulated activation of Caspase 3 may be critical to the development of luteolytic sensitivity.
C. Role of Toll-like receptors (TLR) in the corpus luteum
We have recently begun a collaboration with Prof. Martin Sheldon, Swansea University, Wales, UK, examining the Role of Toll-like receptors (TLR) in the corpus luteum expression on of the cow and their role in regulation of luteal function in animals which have a uterine infection resulting in endometritis. These studies are in progress but thus far have revealed the presence of multiple TLR’s (1-7, 9-10). Future studies will be directed towards determining the role of TLR’s in luteal physiology in cattle with infectious uterine disease.
In our laboratory we employ a wide variety of techniques which allow us to study the corpus luteum, all the way from molecule to animal…and back again…!
Animal: In vivo studies, ovarian infusion of inhibitors/agonists/antagonists. Ovary collection by surgical laparotomy.
Tissue/cell: corpus luteum dissociation, luteal cell isolation by centrifugal elutriation and lectin-coated magnetic bead separation (for endothelial cells), granulosa cell isolation, granulosa and luteal cell culture, histological techniques such as immunocytochemistry, immunofluorescence, in situ hybridization and apoptosis (Apo-Tag) staining.
Molecule: DNA/RNA analysis using Northern, Southern and slot blots, ribonuclease protection assay, semi-quantitative and Real Time PCR, differential-display PCR, DNA sequencing, gene expression microarray analysis; protein analysis by imunoprecipitation, electrophoresis, Western and ligand-blotting, enzyme-activity assays. Hormone/peptide assay by radiomimmunoasay or ELISA.
A. Growth factors in luteinization
1. Gadsby, J.E., Lovdal, J.A., Samaras, S., Barber, J. and Hammond, J.M. (1996). Expression of messenger ribonucleic acids for Insulin-like Growth Factor-I and Insulin-like Growth Factor Binding Proteins in porcine corpora lutea. Biol. Reprod. 54: 339-346.
2. Nicholson, W.E., Plotner, D.M., Farin, C.E. and Gadsby, J.E. (1999). Insulin-like growth factor-I, insulin-like growth factor-I receptor and insulin-like growth factor binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F2α -treated gilts. Biol. Reprod. 61: 1527-1534.
3. Ge, Z., Nicholson, W.E., Plotner, D.M., Farin, C.E. and Gadsby, J.E. (2000). Expression of the messenger ribonucleic acid and protein for insulin-like growth factor-I receptor in porcine corpora lutea . J. Reprod. Fert. 120: 109-114.
4. Wandji, S., Gadsby, J.E. , Simmen, F.A., Barber, J.A., and Hammond, J.M. (2000). Messenger ribonucleic acids for mac25 and CTGF are inversely regulated during folliculogenesis and early luteogenesis. Endocrinology 141: 2648-2657.
5. Wandji, S., Gadsby, J.E. , Simmen, F.A., Barber, J.A., and Hammond, J.M. (2000). Porcine ovarian cells express messenger ribonucleic acids for the acid-labile subunit and insulin-like growth factor binding protein-3 during follicular and luteal phases of the estrous cycle Endocrinology 141: 2638-2647.
6. Boonyaprakob,U., Gadsby, J.E., Hedgpeth, V., Routh, P. and Almond, G. W. (2003). Expression and localization of vascular endothelial growth factor and its receptors in porcine corpus luteum during the estrous cycle. Reproduction 126, 393-405.
7. Ge, Z., Miller, E. A., Nicholson, W.E., Hedgpeth, V. and Gadsby, J.E. (2003). Insulin-Like Growth Factor (IGF)-I and IGF Binding Proteins -2, -3, -4, -5 in Porcine Corpora Lutea During the Estrous Cycle; Evidence for inhibitory actions of IGFBP-3. Domestic Animal Endocrinology 25 , 183-197.
8. Miller, E.A., Ge, Z, Hedgpeth, V., and Gadsby, J.E. (2003). Steroidogenic responses of porcine corpora lutea to IGF-I in vitro and in vivo: involvement of the PI-3-kinase pathway. Reproduction125, 241-249.
9. Boonyaprakob, U., Gadsby, J.E., Hedgpeth, V., Routh, P. and Almond, G. W. (2005). Expression and localization of hypoxia inducible factor-1α in Porcine Corpora Lutea during the estrous cycle. Can. J. Vet. Med. 69, 215-222.
10. Sriperumbudur, R., Zorrilla, L.M., and Gadsby, J.E. (2010). Transforming Growth Factor-beta (TGFB) and its signaling components in peri-ovulatory porcine follicles. Animal Reproduction Science (in press).
B. Control of CL sensitivity to prostaglandin F-2α
1. Gadsby, J.E. , Balapure, A.K., Britt, J.H. and Fitz, T.A. (1990). PGF2α "receptors" on enzyme-dissociated pig luteal cells throughout the estrous cycle. Endocrinology 126: 787-795.
2. Estill, C.T., Britt, J.H. and Gadsby, J.E. (1993). Repeated administration of prostaglandin F2α during the early luteal phase causes premature luteolysis in the pig. Biol Reprod. 49: 181-185.
3. Gadsby, J.E. , Lovdal, J., Britt, J.H. and Fitz, T.A. (1993). Prostaglandin F2α receptor concentrations in corpora lutea of cycling, pregnant and pseudopregnant pigs. Biol. Reprod. 49: 604-608.
4. Gadsby, J.E . and Earnest, K.L. (1994). Prostaglandin F2α stimulates progesterone secretion by porcine luteal cells in vitro throughout the estrous cycle. Prostaglandins 48: 109-125.
5. Estill, C.T., Britt, J.H. and Gadsby, J.E. (1995). Does increased PGF2α receptor concentrations mediate PGF2α -induced luteolysis during early diestrus in the pig? Prostaglandins49: 255-267.
6. Gadsby, J.E. , Grafinger, M., and Almond, G. (1996). Short-cycling of gilts with prostaglandin analogs. In: Proceedings of the 14th International Pig Veterinary Society Congress, Bologna, Italy, pp.564.
7. Boonyaprakob,U., Gadsby, J.E., Hedgpeth, V., Routh, P. and Almond, G. W. (2003). Cloning of a Porcine Prostaglandin F2α (FP) Receptor cDNA and the Expression of its mRNA in the Corpora Lutea. Reproduction125, 53-64.
8. Rose, L., Sriperumbudur, R. and Gadsby, J. (2003). TNF-α sensitizes porcine luteal cells to PGF2a in vitro. Biol. Reprod.68, Suppl. 1: Abs. no. 75, p. 143.
9. Gadsby, J., Rose, L., Sriperumbudur, R and Ge, Z. (2006). The role of intra-luteal factors in the control of the porcine corpus luteum: In: Control of Pig Reproduction VII, Reproduction Supplement 62. Eds. C.J. Ashworth and R.R. Kraeling. Nottingham University Press, Nottingham, U.K. pp. 69-83.
10. Whang, H.S., Vendeix, F.A.P., Gracz, H.S., Gadsby, J.E., and Tonelli, A. E. (2008). NMR Studies of the Inclusion Complex of Cloprostenol Sodium Salt with beta-cyclodextrin in Aqueous Solution. Pharm. Res. 2008 May; 25(5):1142-9. Epub 2007 Dec 5.
11. Zorrilla, L.M., Irvin, M.S. and Gadsby, J.E. (2009). Protein Kinase C Isoforms in the Porcine Corpus Luteum: Temporal and Spatial Expression Patterns. Domestic Animal Endocrinology 36:173-185. Epub. Dec. 2008.
12. Gadsby, J., D’Annibale, M., Gannon, M., Albers, L. and Zorrilla, L. (2009). Tumor Necrosis Factor (TNF)-ainduces luteolytic sensitivity in porcine luteal cells by activating the intra-luteal prostaglandin (PG) pathway. Presented at the 42nd SSR Annual Meeting, Pittsburgh, July 2009. Abstr.# 519.
13. Zorrilla, L.M., Sriperumbudur, R. and Gadsby, J.E. (2010). Endothelin (ET) -1, Endothelin-Converting Enzyme-1 and ET- Receptors in the Porcine Corpus Luteum: Temporal and Spatial Expression Patterns. Domestic Animal Endocrinology, 38:75-85. E. pub. Dec 29 2009
Sriperumbudur, R., Zorrilla, L.M., and Gadsby, J.E. (2010). Transforming Growth Factor-beta (TGFB) and its signaling components
in peri-ovulatory porcine follicles. Animal Reproduction Science. 120: 84-94. Epub. Mar 7 2010.
C. Role of Toll-like receptors (TLR) in the corpus luteum
1. Sheldon, I.M., Price, S.B., Cronin, J., Gilbert, R.O., White, J.O. and Gadsby, J.E. (2009). Mechanisms of infertility associated with clinical and sub-clinical endometritis in dairy cattle. Reproduction in Domestic Animals 44 (Suppl. 3), 1–9.
Melissa D'Annibale-Tolhurst, Research Technician
Raul Salinas-mondragon, Research Assistant