Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: The overall goal of this project was to assess the physiological role of the insulin-like growth factors (IGF) system, including binding proteins (IGFBP) and their proteases, in ovarian follicular development in cattle. Specific goals were to determine the hormonal and developmental control of cell proliferation, steroidogenesis, gene expression, and IGFBP protease activity in bovine granulosa and thecal cells using both in vivo and in vitro approaches. IGF2 increased steroidogenesis and proliferation of granulosa cells by 2- to 3-fold as determined by increased cell proliferation. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for iodinated-IGF2 existed in granulosa cells and two-day treatment with estradiol, follicle-stimulating hormone (FSH) or cortisol had no significant effect on specific iodinated-IGF2 binding. Also, FSH treatment of small- and large- follicle granulosa cells had no effect on IGF2R mRNA levels whereas IGF1 decreased IGF2R mRNA and specific iodinated-IGF2 binding. Granulosa cell IGF2R mRNA abundance was threefold greater in small than large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R. Insulin alone and in combination with estradiol decreased thecal PAPP-A mRNA levels which would likely reduce the bioavailable IGFs in the theca layer during growth and selection of follicles. Granulosa cell PAPP-A mRNA was not affected by any of the hormones tested. Also, in vivo experiments in dairy cows found that PAPP-A mRNA levels did not differ between dominant and subordinate follicles or change between 24 h and 48 h after luteal regression. Thus, estradiol may act as a negative feedback regulator to prevent excessive hormone-induced androgen production, and hence prevent excessive estradiol production by granulosa cells via decreased thecal PAPP-A production and therefore maintain desirable intrafollicular levels of IGFBP and subsequently free IGF1 and IGF2 during follicular development. Furthermore, results from our studies indicate that IGFBP production and PAPP-A gene expression are differentially regulated in theca and granulosa cells by specific hormones and therefore modulate the amount of bioavailable IGF-I to these cells depending upon the specific hormonal stimuli. Increased free IGFs levels (driven by decreased IGFBP) in ovulatory follicles are likely responsible for increased theca and granulosa cell steroid production. PARTICIPANTS: Individuals: PI: L. J. Spicer; graduate students: P. Y. Aad, J. A. Grado-Ahuir, C. A. Santiago and J. L. Voge; Training: undergraduate students: L. B. Hulsey and D. V. Lagaly; TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. For every day past 120 days, about $5 per cow is lost. Thus, with 75,000 dairy cows in Oklahoma, $26 million is lost each year due to reduced reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.1 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Grado-Ahuir, J.A., P.Y. Aad, G. Ranzenigo, F. Caloni, F. Cremonesi and L. J. Spicer. 2009. Microarray analysis of insulin-like growth factor-I-induced changes in mRNA expression in cultured porcine granulosa cells: Possible role of IGF-I in angiogenesis. J. Anim. Sci. 87:1921-1933.
- Spicer, L.J., S. Sudo, P. Y. Aad, L. S. Wang, S.-Y. Chun, I. Ben-Shlomo, C. Klein, and A. J. W. Hsueh. 2009. The hedgehog-patched signaling pathway and function in the mammalian ovary: A novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells. Reproduction 138:329-339.
- Velazquez, M.A., L. J. Spicer, and D. C. Wathes. The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction. Domest. Anim. Endocrinol. 35:325-342, 2008.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF), their binding proteins (IGFBP) and proteases in ovarian follicular development in cattle. Specifically, we determined the effects of adiponectin on IGF-I-induced proliferation, steroidogenesis and gene expression of large-follicle theca and granulosa cells. RT-PCR was used to elucidate the effects of adiponectin on gene expression. Adiponectin decreased insulin-induced progesterone and androstenedione production as well as attenuated IGF-I-induced LHR, CYP11A1, and CYP17A1 gene expression in theca cells. In contrast, adiponectin decreased LHR mRNA abundance in granulosa cells but did not affect steroidogenic enzyme gene expression in granulosa cells. Adiponectin had no effect on proliferation of large-follicle theca cells. RT-PCR also revealed that abundance of mRNA for the adiponectin receptor (ADIPOR2) was greater in large- than small-follicle theca cells and did not significantly differ between small- and large-follicle granulosa cells. In cultured theca cells, LH increased and IGF-I decreased ADIPOR2 mRNA abundance. These results indicate that the inhibitory effects of adiponectin on steroidogenesis are primarily localized to theca cells and that the response of theca cells to adiponectin (i.e., ADIPOR2) may be regulated by LH and IGF-I. In another series of experiments, small-follicle theca cells cultured in the presence of both LH and IGF-I, GDF9 increased cell numbers and DNA synthesis as measured by thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF-I. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Other studies were conducted to determine the effect of feeding propionibacteria (P169) and yeast on metabolic indicators during lactation. Daily uncorrected milk, solids-corrected milk, and fat-corrected milk production for cows fed P169+yeast was greater than Control cows. Milk glucose and plasma insulin responses to P169+yeast feeding suggest that P169+yeast supplementation might have enhanced gluconeogenesis and increased glucose uptake by the mammary gland in Holstein cows. In a steer study, ruminal digestibility, duodenal flow, microbial N synthesis, microbial efficiency, and fluid and particulate passage rates were not affected by dietary treatments. Feeding P169 tended to decrease molar proportion of acetate, increased molar proportion of propionate, and tended to decrease acetate:propionate ratio compared with Control steers. Our results suggest that feeding P169 alters ruminal metabolism towards increased propionate without affecting feed intake or ruminal kinetics. Thus, a combined feed supplement of P169 and yeast may hold potential as a natural feed supplement to enhance lactational performance. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. For every day past 120 days, about $5 per cow is lost. Thus, with 75,000 dairy cows in Oklahoma, $26 million is lost each year due to reduced reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.1 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Spicer, L.J., P.Y. Aad, D. Allen, S. Mazerbourg, A. Payne, and A.J. Hsueh. Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells: Influence of follicle size on responses to GDF9. Biol. Reprod. 78:243-253, 2008.
- Lagaly, D.V., P.Y. Aad, A. Grado, L.B. Hulsey, and L.J. Spicer. Role of adiponectin in regulating granulosa and theca cell function in cattle. Mol. Cell. Endocrinol. 284:38-45, 2008.
- Lehloenya, K.V., D. R. Stein, D. T. Allen, G. E. Selk, D. A. Jones, M. M. Aleman, T. G. Rehberger, K. J. Mertz, and L. J. Spicer. Effects of feeding yeast and propionibacteria to dairy cows on milk yield and components, and reproduction. J. Anim. Physiol. & Anim. Nutr. 92:190-202, 2008.
- Lehloenya, K.V., C. R. Krehbiel, K.J. Mertz, T.G. Rehberger, and L.J. Spicer. Effects of propionibacteria and yeast culture fed to steers on nutrient intake and site and extent of digestion. J. Dairy Sci. 91:653-662, 2008.
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Progress 10/01/06 to 09/30/07
Outputs
The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF), their binding proteins (IGFBP) and proteases in ovarian follicular development in cattle. Specific goals were: To determine the biological function of IGF2 and its receptor in bovine granulosa cells, we set out first to evaluate the dose-response of IGF2 on granulosa cell proliferation and estradiol and progesterone production in the absence and presence of follicle stimulating hormone (FSH), and then to evaluate the effect of various hormones on the IGF2 responses as well as on specific iodinated-IGF2 binding sites and IGF2 receptor (IGF2R) mRNA in bovine granulosa cells. IGF2 increased steroidogenesis and proliferation of granulosa cells by 2- to 3-fold as determined by increased cell numbers and tritiated-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell
proliferation. Specific receptors for iodinated-IGF2 existed in granulosa cells and two-day treatment with estradiol, FSH or cortisol had no significant effect on specific iodinated-IGF2 binding. Also, FSH treatment of small and large follicle granulosa cells had no effect on IGF2R mRNA levels whereas IGF1 decreased IGF2R mRNA and specific iodinated-IGF2 binding. Granulosa cell IGF2R mRNA abundance was threefold greater in small than large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R. Another study was conducted to determine the effect of feeding propionibacteria (P169) on metabolic indicators during lactation: plasma concentrations of glucose, insulin, IGF-I, leptin,
nonesterified fatty acids (NEFA) and cholesterol were measured with and without bovine somatotropin (bST) treatment. Without bST, IGF-I and leptin were not affected. During bST, high-dose P169 multiparous cows and low-dose P169 primiparous cows had lower IGF-I levels than their respective controls, and regardless of parity, high-dose P169 cows had greater NEFA than control cows. Although supplemental feeding of P169 altered plasma hormones and metabolites, the particular effects were dependent on dose of P169 and parity of cows. Collectively, results from our studies indicate that increased free IGFs levels (driven by decreased IGFBP) in ovulatory follicles are likely responsible for increased thecal and granulosal steroid production.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. For every day past 120 days, about $5 per cow is lost. Thus, with 75,000 dairy cows in Oklahoma, $26 million is lost each year due to reduced reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.1 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Aleman, M.M., D. R. Stein, D. T. Allen, E. Perry K. V. Lehloenya, T. G. Rehberger, K. J. Mertz, D. A. Jones, and L. J. Spicer. 2007. Effects of feeding two levels of propionibacteria to dairy cows on plasma hormones and metabolites. J. Dairy Res. 74:146-153.
- Spicer, L.J., and P.Y. Aad. 2007. Insulin-like growth factor (IGF) 2 stimulates steroidogenesis and mitosis of bovine granulosa cells through the IGF1 receptor: Role of follicle-stimulating hormone and IGF2 receptor. Biol. Reprod. 77:18-27.
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Progress 10/01/05 to 09/30/06
Outputs
The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF), their binding proteins (IGFBP) and proteases in ovarian follicular development in cattle. Specific goals were: To determine the effect of growth differentiation factor-9 (GDF-9), gonadotropins, estradiol and IGFs on ovarian cell function in vitro including effects on pregnancy associated plasma protein-A (PAPP-A) mRNA abundance. We have determined that granulosa cells from small and large follicles are responsive to GDF-9. Because GDF-9 increased granulosa cell proliferation and decreased granulosa cell steroidogenesis, oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation induced by IGF-I and prevent premature differentiation of the granulosa cells during follicle development. In addition, we have determined that estradiol and insulin each significantly stimulates PAPP-A gene expression in bovine thecal cells whereas various hormones
tested had no effect on PAPP-A gene expression in bovine granulosa cells. We conclude that PAPP-A gene expression is differentially regulated in granulosa and theca cells. Insulin alone and in combination with estradiol decreased thecal PAPP-A mRNA levels which would likely reduce the bioavailable IGFs in the theca layer during growth and selection of follicles. Thus, estradiol and GDF-9 may act as a negative feedback regulators to prevent excessive hormone-induced androgen production, and hence prevent excessive estradiol production by granulosa cells via decreased thecal PAPP-A production and therefore maintain desirable levels of IGFBP-4 and -5 and subsequently free IGF-I /-II within the follicle during follicular development. Collectively, results from our studies indicate that increased free IGFs levels (driven by decreased IGFBP) in ovulatory follicles are likely responsible for increased thecal and granulosal steroid production.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. For every day past 120 days, about $4 per cow is lost. Thus, with 90,000 dairy cows in Oklahoma, $25 million is lost each year due to reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.5 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Aad, P.Y., C.A.T. Santiago, J.L. Voge, J.R. Malayer and L.J. Spicer. Real-time RT-PCR quantification of pregnancy-associated plasma protein-A gene expression in bovine granulosa and theca cells. Dom. Anim. Endocr. 31:357-72, 2006.
- Spicer, L.J., P.Y. Aad, D. Allen, S. Mazerbourg, and A.J. Hsueh. Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells. J. Endocrinol. 189:329-339, 2006.
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Progress 10/01/04 to 09/30/05
Outputs
The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF), their binding proteins (IGFBP) and proteases in ovarian follicular development in cattle. Specific goals were: To determine the effect of growth hormone, gonadotropins, estradiol and IGFs on ovarian cell function in vitro, and to determine the intrafollicular changes in pregnancy associated plasma protein-A (PAPP-A) and IGFBP mRNA and free IGF-I during follicular development in domestic animals. We have determined that estradiol and IGF-II each significantly stimulates gonadotropin-induced androstenedione production by bovine thecal cells. Follicle-stimulating hormone (FSH) decreased IGF-I production by granulosa cells, but GH had no effect on IGF-I or IGFBP production by granulosa cells. In vivo experiments in mares found that free IGF-I levels did not differ among various sized follicles during the luteal phase of an estrous cycle, whereas large dominant
follicles during estrus have increased levels of estradiol and free IGF-I concentrations, and decreased levels of IGFBPs. Also, in dairy cows it was found that PAPP-A mRNA levels did not differ between dominant and subordinate follicles or change between 24 h and 48 h after luteal regression, but free IGF-I and estradiol levels were greater, and IGFBP-2 and -5 protein and mRNA levels were lower in dominant versus subordinate follicles. Collectively, results from our studies indicate that increased free IGF levels (driven by decreased IGFBP) in dominant follicles are likely important for increased thecal and granulosal steroid production.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. For every day past 120 days, about $4 per cow is lost. Thus, with 90,000 dairy cows in Oklahoma, $25 million is lost each year due to reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.5 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Spicer, L.J., J.L. Voge and D.T. Allen. 2004. Insulin-like growth factor-2 stimulates steroidogenesis in cultured bovine thecal cells: Role of insulin-like growth factor receptors. Mol. Cell. Endocrinol. 227:1-7.
- Spicer, L.J. 2004. Effect of Growth Hormone on Insulin-Like Growth Factor (IGF)-I and IGF Binding Protein Production by Bovine Ovarian Granulosa Cells. Okla. Ag. Exp. Res. Rep. P-1008: http://www.ansi.okstate.edu/research/2004rr/29/29.htm
- Santiago, C.A.T., J.L. Voge, P.Y. Aad, D.T. Allen, D. Stein, J.R. Malayer and L.J. Spicer. 2005. Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle. Domest. Anim. Endocrinol. 28:46-63.
- Spicer, L.J., C.A. Santiago, T.R. Davidson, T.S. Bridges and C.S. Chamberlain. 2005. Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares. Domest. Anim. Endocrinol. 29:573-581.
- Spicer, L.J. 2005. Effects of estradiol on bovine thecal cell function in vitro: dependence on insulin and gonadotropins. J. Dairy Sci. 88:2412-2421.
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Progress 10/01/03 to 09/30/04
Outputs
The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF) binding proteins and their proteases in ovarian follicular development in cattle. Specific goals were: To determine the hormonal control of insulin-like growth factor binding protein (IGFBP) mRNA levels and IGFBP protease activity by bovine granulosa and thecal cells in vitro; to associate metabolic hormone changes with production and reproductive measures, and to determine the changes in pregnancy associated plasma protein-A (PAPP-A), kallikreins and IGFBP mRNA within granulosa and thecal cells during follicular development in cattle. We have determined that thecal PAPP-A mRNA levels were decreased by estradiol and insulin but not affected by FSH, LH or IGF-I. Granulosa cell PAPP-A mRNA was not affected by any of the hormones tested. Also, in vivo experiments in dairy cows found that PAPP-A mRNA levels did not differ between dominant and subordinant follicles or
change between 24 h and 48 h after luteal regression. Collectively, results from our studies indicate that thecal and granulosal IGFBP production and PAPP-A gene expression are differentially regulated by specific hormones and therefore modulate the amount of bioavailable IGF-I to these cells depending upon the specific hormonal stimuli.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. for every day past 120 days about $4 per cow is lost. Thus, with 90,000 dairy cows in Oklahoma, $25 million is lost each year due to poor reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.5 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- Spicer, L.J. Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles: A control mechanism for selection of dominant follicles. Biol. Reprod. 70:1223-1230, 2004.
- Voge, J.L., P.Y. Aad, C.A.T. Santiago, D.W. Goad, J.R. Malayer, and L.J. Spicer. Effects of insulin-like growth factor (IGF), FSH, and leptin on IGF-binding protein mRNA expression in bovine granulosa and theca cells: Quantitative detection by real-time PCR. Peptides 25:2195-2203, 2004.
- Voge, J.L., C.A.T. Santiago, P.Y. Aad, D.W. Goad, J.R. Malayer, and L.J. Spicer. Quantification of insulin-like growth factor binding-protein mRNA using real-time PCR in bovine granulosa and theca cells: Effect of estradiol, insulin and gonadotropins. Domest. Anim. Endocrinol. 26:241-258, 2004.
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Progress 10/01/02 to 09/30/03
Outputs
The overall goal of this project was to assess the physiological role of insulin-like growth factors (IGF) binding proteins and their proteases in ovarian follicular development in cattle. Specific goals were: To determine the hormonal control of insulin-like growth factor binding protein (IGFBP) mRNA levels and IGFBP protease activity by bovine granulosa and thecal cells in vitro; to associate metabolic hormone changes with production and reproductive measures, and to determine the changes in pregnancy associated plasma protein-A (PAPP-A), kallikreins and IGFBP mRNA within granulosa and thecal cells during follicular development in cattle. We have determined that estradiol and LH inhibits IGFBP production by bovine granulosa cells, and that IGF-I, IGF-2, insulin and LH increases IGFBP production by bovine thecal cells. In contrast, IGF-I increased IGFBP-2 mRNA levels in granulosa cells while IGF-2 decreased IGFBP-2 mRNA levels in cells. Thecal PAPP-A mRNA levels were
decreased by IGF-2 but not affected by IGF-I. Leptin and FSH had no effect on IGFBP-2, -3, -4 or -5 mRNA levels in granulosa cells of large bovine follicles. In vivo, granulosal IGFBP-5 mRNA levels were several-fold greater in large subordinate versus preovulatory dominant follicles, whereas granulosal PAPP-A, IGFBP-2, -3 and -4 mRNA levels did not differ between dominant and subordinate follicles. Collectively, results from our studies indicate that thecal and granulosal IGFBP production and PAPP-A gene expression are differentially regulated by specific hormones and therefore modulate the amount of bioavailable IGF-I to these cells depending upon the specific hormonal stimuli.
Impacts
It is hoped that new insights regarding techniques to improve fertility in dairy and beef cattle will be made from these studies. In Oklahoma the average calving interval for dairy cattle is 14.5 months, average days open is 190, and services per conception is 2.6. for every day past 120 days about $4 per cow is lost. Thus, with 90,000 dairy cows in Oklahoma, $25 million is lost each year due to poor reproductive efficiency. A concomitant reduction in services per conception of 1.0 per cow would reduce the economic loss in Oklahoma by an additional $1.5 million. These studies may be used to design new methods to regulate reproductive efficiency in cattle.
Publications
- White, F.J., I. Rubio, C.A. Lents, N.H. Ciccioli, R.P. Wettemann, and L.J. Spicer. Insulin like growth factor (IGF-I), insulin like growth factor binding proteins (IGFBP), and steroids in dominant follicles of postpartum beef cows. J. Anim. Sci. 81(Suppl. 1):119, 2003.
- Sparks, A.L., L.J. Spicer, J. Kirkpatrick, and D. Waldner. Insulin-like growth factor-I and its binding proteins in colostrum compared to measures in serum of Holstein neonates. J. Dairy Sci. 86:2022-2029, 2003.
- Francisco, C.C., L.J. Spicer, and M.E. Payton. Predicting cholesterol, progesterone, and days to ovulation using metabolic and endocrine measures. J. Dairy Sci. 86:2852-2863, 2003.
- Spicer, L.J. The effect of leptin on ovarian steroidogenesis (Chapter 6), In: Leptin and Reproduction, Ed. M.C. Henson and D. Castracane, Klewer academic/Plenum publishers, 2003.
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