Progress 10/01/05 to 09/30/09
Outputs
OUTPUTS: Towards better understanding the role of biological clocks in reproduction, Dr. Bartell's laboratory investigates expression of clock gene transcription factors, such as such as period, Bmal, clock, and cryptochrome. Using real-time polymerase chain reaction, the expression of circadian clock genes were observed in the shell gland, medullary bone, and granulosa and thecal cells of the ovarian follicle. Results indicated that phasing and amplitude of the clock genes were not consistent in these tissues. For example, during maturation of ovarian follicle, circadian clock gene expression varied in the granulosa cells but not in the thecal cells, suggesting an E-box mediated transcriptional regulation in granulosa cells. Proving this hypothesis, increased expression of E-box containing genes such as peroxisome proliferator activating receptor-gamma, glycogen synthase kinase-beta, nuclear translocating factor X-1, and sirtuin1 were found during follicle maturation. In collaboration with Drs. Siopes and El Halawani, Dr. Bartell found that disruption of the normal circadian pattern of dopamine neurotransmission completely abolished egg-laying in Japanese quail. Studies examining the role of dopamine in egg laying of chickens are currently underway. With regard to better understanding the hypothalamic control of reproduction, Dr. Ramachandran's laboratory studied a novel hypothalamic neuropepetide, gonadotropin-inhibitory hormone (GnIH), that is likely to inhibit gonadotropin secretion in avians. His laboratory cloned and characterized the complementary deoxyribonucleic acids (cDNA) as well as the proteins that encode for GnIH and its receptor (GnIHR). In vitro studies revealed that GnIH treatment significantly decreased luteinizing hormone (LH) secretion in the pituitary glands from sexually immature chickens, but not from sexually mature chickens. Intravenous administration of GnIH to Leghorn chickens did not change plasma levels of FSH or LH. Affinity purified anti-GnIHR antibody was developed and utilized to identify GnIHR-immunoreactive (ir) cells in the anterior pituitary gland. Several GnIH-ir cells were colocalized in LH mRNA or FSH mRNA-expressing cells. GnIHR mRNA quantity in total ovary was significantly greater in sexually immature chickens versus sexually mature hens. The viability of granulosa cells was significantly decreased by treatment with GnIH. In summary, GnIH and GnIHR are likely to exert anti-gonadal effects at pituitary and ovarian levels. Towards better understanding ovarian follicular development, Dr. Diaz's laboratory has identified that follicle growth from 0.5 to 4 mm is an important transition event during ovarian folliculogenesis. The abundance of transcripts encoding the steroidogenic proteins, such as Star, Hsd3b, and Cyp11a1, increased dramatically in granulosa cells of 4 mm follicles compared to 0.5 mm follicles. Other transcripts including Amh, Bmp2, Bmp4, Bmp6, Wnt4, Foxl2, Gata4, and Wt1 mRNA decreased during the same period. These genes will serve as useful markers of granulosa cell differentiation in small avian follicles that may have important functional significance during early avian follicular development. PARTICIPANTS: Dr. Bartell spearheaded the design and execution of the research program in his laboratory. A portion of his research project was conducted by Dr. Maria Horvat-Gordon (Research Associate). Additionally, his research project has contributed to the training of a graduate student (Brittany Smith) and to the training of several undergraduate students (Erica Stuber, Patrick Buckley, Elizabeth Ebert-Zavos and Melissa Handler). External collaborators on his Japanese quail studies included Dr. Thomas Siopes (North Carolina State University) and Dr. Mohamed E. El Halawani (University of Minnesota). Dr. Ramachandran contributed to the design and execution of all of the projects in his laboratory. A portion of this work was conducted by Dr. G. L. Hendricks III (Research Support Associate). In addition, this research project trained graduate students (Dr. Sreenivasa R. Maddineni, Dr. Olga Ocon-Grove, and Ms. Susan Krzysik-Walker). External collaborators included Dr. J. Proudman (USDA, Beltsville, MD), Dr. G. Bedecarrats (University of Guelph, Ontario, Canada), and Dr. G. Bentley (University of California, Berkeley). Dr. Diaz contributed to the design and execution of all of the projects in his laboratory. The majority of the laboratory work was conducted by Ms. Kate Anthony (Research Technician). TARGET AUDIENCES: Scientists in academic research laboratories and commercial broiler and egg producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research findings from the Bartell laboratory have advanced our understanding of the role of circadian clocks in avian reproduction. Specifically, they have established that circadian clocks exist within all tissues of the avian reproductive system and that these clocks likely work to mediate metabolism, apoptosis, and transcription during ovarian follicle development and post-ovulation (i.e., in egg formation). Therefore, aberrations in the timing of various developmental processes can potentially result in altered internal and external egg quality. Thus, studies from the Bartell laboratory are envisioned to help advance our current understanding of how biological clocks integrate multiple molecular signaling pathways involved in ovarian follicular maturation. By understanding how clocks interact with photoperiod, it also may be possible to break photorefractoriness, a process which limits some poultry species (turkey, duck, and quail) to shorter egg laying periods. Therefore, if the number of pause days that occur during the egg laying cycle were decreased, egg production would be concomitantly increased. Research findings from the Ramachandran laboratory have advanced our knowledge of the role of GnIH, a novel hypothalamic peptide, and its receptor in chicken reproduction. In particular, they have established that GnIHR expressed in the chicken gonads and pituitary gland is likely to affect ovarian follicular maturation, which is an important process that determines ovulation and egg production in the laying hen. Their research results will aid in the further characterization of molecular signaling pathways involved in ovarian follicular maturation. Research findings from the Diaz laboratory have advanced our knowledge of the mechanisms of early avian follicular development. They have characterized the changes in several transcripts during growth of avian follicles from 0.5 to 4 m in diameter. This information can be used to monitor, and possibly modulate, small avian follicle development in order to attenuate hyperactivation and decreased fertility in broiler breeder females.
Publications
- Anthony, K., and F.J. Diaz. 2009. Early avian follicular development is associated with changes in mRNA abundance in vivo and in vitro. Biol Reprod. 81(1 Supplement):508. (Abstract).
- Bartell, P. A. 2009. Clock genes and their impact on hens and eggs. Proceedings of the Pennsylvania Poultry Sales and Service Conference and 81st Northeastern Conference on Avian Diseases. Grantville, PA. 7 pp.
- Bartell, P. A., and M. Horvat-Gordon. 2009. Circadian clocks control ovarian development and oviposition. Proceedings of the 13th Annual Meeting of the Society for Behavioral Neuroendocrinology. East Lansing, MI. p. 130. (Abstract).
- Krzysik-Walker, S. M., O. M. Ocon-Grove, S. R. Maddineni, G. L. Hendricks III, and R. Ramachandran. 2007. Identification of calcitonin expression in the chicken ovary: Influence of gonadal steroids. Biol. Reprod. 77:626-635.
- Maddineni, S. R., S. M. Krzysik-Walker, O. M. Ocon-Grove, G. L. Hendricks III, and R. Ramachandran. 2007. Calcitonin is expressed in the chicken pituitary gland: Influence of gonadal steroids and sexual maturation. Cell Tiss. Res. 327:521-528.
- Maddineni S. R., O. M. Ocon, S. M. Kryzsik-Walker, G. L. Hendricks III, and R. Ramachandran. 2006. Expression of gonadotropin inhibitory hormone receptor (GnIHR) in the chicken: Regulation by ovarian steroids. Poult. Sci. 85(Suppl. 1):42. (Abstract).
- Maddineni, S.R., and R. Ramachandran. 2004. Cloning of chicken gonadotropin-inhibitory hormone precursor protein cDNA. Proceedings of the VIII International Symposium on Avian Endocrinology. Scottsdale, AZ. p. 88. (Abstract).
- Maddineni, S. R., and R. Ramachandran, R. 2003. Gallus gallus gonadotropin-inhibiting hormone precursor, mRNA, complete CDs. GenBank Accession # AY442186. http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgidb=nucleotide&val=3823 0118.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Our laboratory is elucidating the influence of hypothalamic and adipose tissue hormones on avian reproduction. A novel hypothalamic hormone, termed as gonadotropin inhibitory hormone (GnIH), has been found to inhibit gonadotropin secretion both in vitro and in vivo in several avian and mammalian species. We cloned and characterized the complementary deoxyribonucleic acids (cDNA) as well as the proteins that encode for GnIH and its receptor (GnIHR). Affinity purified anti-chicken GnIH antibody was raised and utilized for the immunohistochemical detection of GnIH-immunoreactive (ir) cells in the chicken brain. GnIH-ir cell bodies were exclusively found in the hypothalamic paraventricular nucleus. An extensive network of GnIH-ir neuronal fibers were found in the tuberal hypothalamus including median eminence pars tuberalis. GnIH-ir cell numbers in the hypothalamus were found to be not different in laying hens compared to non-laying hens. Similarly, hypothalamic GnIH mRNA quantity was not different in the laying and non-laying hens. In vitro studies revealed that GnIH treatment significantly decreased luteinizing hormone (LH) secretion in the pituitary glands from sexually immature, but not from sexually mature chickens. In contrast, GnIH treatment did not change follicle stimulating hormone (FSH) secretion from both sexually immature and laying hen pituitary glands in vitro. Similarly, in vivo administration of GnIH to Leghorn chickens did not change plasma levels of FSH or LH. GnIHR mRNA quantity was found to be the greatest in the diencephalon compared to that of anterior pituitary gland or ovary. Affinity purified anti- GnIHR antibody was developed and utilized to identify GnIHR-ir cells in the anterior pituitary gland. Several GnIH-ir cells were colocalized in LH mRNA or FSH mRNA-expressing cells. GnIHR mRNA quantity in total ovary was significantly greater in sexually immature chicken versus sexually mature hen. GnIHR mRNA expression was significantly reduced in total ovary and pituitary gland of chickens treated with estradiol or estradiol + progesterone. GnIHR mRNA quantity was significantly reduced in the ovary of chickens treated with progesterone alone. Within the ovary, greater GnIHR mRNA quantity was found in theca cells of prehierarchial follicles compared to that of preovulatory follicles. Granulosa cells from 6-8 mm prehierarchial follicles were cultured for 12 h with various concentrations of chicken GnIH with or without recombinant human FSH. The viability of granulosa cells was significantly decreased by treatment with GnIH. No significant changes in cellular viability were observed in response to a combination of FSH and GnIH treatments. Similarly, treatment of granulosa cells dispersed from the F1 preovulatory follicle with various amounts of GnIH did not affect basal or ovine LH-induced progesterone secretion. In summary, GnIH and GnIHR are likely to exert anti-gonadal effect at pituitary and ovarian levels by affecting gonadotropin secretion, gonadal steroidogenesis, and ovarian follicular development. Such inhibitory role of GnIH is likely to be controlled at the receptor (GnIHR) level. PARTICIPANTS: Dr. Ramachandran contributed to the design and execution of the project. A part of this project was conducted by research technician (Dr. Gilbert Hendricks III) in Dr. Ramachandran's laboratory. In addition, this research project trained several graduate students (Dr. Sreenivasa R. Maddineni, Dr. Olga Ocon-Grove, and Ms. Susan Krzysik-Walker). TARGET AUDIENCES: Scientists in academic research laboratories and commercial egg producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The present research findings have advanced our knowledge of the role of GnIH, a novel hypothalamic peptide, and its receptor in chicken reproduction. In particular, we have established that GnIHR expressed in the chicken gonads and pituitary gland is likely to affect ovarian follicular maturation. Follicular maturation in the chicken ovary is an important process that determines ovulation and egg production in the laying hen. Our research results will aid in the further characterization of molecular signaling pathways involved in ovarian follicular maturation and therefore may help to devise methods for prolonging or maintaining economic reproductive cycle of laying hens beyond the present 72 weeks-of-age.
Publications
- Maddineni, S. R., O. M. Ocon-Grove, S. M. Krzysik-Walker, G. L. Hendricks III, and R. Ramachandran. 2008. Gonadotropin-inhibitory hormone (GnIH) receptor gene is expressed in the chicken ovary: Potential role of GnIH in follicular maturation. Reproduction 135:267-274.
- Maddineni, S. R., O. M. Ocon-Grove, S. M. Krzysik-Walker, G. L. Hendricks III, and R. Ramachandran. 2008. Gonadotrophin-inhibitory hormone receptor expression in the chicken pituitary gland: Potential influence of sexual maturation and gonadal steroids. J. Neuroendocrinol. 20:1078-1088.
- Maddineni, S. R. 2008. Characterization of gonadotropin-inhibitory hormone and its receptor in the chicken reproductive system. Ph. D. Thesis. The Pennsylvania State University, University Park, PA. 123 pp.
- Ocon-Grove, O. M., S. M. Krzysik-Walker, S. R. Maddineni, G. L. Hendricks III, and R. Ramachandran. 2008. Adiponectin and its receptors are expressed in the chicken testis: Influence of sexual maturation on testicular AdipoR1 and AdipoR2 mRNA abundance. Reproduction 136:627-638.
- Krzysik-Walker, S. M., O. M. Ocon-Grove, S. R. Maddineni, G. L. Hendricks III, and R. Ramachandran. 2008. Changes in cellular localization of Nampt/PBEF/visfatin in the chicken testis: Influence of sexual maturation. Poult. Sci. 87 (Suppl.1):17. (Abstract).
- Ocon-Grove, O. M., S. M. Krzysik-Walker, G. L. Hendricks III, J. Giles, P. A. Johnson, and R. Ramachandran. 2008. Expression of adiponectin and its receptors, AdipoR1, AdipoR2 and T-cadherin, in chicken ovarian tumors. Proceedings of the 41st Annual meeting of the Society for Study of Reproduction. Abstract #363. p. 151.
- Bedecarrats, G. Y., H. McFarlane, S. Maddineni, and R. Ramachandran. 2008. Gonadotropin inhibitory hormone receptor signalling and its impact on reproduction in chickens. Gen. Comp. Endocrinol. (Pending).
- Shimizu, M., S. R. Maddineni, G. Y. Bedecarrats, and R. Ramachandran. 2008. Chicken gonadotropin inhibitory hormone acts via the adenylate cyclase / cAMP pathway to inhibit gonadotropin releasing hormone receptor II signalling. Proceedings of the IX International Symposium on Avian Endocrinology, Leuven, Belgium. p. 28. (Abstract).
- McGuire, N. L., T. Ubuka, K. Tsutsui, R. Ramachandran, N. Perfito, and G. E. Bentley. 2008. Neuropeptides in the gonad. Proceedings of the 90th Annual Meeting of The Endocrine Society, San Francisco, CA. Abstract #P2-675.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have previously reported the dose-response relationship for nicarbazin, a coccidiostatic drug, on egg production and hatchability in White Pekin ducks. In those studies, female ducks receiving either 250 or 500 ppm nicarbazin had reduced egg production within two days of treatment. The remaining treatment groups (fed 31.25, 62.5, or 125 ppm nicarbazin) began to exhibit decreased egg production within 3 to 8 days. By the end of the 14-day study, all nicarbazin groups had significantly lower rates of egg production compared to the control group. Recovery of egg production after withdrawal of nicarbazin from the diets occurred in reverse order of treatment dose beginning 2 days after drug withdrawal, with complete recovery being attained by all but the 125 ppm group by 4 days. All nicarbazin groups had significantly lower fertility than the control group after only 3 days of treatment; by day 7, the 500 ppm group had 100% infertile eggs and a hen-day egg production rate of
only 20%. The 125 and 250 ppm treatments exhibited 100 % infertility by day 27 (12 days post-treatment). As was the case with egg production, recovery of fertility occurred at a rate that was inversely related to the dietary dose. Western blot analysis clearly demonstrated a reduction in the presence of immunoreactive zona pellucida protein 3 (ZP3), the putative sperm receptor, in the perivitelline membrane of eggs laid by nicarbazin treated ducks. Furthermore, there were far fewer (<5%) sperm trapped in the perivitelline membrane of eggs from nicarbazin-treated ducks than in those from controls. The decreased fertility, Western blot results, and dearth of trapped sperm in eggs from nicarbazin-treated ducks implied a direct and negative effect of nicarbazin on ZP3.
PARTICIPANTS: Guy F. Barbato is the prinicpal investigator and founding author of this project. Ms. R. Katani, research aide, performed critical laboratory and statistical analyses involved in the process. Ms. Katani attended several molecular biology and bioinformatics workshops to improve her skills in these areas. Further, she also enrolled in BMMB/IBIOS 551 (Comparative Genomics) for credit to pursue advanced concepts in this area. The workshops were: (1) Techniques in Molecular Biology, (2) Bioinformatics, (3) PCR analysis, (4) CRYO and (5) 3D Electron Microscopy Workshop & Ultra-microtome workshop. Ms. V. Reinoso, a minority graduate student, performed several experiments as a portion of her Masters thesis and contributed to this project. Ms. Reinoso presented a subset of results at the Poultry Science Association's 2007 Joint Annual Meeting in San Antonio, Texas.
TARGET AUDIENCES: Poultry geneticists and poultry companies that own, or have interest in poultry breeding are the primary targets for this project. In addition, evidence that nicarbazin can be effectively used as a contraceptive for avian pest species will be of interest to pest control companies. Governmental agencies such as the EPA and state DEP will also have an interest.
Impacts
These experiments add to the body of data demonstrating the utility of the Pekin duck model to investigate the reproductive toxicology of potential environmental pollutants and/or drug residues. Further, our work suggests that nicarbazin holds great promise as a contraceptive agent for other waterfowl (e.g. the Black duck in southern Florida or resident Canadian Geese distributed along the entire east coast of the U.S.) by inhibiting the expression of the zona pellucida protein 3 (ZP3), the major sperm-binding protein on the avian vitelline membrane. Moderate inhibition of ZP3 decreases the possibility of sperm fertilizing the egg, thereby reducing fecundity. More complete inhibition of ZP3 prevents the yolk membrane from forming, which results in a total inhibition of egg production.
Publications
- Reinoso, V. P., R. Katani, and G. F. Barbato. 2007. Nicarbazin reduces egg production and fertility in White Pekin ducks via reducing ZP3 in the perivitelline membrane. Poultry Sci. 86 (Suppl. 1):536.
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Progress 01/01/06 to 12/31/06
Outputs
A study determined the dose-response relationship for the effect of nicarbazin in reducing egg production and hatchability in White Pekin ducks. Six doses of nicarbazin were used - 0 ppm (control), 31.25 ppm, 62.5 ppm, 125 ppm, 250 ppm and 500 ppm. Ducks receiving either 250 or 500 ppm nicarbazin had attenuated egg production within 2 days of treatment, followed in the next 2 days by the 125 ppm group. The remaining groups (31.25 and 62.5 ppm) began to exhibit decreased egg production within 8 days. By the end of the treatment period, all nicarbazin groups had significantly lower rates of egg production than the control group. There were no significant differences across treatments in early dead embryos (defined as undeveloped embryos at less than 7 days incubation - 1.6%), late dead embryos (defined as any dead embryo greater than 7 but less than 21 days development - 3.4%), or un-pipped eggs (usually greater than 21 days development - 1.5%). Furthermore, no
observation of teratogenic activity was noted in any of the treatment groups. Within 2 days of drug treatment, average fertility rates began to decrease. All nicarbazin groups had significantly lower fertility than the control group within 3 days. By seven days of nicarbazin treatment, the 500 ppm group produced no fertile eggs (although they were only laying at 20%). The 125 and 250 ppm treatments had zero fertility after 12 days post-treatment. The 31.25 and 62.5 ppm groups declined to 30% fertility by the 14th day of treatment.
Impacts
These experiments have demonstrated the utility of the Pekin duck model to investigate the reproductive toxicology of potential environmental pollutants and/or drug residues. Further, we have specifically demonstrated the value of nicarbazin as a potential contraceptive agent for waterfowl. This technology may prove to be effective in controlling the populations of nuisance birds in urban environments.
Publications
- No publications reported this period
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