Progress 11/25/14 to 09/30/18
Outputs
Target Audience:Research results from the Johnson lab have been reported at the Society for the Study of Reproduction (SSR) and the Poultry Science Association annual meetings in 2018. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One Ph.D. student (expected to complete program in October 2018) trained by ALJ. How have the results been disseminated to communities of interest?Both PIs publish in appropriate scientific journals and communicate recent results to undergaduate and graduate students both informally (e.g., lab meetings) and formally (in seminars and formal teaching). In addition, both PIs and their students attend annual Society meetings to present their research findings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal for this project (directed by ALJ) is to understand the cellular mechanisms responsible for the daily recruitment of a single ovarian follicle into the rapid growth (preovulatory) stage of development using the commercial laying hen and turkey hen as a primary model systems. The regulation of such selection is prerequisite for the approximate daily ovulation, and determines the duration of egg production in both chickens and turkeys. Published data from the laying hensupport the proposal that the initiation of cell differentiation within the granulosa cell (GC) layer is initiated at the time of folliclerecruitment into the preovulatory hierarchy. This process is characterized by the initial capacity for FSH-induced cell signalingvia the protein kinase A/cyclic adenosinemonophosphate (cAMP) pathway. Critical consequences of such signaling includethe initial capacity for GC steroidogenesis, the initiation of rhythmic clock gene expression and the enhancement of follicle ultimately escapesinhibitory signaling to initiate FSH-responsiveness remains the focus of our investigations. Importantly, we have determined that this process is regulated at the level of G-protein coupled receptors (GPCRs), including the follicle stimulating hormone receptor FSHR) and vasoactive intestinal peptide receptors (VPAC1 and VPAC2) by a beta arrestin-mediated event. In turn, beta arrestin-regulated receptor signaling is regulated by mitogen-activated protein kinase (MAPK) signaling. Overall, the results of these studies are intended to help direct the genetic selection of broiler breeder hens, which inherently are predisposed to multiple follicles recruited into the preovulatory hierarchy resulting in double ovulations. Moreover, the results may eventually be used to assist in the selection of turkeys for the purpose of enhancing overall egg production. Finally, we conducted studies of bovine GCs in an effort to study comparable processes from a comparative perspective.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Ghanem, K. and Johnson, A.L. Follicle Dynamics and Granulosa Cell Differentiation in the Turkey Hen Ovary. Poultry Science http://dx.doi.org/10.3382/ps/pey224
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kim, D. and Johnson, A.L. Differentiation of hen granulosa cells from prehierarchal follicles following the initiation of follicle stimulating hormone receptor signaling. Mol. Reprod. & Develop., in press. DOI: http://dx.10.1002/mrd.23042
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kim, D. and Johnson, A.L. Regulation of Vasoactive Intestinal Peptide Receptor (VPAC) Signaling in Undifferentiated Hen Granulosa Cells. Mol. Reprod. & Develop. DOI: 10.1002/mrd.23054
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Ghanem, K. and Johnson, A.L. Time of follicle cyclic recruitment is not temporally linked to ovulation in the laying hen ovary. Submitted to Poultry Science.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Ghanem, K. and Johnson, A.L. Differential Response of Hen Ovarian Pre-recruitment Follicles to Follicle Stimulating Hormone. Submitted to Gen. Comp. Endocrinol.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Both scientists (ALJ & RR) have listed publications in high quality professional journals originating from related work, and have provided summaries of their research at International Symposia and to undergraduate Animal Science majors via formal courses (graduate and undergraduate), independent research projects, and the Poultry Science club at Penn State. Changes/Problems:No significant changes in the direction of either Investigator's lab are anticipated. What opportunities for training and professional development has the project provided?One Ph.D. student (fourth year) and a senior undergraduate conducting an independent research project have been supervised by ALJ during the past year. How have the results been disseminated to communities of interest?Both PIs routinely publish in appropriate scientific journals and communicate recent results to undergaduate and graduate students. In addition, both PIs attend annual Society meetings, including the Poultry Science Association and Society for the Study of Reproduction annual meetings. What do you plan to do during the next reporting period to accomplish the goals?Both the Johnson and Ramachandran labs will continue to conduct related studies in the laying hen, broiler breeder hens and turkey hens. In particular, studies in the Ramachandran lab will compare the beneficial effects of metformin on egg production in broiler breeder hens. This project was recently funded by NIFA . The Johnson lab continues to focus on identifying the most proximal cellular mechanism that results in follicle recruitment. We continue to focus on the granulosa layer as recently published studies from our lab have established that the most proximal event associated with the process of follicle recruitment into the preovulatory hierarchy is the initial capacity for follicle stimulating hormone (FSH)-induced accumation of cAMP specifically within this layer. This mechanism/event represents that which determines the sequence/clutch length (thus overall egg production) in domesticated as well as wild species. A proteomics study currently in progress to identify specific proteins that become initially expressed or differentially expressed specifically within the granulosa layer at follicle recruitment. In the coming year we will follow up with a phosphoproteomics study to establish the relevant cell signaling pathways directly associated with follicle recruitment. Ongoing studies in the Ramachandran lab will focus on the molecular events underlying increased egg production in metformin-supplemented chickens. From a practical perspective, the results from both investigators' laboratories contribute most directly to understanding the misregulation of the preovulatory hierarchy and reduced egg production as occurs in broiler breeder hens.
Impacts
What was accomplished under these goals?
The over-riding goal for the studies directed by ALJ is to identify the cellular mechanisms responsible for the daily recruitment of a single small, slow-growing ovarian follicle into the rapid growth (preovulatory) stage of development using the commercial laying hen as a primary model system. The regulation of such recruitment is prerequisite to maintain the approximate daily ovulation, and determines the duration of egg production in both chickens (laying hens and broiler breeder hens) and turkeys. Published data from the laying hen support the proposal that cell differentiation within the granulosa cell (GC) layer is initiated at the time of follicle recruitment into the preovulatory hierarchy. This process is mediated by the initial capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. Among the critical outcomes of such signaling include the initial capacity for GC steroidogenesis, the initiation of rhythmic clock gene expression and the enhancement of follicle vascularization. The process by which the GC layer from the single follicle selected each ovulatory cycle ultimately escapes inhibitory signaling to initiate FSH-responsiveness remains the focus of our investigations. Overall, the results of these studies are intended to help direct the genetic selection of broiler breeder hens, which inherently are predisposed to more than one follicle recruited into the preovulatory hierarchy resulting in double ovulations. Moreover, the results may eventually be used to assist in the selection of turkeys for the purpose of enhancing overall egg production by increasing clutch length. Dr. Ramachandran's lab is focused on broiler breeder hens which have the poorest reproductive efficiency of all commercial avian species primarily due to excessive feed intake and adiposity. Poor egg production in broiler breeder hens results from either insufficient or excessive ovarian follicular recruitment. Several pilot studies were conducted to determine if dietary supplementation of metformin (an anti-diabetic drug) to broiler breeder hens corrects aberrant recruitment of ovarian follicles thereby increasing egg production. The pullets were photostimulated with a gradual increase in daylight culminating in a photoperiod of 16h light: 8h dark by the time the chickens reached a critical body mass (22 weeks). The hens were reared individually in wire cages to record egg production every day. Starting at 35-weeks of age, the hens were fed a diet containing metformin (0, 25, 50, or 75 mg/kg bw) until 57-weeks of age. Dietary metformin supplementation at 50 or 75 mg/kg bw level was found to sustain a significantly higher level of egg production beyond 18 weeks of treatment. Consequently, the cumulative egg production was significantly greater (by at least 15 eggs/hen) in chickens fed with a diet containing metformin at 50 or 75 mg/kg bw level compared to 0 or 25 mg/kg bw level. Ongoing studies will determine the molecular events underlying increased egg production in metformin-supplemented chickens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kim, D., Lee, J. and Johnson, A.L. (2016) Expression of Vascular Endothelial Growth Factor and Angiopoietins during Hen Follicle Development. Gen. Comp. Endocrinol., 232:25-31.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kim, D. and Johnson, A.L. (2016) Vasoactive Intestinal Peptide Promotes Differentiation and Clock Gene Expression in Granulosa Cells from Prehierarchal Follicles. Mol. Reprod. Develop., 83:455-463.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Johnson, A.L. and Ghanem, K., Turkey ovarian follicle dynamics and granulosa cell responsiveness to follicle-stimulating hormone.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Ghanem, K. and Johnson, A.L. Hen Ovarian Pre-recruitment Follicles Respond Differentially to Follicle Stimulating Hormone.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Both scientists (ALJ & RR) have listed publications originating from related work, and have provided summaries of their research to undergraduate Animal Science majors via formal courses (graduate and undergraduate), independent research projects and the Poultry Science club at Penn State. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One Ph.D. student (third year) and a senior undergraduate conducting an independent research project are being trained, and one postdoctoral research associate worked (from July 2014 through June 2016) on the objectives directed by ALJ. How have the results been disseminated to communities of interest?Both PIs routinely publish in appropriate scientific journals and communicate recent results to undergaduate and graduate students. In addition, both PIs attend annual Society meetings and attended the XI International Symposium on Avian Endocrinology (ISAE) in Niagra-on-the-Falls, Canada in October, 2016. What do you plan to do during the next reporting period to accomplish the goals?Both the Johnson and Ramachandran labs will conduct related studies in the laying hen, broiler breeder hens and turkey hens. In particular, studies in the Ramachandran lab will compare the beneficial effects of metformin and adipoRon on egg production in broiler breeder hens while the Johnson lab will focus on identifying the most proximal cellular mechanism that results in follicle recruitment. This mechanism represents that which determines the sequence/clutch length (thus overall egg production) in domesticated and wild species. From a practical perspective, the results from both investigators' laboratories will contribute to understanding the misregulation of the preovulatory hierarchy and reduced egg production as occurs in broiler breeder hens.
Impacts
What was accomplished under these goals?
Adiponectin, a hormone secreted from adipose tissue, plays a major role in adipose tissue deposition and energy metabolism. The Ramachandran lab has previously determined that plasma adiponectin levels decreases as broiler breeder chickens accumulate visceral adipose tissue. Augmenting circulating levels of adiponectin with recombinant adiponectin is impractical as adiponectin circulates at a very high level (4-18 ug/ml) and has short biological half-life (45-60 minutes). Metformin, a biguanide anti-diabetic drug, affects glucose and lipid metabolism similar to adiponectin and corrects dysfunctional follicular development in women with polycystic ovarian syndrome. Utilizing real-time quantitative polymerase chain reaction and/or Western blotting, metformin treatment was found to increase the abundance of phospho-extra cellular receptor kinase 1/2 phospho-acetyl Co-A carboxylase, phospho-adenosine monophosphate-activated protein kinase in theca cells isolated from both prehierarchical and preovulatory follicles in a dose-dependent manner. Both basal and follicle stimulating hormone (FSH)-stimulated steroidogenic acute regulatory protein (STAR) gene expression in granulosa cells isolated from the 9-12mm follicles was found to be significantly decreased by metformin treatment in a dose-dependent manner. Ovarian cortical follicles (400 micrometer in size) were isolated from broiler breeder hen ovaries and treated with metformin (0 or 10 mM) with or without addition of chicken vasoactive intestinal polypeptide (VIP; 1 ng/ml). VIP treatment resulted in a 12-fold increase in STAR abundance that was completely abolished by addition of metformin suggesting that metformin. It is concluded that metformin treatment is likely to affect steroidogenesis and nutrient metabolism in ovarian follicular cells. A second goal for the project (directed by ALJ) are to understand the cellular mechanisms responsible for the daily recruitment of a single ovarian follicle into the rapid growth (preovulatory) stage of development using the commercial laying hen and turkey hen as a primary model systems. The regulation of such selection is prerequisite for the approximate daily ovulation, and determines the duration of egg production in both chickens and turkeys. Published data from the laying hen support the proposal that the initiation of cell differentiation within the granulosa cell (GC) layer is initiated at the time of follicle recruitment into the preovulatory hierarchy. This process is characterized by the initial capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. Critical consequences of such signaling include the initial capacity for GC steroidogenesis, the initiation of rhythmic clock gene expression and the enhancement of follicle vascularization. The process by which the GC layer from the single follicle selected each ovulatory cycle ultimately escapes inhibitory signaling to initiate FSH-responsiveness remains the focus of our investigations. In addition, recently completed studies using the turkey hen confirm that ovarian follicle dynamics and the expression of critical endocrine signaling components (e.g., FSH receptor, STAR, BMP4, BMP6, AMH), together with the mechanisms regulating the initiation of GC differentiation are highly conserved between the two species. Overall, the results of these studies are intended to help direct the genetic selection of broiler breeder hens, which inherently are predisposed to multiple follicles recruited into the preovulatory hierarchy resulting in double ovulations. Moreover, the results may eventually be used to assist in the selection of turkeys for the purpose of enhancing overall egg production. Finally, we conducted studies of bovine GCs in an effort to study comparable processes from a comparative perspective.
Publications
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Johnson, A.L., Woods, D.C. and Truman, A.M. (2016) Granulosa Cell Tumors. In: Encyclopedia of Cancer, M. Schwab, Ed., fourth Ed., doi:10.1007/978-3-642-27841-9_2508-2 .
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Johnson, A.L. (2015) Reproduction in the female. In: Sturkies Avian Physiology, 6th Ed., C.G. Scanes, Ed., New York: Elsevier, Chapter 28, pp. 635-665.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Johnson, A.L. (2016) Hormonal Mechanisms Associated with Cyclic Recruitment of Hen Ovarian Follicles. Presented at the 11th International Symposium on Avian Endocrinology (ISAE), Niagara-on-the-Lake, Canada.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Ghanem*, K and A.L. Johnson. (2016) Hen Ovarian Prehierarchal Follicles Demonstrate a Differential Sensitivity to Follicle Stimulating Hormone, In Vivo. Society for the Study of Reproduction Annual Meetings, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Anthony KA, Hadley JA, Johnson AL, Ramachandran R, Diaz FJ (2016) Vasoactive intestinal polypeptide plays a role in ovarian cortical follicle steroidogenesis in broiler breeder hens. Presented at the 49th Annual Meeting of the Society for Study of Reproduction, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Hadley JA, Ramachandran R (2016) Adiponectin and metformin affects chicken ovarian follicular cell metabolism. Presented at the 11th International Symposium on Avian Endocrinology (ISAE), Niagara Falls, Canada.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Poole, D.H., Oc�n-Grove, O.M., Johnson, A.L. (2016) Anti-M�llerian Hormone Receptor Type II Expression and Anti-M�llerian Hormone Activity in Bovine Granulosa Cells. Theriogenology, 86:(5):1353-1360.
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Progress 11/25/14 to 09/30/15
Outputs
Target Audience:Both scientists (ALJ & RR) have listed publications originating from related work, and have provided summaries of their research to undergraduate Animal Science majors via formal courses, independent research projects and the Poultry Science club at Penn State. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One Ph.D. student and a senior undergraduate conducting an independent research project are being trained, and one postdoctoral research associate is currently working on the objectives directed by ALJ. How have the results been disseminated to communities of interest?Both PIs routinely publish in appropriate scientific journals and communicate recent results to undergaduate and graduate students. What do you plan to do during the next reporting period to accomplish the goals?We (ALJ group) will continue related studies in the laying hen and extend such studies to include broiler breeder and turkey hens.
Impacts
What was accomplished under these goals?
One set of goals for the project (directed by ALJ) are to understand the cellular mechanisms responsible for the approximate daily selection of a single ovarian follicle into the rapid growth (preovulatory) stage of development in the commercial laying hen. The regulation of such selection is prerequisite for the approximate daily ovulation, and determines the duration of egg production in both chickens and turkeys. Published data support the proposal that final differentiation of the granulosa cell (GC) layer commences at the time of follicle selection. This process is characterized by the enhanced capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. Critical consequences of such signaling within the GC layer include the initial capacity for steroidogenesis and the enhancement of follicle vascularization observed at selection. Recently completed studies determined that Bone Morphogenetic Protein 6 (BMP6) enhances both CYP11A expression and FSH responsiveness in GC collected from 1-2mm, 3-5mm and 6-8mm follicles, with the greatest response again occurring in GCs from 6-8mm follicles. Significantly, factors that activate mitogen activated protein kinase (MAPK) or protein kinase C (PKC) signaling prevent BMP6 from initiating FSH-responsiveness and the initiation of GC differentiation at each stage of development. The process by which the GC layer from the single follicle selected each ovulatory cycle ultimately escapes inhibitory signaling to initiate FSH-responsiveness is currently under investigation. Overall, the results of these studies are intended to help direct the genetic selection of broiler breeder hens, which inherently are predisposed to multiple selection/double ovulations. Moreover, the results may assist in the selection of turkeys for the purpose of enhancing overall egg production. A second goal (directed by RR) is to enhance the reproductive efficiency of the broiler breeder hen. Adiponectin, a hormone secreted from adipose tissue, plays a major role in adipose tissue deposition and energy metabolism. We have earlier determined that plasma adiponectin levels decreases as broiler breeder chickens accumulate visceral adipose tissue. Augmenting circulating levels of adiponectin with recombinant adiponectin is impractical as adiponectin circulates at a very high level (4-18 ug/ml) and has short biological half-life (45-60 minutes). We have tested an orally active compound, adipoRon, that is likely to stimulate adiponectin receptors (AdipoR1 and AdipoR2). AdipoRon has been found to ameliorate type 2 diabetes, improve myocyte functions and prevent obesity in murine models. We found that adipoRon treatment at 0-50 micromolar concentrations resulted in an increase in phospho mitogen activated protein kinase 1/2 and phospho-protein kinase B (Akt) abundance levels in a dose-dependent manner in granulosa cells of preovulatory and prehierarchical follicles. Both adipoRon and recombinant chicken adiponectin dramatically reduced the expression of steroidogenic acute regulatory protein (StAR) in granulosa cells isolated from preovulatory and prehierarchical follicles suggesting a possible downregulation of steroidogenesis in ovarian follicular cells. Future studies will compare the beneficial effects of metformin and adipoRon on egg production in broiler breeder hens.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Kim, D., Lee, J. and Johnson, A.L. (2015) Expression of Vascular Endothelial Growth Factor and Angiopoietins during Hen Follicle Development. General and Comparative Endocrinology. (Under Review)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Johnson, A.L. and Lee, J. (2015). Granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles. Poultry Science, (In press)
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Johnson, A.L. (2014) Ovarian follicle selection, and granulosa cell differentiation. Poultry Science 94(4):781-785.
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