Source: CORNELL UNIVERSITY submitted to NRP
OVARIAN FOLLICULAR DEVELOPMENT IN CATTLE: ACTIVATION AND GROWTH IN VITRO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0196512
Grant No.
2003-35203-13532
Cumulative Award Amt.
$260,000.00
Proposal No.
2003-02730
Multistate No.
(N/A)
Project Start Date
Sep 1, 2003
Project End Date
Aug 31, 2008
Grant Year
2003
Program Code
[41.0]- (N/A)
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
BIOMEDICAL SCIENCES
Non Technical Summary
In mammalian ovaries, most oocytes reside in non-growing, primordial follicles. Virtually nothing is known about the mechanisms that regulate movement of these follicles into the growing pool (activation). The pool of resting primordial follicles is a resource, as yet untapped, that could be exploited as a source of material for in vitro fertilization to hasten genetic improvement of domestic species, preserve endangered species, and provide alternative methods for alleviating infertility in women. This proposal has two complementary goals: 1) to investigate the signals that regulate the activation of primordial follicles and 2) to develop methods for inducing further growth of follicles activated in vitro. When pieces of bovine ovarian cortex are cultured in vitro, almost all the primordial follicles begin to grow, but when bovine cortical pieces are grafted beneath the chorioallantoic membrane of chick embryos, the wholesale, spontaneous activation observed in vitro is completely absent. We propose to use these two complementary experimental systems to address fundamental questions about the signals that control activation of primordial follicles and subsequent follicle growth.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30134101020100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
In mammalian ovaries, most oocytes reside in primordial follicles in a resting stage. Little is known about the mechanisms that regulate the movement of these follicles into the growing pool (activation). The pool of resting primordial follicles is a resource, as yet untapped, that could be exploited as a source of material for in vitro maturation (IVM) and in vitro fertilization (IVF) to hasten genetic improvement of domestic species, preserve endangered species, and provide alternative methods for alleviating infertility in women. This proposal has two complementary goals: 1) to investigate the signals that regulate the entry of resting primordial follicles into the cohort of growing follicles and 2) to develop methods for inducing growth and development in vitro of primordial follicles to a stage where their oocytes are capable of IVM, IVF and embryonic development.
Project Methods
Two complementary experimental systems will be used: 1) culture of pieces of bovine ovarian cortex; in this situation almost all primordial follicles in the cortex activate, 2) grafting ovarian cortical pieces beneath the chorio-allantoic membrane (CAM) of chick embryos; in this situation follicle growth arrest is maintained. In Specific Aim #1 the effects of a potential inhibitor (anti-Mullerian hormone) on activation of bovine primordial follicles will be determined, using the in-vitro and in-ovo experimental systems. In Specific Aim #2, we will test the hypothesis that one or more of five specific hormones/factors that have been implicated in development of preantral follicles (androgens, growth hormone, basic fibroblast growth factor, vascular endothelial growth factor, and growth differentiation factor-9) will enhance bovine follicular development in vitro by promoting the primary to secondary follicle transition. These experiments are crucial steps towards the ultimate goal of using the large numbers of primordial follicles in mammalian ovaries as a resource for genetic improvement of domestic animals, preservation of endangered species, and alleviation of human infertility.

Progress 01/01/06 to 12/31/06

Outputs
The signals that allow or stimulate bovine primordial follicles to leave the resting pool were unknown. It is also unclear whether a stimulator, an inhibitor, or a balance between stimulator/inhibitor is the primary factor involved. We have made significant progress on Specific Aim #1, to determine the effects of potential stimulators and inhibitors of the activation of bovine primordial follicles. Using a culture system for small pieces of ovarian cortex (where primordial follicles are located) from third-trimester bovine fetal ovaries, we identified two stimulators of follicle activation in vitro. 1) Insulin increased the number of follicles that activate in a dose-dependent fashion, with the highest effective doses promoting the activation of 80-90% of primordial follicles. In contrast, addition of kit ligand (KL) to the cultures activated a maximum of about 50% of the primordial follicles. Using a combination of in vitro and in ovo (ovarian cortical pieces transplanted beneath the chorioallantoic membrane of chick embryos) approaches with whole mouse ovaries and pieces of bovine ovarian cortex, we showed that anti-Mullerian hormone (AMH), secreted by the developing male and female gonads of chick embryos, inhibits follicle activation in whole mouse ovaries (direct evidence) and in bovine ovarian cortex (indirect evidence). Thus, we have identified insulin and KL as factors that stimulate the activation of bovine primordial follicles and hypothesize that KL's effects are more physiological than those of insulin. And we have provided the first evidence for AMH as an inhibitor of bovine follicle activation. The next step is to determine the interrelationships among insulin, KL, and AMH. Specific Aim #2 is to determine factors/conditions that promote the growth of bovine ovarian follicles that have been activated in vitro, specifically the primary to secondary follicle transition. We tested the effects of different oxygen concentrations (2, 5, 20, and 60%) and supplements to the culture medium (various concentrations of fetal bovine serum and ITS+ [insulin, transferrin, and selenium + linoleic acid and BSA], alone and in combination) on the primary to secondary follicle transition. Oxygen concentrations of 2 and 5% were no more effective than the usual concentration (20%) and 60% oxygen was detrimental to follicular health. In the presence of 0.5X ITS+, 5 or 10% serum increased the number of secondary follicles about 9-fold, compared with 0.5X ITS+ alone. However, the most striking results for Specific Aim #2 were dose-dependent increases in the number of secondary follicles when ovarian cortical pieces were cultured with testosterone or vascular endothelial growth factor (VEGF). In contrast, neither bFGF nor GDF9 affected the number of secondary follicles, but one or both of these factors may enhance the effects of testosterone and/or VEGF or may facilitate the primary to secondary transition when follicles are activated with KL. We would like to test those hypotheses in the next year.

Impacts
In the ovaries of mammals, most potential female gametes (oocytes) reside within ovarian follicles that are not growing (primordial follicles). The pool of resting, primordial follicles is a resource that could be exploited to hasten genetic improvement of domestic species, preserve endangered species, and provide alternative methods for alleviating some types of infertility in women. However, progress towards those practical goals is currently hampered by our lack of understanding of the mechanisms that regulate the movement of resting follicles into the growing pool and the factors necessary to support their further growth to the stage when the oocyte is capable of completing meiosis, being fertilized, and producing live young. Our laboratory developed two experimental systems for studying the initial stages of follicular development in cattle. During the current grant period, we have thus far used those methods to determine that a hormone (insulin) and a growth factor (kit ligand) stimulate primordial follicles to leave the resting pool and begin to grow and that anti-Mullerian hormone inhibits the initiation of growth. In addition, we have shown that a hormone (testosterone) and a growth factor (vascular endothelial growth factor) can promote the continued growth of follicles. Thus, we have made progress towards the ultimate goal of understanding the signals that regulate follicle growth so that primordial follicles from valuable domestic animals can be stimulated to grow in organ culture and produce female gametes that can be fertilized and produce live young.

Publications

  • Jo, M. and J.E. Fortune, 2003. Changes in oxytocin receptor in bovine preovulatory follicles between the gonadotropin surge and ovulation. Molecular & Cellular Endocrinology 200: 31-43.
  • Fortune, J.E., G.M. Rivera, and M.Y. Yang, 2004. Follicular development: the role of the follicular microenvironment in selection of the dominant follicle. Animal Reproduction Science 82-83:109-126. (This review article contains original data on the role of insulin vs. IGF in early follicular development)
  • Gigli, I., R.A. Cushman, C.M. Wahl, and J.E. Fortune, 2004. Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane. Molecular Reproduction and Development 71:480-488.
  • Gigli, I., D.D. Byrd, and J.E. Fortune, 2006. Effects of culture medium and oxygen tension on activation and growth of bovine follicles in vitro. Theriogenology 66: 344-353.
  • Fortune, J.E., 2003. The early stages of follicular development: activation of primordial follicles and growth of preantral follicles. Animal Reproduction Science 78:135-163. (This review article of the regulation of early follicular development includes our USDA-supported results; no new, original data are presented)
  • Yang, M.Y. and J.E. Fortune, 2005. Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro. Biology of Reproduction 75:924-32.
  • Yang, M.Y. and J.E. Fortune, 2005. Vascular endothelial growth factor (VEGF) stimulates the primary to secondary follicle transition in bovine follicles in vitro. Molecular Reproduction and Development 74:1095-1104.