In female vertebrate animals that ovulate a limited number of oocytes (eggs) per ovulation cycle (e.g., mammals, birds, some reptiles), it is the process of ovarian follicle selection that represents the rate-limiting step to fecundity. Follicle selection entails the selective recruitment of one (or more) undifferentiated follicle(s) into the final stages of maturation prior to ovulation, yet the underlying mechanisms of this process are currently undefined in any vertebrate species. Ultimately, the selection mechanism determines the maximum number of offspring that can be produced within any given reproductive cycle. The present studies will test the hypothesis that the inability of undifferentiated follicles to spontaneously undergo follicle selection is normally due to the active suppression of one or more genes (e.g., the Follicle Stimulating Hormone receptor; FSHR) that are required for the differentiation of the follicle's granulosa cell layer. Importantly, it is the granulosa cell layer that nurtures the oocyte throughout follicle development. A second hypothesis to be tested is that at the time of follicle selection there is a switch from a negative regulation of FSHR gene expression to an upregulation of expression. This event is widely assumed to be prerequisite for the maturation of ovarian follicles to the preovulatory stage. The experimental design incorporates cellular and molecular techniques directed at identifying genomic mechanisms by which the expression of genes critical to this selection process in chickens is repressed or enhanced. Results from these studies are predicted to impact our ability to enhance female fertility in endangered species and domesticated food animals, and to better understand select causes of infertility in women. More broadly, the proposed research will serve to provide relevant and cutting-edge training to undergraduate and graduate students (including those from underrepresented populations) whose career goals include teaching and/or research.
In vertebrate females that ovulate a limited and species-specific number of eggs (1 to perhaps as many as 2 dozen) each ovulation cycle, it is the process of ovarian ‘follicle selection’ that represents the rate limiting step to producing mature gametes for fertilization. This entails a process by which one or at most a few undifferentiated follicle(s) are recruited from a cohort of slow-growing follicles to initiate final differentiation. The process of selection initiates rapid follicle growth and oocyte maturation, and determines the reproductive capacity in those species whose strategy emphasizes a limited number of offspring associated with significant parental investment (e.g., mammals, birds, some reptiles). Unfortunately, the exact mechanism that establishes which and how many follicles are selected each fertile cycle currently remains undefined in any vertebrate species. Among the unique morphological and functional characteristics of the avian ovary, as compared to their mammalian counterparts, is that ovarian follicles at all stages of development, from resting primordial and primary follicles to the fully differentiated preovulatory stage, exist simultaneously during egg-laying. The selection of a single, follicle for final growth and maturation occurs once daily. In the laying hen, the four to six largest preovulatory follicles (e.g., F6 to F1; Fig. 1) represent those that have already undergone the process of follicle selection. This progressively more mature group of follicles enables a single egg to be laid per day to establish a characteristic clutch size before initiating incubation. By all standards, these ‘preovulatory’ follicles are considered actively differentiating (e.g., the most recently selected F6; measuring 9-12 mm) or terminally differentiated (e.g., F1 follicle). By contrast, follicles smaller than 9 mm are considered undifferentiated (Fig. 1, arrows), and it is from this cohort of 'prehierarchal follicles' that a single follicle per day is selected for rapid growth, final maturation, and eventually, ovulation. Our studies have focused on identifying the cell signaling and molecular mechanisms that define the process of 'selecting' a single follicle each day. Our results demonstrate that in the avian ovary, prehierarchal follicles are maintained in an undifferentiated state (e.g., prevented from being selected prematurely) by inhibitory Mitogen Activated Protein Kinase cell signaling (MAPK, also called ERK1/2, signaling), specifically within the granulosa layer. In turn, this inhibitory signaling initiates the production of Inhibitor of Differentiation (Id) proteins that act to regulate gene expression and prevent cell differentiation. It follows then, that the selection of a follicle each day can only occur following the removal of inhibitory MAPK signaling. We have established that the removal of this inhibitory signaling is directly associated with an initiation of cell signaling that promotes differentiation. The stimulatory cell signaling is generated by Follicle Stimulating Hormone (FSH) hormone acting through its FSH receptor and the production of the cellular messenger, cyclic adenosine monophosphate (cAMP). We also demonstrate that cell signaling through this receptor is supported by Bone Morphogenetic Proteins 4 and 6 (BMP4, BMP6) but negatively regulated by BMP2. In particular, BMP4 and BMP6 maintain FSH receptor expression in the inner most granulosa cell layer, while BMP2 promotes expression of Id proteins. While we now know that selection and the initiation of follicle growth and differentiation directly results from the positive actions of FSH receptor signaling and cAMP, we have yet to identify the most proximal mechanism that terminates the inhibitory MAPK signaling or how only a single follicle is selected each ovulatory cycle. Nevertheless, results from this funding period have now provided us with critical information about the nature and characteristics of this proximal mechanism. These data have formed the basis of our ongoing studies. Ultimately, our objectives are to compare mechanisms responsible for follicle selection in avian species to those in eutherian mammals. Importantly, results from these studies have practical application towards enhancing fecundity in threatened/endangered birds and mammals plus food-producing animals, and even pertain to understanding causes of multiple (fraternal) births in humans.
