The absolute requirement for reproduction implies that the hypothalamo-pituitary-gonadal axis, controlling fertility, is an evolutionary robust mechanism. The GnRH neurons of the hypothalamus represent the key cell type within the body dictating fertility. However, the level of functional redundancy within the GnRH neuron population is unknown. As a result of a fortuitous transgene insertion event, GNR23 mice exhibit a marked allele-dependent reduction in GnRH neuron number within their brain. Wild-type mice have approximately 600 GnRH neurons, compared with approximately 200 (34%) and approximately 70 (12%) in GNR23(+/-) and GNR23(-/-) mice, respectively. Using these mice, we examined the minimal GnRH neuron requirements for fertility. Male GNR23(-/-) mice exhibited normal fertility. In contrast, female GNR23(-/-) mice were markedly subfertile, failing to produce normal litters, have estrous cycles, or ovulate. The failure of ovulation resulted from an inability of the few existing GnRH neurons to generate the LH surge. This was not the case, however, for the first cycle at puberty that appeared normal. Together, these observations demonstrate that 12% of the GnRH neuron population is sufficient for pulsatile gonadotropin secretion and puberty onset, whereas between 12 and 34% are required for cyclical control in adult female mice. This indicates that substantial redundancy exists within the GnRH neuronal population and suggests that the great majority of GnRH neurons must be dysfunctional before fertility is affected.

In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.

Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS.