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.

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth.

The obligatory role of follicle-stimulating hormone (FSH) in normal development and function of ovarian antral follicles is well recognized, but its function in preantral growth is less clear. The specific objective of this study was to investigate the response, in culture, to FSH of mouse preantral follicles of increasing size, focusing particularly on growth rate and gene expression. Preantral follicles were mechanically isolated from ovaries of C57BL/6 mice, 12 to 16 days postpartum, and single follicles cultured for up to 96 hours in medium alone (n = 511) or with recombinant human FSH 10 ng/mL (n = 546). Data were grouped according to initial follicle diameter in 6 strata ranging from <100 to >140 μm. Follicles of all sizes grew in the absence of FSH (P < 0.01, paired t test). All follicles grew at a faster rate (P < 0.0001) in the presence of 10 ng/mL FSH but larger follicles showed the greatest change in response to FSH. Even the smallest follicles expressed FSH receptor messenger RNA (mRNA). FSH-induced growth was inhibited by KT5720, an inhibitor of protein kinase A (PKA), implicating the PKA pathway in FSH-induced follicle growth. In response to FSH in vitro, FSH receptor mRNA (measured by quantitative polymerase chain reaction) was reduced (P < 0.01), as was Amh (P < 0.01), whereas expression of StAR (P < 0.0001) and the steroidogenic enzymes Cyp11a1 (P < 0.01) and Cyp19 (P < 0.0001) was increased. These results show heterogeneous responses to FSH according to initial follicle size, smaller follicles being less FSH dependent than larger preantral follicles. These findings strongly suggest that FSH has a physiological role in preantral follicle growth and function.

Kisspeptin plays a pivotal role in pubertal onset and reproductive function. In rodents, kisspeptin perikarya are located in 2 major populations: the anteroventral periventricular nucleus and the hypothalamic arcuate nucleus (ARC). These nuclei are believed to play functionally distinct roles in the control of reproduction. The anteroventral periventricular nucleus population is thought to be critical in the generation of the LH surge. However, the physiological role played by the ARC kisspeptin neurons remains to be fully elucidated. We used bilateral stereotactic injection of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC of adult female rats to investigate the physiological role of kisspeptin neurons in this nucleus. Female rats with kisspeptin knockdown in the ARC displayed a significantly reduced number of both regular and complete oestrous cycles and significantly longer cycles over the 100-day period of the study. Further, kisspeptin knockdown in the ARC resulted in a decrease in LH pulse frequency. These data suggest that maintenance of ARC-kisspeptin levels is essential for normal pulsatile LH release and oestrous cyclicity.