Although hormonal regulation of ovarian follicle development has been extensively investigated, most studies concentrate on the development of early antral follicles to the preovulatory stage, leading to the successful use of exogenous FSH for infertility treatment. Accumulating data indicate that preantral follicles are under stringent regulation by FSH and local intraovarian factors, thus providing the possibility to develop new therapeutic approaches. Granulosa cell-derived C-type natriuretic factor not only suppresses the final maturation of oocytes to undergo germinal vesicle breakdown before ovulation but also promotes preantral and antral follicle growth. In addition, several oocyte- and granulosa cell-derived factors stimulate preantral follicle growth by acting through wingless, receptor tyrosine kinase, receptor serine kinase, and other signaling pathways. In contrast, the ovarian Hippo signaling pathway constrains follicle growth and disruption of Hippo signaling promotes the secretion of downstream CCN growth factors capable of promoting follicle growth. Although the exact hormonal factors involved in primordial follicle activation has yet to be elucidated, the protein kinase B (AKT) and mammalian target of rapamycin signaling pathways are important for the activation of dormant primordial follicles. Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, promote the growth of preantral follicles in patients with primary ovarian insufficiency, leading to a new infertility intervention for such patients. Elucidation of intraovarian mechanisms underlying early folliculogenesis may allow the development of novel therapeutic strategies for patients diagnosed with primary ovarian insufficiency, polycystic ovary syndrome, and poor ovarian response to FSH stimulation, as well as for infertile women of advanced reproductive age.

Intraovarian control of early folliculogenesis

Testosterone and follicle-stimulating hormone (FSH) are required to obtain full reproductive potential. In the testis, somatic Sertoli cells transduce signals from testosterone and FSH into the production of factors that are required by germ cells as they mature into spermatozoa. Recent advances in identifying new signaling pathways that are regulated by FSH and testosterone have allowed for refinement in the understanding of the independent, overlapping and synergistic actions of these hormones. In this review, we discuss the signaling pathways that are regulated by FSH and testosterone as well as the resulting metabolic and gene expression changes that occur as related to Sertoli cell proliferation, differentiation and the support of spermatogenesis.

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FSH and testosterone signaling in Sertoli cells

Successful ovulation requires that developmentally competent oocytes are released with appropriate timing from the ovarian follicle. Somatic cells of the follicle sense the ovulatory stimulus and guide resumption of meiosis and release of the oocyte, as well as structural remodelling and luteinization of the follicle. Complex intercellular communication co-ordinates critical stages of oocyte maturation and links this process with release from the follicle. To achieve these outcomes, ovulation is controlled through multiple inputs, including endocrine hormones, immune and metabolic signals, as well as intrafollicular paracrine factors from the theca, mural and cumulus granulosa cells and the oocyte itself. This review focuses on the recent advances in understanding of molecular mechanisms that commence after the gonadotrophin surge and culminate with release of the oocyte. These mechanisms include intracellular signalling, gene regulation and remodelling of tissue structure in each of the distinct ovarian compartments. Most critical ovulatory mediators exert effects through the cumulus cell complex that surrounds and connects with the oocyte. The convergence of ovulatory signals through the cumulus complex co-ordinates the key mechanistic processes that mediate and control oocyte maturation and ovulation.

Molecular mechanisms of ovulation: co-ordination through the cumulus complex

FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A.

Aromatase Expression in the Ovary: Hormonal and Molecular Regulation

Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSHmediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human Gs Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active Gs Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and Gs signaling pathways are required. (Endocrinology 144: 3985–3994, 2003)

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Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation.

Granulosa cells express the closely related orphan nuclear receptors steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). To determine whether SF-1 and LRH-1 have differential effects on steroid production, we compared the effects of overexpressing LRH-1 and SF-1 on estrogen and progesterone production by undifferentiated rat granulosa cells. Adenovirus mediated overexpression of LRH-1 or SF-1 had qualitatively similar effects. Neither LRH-1 nor SF-1 alone stimulated estrogen or progesterone production, but when combined with FSH and testosterone, each significantly augmented progesterone production and mRNAs for cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase above that observed with FSH alone, with SF-1 being more effective than LRH-1. LRH-1 did not augment FSH-stimulated estrogen production, whereas SF-1 produced only a slight ( approximately 30%) augmentation of FSH-stimulated estrogen production. The stimulatory actions of both were reduced by overexpression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1. Expression of either LRH-1 or SF-1 together with constitutively active protein kinase B in the absence of FSH stimulated progesterone production and mRNAs for 3beta-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme but did not stimulate estrogen production or mRNA for aromatase. These findings demonstrate that LRH-1 and SF-1 have qualitatively similar actions on FSH-stimulated estrogen and progesterone production, which would suggest that these factors may have overlapping actions in the regulation of steroidogenesis that accompanies granulosa cell differentiation.

Liver receptor homolog-1 and steroidogenic factor-1 have similar actions on rat granulosa cell steroidogenesis.

FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and P450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSHstimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for P450 side-chain cleavage and 3-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with AdLRH-1 did not stimulate estradiol production or increases in mRNA for P450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation. (Endocrinology 145: 3821–3829, 2004)

Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation.

Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models ...

Deeksha Saxena

Papers

Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation.

Liver receptor homolog-1 and steroidogenic factor-1 have similar actions on rat granulosa cell steroidogenesis.

Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation.

Defective parasympathetic innervation is associated with airway branching abnormalities in experimental CDH