Primordial ovarian follicles in mice form when somatic cells surround individual oocytes. We show that lack of Nobox, an oocyte-specific homeobox gene, accelerates postnatal oocyte loss and abolishes the transition from primordial to growing follicles in mice. Follicles are replaced by fibrous tissue in female mice lacking Nobox in a manner similar to nonsyndromic ovarian failure in women. Genes preferentially expressed in oocytes, including Oct4 and Gdf9, are down-regulated in Nobox-/- mice, whereas ubiquitous genes such as Bmp4, Kit, and Bax remain unaffected. Therefore, Nobox is critical for specifying an oocyte-restricted gene expression pattern essential for postnatal follicle development.

PTGS2, HAS2 and GREM1 gene expression correlates to morphological and physiological characteristics and provides a novel approach to predict human embryo development. Ultimately, with better predictors of follicular and embryonic health, higher quality embryos can be selected and transferred, reducing higher order pregnancy rates.

Mammalian oogenesis requires oocyte-specific transcriptional regulators. The full complement of oocyte-specific transcription factors is unknown. Here, we describe the finding that Sohlh1, a spermatogenesis and oogenesis basic helix-loop-helix transcription factor in females, is preferentially expressed in oocytes and required for oogenesis. Sohlh1 disruption perturbs follicular formation in part by causing down-regulation of two genes that are known to disrupt folliculogenesis: newborn ovary homeobox gene (Nobox) and factor in the germ-line alpha (Figla). In addition, we show that Lhx8 is downstream of Sohlh1 and critical in fertility. Thus, Sohlh1 and Lhx8 are two germ cell-specific, critical regulators of oogenesis.infertility ͉ oocyte ͉ ovary ͉ reproduction ͉ folliculogenesis M ouse primordial germ cells, at embryonic days 9.5-11.5 (E9.5-E11.5), migrate to the urogenital ridges from the proximal epiblast. Mitotic division of primordial germ cells, coupled with incomplete cytokinesis, results in oocytes in clusters (also called cysts) at ϷE10.5 (1, 2). Female germ cells, at ϷE13.5, begin entry into prophase I of meiosis and arrest in the diplotene stage of the first meiotic division. Oocytes arrested in meiosis I are thought to remain arrested until the time of ovulation. Germ cell clusters formed in the embryonic gonad break down shortly after birth in the mouse. Breakdown of germ cell cysts results in oocytes enveloped by somatic pregranulosa cells (now called primordial follicles) (1). Primordial follicles represent a reservoir of follicles that are recruited periodically to grow into primary and more advanced follicular structures.The breakdown of germ cell clusters, formation of primordial follicles, and transition to primary follicles represents a critical period of follicle formation. Transcription of numerous oocytespecific genes, such as growth and differentiation factor 9 (Gdf9), bone morphogenetic protein 15 (Bmp15), and zona pellucida genes 1-3 (Zp1-3) (3-6), commences during early folliculogenesis. Regulation of these genes is in part due to expression of two known oocyte-specific transcription factors, Figla (7) and Nobox (3). FIGLA is a basic helix-loop-helix transcription factor that regulates expression of the zona pellucida genes (7,8). Nobox is a homeobox gene that is necessary for expression of several key oocyte-specific genes, including Gdf9, Bmp15, and Pou5f1 but not Figla or Zp1-3 (3). However, other oocyte-specific transcriptional regulators likely exist, because numerous oocyte-specific genes are not affected by the lack of Nobox and Figla. Here, we describe our finding that Sohlh1 and Lhx8, which are both preferentially expressed during oogenesis in females, are critical in early folliculogenesis. Results and DiscussionWe identified Sohlh1 by an in silico subtraction strategy to identify genes that are preferentially expressed during early folliculogenesis (9). Sohlh1 encodes a basic helix-loop-helix transcription factor with homologues in humans and other placental mammals (Fig. 8,...

*Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15-/-and Bmp15 -/-Gdf9 +/-double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production.

The classical view of ovarian follicle development is that it is regulated by the hypothalamic-pituitary-ovarian axis, in which gonadotropin-releasing hormone (GnRH) controls the release of the gonadotropic hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and that ovarian steroids exert both negative and positive regulatory effects on GnRH secretion. More recent studies in mice and humans indicate that many other intra-ovarian signaling cascades affect follicular development and gonadotropin action in a stage-and context-specific manner. As we discuss here, mutant mouse models and clinical evidence indicate that some of the most powerful intraovarian regulators of follicular development include the TGF-β/SMAD, WNT/FZD/β-catenin, and RAS/ERK1/2 signaling pathways and the FOXO/FOXL2 transcription factors. IntroductionThe ovary is a highly organized composite of germ cells (oocytes or eggs) and somatic cells (granulosa cells, thecal cells, and stromal cells) whose interactions dictate formation of oocyte-containing follicles, development of both oocytes and somatic cells as follicles, ovulation, and formation of the corpus luteum (the endocrine structure that forms from the ovarian follicle after ovulation and is required for establishing and maintaining pregnancy) (Figure 1). Many events in the adult ovary are controlled by two hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), secreted from the anterior pituitary gland under the control of pulses of gonadotropin-releasing hormone (GnRH) from the hypothalamus (Figure 1). Low-frequency GnRH pulses stimulate a slight increase in FSH levels early in a woman's menstrual cycle, enhancing follicle growth, while high-frequency GnRH pulses lead to a sharp rise in levels of LH just before mid-cycle (an event known as the "LH surge"), triggering ovulation and formation of the corpus luteum (Figure 1). The ovary also has a key role in these processes, ensuring the timely release of fertilizable oocytes and the maintenance of luteal cell function, a necessity for pregnancy, by directing feedback mechanisms to the hypothalamus and pituitary. For example, estrogen produced by the cells of the developing follicle both inhibits GnRH production in the hypothalamus and elicits elevated GnRH pulses, which trigger the mid-cycle LH surge that initiates ovulation. Thus, fertility depends on highly orchestrated endocrine events involving multiple organ systems.Disruption of this finely controlled network can lead to many clinical syndromes including premature ovarian failure (POF), polycystic ovarian syndrome (PCOS), ovarian hyperstimulation syndrome, ovulation defects, poor oocyte quality, and cancer. The goal of this Review is to cover some of the advances in our understanding of the basic biology of the mammalian ovary that may shed light on specific ovarian dysfunctions that lead to infertility. We primarily focus on recent developments using mouse genetic models to study follicle growth and ovarian function because of the inherent di...

The transforming growth factor ␤ (TGF␤) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGF␤ function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGF␤ pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.

SMAD4 is a central component of the TGFbeta superfamily signaling pathway. Within the ovary, TGFbeta-related proteins play crucial roles in controlling granulosa cell growth, differentiation, and steroidogenesis. To study the in vivo roles of SMAD4 during follicle development, we generated an ovarian conditional knockout of Smad4 using the cre/loxP recombination system. Smad4 ovarian-specific knockout mice are subfertile with decreasing fertility over time and multiple defects in folliculogenesis. Regulation of steroidogenesis is disrupted in the Smad4 conditional knockout, leading to increased levels of serum progesterone. In addition, severe cumulus cell defects are present both in vivo and when assayed in vitro. These findings demonstrate that disrupting signaling through SMAD4 in the ovarian granulosa cells leads to premature luteinization of granulosa cells and eventually premature ovarian failure, thereby demonstrating key in vivo roles of TGFbeta superfamily signaling in the timing of granulosa cell differentiation.

The in vitro culture of immature ovarian follicles is used to examine the factors that regulate follicle development and may ultimately provide options for reproductive infertility. The objective of this study was to develop a three-dimensional in vitro culture system for the growth and development of individual granulosa cell-oocyte complexes. An alginate hydrogel was used to encapsulate immature mouse granulosa cell-oocyte complexes (GOCs) that were subsequently maintained in a serum-free in vitro culture. An overall incorporation efficiency of 50% was achieved. The complexes were assessed by transmission electron microscopy for changes in ultrastructure during in vitro growth. The architecture of the follicular complex was maintained during the encapsulation and the subsequent culture. The granulosa cells proliferated, and the oocytes also grew in volume and obtained the structural characteristics of mature oocytes including cortical granule formation, a well-developed zona pellucida with microvilli, normal mitochondria, and lattice-like structures in the cytoplasm. Oocytes retrieved and matured were able to resume meiosis, a necessary step for proper development. Thus, this system represents a new in vitro methodology for growth of individual granulosa cell-oocyte complexes.