Molecular Human Reproduction - current issue

The ovary: from basic research to clinic

Hillier, S. G. — Thu, 12 Nov 2009 07:49:48 PST

Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles

Thu, 12 Nov 2009 07:49:49 PST

To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1–Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

The forkhead transcription factor FOXL2 is expressed in somatic cells of the human ovary prior to follicle formation

Duffin, K., Bayne, R.A.L., Childs, A.J., Collins, C., Anderson, R.A. — Thu, 12 Nov 2009 07:49:49 PST

Interactions between germ cells and surrounding somatic cells are central to ovarian development as well as later function. Disruption of these interactions arising from abnormalities in either cell type can lead to premature ovarian failure (POF). The forkhead transcription factor FOXL2 is a candidate POF factor, and mutations in the FOXL2 gene are associated with syndromic and non-syndromic ovarian failure. Foxl2-deficient mice display major defects in primordial follicle activation with consequent follicle loss, and earlier roles in gonadal development and sex determination have also been suggested. However, despite its importance no data presently exist on its expression in the developing human ovary. Expression of FOXL2 mRNA was demonstrated in the human fetal ovary between 8 and 19 weeks gestation, thus from soon after sex determination to primordial follicle development. Expression in the ovary was higher after 14 weeks than at earlier gestation weeks and was very low in the fetal testis at all ages examined. Immunolocalization revealed FOXL2 expression to be confined to somatic cells, both adjacent to germ cells and those located in the developing ovarian stroma. These cells are the site of action of oocyte-derived activin signalling, but in vitro treatment of human fetal ovaries with activin failed to reveal any regulation of FOXL2 transcription by this pathway. In summary, the expression of FOXL2 in somatic cells of the developing human ovary before and during follicle formation supports a conserved and continuing role for this factor in somatic/germ cell interactions from the earliest stages of human ovarian development.

Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15

Li, Q., Rajanahally, S., Edson, M. A., Matzuk, M. M. — Thu, 12 Nov 2009 07:49:49 PST

Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.

Oocyte peptides as paracrine tools for ovarian stimulation and oocyte maturation

Mottershead, D. G., Watson, A. J. — Thu, 12 Nov 2009 07:49:49 PST

Recent studies report the production and isolation of a stable bioactive recombinant human bone morphogenetic protein 15 (rhBMP15) that is appropriately processed in HEK-293 cells and activates the SMAD 1/5/8 pathway in mouse granulosa cell cultures. Further, the purified rhBMP15 induces the expression of genes associated with cumulus expansion. Thanks to recent research, we have a greater understanding of the importance of the dialogue that occurs between the oocyte and the granulosa cell layer with regard to regulating folliculogenesis and the acquisition of oocyte developmental competence and maturation. BMP15 is one of the critical components of these intra-follicular communication pathways. The production of recombinant human BMP15 is important for understanding the biochemistry of this specific pathway and for also fully understanding its functional contributions to mediating oocyte development. The production of a stable recombinant human BMP15 is also important for use in experiments aimed at optimizing ovarian stimulation protocols and in vitro oocyte maturation methods. This is required to improve oocyte and embryonic developmental competence and increase our ability to effectively use in vitro methods for animal production and the treatment of human infertility.

The primordial pool of follicles and nest breakdown in mammalian ovaries

Tingen, C., Kim, A., Woodruff, T. K. — Thu, 12 Nov 2009 07:49:49 PST

The creation of the pool of follicles available for selection and ovulation is a multi-faceted, tightly regulated process that spans the period from embryonic development through to the first reproductive cycle of the organism. In mice, this development can occur in mere weeks, but in humans, it is sustained for years. Embryonic germ cell development involves the migration of primordial germs cells to the genital ridge, and the mitotic division of germ cell nuclei without complete cytokinesis to form a multi-nucleated syncytia, or germ cell nest. Through combined actions of germ cell apoptosis and somatic cell migration, the germ cell nuclei are packaged, with surrounding granulosa cells, into primordial follicles to form the initial follicle pool. Though often dismissed as quiescent and possibly uninteresting, this initial follicle pool is actually quite dynamic. In a very strictly controlled mechanism, a large portion of the initial primordial follicles formed is lost by atresia before cycling even begins. Remaining follicles can undergo alternate fates of continued dormancy or selection leading to follicular growth and differentiation. Together, the processes involved in the fate decisions of atresia, sustained dormancy, or activation carve out the follicle pool of puberty, the pool of available oocytes from which all future reproductive cycles of the female can choose. The formation of the initial and pubertal follicle pools can be predictably affected by exogenous treatment with hormones or molecules such as activin, demonstrating the ways the ovary controls the quality and quantity of germ cells maintained. Here, we review the biological processes involved in the formation of the initial follicle pool and the follicle pool of puberty, address the alternate models for regulating germ cell number and outline how the ovary quality-controls the germ cells produced.

Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection?

Hartshorne, G.M., Lyrakou, S., Hamoda, H., Oloto, E., Ghafari, F. — Thu, 12 Nov 2009 07:49:49 PST

Prenatal oogenesis produces hundreds of thousands of oocytes, most of which are discarded through apoptosis before birth. Despite this large-scale selection, the survivors do not constitute a perfect population, and the factors at the cellular level that result in apoptosis or survival of any individual oocyte are largely unknown. What then are the selection criteria that determine the size and quality of the ovarian reserve in women? This review focuses on new data at the cellular level, on human prenatal oogenesis, offering clues about the importance of the timing of entry to meiotic prophase I by linking the stages and progress through MPI with the presence or absence of apoptotic markers. The characteristics and responsiveness of cultured human fetal ovarian tissue at different gestational ages to growth factor supplementation and the impact of meiotic abnormalities upon apoptotic markers are discussed. Future work will require the use of a tissue culture model of prenatal oogenesis in order to investigate the fate of individual live oocytes at different stages of development.

Control of ovulation in mice by progesterone receptor-regulated gene networks

Kim, J., Bagchi, I. C., Bagchi, M. K. — Thu, 12 Nov 2009 07:49:49 PST

The mid-cycle surge of luteinizing hormone (LH) induces ovulation, a process during which a fertilizable oocyte is released from a mature ovarian follicle. Although ovulation is a physiologically well-characterized event, the underlying molecular pathways remain poorly understood. Progesterone receptor (PGR), which mediates the biological effects of the steroid hormone progesterone, has emerged as a key regulator of ovulation in mice. The development of a progesterone-receptor-null (Pgr-null) mouse model confirmed a critical role of this hormone in ovulation because in these mutant mice, mature pre-ovulatory follicles fail to release the oocytes. This animal model has thus presented a unique opportunity to study the molecular pathways underlying ovulation. Gene-expression profiling experiments by several groups, using the ovaries of Pgr-null mice, revealed novel gene networks, which act downstream of PGR to control ovulation. These genes encode diverse molecules such as proteases, transcription factors, cell-adhesion molecules, modulators of vascular activities and regulators of inflammation. Functional analyses using gene-knockout mouse models have confirmed that some of these factors play critical roles during ovulation. The knowledge gained from these studies has helped us to understand better the molecular mechanisms that facilitate the release of oocytes from pre-ovulatory follicles. Further analysis of the role of molecular regulators of ovulation will help identify useful molecular targets that would allow the development of improved contraceptives and new therapeutics for anovulatory infertility.

Genetic and gene expression analyses of the polycystic ovary syndrome candidate gene fibrillin-3 and other fibrillin family members in human ovaries

Thu, 12 Nov 2009 07:49:49 PST

Several studies have demonstrated an association between polycystic ovary syndrome (PCOS) and the dinucleotide repeat microsatellite marker D19S884, which is located in intron 55 of the fibrillin-3 (FBN3) gene. Fibrillins, including FBN1 and 2, interact with latent transforming growth factor (TGF)-β-binding proteins (LTBP) and thereby control the bioactivity of TGFβs. TGFβs stimulate fibroblast replication and collagen production. The PCOS ovarian phenotype includes increased stromal collagen and expansion of the ovarian cortex, features feasibly influenced by abnormal fibrillin expression. To examine a possible role of fibrillins in PCOS, particularly FBN3, we undertook tagging and functional single nucleotide polymorphism (SNP) analysis (32 SNPs including 10 that generate non-synonymous amino acid changes) using DNA from 173 PCOS patients and 194 controls. No SNP showed a significant association with PCOS and alleles of most SNPs showed almost identical population frequencies between PCOS and control subjects. No significant differences were observed for microsatellite D19S884. In human PCO stroma/cortex (n = 4) and non-PCO ovarian stroma (n = 9), follicles (n = 3) and corpora lutea (n = 3) and in human ovarian cancer cell lines (KGN, SKOV-3, OVCAR-3, OVCAR-5), FBN1 mRNA levels were approximately 100 times greater than FBN2 and 200–1000-fold greater than FBN3. Expression of LTBP-1 mRNA was 3-fold greater than LTBP-2. We conclude that FBN3 appears to have little involvement in PCOS but cannot rule out that other markers in the region of chromosome 19p13.2 are associated with PCOS or that FBN3 expression occurs in other organs and that this may be influencing the PCOS phenotype.

Paracrine support of ovarian stimulation

Hillier, S. G. — Thu, 12 Nov 2009 07:49:49 PST

Assisted reproductive technology has evolved on the back of blunderbuss ovarian stimulation regimes designed to maximize the number of oocytes recoverable for treatment purposes. However, oocyte ‘quality’ is finely programmed by local paracrine and autocrine signalling events during folliculogenesis and can be adversely affected by inappropriate gonadotrophic stimulation. This brief review traces the full follicular lifespan—from initiation to ovulation—to identify gonadotrophin-responsive checkpoints likely to impact oocyte quality. It is argued that these might be targeted during controlled ovarian stimulation therapy to (i) increase responsiveness to FSH through follicular priming with LH or hCG, (ii) improve follicular synchrony and oocyte quality through conditioning with FSH and (iii) promote ‘gold standard’ pre-ovulatory maturation through follicular coasting with LH or hCG. It is concluded that whereas there can be no one-size-fits-all approach to ovarian stimulation, treatment regimes based on paracrine principles and tailored to personal needs will always be more likely to achieve the desired outcome.