Lhx8 ablation leads to massive autophagy of mouse oocytes associated with DNA damage.

Following proliferation of oogonia in mammals, great numbers of germ cells are discarded, primarily by apoptosis, while the remainder form primordial follicles (the ovarian reserve) that determine fertility and reproductive lifespan. More massive, rapid, and essentially total loss of oocytes, however, occurs when the transcription factor Lhx8 is ablated-though the cause and mechanism of germ cell loss from the Lhx8-/- ovaries has been unknown. We found that Lhx8-/- ovaries maintain the same number of germ cells throughout embryonic development; rapid decrease in the pool of oocytes starts shortly before birth. The loss results from activation of autophagy, which becomes overwhelming within the first postnatal week, with extracellular matrix proteins filling the space previously occupied by follicles to produce a fibrotic ovary. Associated with this process, as early as a few days before birth, Lhx8-/- oocytes failed to repair DNA damage-which normally occurs when meiosis is initiated during embryonic development; and DNA damage repair genes were downregulated throughout the oocyte short lifespan. Based on gene expression analyses and morphological changes, we propose a model in which lineage-restricted failure of DNA repair triggers germ cell autophagy, causing premature depletion of the ovarian reserve in Lhx8-/- mice.

Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature.

Leri-Weill dyschondrosteosis is a pseudoautosomal dominantly-inherited skeletal dysplasia ascribed to haploinsufficiency of the SHOX gene caused by deletions, point mutations, or partial duplications of the gene, or to heterozygous deletions upstream or downstream of the intact SHOX gene involving conserved non-coding cis-regulatory DNA elements that show enhancer activity. Recently, two SHOX conserved non-coding element duplications, one upstream and one downstream, were reported in patients referred with idiopathic short stature. To further evaluate the role of these duplications in SHOX-related disorders, we describe seven patients (five with Leri-Weill dyschondrosteosis and two with short stature) all of whom have duplications of part of the upstream or downstream conserved non-coding element regions, identified by multiplex ligation-dependent probe amplification. In addition, we show data from 32 patients with an apparently identical downstream duplication that includes a proposed putative regulatory element (identified by multiplex ligation-dependent probe amplification or array comparative genome hybridization), which results in a variable phenotype from normal to mild Leri-Weill dyschondrosteosis. These additional data provide further evidence that duplications of upstream and downstream long range cis-regulatory DNA elements can result in a SHOX-related phenotype.

FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice.

BACKGROUND: Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation. METHODS: Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR. RESULTS: Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus. CONCLUSIONS: Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.

Dynamics of the ovarian reserve and impact of genetic and epidemiological factors on age of menopause.

The narrow standard age range of menopause, ∼50 yr, belies the complex balance of forces that govern the underlying formation and progressive loss of ovarian follicles (the "ovarian reserve" whose size determines the age of menopause). We show here the first quantitative graph of follicle numbers, distinguished from oocyte counts, across the reproductive lifespan, and review the current state of information about genetic and epidemiological risk factors in relation to possible preservation of reproductive capacity. In addition to structural X-chromosome changes, several genes involved in the process of follicle formation and/or maintenance are implicated in Mendelian inherited primary ovarian insufficiency (POI), with menopause before age 40. Furthermore, variants in a largely distinct cohort of reported genes-notably involved in pathways relevant to atresia, including DNA repair and cell death-have shown smaller but additive effects on the variation in timing of menopause in the normal range, early menopause (age <45), and POI. Epidemiological factors show effect sizes comparable to those of genetic factors, with smoking accounting for about 5% of the risk of early menopause, equivalent to the summed effect of the top 17 genetic variants. The identified genetic and epidemiological factors underline the importance of early detection of reproductive problems to enhance possible interventions.

Missense mutations in EDA and EDAR genes cause dominant syndromic tooth agenesis.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is the most common form of ectodermal dysplasia and is mainly associated with mutations in the EDA, EDAR, and EDARADD responsible for the development of ectodermal-derived structures. HED displays different modes of inheritance according to the gene that is involved, with X-linked EDA-related HED being the most frequent form of the disease. METHODS: Two families with tooth agenesis and manifestations of HED underwent clinical examination and EDA, EDAR, and EDARADD genetic analysis. The impact of the novel variant on the protein was evaluated through bioinformatics tools, whereas molecular modeling was used to predict the effect on the protein structure. RESULTS: A novel missense variant was identified in the EDAR (c.287T>C, p.Phe96Ser) of a female child proband and her mother, accounting for autosomal dominant HED. The genetic variant c.866G>A (p.Arg289His) in EDA, which has been previously described, was observed in the male proband of another family confirming its role in X-linked HED. The inheritance model of the missense mutation showed a different relationship with X-linked HED and non-syndromic tooth agenesis. CONCLUSION: Our findings provide evidence of variable expression of HED in heterozygous females, which should be considered for genetic counseling, and different modes of inheritance related to tooth development.

Unexpected phenotype in a boy with trisomy of the SHOX gene.

The assessment that heterozygous SHOX mutations leading to SHOX haploinsufficiency play a role in patients with idiopathic short stature (ISS) is already documented in the literature as well as the suggestion that additional copies of SHOX are strongly implicated in a condition of tall stature. However, we report the first case of short stature in a male associated with the presence of three copies of the SHOX gene. Through chromosomal analysis, using Multiplex Ligation-dependent Probe Amplification method of SHOX salsa P018B kit and microsatellite analysis, we identify a new interstitial isolated duplication of the SHOX gene and its enhancer caused by a larger duplication of the PAR1 region in a boy with ISS. Consequently, we propose the hypothesis that this chromosome re-arrangement disrupts the regular interaction between the enhancer and promoter, resulting in a transcription block, thus producing a lack of gene activation, causing the clinical feature of short stature.

Foxl2 functions in sex determination and histogenesis throughout mouse ovary development.

BACKGROUND: Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit. RESULTS: Following Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers. CONCLUSION: The results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.

Transcriptional control of ovarian development in somatic cells.

Developmental transitions of the bipotential gonad to the embryonic ovary and thence to the follicle-filled mature ovary are expected to be coordinated by sets of transcription factors. We infer candidate lists here, focusing on somatic cell fate and function. For the mouse, developmental stages of ovary differentiation are relatively discretely phased, and provide a unique tool to investigate the intricate mechanisms that lead to the acquisition of female reproductive competence. Cross-platform gene expression profiles supplement functional studies of specific genes and comparative information about human biology. Available data suggest that: (1) peak transcription activity just precedes the two most decisive steps of early ovary differentiation (i.e., entry into meiosis and follicle formation); (2) alternating peak gene activities in oocytes and somatic cells may reflect reciprocal interactions; and (3) in addition to stable states of chromatin modification associated with morphogenesis, some features of differentiation are labile, contingent on the expression state of critical factors. Examples are the maintenance of somatic sex determination by continued Foxl2 action and the reversible maintenance of follicles in a quiescent state by nuclear Foxo3.

Genetics of the ovarian reserve.

Primordial follicles or non-growing follicles (NGFs) are the functional unit of reproduction, each comprising a single germ cell surrounded by supporting somatic cells. NGFs constitute the ovarian reserve (OR), prerequisite for germ cell ovulation and the continuation of the species. The dynamics of the reserve is determined by the number of NGFs formed and their complex subsequent fates. During the reproductive lifespan, the OR progressively diminishes due to follicle atresia as well as recruitment, maturation, and ovulation. The depletion of the OR is the major determining driver of menopause, which ensues when the number of primordial follicles falls below a threshold of ∼1,000. Therefore, genes and processes involved in follicle dynamics are particularly important to understand the process of menopause, both in the typical reproductive lifespan and in conditions like primary ovarian insufficiency, defined as menopause before age 40. Genes and their variants that affect the timing of menopause thereby provide candidates for diagnosis of and intervention in problems of reproductive lifespan. We review the current knowledge of processes and genes involved in the development of the OR and in the dynamics of ovarian follicles.

Genes and translocations involved in POF.

Changes at a single autosomal locus and many X-linked loci have been implicated in women with gonadal dysgenesis [premature ovarian failure (POF) with deficits in ovarian follicles]. For the chromosome 3 locus, a forkhead transcription factor gene (FOXL2) has been identified, in which lesions result in decreased follicles by haploinsufficiency. In contrast, sporadic X; autosomal translocations are distributed at many points on the X, but concentrate in a critical region on Xq. The association of the breakpoints with genes involved in ovarian function is thus far weak (in four analyzed cases) and has not been related to pathology in other POF patients. While many more translocations can be analyzed in detail as the human genome sequence is refined, it remains possible that translocations like X monosomy (Turner syndrome) lead to POF not by interrupting specific genes important in ovarian development, but by causing aberrations in pairing or X-inactivation during folliculogenesis. It is noted that the critical region has unusual features, neighboring the X-inactivation center and including an 18 Mb region of very low recombination. These suggest that chromosome dynamics in the region may be sensitive to structural changes, and when modified by translocations might provoke apoptosis at meiotic checkpoints. Choices among models for the etiology of POF should be feasible based on studies of ovarian follicle development and attrition in mouse models. Studies would prominently include gene expression profiling of developmental-specific pathways in nascent ovaries with controlled levels of Foxl2 and interacting proteins, or with defined changes in the X chromosome.

Aging of oocyte, ovary, and human reproduction.

We review age-related changes in the ovary and their effect on female fertility, with particular emphasis on follicle formation, follicle dynamics, and oocyte quality. The evidence indicates that the developmental processes leading to follicle formation set the rules determining follicle quiescence and growth. This regulatory system is maintained until menopause and is directly affected in at least some models of premature ovarian failure (POF), most strikingly in the Foxl2 mouse knockout, a model of human POF with monogenic etiology (blepharophimosis/ptosis/epicanthus inversus syndrome). Several lines of evidence indicate that if the ovarian germ cell lineage maintains regenerative potential, as recently suggested in the mouse, a role in follicle dynamics for germ stem cells, if any, is likely indirect or secondary. In addition, age-related variations in oocyte quality in animal models suggest that reproductive competence is acquired progressively and might depend on parallel growth and differentiation of follicle cells and stroma. Genomewide analyses of the mouse oocyte transcriptome have begun to be used to systematically investigate the mechanisms of reproductive competence that are altered with aging. Investigative and therapeutic strategies can benefit from considering the role of continuous interactions between follicle cells and oocytes from the beginning of histogenesis to full maturation.

Growth patterns of human ovarian volume during intrauterine and postnatal organogenesis.

BACKGROUND: Ovarian volumes during pre-pubertal, reproductive or menopausal life in a healthy female have been studied by in vivo and anatomical methods. In contrast, conclusive studies on normal human ovarian volume during intrauterine and postnatal organogenesis, when the folliculogenesis occurs, do not exist. AIM: To assess the growth patterns of ovarian volume during human ovarian definitive histogenesis using morphometric methods. SUBJECTS: Twenty-five left ovaries removed from fetuses, newborns and children with karyotype 46,XX, ranging from 15 to 74 weeks of development. OUTCOME MEASURES: The ovaries were completely cut obtaining serial sections. Ovarian volume was calculated both with the ellipsoid method, using its length, maximum and minimum diameters, and the Cavalieri quantitative morphometric method. The age-related pattern of ovarian volume was studied using linear regression analyses. RESULTS: Ovarian volume calculated with the Cavalieri method increased from 16 mm3 at the 15th week of development to over 300 mm3 at the end of the process, with a 20-fold increase compared to the initial volume. Ovarian volume calculated with the ellipsoid method ranged between 25 to over 380 mm3, with a 15-fold increase. On average, ellipsoidal volume over-estimated the true Cavalieri volume (mean difference -14%, S.D. 29, p=0.014, Student's t-test for paired samples). Linear regression curves were developed to predict ovarian volume at any age. Overall, the approximation was better for Cavalieri volume (approximately, 72% of variance explained by the linear regression) than for ellipsoid volume (59% of explained variance). CONCLUSIONS: During the intrauterine and postnatal organogenesis, the normal growth rate of human ovary was almost linear. The calculation of ovarian volume using Cavalieri method does not depend on assumptions about the organ shape and it appears to be more appropriate.

Position of follicles in normal human ovary during definitive histogenesis.

BACKGROUND: Follicular growth in the mammalian ovary follows a geographically determined pattern, but no exhaustive data about their spatial localization in the cortex of human ovary exists. AIM: The aim of this study is to assess the spatial position of the follicles during human ovarian definitive histogenesis using morphometric methods. SUBJECTS: Ovaries removed from four fetuses, five newborns and one 8-month-old child with 46,XX karyotype was used. OUTCOME MEASURES: The position of the different follicle categories (primordial, primary, secondary and antral) was estimated as percentage distance of the centroid of the follicle from the cortico-medullary boundary. RESULTS: In normal ovaries, during definitive histogenesis, the primordial follicles progressively occupy more peripheral parts of the cortex (on average, 41% of cortical length at 20 weeks of fetal development, 53% at birth, 68% at 8 postnatal months). Primary follicles were crowded in the inner part of the cortex (newborn: 30-40% of cortical length; 8 months: 52% of cortical length). Secondary and antral follicles were sampled in all neonatal specimens and in the 8-month old specimen; they were grouped next to the cortico-medullary boundary. CONCLUSIONS: Ovarian development corresponded to a migration of the maximum crowding of follicles from a position next to the medulla towards a more peripheral location. The control of the primordial follicle assembly, recruitment and development are coordinated by locally produced paracrine factors. The action of these factors seems to follow a negative gradient from the cortex toward the medulla.

Genotype/phenotype correlations of males affected by Simpson-Golabi-Behmel syndrome with GPC3 gene mutations: patient report and review of the literature.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome with associated visceral and skeletal anomalies. Deletions or point mutations involving the glypican-3 (GPC3) gene at Xq26 are associated with a relatively milder form of this disorder (SGBS1). GPC3 encodes a putative extracellular proteoglycan, glypican-3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. It appears to form a complex with insulin-like growth factor-II (IGF-II), and might thereby modulate IGF-II action. We reviewed the clinical findings of all published patients with SGBS1 with GPC3 mutations to confirm the clinical specificity for the SGBS1 phenotype. Moreover, we report on a new patient with a GPC3 deletion and IGF-II evaluation.

Constitutively active Foxo3 in oocytes preserves ovarian reserve in mice.

During female reproductive life, ovarian follicle reserve is reduced by maturation and atresia until menopause ensues. Foxo3 is required to maintain the ovarian reserve in mice. Here we show that overexpression of constitutively active FOXO3 can increase ovarian reproductive capacity in mice. We find increased follicle numbers and decreased gonadotropin levels in aging FOXO3-transgenic mice compared with wild-type littermates, suggesting maintenance of a greater ovarian reserve. Based on cumulative progeny in aging animals, we find 31-49% increased fertility in transgenic females. The gene expression profile of Foxo3-/- knockout ovaries appears older than that of wild-type littermates, and the transgene induces a younger-looking profile, restoring much of the wild-type transcriptome. This is the first gain-of-function model of augmented reproductive reserve in mice, thus emphasizing the role of Foxo3 as a guardian of the ovarian follicle pool in mammals and a potential determinant of the onset of menopause.

Germ cell formation from embryonic stem cells and the use of somatic cell nuclei in oocytes.

Embryonic stem cells (ESCs) have remarkable properties of pluripotency and self-renewal, along with the retention of chromosomal integrity. Germ cells function as a kind of "transgenerational stem cells," transmitting genetic information from one generation to the next. The formation of putative primordial germ cells (PGCs) and germ cells from mouse and human ESCs (hESCs) has, in fact, been shown, and the apparent derivation of functional mouse male gametes has also been described. Additionally, investigators have successfully reprogrammed somatic nuclei into a pluripotent state by inserting them into ESCs or oocytes. This would enable the generation of ESCs genetically identical to the somatic cell donor and their use in cell therapy. However, these methodologies are still inefficient and their mechanisms poorly understood. Until full comprehension of these processes is obtained, clinical applications remain remote. Nevertheless, they represent promising tools in the future, enhancing methods of therapeutic cloning and infertility treatment.

Determination and stability of gonadal sex.

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. But females are proving to be more complicated. Several master genes appear to be autonomously involved, and female sex determination seems to remain relatively labile. Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women; and in animal models, Foxl2 is required for folliculogenesis as well as for maintenance, and possibly induction, of female sex determination. In the germ line, oocytes apparently form normally even in the absence of Foxl2, dependent on genes that include female-specific factors such as Fig-alpha, Nobox, etc. In the soma, ablation of Foxl2 or the independently expressed gene Wnt4 (likely downstream of Rspo1) can produce partial testis differentiation in XX mice, and the double knockout results in the formation of tubules and spermatogonia. This indicates that at least 2 autonomous ovarian pathways are required to antagonize testis differentiation in females, a finding that is being increasingly corroborated by studies in goats and nonmammalian vertebrates. In recent expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit, we found that following Foxl2 loss, early testis genes (including the downstream effector of Sry, Sox9) and several novel ovarian genes were consistently dysregulated during embryo-fetal development. The results support the proposal of dose-dependent Foxl2 function and antitestis action. A partial working model for somatic development and sex determination is presented in which Sox9 is a direct antagonist of Foxl2 in the supporting cell lineage.

Incidence of non-age-dependent chromosomal abnormalities: a population-based study on 88965 amniocenteses.

Current knowledge about the incidence of chromosomal abnormalities in the general population comes from studies in newborns carried out in the 70s, before the era of widespread prenatal diagnosis. In the following years, data on frequency of chromosomal abnormalities in the second trimester of pregnancy have been used in conjunction with the data on the natural history of chromosomally abnormal fetuses to infer maternal age-specific rates of cytogenetic abnormalities in live-born infants. Starting from the data gathered in 1995-1996 from all Italian cytogenetic laboratories (with 92% compliance to the study), we have compared the frequency of chromosomal abnormalities at amniocentesis in cases with maternal age of >or=35 years (51,758 individuals) and cases with maternal age of <35 years (37,207 cases). The comparison confirmed the age-dependency of aneuploidies, whereas none of the structural abnormalities showed age-related differences. Furthermore, among the mosaic aneuploidies, trisomy 21 and 45,X/46,XX were found with a significantly higher incidence in older women. Chromosomal abnormalities that showed no significant difference between the two groups were summed for the overall national cohort, providing a general estimate of the incidence in the second trimester of pregnancy. The data provide critical background information for prenatal genetic counseling and for the planning of health care policy.

Short stature homeoboxcontaining gene and idiopathic short stature.

The term idiopathic short stature (ISS) refers to patients who are short due to various unknown reasons. Although it is clear that multiple factors contribute to final height, genetic factors play a crucial role. Mutations of a human homeobox gene, short stature homeobox-containing (SHOX) gene, have been shown to be associated with the short stature phenotype in patients with Turner syndrome, most patients with Leri-Weill dyschondrosteosis and some cases of ISS. The prevalence of SHOX anomalies in subjects previously recognized as having ISS has been estimated at 2.4% in a large series of ISS individuals. This review focuses on the functional properties of the SHOX gene and its linkage to ISS.

Establishment of ovarian reserve: a quantitative morphometric study of the developing human ovary.

OBJECTIVE: To assess directly the dynamics of the formation of the ovarian reserve in the normal human ovary by evaluating the total number of follicles in developing ovaries when folliculogenesis occurs. DESIGN: Histomorphometry-based follicle counts in complete serial tissue sections. SETTING: Functional Anatomy Research Center, University of Milano. PATIENT(S): Thirteen fetuses, neonates, and one 8-month-old infant. INTERVENTION(S): Fifteen ovaries were completely cut, obtaining serial sections. Ovarian volume, volume fractions, density and total number of follicles per ovary were calculated using quantitative morphometric methods. MAIN OUTCOME MEASURE(S): Age-related dynamics of the establishment of ovarian reserve in human developing ovary at the end of the organogenesis. RESULT(S): The ovarian reserve (100,000 follicles at 15 weeks of postconceptional age) increased progressively to 680,000 follicles at 34 weeks. At 8 months of postnatal age the pool was still about 680,000 primordial follicles. CONCLUSION(S): The consistence of the primordial follicle pool during organogenesis shows an exponential increase until month 8 of prenatal life and it is subsequently maintained without modifications at least until month 8 of postnatal life.