"Genome-Wide Linkage in a Highly Consanguineous Pedigree Reveals Two Novel Loci on Chromosome 7 for Non-Syndromic Familial Premature Ovarian Failure"

Background: The human condition known as Premature Ovarian Failure (POF) is characterized by loss of ovarian function before the age of 40. A majority of POF cases are sporadic, but 10–15% are familial, suggesting a genetic origin of the disease. Although several causal mutations have been identified, the etiology of POF is still unknown for about 90% of the patients. Methodology/Principal Findings: We report a genome-wide linkage and homozygosity analysis in one large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance. We identified two regions with a LODmax of 3.26 on chromosome 7p21.1-15.3 and 7q21.3-22.2, which are supported as candidate regions by homozygosity mapping. Sequencing of the coding exons and known regulatory sequences of three candidate genes (DLX5, DLX6 and DSS1) included within the largest region did not reveal any causal mutations. Conclusions/Significance: We detect two novel POF-associated loci on human chromosome 7, opening the way to the identification of new genes involved in the control of ovarian development and function.

Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelialmesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.

Peptides generated ex vivo from serum proteins by tumor-specific exopeptidases are not useful biomarkers in ovarian cancer

BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum proteins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if similar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and processed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 centers. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak intensities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approximately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross-validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diagnostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses.

Association between ovarian follicle development and pregnancy rates in dairy cows undergoing spontaneous oestrous cycles

Ovarian follicle development continues in a wave-like manner during the bovine oestrous cycle giving rise to variation in the duration of ovulatory follicle development. The objectives of the present study were to determine whether a relationship exists between the duration of ovulatory follicle development and pregnancy rates following artificial insemination (AI) in dairy cows undergoing spontaneous oestrous cycles, and to identify factors influencing follicle turnover and pregnancy rate and the relationship between these two variables. Follicle development was monitored by daily transrectal ultrasonography from 10 days after oestrus until the subsequent oestrus in 158 lactating dairy cows. The cows were artificially inseminated following the second observed oestrus and pregnancy was diagnosed 35 days later. The predominant pattern of follicle development was two follicle waves (74.7%) with three follicle waves in 22.1% of oestrous cycles and four or more follicle waves in 3.2% of oestrous cycles. The interval from ovulatory follicle emergence to oestrus (EOI) was 3 days longer (P < 0.0001) in cows with two follicle waves than in those with three waves. Ovulatory follicles from two-wave oestrous cycles grew more slowly but were approximately 2 mm larger (P < 0.0001) on the day of oestrus. Twin ovulations were observed in 14.2% of oestrous cycles and occurred more frequently (P < 0.001) in three-wave oestrous cycles; consequently EOI was shorter in cows with twin ovulations. Overall, 57.0% of the cows were diagnosed pregnant 35 days after AI. Linear logistic regression analysis revealed an inverse relationship between EOI and the proportion of cows diagnosed pregnant, among all cows (n = 158; P < 0.01) and amongst those with single ovulations (n = 145; P < 0.05). Mean EOI was approximately I day shorter (P < 0.01) in cows that became pregnant than in non-pregnant cows; however, pregnancy rates did not differ significantly among cows with different patterns of follicle development. These findings confirm and extend previous observations in pharmacologically manipulated cattle and show, for the first time, that in dairy cows undergoing spontaneous oestrous cycles, natural variation in the duration of post-emergence ovulatory follicle development has a significant effect on pregnancy rate, presumably reflecting variation in oocyte developmental competence.

Endocrine regulation of ovarian antral follicle development in cattle

Antral follicle growth in cattle occurs in two distinct phases; the first 'slow' growth phase spans the time from antrum acquisition to a size of approximately 3 mm detectable by transrectal ultrasound, and the second 'fast' phase is gondadotrophin-dependent and includes cohort growth, dominant follicle (DF) selection, and DF growth. This review summarises current concepts of the relative roles FSH and LH, ovarian and metabolic hormones play mainly in the second phase of antral follicle growth in animals of different reproductive and nutritional states. It is proposed that differential FSH response may enable one cohort follicle to become selected, and that follicular secretions, particularly inhibin, suppress FSH and thus are responsible for DF selection and dominance. Acute dependence of the DF on LH pulses will determine DF lifespan, and the LH pulse profile can be influenced by metabolic hormones such as leptin, providing one possible link for nutritional state and reproduction. Direct ovarian effects of acute and chronic changes in growth hormone, insulin and insulin-like growth factor (IGF)-I have been described on cohort follicles, DF oestrogen activity and on DF growth. Influences of metabolic hormones on early antral follicles undergoing their first 'slow' growth phase are less well described, yet metabolic hormones appear to enhance growth into the cohort available for FSH-induced emergence, and may influence subsequent developmental competence of oocytes. (C) 2003 Elsevier Science B.V. All rights reserved.

First lactation ovarian function in dairy heifers in relation to prepubertal metabolic profiles

The aim of this study was to determine whether any differences in the GH-IGF-I axis in juvenile calves were predictive of fertility problems as adult cows. Endogenous metabolic hormone profiles before and after feeding and the response to a GH-releasing factor (GRF) challenge were measured in prepubertal (6 month) dairy calves. These metabolic parameters were subsequently related to physical characteristics at puberty and to ovarian function during the first lactation. Milk progesterone analysis was used to categorize the animals into those with normal progesterone profiles following calving (n = 17) and those that developed delayed ovulation (DOV1, n = 9) or persistent corpus luteum (PCL1, n = 6) profiles. There were associations between prepubertal GH parameters, glucose and non-esterified fatty acid (NEFA) concentrations and the body condition score at which the animals attained puberty. The calves which subsequently developed DOV1 profiles as cows tended to have a higher GH pulse amplitude during fasting than normal profile animals, they did not show the anticipated decrease in circulating glucose concentrations following a post-prandial rise in insulin and they also had the lowest IGF-I concentrations. The calves that later developed PCL1 had a significantly larger GH pulse amplitude and pulse area than normal profile animals in the fed period and had the highest IGF-I concentrations. There were no differences in prepubertal insulin or NEFA concentrations or in the GH response to a GRF challenge between the different progesterone profile categories. Plasma IGF-I concentrations in prepubertal animals were positively correlated with their post-calving concentrations, whereas glucose concentrations had a negative correlation between these time-periods. These results suggested that the different juvenile endocrine profiles of the DOV1 cows may predispose them to a higher rate of tissue mobilization during lactation and a consequent reduction in fertility, while altered GH and IGF-I levels in PCL1 cows may later contribute to the maintenance of the persistent corpus luteum. Therefore metabolic differences in prepubertal calves were later reflected by altered reproductive function during the first lactation.

Variation in the ovarian reserve Is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

BACKGROUND: The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo. METHODS: Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later. RESULTS: We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo. CONCLUSION: These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

Association between ovarian follicle development and pregnancy rates in dairy cows undergoing spontaneous oestrous cycles

Ovarian follicle development continues in a wave-like manner during the bovine oestrous cycle giving rise to variation in the duration of ovulatory follicle development. The objectives of the present study were to determine whether a relationship exists between the duration of ovulatory follicle development and pregnancy rates following artificial insemination (AI) in dairy cows undergoing spontaneous oestrous cycles, and to identify factors influencing follicle turnover and pregnancy rate and the relationship between these two variables. Follicle development was monitored by daily transrectal ultrasonography from 10 days after oestrus until the subsequent oestrus in 158 lactating dairy cows. The cows were artificially inseminated following the second observed oestrus and pregnancy was diagnosed 35 days later. The predominant pattern of follicle development was two follicle waves (74.7%) with three follicle waves in 22.1% of oestrous cycles and four or more follicle waves in 3.2% of oestrous cycles. The interval from ovulatory follicle emergence to oestrus (EOI) was 3 days longer (P < 0.0001) in cows with two follicle waves than in those with three waves. Ovulatory follicles from two-wave oestrous cycles grew more slowly but were approximately 2 mm larger (P < 0.0001) on the day of oestrus. Twin ovulations were observed in 14.2% of oestrous cycles and occurred more frequently (P < 0.001) in three-wave oestrous cycles; consequently EOI was shorter in cows with twin ovulations. Overall, 57.0% of the cows were diagnosed pregnant 35 days after AI. Linear logistic regression analysis revealed an inverse relationship between EOI and the proportion of cows diagnosed pregnant, among all cows (n = 158; P < 0.01) and amongst those with single ovulations (n = 145; P < 0.05). Mean EOI was approximately I day shorter (P < 0.01) in cows that became pregnant than in non-pregnant cows; however, pregnancy rates did not differ significantly among cows with different patterns of follicle development. These findings confirm and extend previous observations in pharmacologically manipulated cattle and show, for the first time, that in dairy cows undergoing spontaneous oestrous cycles, natural variation in the duration of post-emergence ovulatory follicle development has a significant effect on pregnancy rate, presumably reflecting variation in oocyte developmental competence.

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4,-6 and-7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed RMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K-d 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.(C) 2003 Elsevier Science B.V. All rights reserved.