Ovarian Gene Expression Research Articles

Effects of genistein and estrogen receptor subtype-specific agonists in ArKO mice following different administration routes

We have scrutinized the effects of the phytoestrogen genistein and three synthetic estrogen receptor agonists, 17α-ethynylestradiol (EE), propylpyrazole-triol (PPT) and diarylpropionitrile (DPN) in the completely estrogen-free background of aromatase knockout (ArKO) mice by means of two routes of substance administration: oral via diet ( per os; po) or subcutaneous injection (sc) with the intention to evaluate the ArKO mice as sensitive model organism for uterotrophic assays. Additionally, we were aiming to qualitatively analyze effects resulting from oral administration path, in particular for PPT and DPN. Therefore, we analyzed the resulting uterine wet weights (UWW) and epithelial heights as physiological endpoints of function as well as the gonadotropin levels. Moreover, the gene expression profiles of estrogen receptors as well as important uterine and ovarian estrogen-response genes were investigated by real-time PCR. The uterus of ArKO mice responded very sensitive upon the substitution with EE (sc 5 μg/kg BW; po 50 μg/kg BW) in a proliferative manner. This was evaluated inter alia by increased UWW and by up-regulation of the expression of proliferation-associated and estrogen-response genes. It is important to note, that ERα and ERβ-agonist, PPT and DPN respectively (po 5 mg/kg BW and sc 0.5 mg/kg BW), have only been used for sc applications so far. Here, effects resulting from oral application were qualitatively described and evaluated for their applicability. The UWW and expression of proliferation-associated genes were increased following both po and sc treatment with PPT. In contrast, DPN did not exert an increase of the UWW, but a significant decrease of proliferation-associated gene and protein expression. Additionally, a substantial hypoplasia was detectable in the uterine cross-sections of DPN-treated mice. On the other hand, the phytoestrogen genistein (sc 10 mg/kg BW; po 70 mg/kg BW) did not cause detectable uterotrophic responses or large changes of uterine and ovarian gene expression profiles under the applied experimental conditions, but significantly reduced the elevated gonadotropin levels of ArKO mice. In summary, we showed the utility of ArKO mice to detect ER-specific effects, in particular those of PPT and DPN also when applied orally.

Accelerated Ovarian Aging in the Absence of the Transcription Regulator TAF4B in Mice1

The mammalian ovary is unique in that its reproductive life span is limited by oocyte quantity and quality. Oocytes are recruited from a finite pool of primordial follicles that are usually exhausted from the ovary during midadult life. If regulation of this pool is perturbed, the reproductive capacity of the ovary is compromised. TAF4B is a gonad-enriched subunit of the TFIID complex required for female fertility in mice. Previous characterization of TAF4B-deficient ovaries revealed several reproductive deficits that collectively result in infertility. However, the etiology of such fertility defects remains unknown. By assaying estrous cycle, ovarian pathology, and gene expression changes in young Taf4b-null female mice, we show that TAF4B-deficient female mice exhibit premature reproductive senescence. The rapid decline of ovarian function in Taf4b-null mice begins in early postnatal life, and follicle depletion is completed by 16 wk of age. To uncover differences in gene expression that may underlie accelerated ovarian aging, we compared genome-wide expression profiles of 3-wk-old, prepubescent Taf4b-null and wild-type ovaries. At 3 wk of age, decreased gene expression in Taf4b-null ovaries is similar to that seen in aged ovaries, revealing several molecular signatures of premature reproductive senescence, including reduced Smc1b. One significantly reduced transcript in the young TAF4B-null ovary codes for MOV10L1, a putative germline-specific RNA helicase that is related to the Drosophila RNA interference protein, armitage. We show here that Mov10l1 is expressed in mouse oocytes and that its expression is sensitive to TAF4B level, linking TAF4B to the posttranscriptional control of ovarian gene expression.

WebWatch

WebWatch

Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-beta, which was down-regulated, whereas ERalpha was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 microg/kgxd or 100 mg/kg.d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERalpha and ERbeta genes in postnatal d 50-60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERbeta promoter regions (P < 0.05), whereas the ERalpha promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

Disruption of the Salmon Reproductive Endocrine Axis Through Prolonged Nutritional Stress: Changes in Circulating Hormone Levels and Identification of Differentially Expressed Ovarian Genes.

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49. (poster)

Inhibition of egg production in zebrafish by fluoxetine and municipal effluents: A mechanistic evaluation

This study explored the impact of nominal concentrations of ethinylestradiol (EE 2; 10 ng/L), fluoxetine (FLU; 0.32, 3.2, 32 μg/L), and 1–50% treated municipal effluent on reproduction and liver function in sexually mature female zebrafish over a 7-day period. Compared with the control groups, FLU (32 μg/L) and 50% effluent treatment significantly reduced the average eggs spawned by approximately 4.5 and 2 fold, respectively. FLU also decreased ovarian levels of 17β-estradiol (E 2) without affecting the gonadosomatic indices of the fish. The expression of ovarian genes involved in the production of prostaglandins, steroid biosynthesis, and gonadotropin receptors were quantified to determine a potential mechanism underlying the reduced egg production in FLU and effluent exposed fish. Quantitative real-time PCR analysis determined that ovarian aromatase, follicle stimulating hormone receptor (FSHr), and luteinizing hormone receptor (LHr) gene expression were significantly reduced by FLU suggesting that disruptions to the synthesis of ovarian steroids and the actions of gonadotropins may underlie the negative influence of FLU on ovarian E 2 and spawning levels. Potential mechanisms involved in the modest effects of the effluent on reproduction remain unknown, but the elevated levels of total ammonia and nitrite in the 50% effluent treatment groups compared with the other treatments should not be discounted. Liver expression of CYP3A65 was significantly induced by all treatments of effluent, while EE 2 caused a reduction in the expression levels of CYP1A1, PXR, and CYP3A65. The results of the present study suggest that SSRI may disrupt reproductive functioning at concentrations greater than those found in receiving environments; yet, more research is warranted into to the possible effects of low levels of synthetic estrogens on liver function in exposed fish, particularly the PXR-CYP3A pathway.

Different promoter usage for CYP19 gene expression in buffalo ovary and placenta

Aromatase is the key enzyme for estrogen biosynthesis and is coded by CYP19 gene. The expression of CYP19 gene is regulated in tissue-specific manner by alternate use of different promoters. In this study, we have analyzed tissue-specific expression and their regulation of CYP19 gene in preovulatory and postovulatory stages of buffalo ( Bubalus bubalis) ovary and placenta. RT-PCR analysis showed that the CYP19 gene expression was significantly ( p < 0.05) higher in granulosa cells of large follicles as compared to the other tissues. The transcript analysis and transcriptional start site identified by 5′-RLM RACE for CYP19 expression indicated different transcriptional start sites within the different sized follicles during folliculogenesis. Sequence analysis showed that the transcription start site of transcript isolated from buffalo granulosa cells of small follicles was 37 bases upstream of the transcript isolated from granulosa cells of large follicles. However, both the transcripts were found to be derived from proximal promoter II. Difference in the transcriptional start site indicates the different promoter sequence usage in granulosa cells of different sized follicles. Further, in silico analysis of the difference in promoter sequence based on the 5′-UTRs isolated from granulosa cells of small follicles (151 bases) and large follicles (114 bases) showed that consensus sequence for certain important trans-elements (viz., TATA binding protein, E2F and CAAT binding protein) would lie in the promoter sequence isolated from the granulosa cells of large follicles. These transcription factors may be involved in regulation of CYP19 gene expression in ovary, either directly or indirectly. The difference in the size of 5′-UTR among the granulosa cells of ovary reflects the possible mechanism for the differential regulation of CYP19 gene during development. The transcripts isolated from buffalo corpus luteum and placental cotyledons were having same 5′-UTR comprises of 168 bases and found to be derived from PI.1. Estimates of CYP19 gene transcript concentration in the different tissues revealed that the CYP19 gene expression in granulosa cells is predominantly regulated by PII and to a minor extent by PI.1. However, PI.1 is almost exclusively responsible for CYP19 gene expression in placenta and residual expression in corpus luteum. In order to understand the complex CYP19 gene regulation in these tissues, further studies are needed to elucidate the activity of different promoters and define regulatory elements for binding of transcription factors.

Effect of chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin in female rats on ovarian gene expression

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. Female rats exposed chronically to environmentally relevant doses of the AHR agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) across their lifespan experience accelerated reproductive senescence preceded by ovarian endocrine disruption. The purpose of this study was to determine the changes in ovarian gene expression that accompany the loss of ovarian function caused by chronic exposure to TCDD. Beginning in utero, female Sprague–Dawley rats received TCDD (1, 5, 50, or 200 ng/kg-week; n = 4 per group) or vehicle weekly throughout their lifespan, and were sacrificed on diestrus just prior to loss of reproductive cyclicity at 11 months of age. Microarray analysis was used to determine differences in ovarian gene expression between control and TCDD-treated (200 ng/kg-week) animals. To confirm microarray results, real-time PCR was used to assess changes in gene expression among treatment groups. TCDD treatment decreased ( p < 0.05) proestrus serum estradiol concentrations with no effect on serum progesterone. In ovaries from rats treated with 200 ng/kg-week TCDD compared to controls, 19 genes of known function were found to be up-regulated, while 31 ovarian genes were found to be down-regulated ≥1.5-fold ( p ≤ 0.05). Gene expression of 17α-hydroxylase decreased following chronic TCDD treatment, suggesting the decrease in estradiol biosynthesis may be a consequence of decreased substrate. Taken together with past studies indicating a lack of effect on hypothalamus or pituitary function, the apparent regulation of key ovarian genes support the hypothesis that chronic TCDD exposure directly affects ovarian function.

Gene expression profile of rat ovarian tissue following xenotransplantation into immune-deficient mice

Immune-compromised mice have been used as gonadal tissue recipients to develop gametes of various mammalian species. The aim of this research was to determine gene expression differences between fresh and frozen-thawed rat xenotransplanted (XT) ovaries as well the gene expression differences between XT and sexually mature rat ovaries that were non-transplanted (NT). Ovaries from sexually immature female rats were transplanted under the kidney capsule of ovariectomized athymic nude mice either fresh or after freezing. The XT ovaries were collected approximately 10-12 weeks after xenografting for microarray analysis. The NT ovaries were collected from sexually mature rats. Gene expression was very similar between fresh and cryopreserved XT ovaries: 125 genes were twofold up- or downregulated, but level of regulation was not statistically significant. Overall patterns of gene expression between XT and NT ovaries were very different indicated by the absence of diagonal relationship between XT and NT ovary gene expression. More than 3000 genes were significantly (P<0.01) up- or downregulated between XT and NT ovaries. Genes involved in metabolic processes, lipid metabolism, and growth were downregulated in XT ovaries, whereas genes involved in immune and inflammatory response were upregulated in XT ovaries. The results showed that ovarian tissue xenografting significantly alters genes responsible for ovarian metabolism and function and leads to an upregulation of genes responsible for graft rejection.

Obese Women Exhibit Differences in Ovarian Metabolites, Hormones, and Gene Expression Compared with Moderate-Weight Women

Obese women experience longer times to conception, even if they are young and cycling regularly, which is suggestive of alterations in ovarian function during the periconceptual period. This study sought to determine whether there are alterations in the preovulatory follicular environment that are likely to influence oocyte developmental competence. Women attending a private infertility clinic were categorized into body mass index (BMI) groups of moderate (n = 33; BMI 20-24.9 kg/m(2)), overweight (n = 31; BMI 25-29.9 kg/m(2)), and obese (n =32; BMI >or=30 kg/m(2)). For each patient, follicular fluid was recovered from single follicles at oocyte retrieval, granulosa cells were pooled from multiple follicular aspirates and cumulus cells were pooled after separation from the oocytes. Follicle fluid was assayed for hormones and metabolites. Granulosa and cumulus cells were analyzed for mRNA expression of insulin signaling components (IRS-2 and Glut4), glucose-regulated genes (ChREBP, ACC, and FAS) and insulin-regulated genes (SREBP-1, CD36, and SR-BI) associated with obesity/insulin resistance. Increasing BMI was associated with increased follicular fluid insulin (P < 0.001), lactate (P = 0.01), triglycerides (P = 0.0003), and C-reactive protein (P < 0.0001) as well as decreased SHBG (P = 0.001). IRS-2, Glut4, ChREBP, and SREBP exhibited cell-type-specific expression but were not affected by BMI. CD36 and SRBI mRNA were modestly altered in granulosa cells of obese compared with moderate-weight women. Obese women exhibit an altered ovarian follicular environment, particularly increased metabolite, C-reactive protein, and androgen activity levels, which may be associated with poorer reproductive outcomes typically observed in these patients.

In situ hybridization to detect spatial gene expression in medaka

A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka ( Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with a 7 d exposure to the model aromatase inhibitor, fadrozole. Fadrozole did not affect the magnitude of gene expression in testes, but significantly up-regulated CYP19a gene expression in ovaries. These results were confirmed with quantitative real-time-polymerase chain reaction (RT-PCR). Histological evaluation revealed that females exposed to 100 μg/L fadrozole lacked mature oocytes. Male gonadal morphology was normal in all treatments. The ISH method developed in this study allowed tissue-specific resolution of gene expression in a whole animal model, as well as the ability to analyze cellular morphological detail in the same organism.

Progressive obesity leads to altered ovarian gene expression in the Lethal Yellow mouse: a microarray study

BackgroundLethal yellow (LY; C57BL/6J Ay/a) mice exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. The present study was designed to test the hypothesis that obese LY mice possess differences in expression of ovarian genes relative to age-matched lean mice.Methods90- and 180-day-old LY and lean black (C57BL/6J a/a) mice were suppressed with GnRH antagonist (Antide®), then stimulated with 5 IU eCG. cRNA derived from RNA extracts of whole ovarian homogenates collected 36 h post-eCG were run individually on Codelink Mouse Whole Genome Bioarrays (GE Healthcare Life Sciences).ResultsFifty-two genes showed ≥ 2-fold differential (p < 0.05) expression between 180-day-old obese LY and lean black mice. LY mice exhibited elevated ovarian expression of agouti (350×), leptin (6.5×), and numerous genes involved in cholesterol/lipid transport and metabolism, e.g. lanosterol synthase, Cyp51, and steroidogenic acute regulatory protein (Star). Fewer genes showed lower expression in LY mice, e.g. angiotensinogen. In contrast, none of these genes showed differential expression in 90-day-old LY and black mice, which are of similar body weight. Interestingly, 180-day-old LY mice had a 2-fold greater expression of 11beta-hydroxysteroid dehydrogenase type 1 (Hsd11b1) and a 2-fold lesser expression of 11beta-hydroxysteroid dehydrogenase type 2 (Hsd11b2), differences not seen in 90-day-old mice. Consistent with altered Hsd11b gene expression, ovarian concentrations of corticosterone (C) were elevated in aging LY mice relative to black mice, but C levels were similar in young LY and black mice.ConclusionThe data suggest that reproductive dysfunction in aging obese mice is related to modified intraovarian gene expression that is directly related to acquired obesity.

Ovarian development in mice requires the GATA4-FOG2 transcription complex

We have demonstrated previously that mammalian sexual differentiation requires both the GATA4 and FOG2 transcriptional regulators to assemble the functioning testis. Here we have determined that the sexual development of female mice is profoundly affected by the loss of GATA4-FOG2 interaction. We have also identified the Dkk1 gene, which encodes a secreted inhibitor of canonical beta-catenin signaling, as a target of GATA4-FOG2 repression in the developing ovary. The tissue-specific ablation of the beta-catenin gene in the gonads disrupts female development. In Gata4(ki/ki); Dkk1(-/-) or Fog2(-/-); Dkk1(-/-) embryos, the normal ovarian gene expression pattern is partially restored. Control of ovarian development by the GATA4-FOG2 complex presents a novel insight into the cross-talk between transcriptional regulation and extracellular signaling that occurs in ovarian development.

Developmental methoxychlor exposure affects multiple reproductive parameters and ovarian folliculogenesis and gene expression in adult rats

Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 μg/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity ( P < 0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females ( P < 0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries ( P < 0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor β was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles ( P < 0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles ( P < 0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis.

Obesity-Related Modification of Ovarian Gene Expression and Glucocorticoid Metabolism in Aging Lethal Yellow Mice.

Mice possessing the lethal yellow (LY) agouti mutation (C57BL/6J Ay/a) exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. We have previously reported that aging (180-day-old) LY mice exhibit altered ovarian expression of genes involved in sterol synthesis and metabolism relative to age-matched lean non-mutant black mice (B; C57BL/6J a/a). The present study was designed to test the hypothesis that the altered expression of ovarian genes is related to acquired obesity rather than to a direct consequence of the agouti mutation. Differential gene expression in young (90-day-old) LY and B mice, which have similar body weights, were compared with aging mice in which the LY animals are obese. To synchronize ovarian state and exclude gonadotropin-mediated effects, 90-day-old diestrous LY (n = 3) and B (n = 3) mice were suppressed with GnRH antagonist (Antide, Sigma, St. Louis, MO) for five days, then injected with 5 IU eCG (Sigma). Mice were killed 36 h after eCG, and ovaries were immediately removed, carefully trimmed of adhering tissue, and placed in RNALater (Ambion, Austin, TX ) for subsequent RNA extraction. cRNA derived from RNA extracts were run individually on Codelink Mouse Whole Genome Bioarrays (GE Healthcare Life Sciences). Relative gene expression was compared between B and LY mice by t-test using GeneSpring software. Unnamed genes, EST, genes whose expression was below sensitivity limits, and those with less than a 2-fold difference in expression between groups were excluded from further analysis. After these exclusions, 45 genes were differentially expressed in young B and LY mice, compared to 110 differentially expressed genes in aging mice. Both 90- and 180-day-old LY mice exhibited elevated ovarian expression relative to B mice of the agouti gene (330x,350x) as expected. In contrast, numerous genes whose expression was elevated in aging LY mice relative to B mice, e.g. leptin, HMG-CoA reductase, CYP51, and steroidogenic acute regulatory protein (StAR), were not differentially expressed in young LY and B mice. Notably, aging LY mice had a 2-fold greater expression of 11β-HSD type 1 and a 2-fold lesser expression of 11β-HSD type 2 relative to B mice. There was no difference in expression of these genes in young B and LY mice. Consistent with altered 11β-HSD gene expression, ovarian concentrations of corticosterone (C) were elevated in aging LY mice relative to B mice (1.7 ng/mg vs. 0.8 ng/mg, respectively), but C levels were similar in young B and LY mice (0.8 ng/mg vs. 0.9 ng/mg). Results suggest that differential ovarian gene expression and glucocorticoid metabolism observed in aging LY mice is directly related to acquired obesity. (Supported by NIH INBRE 2P20RR016479, NIH R15 HD044438, and Sanford Research/USD)

Peroxisome Proliferator-Activated Receptor-γ Agonist Rosiglitazone Reverses the Adverse Effects of Diet-Induced Obesity on Oocyte Quality

Obesity and its physiological consequences are increasingly prevalent among women of reproductive age and are associated with infertility. To investigate, female mice were fed a high-fat diet until the onset of insulin resistance, followed by assessments of ovarian gene expression, ovulation, fertilization, and oocyte developmental competence. We report defects to ovarian function associated with diet-induced obesity (DIO) that result in poor oocyte quality, subsequently reduced blastocyst survival rates, and abnormal embryonic cellular differentiation. To identify critical cellular mediators of ovarian responses to obesity induced insulin resistance, DIO females were treated for 4 d before mating with an insulin-sensitizing pharmaceutical: glucose and lipid-lowering AMP kinase activator, 5-aminoimidazole 4-carboxamide-riboside, 30 mg/kg.d; sodium salicylate, IkappaK inhibitor that reverses insulin resistance, 50 mg/kg.d; or peroxisome proliferator activated receptor-gamma agonist rosiglitazone, 10 mg/kg.d. 5-aminoimidazole 4-carboxamide-riboside or sodium salicylate treatment did not have significant effects on the reproductive parameters examined. However, embryonic development to the blastocyst stage was significantly improved when DIO mice were treated with rosiglitazone, effectively repairing development rates. Rosiglitazone also normalized DIO-associated abnormal blastomere allocation to the inner cell mass. Such improvements to oocyte quality were coupled with weight loss, improved glucose metabolism, and changes in ovarian mRNA expression of peroxisome proliferator activated receptor-regulated genes, Cd36, Scarb1, and Fabp4 cholesterol transporters. These studies demonstrate that peri-conception treatment with select insulin-sensitizing pharmaceuticals can directly influence ovarian functions and ultimately exert positive effects on oocyte developmental competence. Improved blastocyst quality in obese females treated with rosiglitazone before mating indicates that peroxisome proliferator activated receptor-gamma is a key target for metabolic regulation of ovarian function and oocyte quality.

Ovarian gene expression is stable after exposure to trichloroethylene

Exposure of female rats to trichloroethylene (TCE), an environmental toxicant commonly found in ground and surface waters throughout the United States, reduces the fertilizability of oocytes produced by these females compared with oocytes from control females. Localization of cytochrome P450 2E1 and glutathione s-transferase α, TCE-metabolizing enzymes, in the ovary suggests TCE metabolism occurs in the ovary. The production of bioactive TCE metabolites in the ovary may alter female reproductive function by altering ovarian gene transcription and/or protein expression and function. The purpose of the present study was to examine ovarian gene transcription after exposure of female rats to 0.45% TCE (v/v) in 3% Tween. Control rats received 3% Tween. Microarray analysis after 1 and 5 days of exposure indicated ovarian gene transcription was maintained during TCE exposure with the possible exception of a very few genes. Although conclusions for these few genes were ambiguous from the microarray analysis due to the minimal but statistically significant reductions, quantitative real time RT-PCR (qRT-PCR) analysis indicated expression of these genes was unaltered after TCE exposure. Protein analysis confirmed qRT-PCR results. This study suggests TCE-induced reductions in oocyte fertilizability are independent of currently detectable alterations in ovarian gene expression.

EFFECT OF CHRONIC EXPOSURE TO THE ARYL HYDROCARBON RECEPTOR AGONIST 2,3,7,8-TETRACHLORODIBENZO- P-DIOXIN IN FEMALE RATS ON OVARIAN GENE EXPRESSION

Abstract The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. Female rats exposed chronically to environmentally relevant doses of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) across their lifespan experience accelerated reproductive senescence preceded by ovarian endocrine disruption. The purpose of this study was to determine the changes in ovarian gene expression that accompany the loss of ovarian function caused by chronic exposure to TCDD. Beginning in utero, female Sprague–Dawley rats received TCDD (1, 5, 50, or 200 ng/kg-week; n = 4 per group) or vehicle weekly throughout their lifespan, and were sacrificed on diestrus just prior to loss of reproductive cyclicity at 11 months of age. Microarray analysis was used to determine differences in ovarian gene expression between control and TCDD-treated (200 ng/kg-week) animals. To confirm microarray results, real-time PCR was used to assess changes in gene expression among treatment groups. TCDD treatment decreased (p

Ovarian gene expression in the absence of FIGLA, an oocyte-specific transcription factor

BackgroundOvarian folliculogenesis in mammals is a complex process involving interactions between germ and somatic cells. Carefully orchestrated expression of transcription factors, cell adhesion molecules and growth factors are required for success. We have identified a germ-cell specific, basic helix-loop-helix transcription factor, FIGLA (Factor In the GermLine, Alpha) and demonstrated its involvement in two independent developmental processes: formation of the primordial follicle and coordinate expression of zona pellucida genes.ResultsTaking advantage of Figla null mouse lines, we have used a combined approach of microarray and Serial Analysis of Gene Expression (SAGE) to identify potential downstream target genes. Using high stringent cutoffs, we find that FIGLA functions as a key regulatory molecule in coordinating expression of the NALP family of genes, genes of known oocyte-specific expression and a set of functionally un-annotated genes. FIGLA also inhibits expression of male germ cell specific genes that might otherwise disrupt normal oogenesis.ConclusionThese data implicate FIGLA as a central regulator of oocyte-specific genes that play roles in folliculogenesis, fertilization and early development.

Genomic and post-genomic approaches to polycystic ovary syndrome--progress so far: Mini Review

Genomic studies in polycystic ovary syndrome (PCOS) have focused on ovarian tissues and gene expression changes related to the gynaecological manifestations of PCOS. These studies have revealed a variety of altered genes that fall into many functional categories. Of these, the genes involved in steroidogenesis, including genes related to retinoic acid biosynthesis and LH-stimulated gene pathways, are generally up-regulated in PCOS samples. Genes involved in the Wnt signalling pathway appear down-regulated. Immune response genes and those involved in apoptosis are altered, but the net effect of these alterations is unclear at present. However, these altered gene expression patterns are yet to produce a defined aetiological basis or diagnostic biomarker for PCOS. The use of proteomic technologies for the study of the PCOS proteome is in its infancy; however, a few pilot studies have been published and the data are reviewed. Proteomics looks directly at the functional units within a cell, the proteins. This approach should thus serve to validate some of the gene expression changes identified and then build on the genomic results collected to date.