Multiple generation distinct toxicant exposures induce epigenetic transgenerational inheritance of enhanced pathology and obesity.

Three successive multiple generations of rats were exposed to different toxicants and then bred to the transgenerational F5 generation to assess the impacts of multiple generation different exposures. The current study examines the actions of the agricultural fungicide vinclozolin on the F0 generation, followed by jet fuel hydrocarbon mixture exposure of the F1 generation, and then pesticide dichlorodiphenyltrichloroethane on the F2 generation gestating females. The subsequent F3 and F4 generations and F5 transgenerational generation were obtained and F1-F5 generations examined for male sperm epigenetic alterations and pathology in males and females. Significant impacts on the male sperm differential DNA methylation regions were observed. The F3-F5 generations were similar in ∼50% of the DNA methylation regions. The pathology of each generation was assessed in the testis, ovary, kidney, and prostate, as well as the presence of obesity and tumors. The pathology used a newly developed Deep Learning, artificial intelligence-based histopathology analysis. Observations demonstrated compounded disease impacts in obesity and metabolic parameters, but other pathologies plateaued with smaller increases at the F5 transgenerational generation. Observations demonstrate that multiple generational exposures, which occur in human populations, appear to increase epigenetic impacts and disease susceptibility.

The role of epigenetics in multi-generational transmission of asthma: An NIAID workshop report-based narrative review.

There is mounting evidence that environmental exposures can result in effects on health that can be transmitted across generations, without the need for a direct exposure to the original factor, for example, the effect of grandparental smoking on grandchildren. Hence, an individual's health should be investigated with the knowledge of cross-generational influences. Epigenetic factors are molecular factors or processes that regulate genome activity and may impact cross-generational effects. Epigenetic transgenerational inheritance has been demonstrated in plants and animals, but the presence and extent of this process in humans are currently being investigated. Experimental data in animals support transmission of asthma risk across generations from a single exposure to the deleterious factor and suggest that the nature of this transmission is in part due to changes in DNA methylation, the most studied epigenetic process. The association of father's prepuberty exposure with offspring risk of asthma and lung function deficit may also be mediated by epigenetic processes. Multi-generational birth cohorts are ideal to investigate the presence and impact of transfer of disease susceptibility across generations and underlying mechanisms. However, multi-generational studies require recruitment and assessment of participants over several decades. Investigation of adult multi-generation cohorts is less resource intensive but run the risk of recall bias. Statistical analysis is challenging given varying degrees of longitudinal and hierarchical data but path analyses, structural equation modelling and multilevel modelling can be employed, and directed networks addressing longitudinal effects deserve exploration as an effort to study causal pathways.

Epigenetic transgenerational inheritance of parent-of-origin allelic transmission of outcross pathology and sperm epimutations.

Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease. Previous observations have demonstrated that the agricultural fungicide vinclozolin and pesticide DDT (dichlorodiphenyltrichloroethane) induce transgenerational sperm epimutations involving DNA methylation, ncRNA, and histone modifications or retention. These two environmental toxicants were used to investigate the impacts of parent-of-origin outcross on the epigenetic transgenerational inheritance of disease. Male and female rats were collected from a paternal outcross (POC) or a maternal outcross (MOC) F4 generation control and exposure lineages for pathology and epigenetic analysis. This model allows the parental allelic transmission of disease and epimutations to be investigated. There was increased pathology incidence in the MOC F4 generation male prostate, kidney, obesity, and multiple diseases through a maternal allelic transmission. The POC F4 generation female offspring had increased pathology incidence for kidney, obesity and multiple types of diseases through the paternal allelic transmission. Some disease such as testis or ovarian pathology appear to be transmitted through the combined actions of both male and female alleles. Analysis of the F4 generation sperm epigenomes identified differential DNA methylated regions (DMRs) in a genome-wide analysis. Observations demonstrate that DDT and vinclozolin have the potential to promote the epigenetic transgenerational inheritance of disease and sperm epimutations to the outcross F4 generation in a sex specific and exposure specific manner. The parent-of-origin allelic transmission observed appears similar to the process involved with imprinted-like genes.

Environmental Toxicant Induced Epigenetic Transgenerational Inheritance of Prostate Pathology and Stromal-Epithelial Cell Epigenome and Transcriptome Alterations: Ancestral Origins of Prostate Disease.

Prostate diseases include prostate cancer, which is the second most common male neoplasia, and benign prostatic hyperplasia (BPH), which affects approximately 50% of men. The incidence of prostate disease is increasing, and some of this increase may be attributable to ancestral exposure to environmental toxicants and epigenetic transgenerational inheritance mechanisms. The goal of the current study was to determine the effects that exposure of gestating female rats to vinclozolin has on the epigenetic transgenerational inheritance of prostate disease, and to characterize by what molecular epigenetic mechanisms this has occurred. Gestating female rats (F0 generation) were exposed to vinclozolin during E8-E14 of gestation. F1 generation offspring were bred to produce the F2 generation, which were bred to produce the transgenerational F3 generation. The transgenerational F3 generation vinclozolin lineage males at 12 months of age had an increased incidence of prostate histopathology and abnormalities compared to the control lineage. Ventral prostate epithelial and stromal cells were isolated from F3 generation 20-day old rats, prior to the onset of pathology, and used to obtain DNA and RNA for analysis. Results indicate that there were transgenerational changes in gene expression, noncoding RNA expression, and DNA methylation in both cell types. Our results suggest that ancestral exposure to vinclozolin at a critical period of gestation induces the epigenetic transgenerational inheritance of prostate stromal and epithelial cell changes in both the epigenome and transcriptome that ultimately lead to prostate disease susceptibility and may serve as a source of the increased incidence of prostate pathology observed in recent years.

Environmental toxicant induced epigenetic transgenerational inheritance of ovarian pathology and granulosa cell epigenome and transcriptome alterations: ancestral origins of polycystic ovarian syndrome and primary ovarian insufiency.

Two of the most prevalent ovarian diseases affecting women's fertility and health are Primary Ovarian Insufficiency (POI) and Polycystic Ovarian Syndrome (PCOS). Previous studies have shown that exposure to a number of environmental toxicants can promote the epigenetic transgenerational inheritance of ovarian disease. In the current study, transgenerational changes to the transcriptome and epigenome of ovarian granulosa cells are characterized in F3 generation rats after ancestral vinclozolin or DDT exposures. In purified granulosa cells from 20-day-old F3 generation females, 164 differentially methylated regions (DMRs) (P < 1 x 10-6) were found in the F3 generation vinclozolin lineage and 293 DMRs (P < 1 x 10-6) in the DDT lineage, compared to controls. Long noncoding RNAs (lncRNAs) and small noncoding RNAs (sncRNAs) were found to be differentially expressed in both the vinclozolin and DDT lineage granulosa cells. There were 492 sncRNAs (P < 1 x 10-4) in the vinclozolin lineage and 1,085 sncRNAs (P < 1 x 10-4) in the DDT lineage. There were 123 lncRNAs and 51 lncRNAs in the vinclozolin and DDT lineages, respectively (P < 1 x 10-4). Differentially expressed mRNAs were also found in the vinclozolin lineage (174 mRNAs at P < 1 x 10-4) and the DDT lineage (212 mRNAs at P < 1 x 10-4) granulosa cells. Comparisons with known ovarian disease associated genes were made. These transgenerational epigenetic changes appear to contribute to the dysregulation of the ovary and disease susceptibility that can occur in later life. Observations suggest that ancestral exposure to toxicants is a risk factor that must be considered in the molecular etiology of ovarian disease.

Atrazine induced epigenetic transgenerational inheritance of disease, lean phenotype and sperm epimutation pathology biomarkers.

Ancestral environmental exposures to a variety of environmental toxicants and other factors have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The current study examined the potential transgenerational actions of the herbicide atrazine. Atrazine is one of the most commonly used herbicides in the agricultural industry, in particular with corn and soy crops. Outbred gestating female rats were transiently exposed to a vehicle control or atrazine. The F1 generation offspring were bred to generate the F2 generation and then the F2 generation bred to generate the F3 generation. The F1, F2 and F3 generation control and atrazine lineage rats were aged and various pathologies investigated. The male sperm were collected to investigate DNA methylation differences between the control and atrazine lineage sperm. The F1 generation offspring (directly exposed as a fetus) did not develop disease, but weighed less compared to controls. The F2 generation (grand-offspring) was found to have increased frequency of testis disease and mammary tumors in males and females, early onset puberty in males, and decreased body weight in females compared to controls. The transgenerational F3 generation rats were found to have increased frequency of testis disease, early onset puberty in females, behavioral alterations (motor hyperactivity) and a lean phenotype in males and females. The frequency of multiple diseases was significantly higher in the transgenerational F3 generation atrazine lineage males and females. The transgenerational transmission of disease requires germline (egg or sperm) epigenetic alterations. The sperm differential DNA methylation regions (DMRs), termed epimutations, induced by atrazine were identified in the F1, F2 and F3 generations. Gene associations with the DMRs were identified. For the transgenerational F3 generation sperm, unique sets of DMRs (epimutations) were found to be associated with the lean phenotype or testis disease. These DMRs provide potential biomarkers for transgenerational disease. The etiology of disease appears to be in part due to environmentally induced epigenetic transgenerational inheritance, and epigenetic biomarkers may facilitate the diagnosis of the ancestral exposure and disease susceptibility. Observations indicate that although atrazine does not promote disease in the directly exposed F1 generation, it does have the capacity to promote the epigenetic transgenerational inheritance of disease.

Ancestral dichlorodiphenyltrichloroethane (DDT) exposure promotes epigenetic transgenerational inheritance of obesity.

BACKGROUND: Ancestral environmental exposures to a variety of environmental factors and toxicants have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The present work examined the potential transgenerational actions of the insecticide dichlorodiphenyltrichloroethane (DDT) on obesity and associated disease. METHODS: Outbred gestating female rats were transiently exposed to a vehicle control or DDT and the F1 generation offspring bred to generate the F2 generation and F2 generation bred to generate the F3 generation. The F1 and F3 generation control and DDT lineage rats were aged and various pathologies investigated. The F3 generation male sperm were collected to investigate methylation between the control and DDT lineage male sperm. RESULTS: The F1 generation offspring (directly exposed as a fetus) derived from the F0 generation exposed gestating female rats were not found to develop obesity. The F1 generation DDT lineage animals did develop kidney disease, prostate disease, ovary disease and tumor development as adults. Interestingly, the F3 generation (great grand-offspring) had over 50% of males and females develop obesity. Several transgenerational diseases previously shown to be associated with metabolic syndrome and obesity were observed in the testis, ovary and kidney. The transgenerational transmission of disease was through both female (egg) and male (sperm) germlines. F3 generation sperm epimutations, differential DNA methylation regions (DMR), induced by DDT were identified. A number of the genes associated with the DMR have previously been shown to be associated with obesity. CONCLUSIONS: Observations indicate ancestral exposure to DDT can promote obesity and associated disease transgenerationally. The etiology of disease such as obesity may be in part due to environmentally induced epigenetic transgenerational inheritance.

Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

Environmentally induced epigenetic transgenerational inheritance of ovarian disease.

The actions of environmental toxicants and relevant mixtures in promoting the epigenetic transgenerational inheritance of ovarian disease was investigated with the use of a fungicide, a pesticide mixture, a plastic mixture, dioxin and a hydrocarbon mixture. After transient exposure of an F0 gestating female rat during embryonic gonadal sex determination, the F1 and F3 generation progeny adult onset ovarian disease was assessed. Transgenerational disease phenotypes observed included an increase in cysts resembling human polycystic ovarian disease (PCO) and a decrease in the ovarian primordial follicle pool size resembling primary ovarian insufficiency (POI). The F3 generation granulosa cells were isolated and found to have a transgenerational effect on the transcriptome and epigenome (differential DNA methylation). Epigenetic biomarkers for environmental exposure and associated gene networks were identified. Epigenetic transgenerational inheritance of ovarian disease states was induced by all the different classes of environmental compounds, suggesting a role of environmental epigenetics in ovarian disease etiology.

Inhibitory actions of Anti-Müllerian Hormone (AMH) on ovarian primordial follicle assembly.

The current study was designed to investigate the actions of Anti-Müllerian Hormone (AMH) on primordial follicle assembly. Ovarian primordial follicles develop from the breakdown of oocyte nests during fetal development for the human and immediately after birth in rodents. AMH was found to inhibit primordial follicle assembly and decrease the initial primordial follicle pool size in a rat ovarian organ culture. The AMH expression was found to be primarily in the stromal tissue of the ovaries at this period of development, suggesting a stromal-epithelial cell interaction for primordial follicle assembly. AMH was found to promote alterations in the ovarian transcriptome during primordial follicle assembly with over 200 genes with altered expression. A gene network was identified suggesting a potential central role for the Fgf2/Nudt6 antisense transcript in the follicle assembly process. A number of signal transduction pathways are regulated by AMH actions on the ovarian transcriptome, in particular the transforming growth factor-beta (TGFß) signaling process. AMH is the first hormone/protein shown to have an inhibitory action on primordial follicle assembly. Due to the critical role of the primordial follicle pool size for female reproduction, elucidation of factors, such as AMH, that regulate the assembly process will provide insights into potential therapeutics to manipulate the pool size and female reproduction.

Progesterone regulation of primordial follicle assembly in bovine fetal ovaries.

Fertility in mammals is dependant on females having an adequate primordial follicle pool to supply oocytes for fertilization. The formation of primordial follicles is called ovarian follicular assembly. In rats and mice progesterone and estradiol have been shown to inhibit follicle assembly with assembly occurring after birth when the pups are removed from the high-steroid maternal environment. In contrast, primordial follicle assembly in other species, such as cattle and humans, occurs during fetal development before birth. The objective of the current study is to determine if progesterone levels regulate primordial follicle assembly in fetal bovine ovaries. Ovaries and blood were collected from bovine fetuses. Interestingly, ovarian progesterone and estradiol concentrations were found to decrease with increasing fetal age and correlated to increased primordial follicle assembly. Microarray analysis of fetal ovary RNA suggests that progesterone membrane receptor and estrogen nuclear receptor are expressed. Treatment of fetal bovine ovary cultures with a higher progesterone concentration significantly decreased primordial follicle assembly. Observations indicate that progesterone affects ovarian primordial follicle assembly in cattle, as it does in rats and mice.

Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease.

Endocrine disruptor exposure during gonadal sex determination was previously found to induce male rat adult onset transgenerational disease (F1-F4 generation), and this was associated with an alteration in the epigenetic (i.e., DNA methylation) programming of the male germ line. The current study was designed to characterize the transgenerational disease phenotypes of the female adult offspring. Pregnant rats (F0 generation) were treated transiently with vinclozolin (i.e., fungicide with anti-androgenic activity) on embryonic (E) days E8-E14 of gestation. F1 control and vinclozolin generation offspring from different litters were mated to produce F2 offspring, and similarly F2 generation animals produced F3 generation offspring. Observations demonstrated that 9 out of 105 pregnant rats (8.6%) from the vinclozolin F1-F3 generations exhibited uterine hemorrhage and/or anemia late in pregnancy. None (0 out of 82) of the control F1-F3 generation females had similar pregnancy problems. Complete blood cell counts and serum chemistry profiles demonstrated that selected vinclozolin generation animals, but not controls, exhibited marked regenerative anemia in late pregnancy. Examination of kidney histology revealed moderate or severe glomerular abnormalities in 67% of the vinclozolin F2 and F3 generation adult females compared with 18% of the controls. Adult female vinclozolin generation animals also developed various types of tumors in 6.5% of the animals (11 out of 170), while 2% of control-line animals (3 out of 151) developed mammary tumors. Observations demonstrate that vinclozolin exposure during gonadal sex determination promotes a transgenerational increase in pregnancy abnormalities and female adult onset disease states.

Regulation of granulosa and theca cell transcriptomes during ovarian antral follicle development.

Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function.

Role of triptolide as an adjunct chemotherapy for ovarian cancer.

BACKGROUND: Triptolide (TPL) has been identified as the active component of the Tripterygium wilfordii hook F plant and demonstrated to possess antitumor properties and induce apoptosis in a variety of tumor cell lines. Since TPL actions are associated with changes in the activities of both p53 and NF kappaB, which are implicated in the chemoresistance of ovarian cancer, the ability of TPL to be a potential chemotherapeutic for ovarian cancer was considered. METHODS: TPL actions on human ovarian cancer cells were investigated in vitro and in vivo with a nude mouse model to monitor tumor burden both in the absence or presence of other chemotherapy agents. RESULTS: TPL was effective as a single agent in inducing apoptosis of ovarian cancer cells in vitro, but not in vivo. TPL enhanced the cytotoxicity of carboplatin in culture and enhanced carboplatin-mediated reduction of tumor burden in nude mice inoculated with human ovarian cancer cells. Previously, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor was found to enhance carboplatin actions on ovarian cancer. Interestingly, the combined treatment of TPL, PI3 kinase inhibitor LY294002 and carboplatin was found to dramatically reduce ovarian tumor progression and burden in nude mice. CONCLUSION: In 44% of the animals tested the combined treatment caused complete regression of ovarian cancer. Combined observations indicate TPL may be an effective adjunct chemotherapy for ovarian cancer.

Interactions between progesterone and tumor necrosis factor-alpha in the regulation of primordial follicle assembly.

Follicle assembly is the process by which groups or "nests" of oocytes break down to form primordial follicles. The size of the primordial follicle pool is the major determinant of the reproductive lifespan of a female. Previously, progesterone (P(4)) has been shown to inhibit follicle assembly, while tumor necrosis factor-alpha (TNFalpha) has been shown to promote the apoptosis that is necessary for follicle assembly. The present study examines how TNFalpha and progesterone interact to regulate primordial follicle assembly. Ovaries were collected from newborn rats and placed in organ culture to examine the actions of P(4) and TNFalpha. P(4) was found to decrease primordial follicle assembly and increase the percentage of unassembled oocytes both in vitro and in vivo. TNFalpha treatment did not change the proportion of assembled follicles in cultured ovaries, but blocked the ability of P(4) to inhibit follicle assembly. Microarray analysis of the ovarian transcriptome revealed that progesterone treatment of the ovaries altered the expression of 513 genes with 132 only expressed after P(4) treatment and 16 only expressed in control ovaries. The majority of genes were up-regulated greater than twofold over control, with a small subset of 16 genes down-regulated. Categories of genes affected by P(4) are described including a group of extracellular signaling factors. The progesterone receptors expressed at the time of follicle assembly included the surface membrane progesterone receptors PGRMC1, PGRMC2, and RDA288. The nuclear genomic P(4) receptor was not expressed at appreciable levels. Progesterone increased the expression of several genes (TANK, NFkappaB, Bcl2l1, and Bcl2l2) involved in a signaling pathway that promotes cell survival and inhibits apoptosis. Observations indicate that P(4) acts through the surface membrane progesterone receptors to regulate primordial follicle assembly, and that TNFalpha can override the inhibitory actions of P(4) on follicle assembly. A major mechanism involved in the actions of P(4) is an increase in cell survival genes and inhibition of the apoptosis pathway. Observations provide insight into the hormonal regulation of primordial follicle assembly and lead to novel approaches to potentially manipulate follicle assembly and reproductive capacity.

Platelet-derived growth factor modulates the primordial to primary follicle transition.

Primordial follicles steadily leave the arrested pool and undergo a primordial to primary follicle transition during the female reproductive lifespan. When the available pool of primordial follicles is depleted reproduction ceases and humans enter menopause. The present study was designed to investigate the actions of several growth factors previously identified as candidate regulatory factors for the primordial to primary follicle transition with a microarray analysis. Ovaries from 4-day-old rats were placed into culture and treated for 2 weeks with platelet-derived growth factor (PDGF), anti-PDGF neutralizing antibody, vascular endothelial growth factor (VEGF), neuregulin (NRG), or kit ligand (KITL) as a positive control. PDGF-treatment resulted in a significant decrease in the percentage of primordial follicles and a concomitant increase in the percentage of developing primary follicles compared to controls. In contrast, ovaries treated with an anti-PDGF neutralizing antibody had a significant increase in the percentage of primordial follicles demonstrating an inhibition of endogenous follicle development. Ovaries incubated in the presence of VEGF or NRG had no change in follicle development. Observations indicate that PDGF, but not VEGF or NRG, promotes the primordial to primary follicle transition. Immunohistochemical localization indicated that the PDGF protein was present in the oocytes of both primordial and developing follicles. PDGF-treatment of cultured ovaries resulted in an increase in KITL mRNA expression. KITL has been previously shown to promote the primordial to primary follicle transition. KITL-treatment of ovaries had no effect on expression of Pdgf or any PDGF homologs or receptors. Therefore, PDGF appears to be produced by the oocyte and acts as one of several extracellular signaling factors that regulate the primordial to primary follicle transition. These observations provide insight into the cell-cell interactions involved in the regulation of primordial follicle development and can be used in the future development of therapies for some forms of infertility.

Alterations in the ovarian transcriptome during primordial follicle assembly and development.

The assembly of the developmentally arrested primordial follicle and subsequent transition to the primary follicle are poorly understood processes critical to ovarian biology. Abnormal primordial follicle development can lead to pathologies such as premature ovarian failure. The current study used a genome-wide expression profile to investigate primordial follicle assembly and development. Rat ovaries with predominantly unassembled, primordial, or primary follicles were obtained. RNA from these ovaries was hybridized to rat microarray gene chips, and the gene expression (i.e., ovarian transcriptome) was compared between the developmental stages. Analysis of the ovarian transcriptome demonstrated 148 genes up-regulated and 50 genes down-regulated between the unassembled and primordial follicle stages. Observations demonstrate 80 genes up-regulated and 44 genes down-regulated between the primordial and primary follicle stages. The analysis demonstrated 2332 genes common among the three developmental stages, 146 genes specific for the unassembled follicles, 94 genes specific for the primordial follicles, and 151 genes specific for the primary follicles. Steroidogenic genes are up-regulated between unassembled and primordial follicles, and then many are again down-regulated between primordial and primary follicles. The hormones inhibin and Mullerian inhibitory substance (MIS) display a similar pattern of expression with the highest levels of mRNA in the primordial follicles. Several novel unknown genes that had dramatic changes in expression during primordial follicle development were also identified. Gene families/clusters identified that were up-regulated from unassembled to primordial follicles include growth factors and signal transduction gene clusters, whereas a down-regulated gene family was the synaptonemal complex genes associated with meiosis. Gene families/clusters that were up-regulated between primordial and primary follicles included immune response genes, metabolic enzymes, and proteases, whereas down-regulated gene families include the globulin genes and some steroidogenic genes. The expression of several growth factors changed during primordial follicle development, including vascular endothelial growth factor and insulin-like growth factor II. Elucidation of how these changes in gene expression coordinate primordial follicle assembly and the primordial to primary follicle transition provides a better understanding of these critical biological processes and allows selection of candidate regulatory factors for further investigation.

Role of transforming growth factor beta in ovarian surface epithelium biology and ovarian cancer.

Ovarian cancers arise out of the ovarian surface epithelium (OSE), which is the single layer of epithelial cells covering the ovary. These cells go through repeated cycles of proliferation with the growth and rupture of ovarian follicles. One growth factor involved in the regulation of OSE is transforming growth factor beta (TGFbeta). The different isoforms of TGFbeta (TGFbeta1, TGFbeta2 and TGFbeta3) and its receptor are all present in both OSE and the underlying ovarian surface stroma. The levels of the TGFbeta isoforms and receptors are regulated independently of each other in these different ovarian tissues. Observations suggest the existence of multiple autocrine/paracrine TGFbeta signalling loops. TGFbeta acts to inhibit proliferation of normal OSE and early stage ovarian carcinomas. Conversely, in later stage ovarian cancer the inhibitory actions of TGFbeta on epithelial proliferation have been overcome, while TGFbeta is able to promote malignant neoplastic behaviours. The regulation of TGFbeta signalling by ovarian steroid hormones may be one mechanism by which the OSE responds to cyclic changes in the underlying follicles.

Cell-cell interactions in primordial follicle assembly and development.

Two critical processes in ovarian biology are the assembly of the primordial follicles early in development and then the subsequent development and transition of the primordial follicle to the primary follicle. These processes directly effect the number of oocytes available to a female throughout her reproductive life. Once the pool of primordial follicles is depleted a series of physiological changes known as menopause begins in humans. The inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure. Studies demonstrate primordial follicle assembly and development are coordinated by locally produced paracrine and autocrine factors. Factors have been identified that influence follicular assembly such as neurotropins. Several local factors that promote the primordial to primary follicle transition have also recently been identified. These include growth factors such as kit-ligand, leukemia inhibitory factor and basic fibroblast growth factor. Interestingly, recent studies demonstrate Müllerian inhibitory substance appears to inhibit the primordial to primary follicle transition. Therefore, observations suggest a mechanism for both positive and negative control of the primordial to primary follicle transition. The studies reviewed regarding the control of primordial follicle assembly and the primordial to primary follicle transition help elucidate these poorly understood aspects of ovarian biology.

Insulin but not insulin-like growth factor-1 promotes the primordial to primary follicle transition.

A critical step in ovarian biology is the transition of the developmentally arrested primordial follicle to the growing primary follicle. The current study utilizes a rat ovarian organ culture system to investigate the role of insulin and insulin-like growth factor-1 (IGF-1) in this process. Four-day-old rat ovaries were cultured and the degree of primordial to primary follicle transition measured. Insulin increased the primordial to primary follicle transition 30% over control with a half maximal effective concentration (EC50) between 2.5 and 5 ng/ml. IGF-1 did not cause an increase in the primordial to primary follicle transition at concentrations up to 100 ng/ml. Ovaries were also treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) and neither had an effect on the primordial to primary follicle transition. Ovaries were treated with insulin in conjunction with other factors known to promote the primordial to primary follicle transition in order to discern any potential synergistic effects. Previous experiments have shown that kit ligand (KL), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) promote the primordial to primary follicle transition. Insulin was shown to have an additive effect with KL and LIF, but not bFGF. The fact that insulin can influence the primordial to primary follicle transition at low concentrations (i.e. 5 ng/ml) and that IGF-1 has no effect suggests that insulin is acting at the insulin receptor, not the IGF-1 receptor. The observation that insulin has an additive effect with KL and LIF, but not bFGF, suggests the insulin's site of action is likely the oocyte. In summary, observations suggest that insulin acts as an endocrine type factor to help coordinate primordial to primary follicle transition at the level of the oocyte. The significance of the observations in relation to diabetes and female infertility is discussed.