Eric E. Nilsson

Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis

The recruitment of primordial follicles to initiate folliculogenesis determines the population of developing follicles available for ovulation and directly regulates female reproductive efficiency. In the current study, a floating organ culture system was used to examine the progression of primordial (stage 0) follicles to developing (stages 1-4) follicles in 4-day-old pre-pubertal rat ovaries. Basic fibroblast growth factor (bFGF) was found to induce primordial follicle development similar to what has been demonstrated for kit ligand/stem cell factor (KL). The bFGF-treated ovaries contained 85% developing follicles compared with 50% developing follicles for control untreated organ cultures. Correspondingly, the number of primordial follicles in bFGF-treated ovaries decreased to 15% of the total compared with 45% for controls. A bFGF neutralizing antibody was found to decrease the small amount of spontaneous follicle development that occurs during the organ culture. Basic FGF was localized to primordial and early developing follicles by immunocytochemistry and was primarily observed in the oocytes. Treatment of bovine ovarian theca cells and stroma cells with bFGF was found to promote cell growth. Basic FGF produced by the oocyte in early stage follicles appears to act on adjacent somatic cells to promote cell growth and development. Basic FGF, like KL, appears to be a primordial follicle-inducing factor. In summary, bFGF can regulate primordial follicle development that directly influences female reproductive efficiency.

Bone Morphogenetic Protein-4 Acts as an Ovarian Follicle Survival Factor and Promotes Primordial Follicle Development

Abstract The growth and development of follicles within the ovary are highly dependent on autocrine and paracrine signaling involving growth factors from granulosa cells, theca cells, stromal interstitial cells, and the oocytes. The growth factor bone morphogenetic protein-4 (BMP-4) and its receptor (BMPR-IB) have been detected in ovaries, and a mutation in BMPR-IB has been associated with abnormal ovulation rate. The objective of the current study was to examine the role that BMP-4 plays in the early stages of primordial follicle development. Ovaries from 4-day-old rats were placed into a whole-ovary organ culture system for 2 wk to investigate the effect that treatment with exogenous BMP-4 has on early follicle development. BMP-4-treated ovaries had a significantly higher proportion of developing primary follicles and fewer arrested primordial follicles than did untreated controls. This indicates that BMP-4 promotes primordial follicle development and the primordial-to-primary follicle transition. Ovaries were also treated with neutralizing antibody against BMP-4 to determine effects of removing endogenously produced BMP-4. Interestingly, ovaries treated with BMP-4 antibody were markedly smaller than controls. This was associated with a progressive loss of oocytes and primordial follicles, a progressive increase in cellular apoptosis, and an accompanying loss of normal ovarian tissue morphology over time. Immunocytochemistry localized BMP-4 protein to isolated stromal cell populations, selected stromal cells (i.e., pretheca cells) associated with developing primordial follicles, and the basement membrane of follicles. Ovaries were treated with BMP-4 and RNA collected after organ culture to determine whether BMP-4 signaling affects expression of other growth factors. Kit ligand and basic fibroblast growth factor expression was unchanged, but TGFα expression was decreased in whole ovaries. Taken together, these data suggest that BMP-4 plays an important role in promoting the survival and development of primordial follicles in the neonatal ovary.

Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries.

In a sexually mature female, primordial follicles continuously leave the arrested pool and undergo the primordial to primary follicle transition. The oocytes increase in size and the surrounding squamous pre-granulosa cells become cuboidal and proliferate to form a layer of cuboidal cells around the growing oocyte. This development of the primordial follicle commits the follicle to undergo the process of folliculogenesis. When the available pool of primordial follicles is depleted reproductive function ceases and humans enter menopause. The current study examines whether leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition that initiates follicular development. Ovaries from 4 day-old rats were cultured in the absence or presence of LIF or neutralizing antibody to LIF. LIF treatment increased the proportion of follicles that initiated the primordial to primary follicle transition to 59%, compared to 45% in untreated cultured ovaries. The ability of LIF to induce primordial follicle development was enhanced to greater than 75% by the presence of insulin in the culture medium. Anti-LIF neutralizing antibody reduced the proportion of spontaneous developing primordial follicles. Immunocytochemical studies demonstrated higher levels of LIF protein in the granulosa and surrounding somatic cells of primordial and primary follicles compared to the oocyte. In contrast, later pre-antral and antral stage follicles showed LIF expression primarily in the oocyte. In granulosa and theca cell cultures LIF had no effect on cell proliferation. However, LIF treatment did increase expression of Kit ligand (KL) mRNA in cultured granulosa cells. KL has been shown to promote ovarian cell growth and induce primordial follicle development. LIF induction of KL expression may be involved in the actions of LIF to promote primordial to primary follicle transition. In summary, LIF treatment increased the primordial to primary follicle transition in cultured ovaries and LIF may interact with KL to promote primordial follicle development.

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.

Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line.

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

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.

Growth and Differentiation Factor-9 Stimulates Progression of Early Primary but Not Primordial Rat Ovarian Follicle Development

Abstract The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.

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 insulins 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.

KIT LIGAND AND BASIC FIBROBLAST GROWTH FACTOR INTERACTIONS IN THE INDUCTION OF OVARIAN PRIMORDIAL TO PRIMARY FOLLICLE TRANSITION

Ovulated eggs during a females reproductive life are derived from a pool of primordial follicles arrested in prophase of the first meiotic division. When follicles leave the resting pool they undergo a primordial to primary follicle transition and will grow and develop until either ovulation occurs or follicles undergo atresia. Several growth factors have been implicated as acting locally within the ovary to regulate the primordial to primary follicle transition. How these growth factors may interact and cooperate to perform this vital function remains to be elucidated. The objective of the current study is to investigate interactions between kit ligand (KL) (i.e. stem cell factor) and basic fibroblast growth factor (bFGF) that promote the primordial to primary follicle transition in rat ovaries. Ovaries were removed from 4-day-old rat pups and cultured for 2 weeks with KL alone or with KL and a neutralizing antibody against bFGF. The ability of KL treatment to increase primordial follicle transition was blocked with a bFGF neutralizing antibody. In addition, ovary cultures were treated with bFGF alone or with bFGF and an anti-c-kit receptor antibody which blocks KL signaling. The ability of bFGF treatment to increase primordial follicle transition was blocked with an anti-c-kit receptor antibody. Observations indicate that both KL and bFGF must be active in order to optimally promote the changes that occur in oocytes, granulosa cells, and stromal/interstitial cells when primordial follicles initiate development. Cultured ovaries were treated with either KL or bFGF for 3 days and then bFGF and KL mRNA expression levels in the whole ovary were measured. KL was not found to regulate bFGF expression. In contrast, bFGF treatment was found to increase KL mRNA expression in cultured ovaries. These observations suggest that one function of the oocyte-derived bFGF is to increase the granulosa derived KL expression and that both KL and bFGF are required to optimally promote primordial to primary follicle transition. Elucidating the cell-cell interactions that mediate this network of specific locally derived growth factors is critical to understanding the physiology of the primordial to primary follicle transition.