Genetic analysis and cAMP measurement : comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups. METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method. RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol). CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.


Introduction
The adipose tissue has recently been recognized as an important endocrine organ.Through the secretion of soluble factors (collectively called adipokines), this tissue may regulate several physiological processes, such as glucose and lipid metabolism, immune response, and reproduction 1 .Among those adipokines, leptin seems to strongly influence on the onset of puberty and ovulatory capacity 2 .
Leptin is a peptide of 146 amino acids encoded by the ob gene.Its expression occurs mainly in the white adipose tissue and, in a lesser extent, in the brown one.Other structures that express this gene include hypothalamus, pituitary gland, gastric epithelium, placenta, mammary glands, and gonads [3][4][5][6][7] .Leptin controls food intake and metabolic rates according to the nutritional status.During a meal, the serum leptin concentration increases and it exerts an inhibitory action on hypothalamus, reducing appetite.The concentration of this hormone still increases during a couple of hours after the meal, and it stimulates thermogenesis to promote energy expenditure and reduce fat stocks.On the other hand, individuals deprived from food have a decrease in leptin serum concentration, which stimulates appetite and restricts energy expenditure.Leptin production is directly proportional to fat mass on the body.Obese individuals produce great amounts of leptin, but they also become hormone-resistant.Tendency of becoming leptin-resistant through chronic overfeeding is observed both in humans and animals 8 .
Female leptin-knockout mice (ob/ob) are sterile, indicating a role for this hormone in the control of reproductive status.They present low gonadotropin concentrations and incomplete development of reproductive organs.This phenotype is reversed by administration of exogenous leptin 9 .In women, the leptin serum concentration is inversely correlated with amenorrhea age 10 .This hormone is also implicated in maintaining other normal female reproductive functions, including lactation, folliculogenesis, ovarian steroidogenesis, endometrial maturation, and receptivity to embrio 11 .Leptin may influence on the reproductive status by acting on hypothalamus and stimulating secretion of GnRH 2,12 .However, this stimulation does not seem to be exerted directly on GnRH-producing neurons.There is a complex interplay among different types of neurons, which produces various kinds of neuropeptides.Kisspeptin might be the strongest candidate to mediate leptin effects on hypothalamus 2,12 .
Although leptin receptors are expressed on ovaries 13 , little is known about how leptin regulates gene expression in these organs.In this work, we evaluated gene expression on ovaries from lean (wild type) and obese (ob/ob) mice, in order to determine the differentially expressed genes.
We focused our discussion on about 10 genes, and we speculated why they are up or downregulated, and how these differences in expression levels can account for the anovulatory phenotype in obese female mice.

Methods
Female C57Bl/6 (Control) and B6.V-Lep ob /J (ob/ob) mice purchased from the Development Center of Medicine and Biology Experimental Models (CEDEME, acronym in Portuguese), at Universidade Federal de São Paulo (UNIFESP), were analyzed in this study.The Ethics Research Committee of UNIFESP approved all the procedures used.
Ovaries from the Control (n=5) and ob/ob (n=6) Groups were aseptically extracted.The organs were maintained at -80oC until they were applied for total RNA extraction, using Trizol reagent (Life Technologies) and following manufacturer's instructions.
Real-time PCR was performed using the ABI prism 7,000 Sequence 10 Detection System (Applied Biosystems) equipped with a 96-well optical reaction plate.The reactions were set up with 16.5 µL Sybr ® Green PCR Master Mix (Applied Biosystems).All real-time experiments were ran in triplicate and a mean value was used for the determination of mRNA levels.Considering the anovulatory phenotype from ob/ob female mice, we attempted to establish how the absence of leptin modulates the gene expression profile in ovaries.Genes differentially expressed between controls (wild type) and obese (ob/ob) mice were determined through PCR array, using RNAs extracted from ovaries.According to the false discovery rate (FDR) approach 14 , nine genes were considered for analysis.The identification of differentially expressed genes between Control and ob/ob Groups was carried out using the ∆∆Ct methodology and statistical analysis, based on unpaired Student's t-test.The FDR approach 14 with 0.05 cutoff was used to select a list of differentially expressed genes 15 .
Some of the ovaries removed for RNA extraction was stored in liquid nitrogen until cyclic AMP quantification, using the cAMP Direct EIA Kit (Amersham Biosciences, NJ, USA), following manufacturer's instructions.
The difference between both groups was analyzed applying the Student's t-test.If data normality was confirmed, through Shapiro-Wilk's test using SPSS software (SPSS Statistics, v. 16.0, SPSS Inc., USA), it was analyzed by Mann-Whitney's test.Statistical significance considered p<0.05.

Cyclic AMP accumulation
In order to determine the cellular activation status in ovaries from control and obese mice, intracellular cAMP accumulation was measured applying ELISA.The cAMP accumulation was statistically higher (p<0.01) in ovaries from obese mice (2,229±52 fMol) compared to controls (1,814±45 fMol, Figure 2).

Discussion
We demonstrated in this report that several genes have their expression levels altered when leptin is absent.We used ob/ob mice, which are genetically deprived from leptin.These mice are obese and anovulatory; they have reduced reproductive hormone levels, revealing a functional defect in the hypothalamic-pituitary axis 16,17 .Although it is clear that leptin stimulates ovogenesis by interfering with LH/estrogen levels, little is known about the effects that this hormone exerts over gene expression on ovaries.
In the absence of leptin, Adcyap1/PACAP, ApoA4, Sst, CLPS, and IL-1β are all downregulated compared to control mice (Figure 1).The most important alteration was in the expression levels of Adcyap1/PACAP, which was downregulated (96-fold) in comparison with controls.PACAP is a neuropeptide produced by mammals that have actions on the central nervous system and peripheral tissues, including intestine, lung, adrenal gland, testicles, and ovaries [18][19][20] .Moreover, its action on the hypothalamic-pituitary-gonad regulates the reproductive function.More specifically, the presence of PACAP and its receptors in the ovary stimulates

Level of expression (A) Difference of expression (M)
Differentialy expressed genes (FDR of 5% and 2-fold) Non-differentialy expressed genes Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice ovarian functions, including steroidoigenesis and cAMP accumulation in granulosa cells rat 21,22 .This peptide also accelerates meiotic maturation in mouse oocytes 23 .Furthermore, PACAP contributes to granulosa cells survival through the inhibition of apoptosis in pre-ovulatory follicles 24 .Thus, the PACAP presence and its receptors in ovaries have marked effects on ovarian physiology, and the reduced expression of this gene may account for the anovulatory phenotype.It is intriguing that cAMP levels were greatly increased in ob/ob ovaries (Figure 2) compared to controls by the same time that PACAP, an adenylate cyclase activator, was reduced.This fact, however, can be explained because other cell signaling pathways that have cAMP as second messenger may be activated in ob/ ob ovaries.CALCR and CRHR1, both upregulated in ob/ob ovaries, may account for this phenomenon.It has already been described that both receptors can transduce signal through cyclic AMP, although it has not been clear if both receptors are coupled do G s in the ovarian tissue 25,26 .
The Sst expression was downregulated (40-fold) in ovaries from obese mice, a fact that reinforces its role in metabolism and reproductive physiology.In vertebrates, the Sst is a multifunctional peptide distributed through the central nervous system and peripheral tissues, wich has a wide physiological role of neuromodulation, osmoregulation, and also affects aspects of growth, development and metabolism 27,28 .Sst secretion is regulated by intra/extra-pancreatic mediators and nutrients 29 .In addition, it inhibits the secretion of glucagon and insulin 29,30 .It has been reported that Sst administered to peripubertal mice blocked the initial gonadotropinindependent process of follicle growth in vivo, suggesting that Sst might directly act on follicular component cells in the ovary 31 .Thus, increased Sst could account for the anovulatory phenotype.Further investigation is necessary to explain this apparent contradiction.
We observed that mRNA expression level of ApoA4 in ob/ob ovaries was reduced (9-fold) compared to controls.This finding is reinforced in the study by Liu et al. 32 , who observed that hypothalamic mRNA levels of ApoA 4 were significantly reduced in obese mice (ob/ob).The anorexigen gene, ApoA4, is involved in controlling food intake and body weight 33,34 .Furthermore, obese animals have increased satiety threshold, contributing to obesity etiology.No effects on the reproductive status exerted by Apo A4 had already been described.
IL-1β is also downregulated in obese mice.The IL-1 system components have several sites of synthesis in the ovary.These factors have been localized in several ovarian cell types, such as the oocyte, granulosa and theca cells and in several mammalian species.IL-1 is involved in the ovulation process, in oocyte maturation and in several other ovulationassociated events like the synthesis of proteases, regulation of plasminogen activator activity, prostaglandin and nitric oxide production.It also regulates ovarian steroidogenesis 35 .
We also found some genes that were marked upregulated in ob/ob ovaries: RAMP3, PPARA, CALCR, and CRHR1.
Adrenomedullin (ADM), calcitonin gene-related peptides (α-and β-CGRPs), and intermedin/adrenomedullin  References 2 (IMD/ADM2) are key regulators for vascular and cardiovascular developments.Chang and Hsu 36 evaluated the role of these peptides and their cognate receptors (CLR/ RAMP) in the reproduction and fetal-maternal blood circulation, gamete development, and gamete movement in the oviduct.Wang et al. 37 , using intrauterine assays in mice, showed that calcitonin promotes pre-implantation and development of embryos in a dose-dependent manner of intracellular Ca 2+ level 38 .The authors correlated these findings to a significant increased gene expression of phospho-P38MAPK.They are in agreement with the concept that CLR/RAMP receptors were recruited to create a myriad of functions during the reproduction in mammals.Regarding this gene, we found that CALR was upregulated on ob/ob mice, showing the novel and important role in the regulation of lipid and glucose metabolisms and inflammation.In this article, it was seen that a closer correlation between leptin lack, obesity and overweight is significantly involved in the declining fertility and the increase of CALR provides an increase of second messengers, i.e. cyclic AMP.
Another gene that was strongly influenced in our model and was found to be upregulated was PPAR, (Peroxisome Proliferator Activated Receptor) which suppresses leptin expression in adipocytes.PPAR has many potential roles in pathological states, including atherosclerosis, inflammation, cancer, and demyelination.Herein, we present the current state of the molecular findings of PPAR action and their relationship involvement between obesity and infertility.The increased expression of this receptor may be associated with reduction of leptin as a compensatory mechanism.The phenotype infertility is associated, in part, due to PPAR effect on blastocyst implantation decrease and decidualization.Recent observations using isoforms of PPARδ ligands unveil that it may also be an important strategy to therapeutic target disorders, including cancer, dyslipidemia and now, infertility.It is known that CRH, a 41-amino acid neuropeptide, synthesized in the hypothalamus that regulates hypothalamus-pituitary-adrenal axis, is strongly associated with stress and inflammation.The effects of CRH are mediated through CRHR1 and CRHR2 receptors that are implicated in several reproductive functions acting as inflammatory components.In the present study, we assessed the relative expression levels of several genes and molecules in healthy and obese/infertile ovaries, revealing the first evidence for a potential role of CRH receptors in infertility profile and their further association with obesity.We have showed that CRH receptor is significantly more expressed in the obese and infertile ovaries mice compared to wild type healthy mice.Such stress and inflammation could be further correlated to obesity, since increased infertility rates, mainly affected by modulation of the decidualization and decrease blastocyst process in ob/ob mice may, contribute to the expression of their receptors to keep a proper function of ovaries 39 .
In summary, this model of obese and anovulatory mice has reduced reproductive hormone levels and altered ovogenesis.Furthermore, we demonstrated in this work that several genes have their expression levels altered when leptin is absent, especially Adcyap1/PACAP.However, more studies are needed to further characterize the effects that this hormone exerts on the ovaries.

Figure 1 .
Figure 1.Schematic plot showing differentially expressed genes between control and obese female mice

Figure 2 .
Figure 2. Determination of cAMP levels in ovaries of control and obese mice female.Enzyme immunoassay was used to determine cAMP production expressed as percentage of basal value.Data are expressed as mean±SEM of triplicate values and are representations of three experiments.The values of cAMP were: Control (n=3): 1,814±45 (fMol) and Obese (n=3): 2,229±52 (fMol).The p<0.01 was significantly different from the Control Group value

Table 1 .
List of genes considered differentially expressed (FDR of 5% and 2-fold or twice the level of expression as the Control Group) A: (ob/ob + Control)/2 and M: (Control-ob/ob); p<0.05 (t-test) was significant.