INTRODUCTION

Progesterone production in the ovary is dependent on the biochemical capacity of the steroidogenic tissue (Niswender et al. 2000), the key proteins involved in the steroidogenesis, and vascularization of the corpus luteum (CL), (Stocco et al. 2007). It is indicated that increased blood !ow in the CL is closely associated with the increased plasma progesterone concentrations, (Beri- sha et al. 2000). Proliferation of the endothelial cells results in the vascularization of the CL during luteal development (Reynolds et al. 2000) and consequent ang- iogenesis is essential for the function of the CL(Hunter et al. 2004). Vascular endothelial growth factor (VEGF) is the main angiogenic factor that stimulates microv- ascular endothelial cell proliferation and migration, as well as vascular permeability, (Ferrara et al. 2003).

Previous study showed that, inhibition of VEGF expression results in decreased vascularization of the CL and consequent decline of post-ovulatory proges- terone secretion in primate (Fraser et al. 2000). The expression of VEGF and its receptor, fetal liver kinase-1/ kinase insert domain-containing receptor (Flk-1/KDR or VEGFR-2), was determined in rodent ovarian folli- cles and CLs (Kashida et al. 2001, Pauli et al. 2005). Our previous studies showed that steroidogenic genes expression and serum progesterone level are affected by lithium chloride (LiCl) treatment, an effective drug for the treatment of bipolar disorder, in gonadortopin- stimulated immature rat ovary (Khodadadi et al. 2012, Khodadadi & Ansari Pirsaraei 2013).

Whereas lithium upregulates VEGF expression in human brain (Guo et al. 2009), previous study demon- strated that blood vessel development in human embryo was affected by high dose of lithium. Also, experiments with other vertebrates showed that lithium affects dor- soventral speci"cation by inhibition of vasculogenesis, (Giles & Bannigan 2006). The aim of this study was to determine whether toxicity effects of lithium observed in the steroidogenesis was associated with any alteration in the angiogenic genes expression in gonadotropin- stim- ulated immature rat ovary.

MATERIALS AND METHODS

ANIMALS

Immature (25-day-old) female albino rats of the Wistar strain were kept under controlled temperature (22 ± 2 ºC) and 12/12-h light-dark cycle in Plexiglas cages with free access to rat chow and water. The procedures were per- formed in accordance with institutional guidelines for animal care and use. The Animal Ethics Committee of

Khodadadi, Abediankenari and Ansari Pirsaraei

Department of Microbiology and Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences approved the experimental protocol.

EXPERIMENTAL DESIGN

Immature female Wistar rats (25-day-old) were injected intraperitoneally (i.p.) with 2.0 mg/kg/day of lithium chloride (Sigma, Germany) or sterile distilled water (0.5 ml) for 15 days. Then, all rats were treated with single i.p. injection of 10 IU pregnant mare’s serum gonadotrophin (PMSG) on the 13th day of experiment to induce fol- licular maturation and followed by single i.p. injection of 10 IU human chorionic gonadotropin (hCG) (Intervet Inc., Germany) 48 hours later to induce ovulation. The last injection of lithium chloride (LiCl) or distilled water was 12 hours post-hCG injection. Rats injected only with distilled water and gonadotropins constituted the control group. In this model, ovulation approximately occurred at 12–14 h post-hCG injection (The oocytes were observed by applying gentle pressure to both ends of the ampulla, and placed on a slide under the ster- eomicroscope). All animals were killed by spinal disloca- tion at 4 h interval from 8 to 24 h post-hCG injection. The ovaries were rapidly removed, washed in the cold saline solution. One ovary from each animal was "xed in Bouin’s solution for histological studies, and the other was snap-frozen in liquid nitrogen and stored at –80 °C for RNA extraction.

HISTOLOGY

The paraf"n sections (5 µm) of the entire ovaries "xed in Bouin’s solution were stained using either hematoxylin and eosin (H&E) or Masson’s trichrome staining accord- ing to a previously described method(Everett & Miller 1973). Then, the stained slides were examined under the microscope. Morphometric analysis of the "brosis areas was performed with a computer-assisted image analyzer, with KS-300 software (Carl Zeiss Co., Germany).

RNA ISOLATION AND REAL TIME RT-PCR

Total RNA was isolated from whole ovaries using the RNeasy mini kit (Qiagen Sciences, Germany) and "rst- strand complementary DNA (cDNA) was synthesized from 2 µg of total RNA using cDNA synthesis kit (Qia- gen, Germany), according to the manufacturer’s proto- col.

The primers were designed using the Gene Runner (Hastings Software Inc, Hastings, NY, USA) for Rattus VEGF and KDR genes as described in Table 1. Gene expression was evaluated in control and LiCl-treated ovary at each time point. Real-time PCR was performed using Quanti-Fast SYBR Green PCR Kit (Qiagen, Ger-

many) according to the manufacturer’s protocol. PCR ampli"cations were performed in the Corbett Rotor- gene 3000 real-time PCR machine (Corbett Research, Australia) with the following conditions: 5 min at 95 °C and then 40 cycles of 10 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. All samples were tested in duplicate. Melt curve analyses were conducted for all target genes and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR products were visualized in an agarose gel (1.5%). Analysis were performed using the Pfaf! method (Pfaf! 2001, Pfaf! et al. 2002). Data are presented as fold change in the mean normalized expression values at each time point relative to the nor- malized expression values at 8 h post-hCG injection, as a baseline expression.

Statistical analysis Experiments were repeated at least two times with eight animals in each group. One-way

ANOVA was used to analyze differences between groups. The P value less than 0.05 were considered statistically signi"cant. Tests were performed using SPSS software (Software program, version 13, SPSS, Inc., Chicago, USA).

RESULTS AND DISCUSSION

FIBROSIS FORMATION IN THE OVARY AND CORPUS LUTEUM

Histological analysis of the ovaries indicated that ovula- tion was induced by hCG treatment and resulted in CL formation (Fig. 1).Masson’s trichrome revealed the "bro- sis formation in some region of ovary and within the corpora lutea (Fig. 2B, D and F). The signi"cant increase

in the amount of "brosis was observed with a 6-fold increase in LiCl-treated group in comparison with con- trol at 20h post-hCG injection (Fig.3).

RELATIVE EXPRESSION OF VEGF AND ITS RECEPTOR, KDR

The expression of genes involved in angiogenesis, VEGF and KDR, was evaluated by real-time RT-PCR in the ovary. Data were presented as fold change in the mean normalized expression values at each time point relative to the normalized expression values at the 8 h post hCG expression (Fig. 4A and B).

Result showed that gonadotropin treatment signi"- cantly (P < 0.05) induced VEGF and its receptor (KDR)

expression in the control groups during luteinization (Fig. 4C). The expression of VEGF and KDR mRNA was higher at 16 h (VEGF, 4.79-fold and KDR, 23.03-fold), 20 h (VEGF, 4.62-fold and KDR, 22.64-fold) and 24 h (VEGF, 2.92-fold and KDR, 8.57-fold) than those of the 8 h post-hCG injection group as shown in "gure 6. However, VEGF mRNA levels were lower in LiCl-treated groups than their respective control groups (Fig. 4A). VEGF expression was signi"cantly decreased with a 4.98- and 2.3- fold (P< 0.01) at 16 h and 20 h, respec- tively, in comparison with respective control. In addi- tion, a signi"cant decrease was observed in KDR mRNA levels with a 1.53-fold decrease (P< 0.05) at 20 h in LiCl-treated rats in comparison with control at the same time (Fig. 4B).

In rodents, the corpus luteum is a substantial source of the progesterone production that is required for main- tenance of early pregnancy (Stocco et al. 2007). Serum levels of progesterone are dependent on the quantum of steroidogenic tissue that is dependent on the number and size of steroidogenic luteal cells, both of which increase during luteal development (Farin et al. 1986). Concen- trations of progesterone in serum are also dependent on the blood !ow in the CL. Serum level of progesterone increases as luteal blood !ow increases (Smith & Fors- man 2012). Luteal blood !ow predicted reliably a func- tional CL and progesterone production in bovine (Her- zog et al. 2010), also low progesterone during diestrus was associated with reduced luteal blood !ow in cow (Luttgenau et al. 2011).

In the laboratory rat, newly formed capillaries are found in the CL at 16 h post-ovulation (Matsushima et al. 1996). Furthermore, experiments in the rodents have demonstrated that VEGF is the primary modulator of angiogenesis in the ovary and its inhibition leads to dis- ruption of the folliculogenesis and CL formation (Doug- las et al. 2005). In the rodent ovary, VEGF is expressed in luteal cells but not in the granulosa cells, suggesting a cause-effect relation between luteinization and growth of capillary vessels (Phillips et al. 1990). Also, it has been shown that LH /hCG stimulate not only the expres- sion of VEGF but also Flk-1/KDR receptors in during luteinization (Fraser et al. 2005). In present study, a sig- ni"cant increase in the VEGF and KDR expression was observed in early stage of luteinization in gonadotropin- stimulated rats. Results also showed that VEGF expres- sion was reduced in the LiCl-treated rat ovary during luteinization. Therefore, it is possible that LiCl inhibits VEGF-induced angiogenesis.

Recently we have showed a rapid decline in plasma progesterone concentration after LiCl treatment in gonadotropin-stimulated rat (Khodadadi & Ansari Pir- saraei 2013). The reduction of VEGF expression and its receptor by LiCl treatment supports the hypothesis that inhibition of angiogenesis could deprive the luteal cells of progesterone production and its transport into the bloodstream, which explains the marked decline in plasma progesterone concentration. Also, results from this study showed that inhibition of VEGF production by LiCl leads to increased "brosis in the ovary which is associated with the loss of blood vessels. Thus, we sug- gest that LiCl plays a dual role in completing the "brotic processes by anti-angiogenic activity against VEGF, diminishing VEGF-induced blood vessel formation and enhancing "broblast proliferation to produce collagens. It is concluded that LiCl effects on the angiogenesis by suppressing expression of VEGF and KDR genes in the rat ovary during luteinization.

Khodadadi, Abediankenari and Ansari Pirsaraei

FUNDING

This research did not receive any speci"c grant from any funding agency in the public, commercial or not- for-pro"t sector.

ACKNOWLEDGEMENTS

The author offers special thanks to Dr. T. Shivanandapa (University of Mysore, India) for his valuable sugges- tions and critical review of the manuscript.

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