INTRODUCTION

It is interesting that communications between hormones and the sympathetic nerve are occurred (Weitz et al., 2001; Krizsan-Agbas and Smith, 2002). For example, the rise in estradiol prior to estrus is sufficient to deplete uterine sympathetic innervation (Zoubina et al., 2001). A second example, the sympathetic denervated ovary of guinea pigs caused a decrease of the serum progesterone concentration but an increase of estradiol concentration (Riboni, 2002) during their first diestrus. Richeri et al. (2005) reported that uterine sympathetic innervations underwent profound remodeling in response to physiological and experimental changes in the circulating levels of sex hormones and neurotrophin receptor-mediated events contributed to regulate sex hormone-induced plasticity in uterine sympathetic nerves. Sympathetic neurite outgrowth on uterine tissue sections reduced in rats treated with estrogen (Richeri et al., 2010). Furthermore, growing sympathetic fibers are more vulnerable to estrogen than mature fibers and nerve fibers that have been in contact for longer periods with their target become less susceptible to estrogen (Chavez-Genaro et al., 2002) and progesterone had the neuroprotective effects on experimental diabetic neuropathy in rats (Sameni et al., 2008). Most data suggested that the sympathetic nerve was regulated by sex hormone. However, few researches explored to the effects of the sympathetic nerve on the maintenance of endocrine balance during pregnancy.

Mast Cells (MCs) are widely dispersed among the tissues and organs (Galli, 1993; Karaca et al., 2006) and perform their function by releasing mediators such as histamine, tryptase, chymase and heparin (Gilfillan and Tkaczyk, 2006). It has been demonstrated that oestradiol affects the density of MCs in several tissues. For example, oestradiol diminishes the density of MCs in possum cul-de-sac tissue (Mahoney et al., 2003), but increases the number of MCs in the Harderian gland (Di Matteo et al., 1995) and testis (Minucci et al., 1997) of a frog. In addition, estradiol receptors have been identified to exist in rat peritoneal mast cells (Vliagoftis et al., 1992) and in aortic tissue mast cells from fertile aged women (Nicovani and Rudolph, 2002). Recently, it was found that histamine may be a novel sympathetic neurotransmitter (Li et al., 2006). Furthermore, it was reported that the noradrenalin (NA) which was released by sympathetic nerve fiber ending inhibited the histamine content. For instance, 10^-11-10^-3 M of NA could inhibit the release of histamine from mast cells in skin (Alm and Bloom, 1981). In addition, Ke et al. (2011) reported that mast cell numbers decreased in whole small intestines when 6-OHDA was intraperitoneally injected into mice. In the pregnant mouse uterus, sympathetic fibers were present the walls of branches from the uterine artery in the mesentery (Sheikhi et al., 2007), which is similar to that of mast cell. Therefore, the communicating effect between MCs and sympathetic nerve during early pregnancy is worth investigating.

However, presently, sufficient data about the regulation of sympathetic nerve on the reproductive hormones and MCs during early pregnancy has not yet been provided in mammals. The neurotoxin 6-hydroxydopamine (6-OHDA) can selectively destroy the sympathetic nerve terminals containing catecholamine in animals. Thus, this study have attempted to demonstrate the role of the sympathetic nervous system on serum progesterone and 17β-estrogen levels and the number of MCs in the uterus during early pregnancy using an experimental model in mice.

MATERIALS AND METHODS

Animals and 6-OHDA treatments: Eight to nine-week-old Kunming species white female (25-35 g) and male (35-45 g) mice obtained from Beijing Biological Products Company (Beijing, China), were used in this study. The mice were housed in conventional conditions and fed a standard diet. This study was carried out in accordance with the Guidelines for Animal Experimentation of China Agriculture University. After an adaptive period of one week, fifty female mice were divided equally into two groups which were treated daily with a 0.01% antioxidant L-ascorbic acid vehicle in sterile saline (0.01 mL g^-1 b.wt.) containing both 6-OHDA (Sigma Chemical Co., St. Louis, MO) doses of 0 (control group, n = 25) or 100 mg kg^-1 b.wt. (6-OHDA treated group, n = 25) by intraperitoneal injection, respectively. After a series of injections for five days, the mice (6-OHDA -treated group, n = 25, control group, n = 25) were immediately tested for estrus detection by vaginal smears with Wright's staining each evening. The estrous mice were mated with male mice.

At pregnant day 1 (E1), E3, E5, E7 and E9, the animal was euthanized by cervical dislocation under Nembutal deep anaesthesia (50 mg kg^-1 b.wt., intraperitoneally). The blood was collected and the serum was separated by centrifugation, then stored at –20°C until the estradiol and progesterone was measured by radioimmunoassay using a commercially available 125I RIA Kit, which operated by the Beijing Huaying Institute of Biological Technology. The uterus tissues were treated with different solutions depending on the purpose.

Histochemistry for mast cells in the uterus: Uterine tissues were immediately immersed in 4% paraformaldehyde in 0.1 M PB (pH 7.4) fixed overnight (4°C) and was dehydrated in a graded ethanol series, then embedded in paraffin. Sections (5 μm) were mounted on gelatinized glass slides. The section was dewaxed in xylene, hydrated in serial ethanol, washed in Distilled Water (DW) and then stained with 0.8% toluidine blue in DW (contain 0.6% potassium permanganate) for 30 sec. Then, it was dehydrated in 95% ethanol and 100% ethanol, cleared in xylene, mounted and finally coverslipped. The number of MCs in the whole section was counted using a light microscope, 5 sections per animal. Each section was photographed using an Olympus digital camera, then measured its area using the Scn Image software. The unit of measurement of the number of MCs in each section was MCs mm^-2.

Fluorospectrophotometry for histamine content in the homogenate of the uterus: Uterine tissues without embryos were homogenized in 0.4 M perchloric acid (containing 0.02% vitamin C, 0.04% EDTA) and then were centrifuged at 1500 g for 10 min at 4°C. The supernatants were collected and used to measure the histamine content by fluorospectrophotometry. First, the samples were treated by 25% w/v trichloroacetic acid and centrifuged at 2800 g for 10 min. Next, in order to extract the histamine from the homogenate, the supernatants (1.6 mL) were added to the tube containing 1.5 g NaCl. Then, 4.0 mL n-butanol and 0.2 mL 2.5 M NaOH were also added and those were put rested after being mixed for 5 min. The 3.6 mL n-butanol dissolving the histamine were taken out and again added to the tube containing 1.2 mL 0.1 M HCl and 2.0 mL skellysolve C and those were put rested after being mixed for 5 min. The 0.8 mL HCl dissolving the histamine was taken out. Thirdly, the histamine dissolved in the HCl solution chemically responded to o-phthalaldehyde and fluorescent material was produced. The fluorescence intensity was determined at Ex346 nm and Em447 nm by fluorospectrophotometer (Hitachi F-4500, Japan). The standard curve was made according to the same procedure. The concentrations of histamine were represented by μg g^-1 tissue.

Data analysis: Data are expressed as Mean±SD. To analyze the results, the independent-sample T test was used. Differences at p<0.05 were considered statistically significant.

RESULTS

Changes in the number of embryos in pregnant mice treated with 6-OHDA: The number of embryos was 13.5 (E7)-12.30 (E9) in the control group, but in the experiment group, it was significantly decreased to 4.8 (E7)-3.7 (E9) (p<0.05) (Table 1, Fig. 1).

Table 1:	Changes of embryos, mast cell and histamine in uterus of both groups
Values are significant at *p<0.05 and **p<0.01 comparing with corresponding control, -: Embryos can not be counted

Fig. 1(a-h):

Appearance of the embryos and mast cells in early pregnancy uterus, (a) and (c) The uterine horns were thicken and about fourteen embryos at (a) E7 and (c) E9 in the control, but just a few embryos at (b) E7 and (d) E9 in treated mice are present, Sections were stained with toluidine blue histochemistry (e, f, g and h), No. of mast cells at E1 in the treated group (g) is less than that in the control group (e), The difference is not showed at E5 between control (f) and treated (h) group in the figure because of the changes of uterine wall area, Scale bar = 1 cm in a, b, c and d, Scale bar = 100 μm in e, f, g and h, EB: Embryo, OV: Ovary

Changes in the serum concentration of 17β-estradiol and progesterone: The concentration of 17β-estradiol and progesterone during early pregnancy were investigated in the 6-OHDA and control groups. As shown in Fig. 2a, the concentration of 17β-estradiol significantly increased from (5.45±0.39) pg mL^-1 at E3 (p<0.05) and (2.51±0.2) pg mL^-1 at E9 (p<0.01) in the control groups to (9.77±2.38) pg mL^-1 and (6.44±0.47) pg mL^-1 in the 6-OHDA-treated group. But it decreased from 8.58±0.52 at E1 and 9.37±0.33 pg mL^-1 at E7 in the control group to 3.14±0.25 and 6.98±0.83 pg mL^-1 in the 6-OHDA-treated group and decreased from 7.99±0.24 pg mL^-1 in the control to 5.14±0.88 pg mL^-1 in the treated at E5. Comparing data in both groups, the serum concentration of 17β-estradiol was larger in the treated group than that in the control group during at E3 (up to 44%) and E9 (up to 61%). However, it decreased by 63% (at E1) and 36% (at E5) in the treated groups.

Fig. 2(a-b):

Frequency histograms demonstrating changes on serum sex hormone concentration, (a) 17β-estrogen and (b) Progesterone in the mice with different treatments and pregnant stages, the concentration of 17β-estrogen was increased at E3 (p<0.05) and E9 (p<0.01), but was lower than that of control at E1 (p<0.05), E5 (p<0.05) and E7, The concentration of progesterone was higher than that of control at E1-E5 and lower than that of control at E7 (p<0.01) and E9, value are significant at *p<0.05 and **p<0.01 comparing with corresponding control

The serum concentration of progesterone changed as shown in Fig. 2b. Comparing data between the treated and the control groups, it significantly increased from 0.58±0.09, 2.06±0.08 and 1.94±0.12 ng May 5, 2012 mL^-1 in the control to 1.14±0.79, 4.10±0.14 and 2.34 ng mL^-1 in the treated group, respectively at E1, E3 and E5. The maximum concentration increased almost by 2 times than that of the control group at E3. Nevertheless, there was a decrease from 6.07±0.39 and 3.77±0.49 ng mL^-1 in the control group to 4.48±0.57 ng mL^-1 at E7 (p<0.05) and 3.19±0.12 ng mL^-1 at E9 (p>0.05) in the treated groups.

Changes in the number of mast cells and the content of histamine in the uterus: The appearance of MCs in the uterus was mostly round or oval-shape and the nucleus was colored blue with toluidine blue staining or vacuole and cytoplasm contained porous granules. MCs mostly distributed in myometrium, especially in mucosa triangular area, but few in endometrium. Table 1 illustrated the changes of MCs and histamine content in the uterus of the control and treated groups during E1-E9. The number of MCs increased at E3 then decreased at E5 in both the control and treated mice. Compared to the number of MCs in the control group, it was significantly increased by 136.03, 24.54 and 177.97% at E5 (p<0.01), E7 (p<0.05) and E9 (p<0.01), respectively. The change of histamine content was different to that of MCs from E1 to E9. It was almost under the limit of the detection at E5 in the both of groups, at E7 and E9 in the control group. Compared with that of the control group, the histamine content was significantly decreased from 222.23±0.80 to 26.02±0.80 μg g^-1 at E3 (p<0.01), but increased to 114.24±0.61 μg g^-1 at E7 and 17.53±0.37 μg g^-1 at E9 (p<0.01), respectively.

DISCUSSION

6-OHDA, the specific neurotoxin for catecholaminergic neurons, can induce the lesion of catecholaminergic neurons and was often used in brain to induce the animal model of Parkinson’s disease (Ardestani, 2010). However, 6-OHDA can not penetrate the blood-brain barrier for the large molecular weight (250.09), so it damages the only periphery catecholaminergic nerve by intraperitoneal injection. The localization and role of autonomic innervation in the rat thymus (Dorko et al., 2000) and ovine ureter (Cherian et al., 2010), even in Decapod Crustaceans (Shuranova et al., 2006) had been studied. We had confirmed that the sympathetic nerve in the uterus of the treated groups disappeared completely after 6-OHDA intraperitoneal injection with the dose of 100 mg kg^-1 b. wt., but not the 50 mg kg^-1 b.wt. (Dong et al., 2007). In this study, it was reported that the number of implanted embryos of the treated mice decreased to 64.4% (at E7). A similar finding was also reported in the rats (MacDonald and Airaksinen, 1981) and rats with amputation of the autonomic nerves showed implantation failure (Yuan et al., 2009). Our experiments showed that there were no significant differences between the 6-OHDA-treated (12-13 ova) and control (14-15 ova) groups in the number of fertilized ova at E1. Additionally, many reports found that the sympathetic nerves would undergo natural axonal degeneration in the middle and later periods of normal pregnancy in rat (from day 15 of pregnancy, Klukovits et al., 2002) and rabbit (at day 18 of pregnancy, Chen et al., 2000), which implied that the sympathetic nerves of the uterus play a more important role in the early period of pregnancy. However, some previous studies recognized that sympathectomy had no significant effect on the fertility in mice (Johns et al., 1975) and in rats (Lara et al., 1989). Latini et al. (2008) suggested that alterations of the biological mechanisms of uterus sympathetic innervation might play significant roles in various pathologies, such as infertility and spontaneous abortion. Maternal distress have negative effect on the fetus growth by stimulating the fetal autonomic nervous system and mediating of heart rate (Shafizadeh and Mehdizadeh, 2009). Therefore, research continues to study the effect of catecholaminergic nerve on pregnancy.

Many papers reported that there was a modulation role of 6-OHDA on endocrine system. For an instance, 6-OHDA significantly increased the luliberin contents in the anterior hypothalamus while preserving its normal level in the medio-basal portion (Savchenko et al., 1983). Another example, in vitro, 6-OHDA significantly decreased mRNA level of gonadotropin releasing hormone in the male rat hypothalamus (Kim et al., 1993). Based on the above examples, it had a regulation effect on endocrine when it was injected in brain. However, it is not clear if it was injected outside brain. In the study, it is reported firstly that the serum 17β-estrogen and progesterone during early pregnancy were changed significantly compared to that of control group after injection intraperitoneally 6-OHDA inducing peripheral sympathetic nerve lesion.

It is well known that the abnormal level of progesterone and estrogen in the uterus can cause early embryonic death (Bajaj and Sharma, 2011). The current manuscript showed that the concentration of serum 17β-estrogen significantly decreased during E1 and E5 (p<0.05), but increased at E3 (p<0.05) and E9 (p<0.01) in the treated mice. It was reported that estrogen stimulated follicular growth in the process of oocyte maturation and prepares it for implantation (Austin and Short, 1982). At present, estrogen level is available to be considered as a critical determinant that specifies the duration of the window of uterine receptivity for implantation and the window of uterine receptivity remains open for an extended period at lower estrogen levels but rapidly closes at higher levels (Ma et al., 2003). The uterus becomes receptive on day 4 of pregnancy or pseudopregnancy and proceeds to the refractory state on day 5 in mice (Paria et al., 2002). Therefore, the elevated level of 17β-estrogen at E3 is harmful to implantation of embryos and it may be one of the reasons for implantation failure and embryo loss.

In this study, it is noted that the concentration of serum progesterone increased before implantation in the treated groups. It is clear that the high level of progesterone is responsible for maintenance of gestation and is necessary for implantation in mice, but it is apparently not essential for development (Vinijsanun and Martin, 1990). The serum progesterone concentrations decreased in all ovarian-denervated guinea pigs during their first diestrus (Riboni, 2002). In this study, the elevated concentration of progesterone during the preimplantation period in mice with chemical sympathectomy suggests that the progesterone balance is probably regulated in a different manner by the peripheral sympathetic nerve.

Previously, paper reported that estrogen modulated uterine sympathetic nerve degeneration and remodeling (Zoubina and Smith, 2002). Furthermore, the metabolite of progesterone has an effect on the regulation of sympathetic outflow to contribute to pregnancy (Heesch and Rogers, 1995), but it does not alter sympathetic activity in brown adipose tissue, pancreas and heart (Puerta et al., 1996). In addition, some experiments suggested that estrogen promotes maternal behavior by enhancing pup-stimulated activity in the brain regions, whereas progesterone could inhibit maternal behavior by inhibiting neural activity in some regions of brain (Sheehan and Numan, 2002). This confirms that a significant interaction exists between sexual hormone and nerve system.

Our previous study found that the number of MCs and the concentration of histamine in rat mammary glands fluctuated with the relationship of the estradiol level during the estrous cycle and ovariectomized rats showed a decrease in MCs and histamine, but elevation after administration of estradiol to ovary-intact rats (non-published results). Previous studies have reported that MCs in the uterus could change the histamine content throughout the estrus cycle (Aydin et al., 1998). Recently, it was found that histamine may be a novel sympathetic neurotransmitter (Li et al., 2006). Therefore, it is interesting to us that the sympathetic nerve plays a crucial effect on histamine level during early pregnancy. It was reported that the number of MCs in myometrium of rats increased significantly (p<0.05) during early pregnancy after autonomic nerves controlling the uterus had been cut (Yuan et al., 2009). In the present study, in the 6-OHDA treated group, the number of MCs was significantly higher than in the control (p<0.0) at E5, which may result in the increase of immune reaction in the treated group and be harmful to early embryo implantation.

In conclusion it is inferred that the 6-OHDA induced lesion of the periphery catecholaminergic nerve may disrupt levels of the estradiol and progesterone, the number of MCs and level of histamine. Furthermore, the effect of 6-OHDA is significant during the implantation period, but not after embryo implantation. The present study suggests that embryo implantation is regulated by catecholaminergic nerve through modulating sex hormones and MCs, but also provides evidences that communication is necessary among nerve, sex hormones and MCs during the implantation. Future studies are required to address the receptor and molecule mechanism of sympathetic nerve on the hormones and MCs.

ACKNOWLEDGMENTS

This work was supported by Grants (30700575, 30471247) from the National Nature of Science Foundation of China and by Grants (200800191004) from the Ph.D. Programs Foundation of Ministry of Education of China. We would like to express this gratitude to Dr. Quinton A. Winger, Assistant Professor Colorado State University, USA, for his critical reading of this manuscript.