Animals
This research was approved by the ethics committee on animal studies, Payame Noor University (Protocol number: IR.PNU.REC.1396.1). In this experimental study, 3-month-old BALB/c pregnant mice were purchased from Razi institute of Karaj in Iran. The mice were housed in plastic cages under controlled conditions (12:12 h light/dark cycle, temperature of 22 ± 1°C and humidity of 55% + 5%) with voluntary access to water and chow.

Preparation of Extract
Fennel Seeds were purchased from the grocery store. The fennel alcoholic extract (FAE) was made based on the World Health Organization (WHO) guidance for provision of an alcoholic extract. Briefly, 100 g of dried seeds were powdered and added to 1000 ml of 70% ethanol (v/v) and left to soak at 25ºC for 20 h. The basin was slowly rotated during this time. Following filtration, the alcohol was turn from liquid into vapor at low pressure at 30°C (14).

Treatment
After the neonates are born naturally, the birthday of the neonatal mice was determined as postnatal day (PND) 1. 48 female neonates were randomly distributed into 4 groups (n=12, the 6 mice in each sub-group were used for the assessment of puberty onset, estrus cycle, graafian follicle (GF) count, corpus luteum (CL), levels of plasma hormones (the 6 mice in each sub-group used only for behavior testing) and were given one of the following neonatal injections (0.05 mL) in neck skinfold (subcutaneous method) during PND 1 to 5: 1: without treatment (control), 2: 10 µg/kg body weight (bw) estradiol benzoate as gold standard group (EB, SigmaAldrich, Steinheim, Germany), 3 and 4: 100 and 200 mg/kg bw FAE (13). The neonates were weighed daily to inject a precise dose of drugs.

Assessment of Puberty and Esturs Cycle
The offspring were weaned on 21 days after birth and were checked daily for VO from 24 days after birth. Upon VO, the estrus cycle was assessed daily from 40 to 70 after birth. The presence of cornified cells as the sign of first estrus happens between 2-10 days after incidence of VO. For this purpose, the smears using a cotton tipped swab (with 0.9% NaCl solution) were collected from vagina of mice in the morning. The smear was spotted on glass slides and dried in the air. Then microscopic slides were stained using methylene blue and observed with a light microscope. In the mice, the mean duration of the estrous cycle happens within between 4 to 5 days and it separated into 4 steps:

• The first step (proestrus) is the period during which conquest of nucleated epithelial cells become visible.

• The second step (estrus) is the period during which cornified squamous epithelial cells in clusters distinguished.

• The third step (metestrus), there is a mixture of all three cell types with a conquest of leukocytes, a few nucleated epithelial and/or cornified squamous epithelial cells.

• The fourth step (diestrus), this step contains mainly of leukocytes (8,15,16).

The diestrus index was assessed using the formula:

Collection of Tissue and Plasma
In the diestrus step at PND 70, 6 mice were deeply anesthetized with ketamine and xylazine mixture. Then mice blood collected by cardiac puncture and was shed in EDTA-coated tubes. The plasma centrifuged at 2500 g for 15 min and kept at -70°C until used in the assay. Ovaries were removed, weighted, and fixed in 10 percent formalin for 7 days at 4°C.

Count of Graafian Follicles and Corpus Luteum
The ovaries were dehydrated by ethanol and embedded in paraffin. Serial sections were provided at thicknesses of 7 µm and samples stained with H&E stain. For each ovary, count of GF and CL was inquired with every 20^th histological section (8).

Levels of Plasma Hormones
Levels of hormones, including E2 and LH determined using commercially available radioimmunoassay (RIA) kits (Radim, Rome, Italy) according to the directions supplied by the manufacturer.

Behavioral Test (Lordosis)
The behavioral test was directed in a glass aquarium (40 cm long, 30 cm high, 25 cm wide) whose floor was lidded with fresh sawdust. First, a sexually experienced vigorous male of the BALB/c mice was put alone in the glass and permitted to reconcile for 30 min. Then a female was put in the glass and we registered the Lordosis behavior to the mounts of the stimulus male. The exam lasted until the female had received 10 mounts or 10 min had elapsed. The test was repeated at 9 pm for three consecutive days. An LQ for each test was distinguished by dividing the number of times a female displayed lordosis by the whole number of mounts by the male sexual partner, and multiplying this value by 100 (17,18). 

Data Analysis
All assessments were analyzed using SPSS 19 (SPSS Inc., Chicago, Illinois, USA) and statistical analyses were performed by one-way analysis of variance (ANOVA). Then Tukey test used as post hoc test and data expressed as the mean ± standard error of the mean (SEM). P-value<0.05 were considered significant.

 

Neonatal treatment of FAE did not show any significant changes in body weight in mice as compared to control mice. Neonatal treatment of the EB group resulted in a significant decrease in body weight in comparison to the control group which denotes weight loss (Figure 1).
 

Figure 1. Body weight of female offspring on PND 70. Control: without treatment, EB: 10 mg/kg, estradiol benzoate, FAE: 100 and 200 mg/kg, fennel alcoholic extract. Data are the mean SEM.
**P < 0.01 compared with control.

The age of VO was significantly advanced in the mice being treated with 10 µg/kg of EB (P<0.001) or 200 mg/kg of FAE (P<0.05) than in the control group. However, the low dose (100 mg/kg) of FAE moderately, but not significantly, advanced the day of VO (Figure 2A). There was no difference in body weight at VO day among FAE-treated groups, and FAE treatment had no effect on either the growth or appearance of health among the mice. However body weight at VO day significantly (P<0.01) decreased in the EB group’s mice (Figure 2B).

The control group exhibited a regular estrus cycle and a normal duration for each stages of the estrus cycle for 30 days (Table 1). The estrus cycle was affected, showing a significant decrease in the number of estrus cycles (P<0.001), in the duration of proestrus (P<0.001), estrus (P<0.01) and metestrus (P<0.05), with a significant increase in the duration of diestrus (P<0.001) and in the diestrus index (P<0.001) in EB-treated mice. In addition, in FAE 200-treated mice, there was a significant decrease in the number of estrus
cycles (P<0.05), the duration of proestrus (P<0.05), with a significant increase in the duration of the diestrus stage (P<0.05) and the diestrus index (P<0.05). However, treatment with FAE 100 has no significant effect on the number cycles, or the duration of each stage of the cycle, or the diestrus index when compared with the control group (Table 1).
In addition, the results in Table 1 showed a significant decrease of LQ for EB (P˂0.001) and FAE 200 (P˂0.01) groups compared with the control group. However, the low dose (100 mg/kg) of FAE moderately, but not significantly, decreased the rate of LQ.
 

Discussion

The findings of the present study indicate that, similar to the EB group, neonatal treatment of FAE especially in higher dose (200 mg/kg) altered the reproductive function in the form of accelerated puberty onset, disrupted estrus cycles, altered estradiol and LH hormone levels, a decrease of ovary FG and CL and decrease of sexual behavior. This data suggests that neonatal FAE treatment may be responsible for defeminizing hypothalamic neural networks, controlling the female reproductive axis, therefore altering hormonal levels, and is subsequently responsible for the deficiency in reproductive function and sexual behavior.
Sex differentiation of the brain is governed by endogenous sex hormones, which are also key players of the neuroendocrine systems controlling puberty onset. Accordingly, both states might be sensitive to the disrupting actions of external substances with sex steroid-like activity that may pose long-lasting outcomes in terms of reproductive safety. It is obvious that the phenomenon of sex differentiation of the brain in laboratory rodents occur during critical periods of development, especially the neonatal period, when key neuronal systems at the hypothalamus become determined in a permanent procedure differentially in different genders. Evidence of rodents have indicated that the key molecular signal responsible for brain feminization is low estrogen input due to passive ovaries and high amounts of circulating α-fetoprotein. According to the above functional aspects, it is acceptable that EDCs with sex hormone-like actions may effect at central levels to interfere with suitable sexual differentiation or later maturation of the reproductive system (4). Exposure to EDCs with sex steroid-like activities and phytoestrogens show that they may not have the ability to bind to alpha-fetoprotein and are therefore able to cross the blood–brain barrier and alter development of a normal female brain (2, 19, 20).
In the present study, the VO age of FAE-treated mice was found to have accelerated and the estrus cycle was disrupted compared to the control group’s mice. These results are consistent with the effects in previous studies on the neonatal administration of phytoestrogens genistein (6), coumestrol (7), mycoestrogen zearalenone (8), tamoxifen (21) and BPA (22) to laboratory rodents. VO is an apoptosis-mediated event used as an exogenous indicator of puberty onset. It happens as a result of increasing E₂ secretion and can be motivated by treatment of E₂ (16) and estrogenic EDCs (6-8, 21) into rodents. In this study, E₂ levels also increased in FAE-treated mice. Puberty onset in many species involves reduced sensitivity of the hypothalamic nuclei controlling the reproductive axis to steroid negative feedback (23-25). In addition, minor changes of gonadal hormone level during the neonatal stage can accelerate development and growth of reproductive system (26).
In rodents, estrogen-sensitive brain nuclei, anteroventral periventricular (AVPV) nucleus and arcuate (ARC) nucleus impose Supervisory control over reproductive system physiology such as the timing of puberty onset and estrus cycle. Previous data from female rodents demonstrated that the lesions on these neuronal nuclei disorder the reproductive physiology (8,21,27-29). These hypothalamic nuclei have been reported to be sexually dimorphic. These sexual differences in reproductive functions are formed by low E₂ level in the neonatal stage in female rodents, because exposure to estrogen or phytoestrogens in neonatal female rodents caused a decrease in LH surge, a decrease in CL and disruption of the estrous cycle (9,30,31). In agreement with this issue, here, LH levels also decreased in FAE-treated mice.
Similar to the EB group, ovary weight and the number of GF and CL were decreased significantly in the mice treated with FAE. This data may be due to hormonal imbalance and a decrease in the LH hormone. Similar findings have been expressed in rodents treated with genistein (6) and coumestrol (7). Weight and ovarian function are controlled by gonadotropins, which functions in a coordinated procedure with a specific mark provided by the ovary through the pituitary gland and is accountable for metabolism of gonadotropins. Gonadotropin hormones in turn perform an important role in the control of folliculogenesis. Accordingly, toxic substances that interfere with the ovarian physiology could do so indirectly by acting on sensitive brain areas (32).
Similar to the EB group, LQ was declined significantly in the mice administered with FAE. The Ventromedial Hypothalamic Nucleus (VMN) is an important site for female sexual response.  Lesions on this hypothalamic area delete the lordosis behavior in female rats, and implanting E2 directly into the VMN induces the behavior. Neurons in the ventrolateral subdivision represent high levels of ER and project to the midbrain central gray, forming an essential link in the neural circuitry regulating female sex response. Defeminization is an E₂-mediated active process that either blocks the organization of the feminine network or somehow overrides it, resulting in a permanent prevention of female sex response (31). Such a difference in the impact of FAE on the behavior of Lordosis in female mice is in agreement with the findings of other studies about other phytoestrogens (6,7).
 It might be possible that during neonatal exposure, estrogenic components of fennel such as trans-anethole bind with ER in the hypothalamus, which has been found to decrease the construction of GnRH, therefore decreasing the secretion of FSH and LH. The low level of FSH and LH may lead to decreased folliculogenesis and ovulation. The feedback blockage of GnRH secretion by estrogens provides the basis for the most widely used form of contraception. Such feedback blockage of GnRH inhibits the midcycle surge of LH and ovulation (33). Trans-anethole (anethole; 1-methoxy-4-(1E)-1-propenylbenzene) is a benzene ring with a single methoxy group para to the double-bonded propenyl group, occurs naturally as a key constituent of the essential oils in fennel. The trans isomer is by far more abundant (>99%) than the cis isomer in natural oils. Anethole is a major component of the essential oils obtained from fennel and anise, which are known to have estrogen-like activities as well as pharmacological properties in traditional medicines (34). Although many natural and synthetic compounds are found everywhere, little is obvious about harmful effects to humans while being exposed to known exogenous estrogens. As a result, remarkable consideration has focused on dietary phytoestrogens bearing hydroxyl groups as EDCs exhibiting weak estrogenic activity in in vivo and in vitro bioassays (34-37). However, further investigations on the mechanism of fennel and its estrogenic components including trans-anethole about this toxicity are necessary.

 

Conclusion

Overall, the results of this study demonstrated that alterations in the FAE-treated mice are like those observed after neonatal treatment of estradiol benzoate. As a result, this data demonstrated that FAE that was injected on days 1–5 may act as an agent with estrogenic function on the hypothalamic nuclei regulating reproductive physiology and behavior in female rodents.

 

Acknowledgements

Financial support of this study (grant, No. 68424) was performed by Payame Noor University, which is greatly appreciated.

 

Conflicts of Interest

Authors declared no conflict of interests.