Nurdiana Nurdiana, Elly Mayangsari, Bayu Lestari, Bambang Setiawan

1Department of Pharmacology, Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia

2Research Center for Toxicolgy, Cancer and Regenerative Medicine, Department of Medical Chemistry and Biochemistry, Faculty of Medicine, University of LambungMangkurat, Banjarmasin, South Kalimantan, Indonesia

Hormonal changes and spermatogenesis of male rat puppies born by mothers consuming soybean extract

Nurdiana Nurdiana1*, Elly Mayangsari1, Bayu Lestari1, Bambang Setiawan2

1Department of Pharmacology, Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia

2Research Center for Toxicolgy, Cancer and Regenerative Medicine, Department of Medical Chemistry and Biochemistry, Faculty of Medicine, University of LambungMangkurat, Banjarmasin, South Kalimantan, Indonesia

ARTICLE INFO

Article history:

Received

Received in revised form

Accepted

Available online

Soybean extract

Reproductive hormones

Male rats

Testis

Spermatogenesis

Objective:To analyze the effect of administering soybean extracts during pregnancy and breastfeeding on hormonal disturbances and impaired spermatogenesis in male rats born to mothers receiving soybean extract.Methods:Twenty-eight male rat puppies were divided into four groups: the control group (no treatment) and the groups male rat puppies whose mothers have been given soy extract of various doses (68.88 μg/mL, 137.76 μg/mL and 275.52 μg/mL). Those male rats were born to female rats given soybean extracts during their pregnancy and lactation until the age of one month. Analysis of the levels of LH, FSH and testosterone was performed by the ELISA technique. Testicular spermatogenesis was analyzed by histopathology.Results:FSH levels were significantly lower for all three doses of soy extract than those of the control group (P＜0.05). FSH levels increased significantly in the group treated with the third dose of soybean extract relative to those of the first and second doses (P＜0.05). LH levels were significantly lower for all three doses of soy extract than those of the control group (P＜0.05). Testosterone levels were significantly lower for the third dose of soybean extract relative to those of the control group (P＜0.05). With regard to histology of seminiferous tubules, it was found that the higher the dose of extract the more severe the constraints to spermatogenesis would be.Conclusion:Administration of soybean extract since the intrauterine period, during lactation and at the age of two months to male rats will lead to hormonal changes and impaired spermatogenesis.

1. Introduction

Endocrine disruptor (ED) is an exogenous substance or mixture capable of altering the function of the endocrine system and has adverse effects on intact organisms and their progeny and subpopulations[1]. Chemical compounds classified as ED are very diverse, such as industrial chemicals, pharmaceutical compounds, cosmetic compounds and heavy metals[2-4]. These compounds can be found by naked eye in the environment and as contaminants in the food chain and exposure in the work environment[5].

Phytoestrogens abundantly found in soybeans and soybean products have properties similar to estrogen or act as an anti-estrogen so as to be considered beneficial. However, the findings regarding phytoestrogen benefits are indirect and inconsistent. Exposure to estrogen compounds, especially in certain periods of life, will lead to malignancies and some anomalies in the reproductive system[6]. Structurally, phytoestrogens are similar to endogenous estrogen and have an affinity for estrogen receptors. There are various types of phytoestrogens, including isoflavones, prenylated flavonoids and coumestants. Genistein and daidzein are the most common of those compounds[7]. Previous studies demonstrated that genistein given in the maternal diet would significantly lead to growth inhibition of male progeny when given during lactation[8].

Impaired spermatogenesis and male infertility are conditions occurring as a result of the action of ED compounds. ED compounds will disturb biosynthesis, metabolism and action of hormones[9,10].Isoflavones may downregulate mRNA expression of follicle-stimulating hormone receptors, inhibinα, INHβB, androgen-binding protein and transferrin in Sertoli cells[11].

To date, there has been controversy over the finding of the effects of soybeans exposure with regard to hormonal disturbances and impaired spermatogenesis in boys born to mothers consuming ED compounds.

Hence, the purpose of present study was to analyze hormonal disturbances and impaired spermatogenesis in male rats born to mothers receiving soybean extract in pregnancy and lactation.

2. Materials and methods

2.1. Laboratory animals

This study was an experimental one. It used male rats born to pregnant female rats that were given soybean extracts during pregnancy and lactation until the age of one month. One-month-old rats were separated from their mothers and given soybean extractusing a feeding tube until 2 months of age. The control group did not receive soybean extract during the period. Twenty-eight male rat puppies treated were randomly divided into four groups: the control group (no treatment) and the groups treated with various doses of soybean extract (68.88 μg/mL, 137.76 μg/mL and 275.52 μg/mL).

2.2. Soybean extraction

The soybean plant used in the present study was of Argomulyo variety. It was obtained from the Indonesian Legumes and Tuber Crops Research Institute Malang of East Java, Indonesia. Dried soybeans were crushed to obtain soybean powder. Soybean powder was macerated in 95% methanol. The procedures for maceration were in accordance with previous studies. Soybean extract was orally administered to pregnant female rats, which was continued until lactation and the puppies were one month of age.

2.3. Analysis of hormone levels

Analysis of the levels of LH, FSH and testosterone was performed by ELISA technique. Analytical procedures were according to the instructions on the kit.

2.4. Histopathology

The testicles were subjected to histopathological analysis. The analytical procedures were in accordance with previous studies. The quality of spermatogenesis was analyzed based on Jonsen’s criteria[12,13].

2.5. Ethics

The present study passed the review of the ethics committee of Medicine Faculty of Brawijaya University Malang, East Java, Indonesia.

2.6. Statistical analysis

All data are presented in mean±SD. Differences in levels among treatment groups were analyzed by ANOVA using SPSS 16.0 statistical package. A P-value＜0.05 was considered statistically significant.

3. Results

Figure 1 shows FSH levels for different treatment groups. FSH levels were significantly lower for all three doses of soybean extract than those of the control group (P＜0.05). FSH levels increased significantly for the group treated with the third dose of soybean extract relative to the first and second doses (P＜0.05). There were no significant differences in FSH levels between the first and second doses (P＞0.05).

LH levels for different treatment groups are presented in Figure 2. LH levels were significantly lower for all three doses of soy extract than those of the control group (P＜0.05). There were no significant differences in LH levels among all three doses of soybean extract (P＞0.05).

Figure 3 shows testosterone levels for different treatment groups. Testosterone levels were significantly lower for the third dose of soybean extract than those of the control group (P＜0.05). There were no significant differences in testosterone levels between the control group and the first and second doses (P＞0.05).

In the control group, there were extremely light spermatogenesis disorder, many advanced spermatids, and the disorganization of the epithelium. On the first-dose group, there were less than five spermatozoa in each tubule and a few advanced spermatids. In the second and third doses groups, there were advanced spermatozoa and spermatid, but only early spermatids were found. At the third dose group, there was only spermatocytes. Hematoxylin eosin staining, magnification of 400 times (Figure 4).deviation;a: P＜0.05 compared with the control group;b: P＜0.05 compared with SE1;c: P＜0.05 compared with SE2. SE: soy extract.

Figure 1. The levels of FSH in the control and treatment groups.

Figure 2. The levels of LH in the control and treatment groups.

Figure 3. Testosterone levels in the control and treatment groups.

Figure 4. Histology of the seminiferous tubules of male rats in the control group (A) and groups treated with the first dose of soy extract (B), the second dose (C), and the third dose (D) (Magnification×400).

4. Discussion

In the present study, FSH levels decreased significantly for all three doses of soybean extract relative to the control group (P＜0.05). With the third dose, there was a significant increase in FSH levels relative to the first and second doses (P＜0.05). This indicates that administration of soybean extract will alter the bonding, release and metabolism of FSH. This is consistent with previous studies that ED compounds would impair endocrine function through altering the bonding, release and metabolism of endogenous hormones[14,15]. Fluctuating levels of FSH is underlain by the feedback mechanisms between the testis and hypothalamus. Decreased levels of FSH indicate hypothalamic impairment and increased levels of FSH indicate testicular impairment[16]. This is supported by histological findings of the present study. Meanwhile, the levels of LH were found to be significantly lower for the three groups of soybean extract treatment (P＜0.05). With the third dose, there was a trend of increasing LH levels relative to the first and second doses, although no significant difference was found.

Interestingly, for testosterone, there was an increasing trend of levels for the group of the first and second dose treatment, although it was not different from the control group. These levels were drastically reduced for the third dose group of soybean extract administration. This indicates that, with the third dose, there was impaired testicular function. Previous studies demonstrated that genistein has a significant role in steroidogenesis in the adrenal glands and testes of rats and lowers testosterone levels. Meanwhile, the levels of FSH remain unchanged and the levels of LH tend to increase[17].

With regard to histology of seminiferous tubules, it was found that the higher the dose of extract the more severe the constraints to spermatogenesis would be. This finding is consistent with the previous ones that genistein delay spermatogenesis in male rats and humans[18,19].

In conclusion, administration of soybean extract since the intrauterine period, during lactation and at the age of two months to male rats will lead to hormonal changes and impaired spermatogenesis.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

We thank to all laboratory assistants at the Laboratory of Pharmacology of Medicine Faculty of Brawijaya University Malang of East Java, Indonesia. This study was funded by the grant of Educational Development Fund of Medicine Faculty of Brawijaya University Malang ofEast Java, Indonesia.

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ment heading

10.1016/j.apjr.2016.10.009

*Corresponding author: Nurdiana Nurdiana, Department of Pharmacology, Faculty of Medicine, University of Brawijaya, Malang, East Java, Indonesia.

Tel: +628123388428

E-mail: farmakodes@gmail.com

Foundation Project: This study was funded by the grant of Educational Development Fund of Medicine Faculty of Brawijaya University Malang ofEast Java, Indonesia.