Reproductive disruption in adult female and male rats prenatally exposed to mesquite pod extract or daidzein

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Abstract

Phytoestrogens are considered to be endocrine disruptors, since they can alter the endocrine system, thus disturbing many reproductive events. The intake of diets containing a high content of phytoestrogens has increased worldwide in human populations and in domestic animals. Phytoestrogens in maternal blood can pass through the placenta to the fetus in high amounts and can have long-term organizational effects. Mesquite (Prosopis sp) is a leguminous plant widely used to feed several livestock species, and is also used in the human diet. In this study we assessed the effects of exposure to mesquite pod extract during the periconception and pregnancy periods on the reproduction of male and female descendants. The females of three experimental groups received one of the following treatments: 1) vehicle injection; 2) mesquite pod extract or 3) the isoflavone daidzein during the periconception and pregnancy periods. Estrous cyclicity, sexual behavior and hormones, as well as uterine and vaginal epithelia were evaluated in the female descendants. In the males, sexual behavior and hormones, apoptosis in testicular cells and sperm quality were evaluated. In females the following was observed: alterations in estrous cycles, decreased sexual behavior, estradiol and progesterone levels, increased uterine and vaginal epithelia. In males, we observed a decrease in sexual behavior, testosterone and sperm quality, and apoptosis increased in testicular cells. All these effects were similar to those caused by daidzein. These results indicate that prenatal exposure to mesquite pod extract or daidzein, administered to females before and during pregnancy, can disrupt normal organizational-activational programming of reproductive physiology in female and male descendants.

Introduction

With the increased intake of legumes in vegan diets in some human populations [1], and in animal diets [2] there has been an increase in the consumption of high amounts of phytoestrogens worldwide. Phytoestrogens are considered endocrine disruptor compounds (EDCs), as these compounds can alter hormonal effects by stimulating or inhibiting the endocrine system, acting as agonists or antagonists of endogenous steroid hormones, or disturbing the biosynthesis, secretion, transportation, binding, action, degradation or elimination of endogenous hormones that are responsible for the maintenance of homeostasis, reproduction, development and/or behavior [3], [4]. Most of the legumes commonly used for feeding livestock or human diets contain anywhere from 5 % to 25 % phytoestrogens, and their concentrations vary depending on the age of the plant, environmental temperature, humidity, light, and pathogens [5].

Phytoestrogens are considered nonharmful products due to their antioxidant properties. Consequently, their ingestion during gestation has increased. However, there is little knowledge regarding their possible deleterious effects on fetal development, considering their xenoestrogenic and epigenetic properties [6]. During pregnancy, the fetus is exposed to substances present in the maternal plasma that can cross the placental barrier and enter fetal circulation, either by active transport or by passive diffusion [7]. Fetal exposure to phytoestrogens is therefore directly related to circulating levels in maternal serum [8].

In women, starting from the second trimester of pregnancy, daidzein and genistein have been detected in amniotic fluid and in umbilical cord blood at concentrations of 1.44 ± 1.34 and 1.69 ± 1.48 ng/mL, with maximum levels of 5.52 and 6.54 ng/mL, respectively [9], [10]. Also, levels of genistein and daidzein were higher in the cord (19.4 and 4.3 ng/mL) than in maternal serum (7.2 and 4.3 ng/mL) [11], indicating fetal exposure. Several reproductive alterations in humans and animals have been associated with prenatal exposure to phytoestrogenic compounds of natural origin present in food [11], [12]. A study in rats showed pregnancy failure, with poorly developed pinopodes and a decreased implantation rate (32 %) due to quercetin, a phytoestrogen [13].

In rats, ingestion during pregnancy of plants containing quercetin during pregnancy increased gestation length (8 %), and induced accelerated vaginal opening (2 days) in female offspring [14]. In mice, perinatal exposure to daidzein can alter estradiol receptor (ER) expression in the bed nucleus of stria terminalis in adulthood, indicating that phytoestrogens can have long-term organizational effects [15]. Perinatal exposure of rats to a mixture of 12 phytoestrogens consisting of lignans, genistein, and daidzein in doses of 1, 10, or 100 times the ingestion of the human population, from gestational day 7 to postnatal day (PND) 13, induced antiestrogenic action, such as accelerated vaginal opening, hypertrophic mammary glands, and decreased estradiol levels, as well as antiandrogenic effects, with reduced anogenital distance [16].

Ingestion of forages with a high content of phytoestrogens by grazing animals is associated with reproductive alterations, especially in ruminants, which have broad economic importance in livestock [17]. Abortions have been observed in young and adult pregnant ewes fed with Medicago sativa containing high quantities of coumestrol [18]. In bovines, phytoestrogens can inhibit the sensitivity of the corpus luteum to gonadotropins, decreasing progesterone and leading to abortion [19].

In several countries, mesquite (Prosopis sp) is a leguminous plant widely used to feed several livestock species (ovine, caprine, bovine). It is also used in the human diet as bread [20], [21], [22], coffee substitute [23], and porridge [24], or in beverages [25]. Mesquite contains protein (26.69–29.84 %), crude lipid (11.89–13.75 %), total crude fiber (8.78–9.89 %), carbohydrates (42.45–46.37 %) [26], minerals, and vitamins [24], [27]; it also possesses high antioxidant activity [28], [29]. Mesquite contains several phytoestrogens: mesquitol (flavonoid, 6.4 μg/g) [30], quercetin, luteolin, isorhamnetin, vitexin [31], genistein (36 μg/g) and daidzein (5.27 μg/g) [32], which possibly contribute to its estrogenic effects in livestock and human beings.

In previous studies, we have reported that mesquite pod extract has disruptive effects on reproductive function in female rats, altering estrous cyclicity, decreasing lordotic quotient and intensity of lordosis [33]. In male rats, it disturbs sexual behavior, increases testicular germ cell apoptosis, decreases sperm quality and serum testosterone. All these effects are similar to those of genistein and daidzein [34]. Also, mesquite pod extract and daidzein decrease female fertility, lowering the number of pups in a litter and increasing the percentage of dead pups [35].

To date, there are no reports on the effects on the offspring of the exposure of females to mesquite pod extract before and during pregnancy. Therefore, this study aimed to evaluate some reproductive events in female and male offspring in adulthood. The effects were compared with those of phytoestrogen daidzein.

Section snippets

Ethics statement

Animal management and experiments were carried out in accordance with Mexican official regulations (NOM-062-ZOO-1999), and the Ethical Conduct Guidelines for Research, Teaching and Outreach of the Health and Biological Sciences Division, Autonomous Metropolitan University (2010). This study was approved by the Ethics Committee of the Health and Biological Sciences Division, Autonomous Metropolitan University.

Animals

Three-month-old adult female (250–300 g) and male (300–350 g) Wistar rats were housed

Dams

All control females treated with vehicle got pregnant, while 90 % of females treated in the periconception and gestational periods with mesquite got pregnant. Similarly, 90 % of females treated with daidzein got pregnant. The number of offspring varied depending on the treatment [F2,28 = 7.25; p = 0.004]. Control dams had 9.25 ± 0.45 pups on average, with no dead pups after birth. In dams treated with mesquite pod extract, there were lower numbers of live offspring (5.12 ± 0.91; p = 0.01), and

Discussion

The increase in the consumption of diets having a high intake of phytoestrogens before and during pregnancy in women and in livestock that are fed forage is well documented [1], [2], [9], [10], [11]. In Japanese women, it has been shown that phytoestrogens are transferred from mother to fetus through the placenta. Genistein, daidzein, and equal are higher in cord serum than in maternal serum, remaining longer in the fetus than in the mother [11]. As phytoestrogens can bind to estrogen

Conclusions

The results obtained in the current study show that mesquite pod extract or daidzein administered to females before and during pregnancy interfere with the reproductive physiology on female and male offspring. In female offspring the results are: disrupted estrous cycles, decreased sexual behavior and sexual hormones, increased uterine and vaginal epithelia; in male offspring: lower testosterone, sperm quality, sexual behavior and relative weight of glands, a high apoptosis index and sperm

CRediT authorship contribution statement

FS participated in the writing of the manuscript, analyzed vaginal smears, sexual behavior evaluation, and performed histological evaluation; EH analyzed vaginal smears and participated in sexual behavior assessment, and performed histological evaluation; LJR performed histological evaluation and TUNEL test; SRM evaluated E2, P4 and testosterone; GL and CR participated in the obtention of mesquite pod extract; FC, MB, AL, RH and MMO participated in the critical review of the manuscript. SR-M:

Funding sources

This study was partially supported by SEP-PROMEP, Grant no. 1035-09-1247.

Conflict of interest

Authors declare no conflict of interest.

Acknowledgments

The authors want to express their gratitude to Edith Monroy for her advice on the English language.

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