When does the sex ratio of offspring of the paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decrease: In the spermatozoa stage or at fertilization?

doi:10.1016/j.reprotox.2009.09.009

Reproductive Toxicology

Volume 29, Issue 1, January 2010, Pages 68-73

https://doi.org/10.1016/j.reprotox.2009.09.009 Get rights and content

Abstract

Recent animal experiments confirmed that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases the sex ratio of offspring at birth without altering litter size. However, the timing of this decrease remained unclear. Male mice were administered TCDD at 7–12 weeks of age and mated with non-treated females. The Y-bearing/X-bearing sperm ratio was examined by real-time PCR and FISH methods, and the sex ratio of the 2-cell embryos collected from non-treated females that had been mated with TCDD-exposed males were investigated by nested PCR. The Y-bearing/X-bearing sperm ratio was not significantly decreased in the TCDD group. However, the sex ratio of the 2-cell embryos of the TCDD group was significantly lower than that of the control group. These results may have resulted from a decrease in fertility of Y-bearing sperm. Thus, the results of this study suggested that the sex ratio of the offspring was decreased at fertilization and not during the spermatozoa stage.

Introduction

In recent years the public has become more aware that exposure of males to certain environmental or occupational agents affects their offspring. Occupational exposure in various industries has led to increased incidences of miscarriage [1] and various birth defects [2]. It has also been shown that paternal cranial irradiation leads to epigenetic alterations in offspring [3]. Prominent among these reports are those by Mocarelli et al. [4], [5] showing that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in Seveso, Italy, led to a decrease in male offspring.

In 1976, TCDD was released in an explosion at a chemical plant near Seveso, Italy, resulting in the highest concentrations of TCDD ever recorded in humans. Subsequent data linked a decrease in male births in Seveso to the increased TCDD concentration in fathers, and the altered sex ratio (the proportion of male offspring) was especially pronounced in the children of fathers exposed to TCDD before age 19 [4], [5]. In addition to the Seveso incident, Ryan et al. [6], in a study conducted in Ufa, Russia, suggested that human exposure to high levels of dioxin is associated with the birth of more girls only in cases of paternal exposure. In our previous study, we exposed young male mice to two concentrations of TCDD (TCDD2/0.4 group and TCDD2000/400 group; an initial loading dose of 2 or 2000 ng TCDD/kg, followed by a weekly maintenance dose of 0.4 or 400 ng TCDD/kg) to re-create the Seveso incident and evaluated the sex ratio of their offspring at birth [7]. The reason why we used young male mice (7 weeks at the start of administration) in that study was that the sex ratio of offspring of males exposed to TCDD during adolescence showed a greater decrease than that of males who were older than that when the incident occurred [4], [5]. The results from the previous study revealed that paternal TCDD administration produced a dose-dependent reduction in the sex ratio of offspring (F1) and a significantly lower proportion of male offspring in the high-dose (TCDD2000/400) group. In addition, the induction intensity of CYP1A1 in the liver varied among individuals in the TCDD group, and the dimensions of the CYP1A1 immunoreactive area were correlated with the sex ratio of the offspring. This means that the high sensitivity subgroup of male parents to TCDD was strongly related to the decrease in male offspring. We also reported that TCDD exposure does not influence litter size; the number of male offspring decreases by TCDD exposure, while the number of female offspring increases. From these data, we presumed that paternal TCDD exposure decreased the sex ratio of offspring and altered it before implantation occurred. However, the mechanisms underlying the reduction in male offspring and the timing of this change have remained unclear.

The purpose of this study was to investigate when the sex ratio of the offspring decreases. We examined the Y-bearing/X-bearing sperm ratio as well as the sex ratio of 2-cell embryos by exposure to the same dose (an initial loading dose of 2000 ng TCDD/kg, followed by a weekly maintenance dose of 400 ng TCDD/kg) as used in our previous study [7], because this dose group showed a significant decrease in the sex ratio of offspring at birth. The effects of TCDD exposure to sperm concentration and motility were also examined.

Section snippets

Chemicals

TCDD was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sesame oil, used for dissolving TCDD and for vehicle treatment as a control, was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

Animals and treatments

Male and female ICR mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and bred at Kobe University (Kobe, Japan). They were maintained under controlled conditions of temperature (23 ± 2 °C) and humidity (50 ± 10%) on a 12 h light, 12 h dark cycle. The animals were given an MR-A1

Epididymal sperm concentration and motility

Epididymal sperm concentrations of five males per group and sperm motility of seven males in the control group and five males in the TCDD group were examined. Fig. 1, Fig. 2 show the effects of TCDD exposure on sperm concentration and motility. Compared with the control group, the TCDD group showed a lower level of caudal epididymal sperm concentration [Control: 4.28 ± 0.79 × 10⁷/ml; TCDD: 2.29 ± 0.70 × 10⁷/ml; P = 0.10], because one of the mice in the TCDD group showed a very low sperm concentration.

Discussion

In theory, the sex allocation ratio of many species of mammals is almost 1:1. However, in reality this is not necessarily true. There have been many reports about changes in the sex ratio of human offspring. Severe periconceptional life events have been found to reduce the sex ratio in offspring [10]. The proportion of male offspring decreases with increasing parental age, and is higher in white people than in black people [11]. The sex ratio varies with the coital rate and with the time taken

Conflicts of interest

The authors have no conflicts of interest that would have inappropriately influenced the work presented in this manuscript.

Acknowledgments

The authors would like to express their heartfelt thanks to Prof. Chiharu Tohyama (The University of Tokyo) for his excellent advice during this research.

This work was supported in part by Grants-in-Aid for Scientific Research (B) (15390510, 18380089) and for Scientific Research on Priority Areas (1) (14042260) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to N.H.

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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins, has a proven reproductive toxicity. Due to the lack of evidence on the multigenerational female reproductive toxicity of TCDD through the maternal exposure, the current study aims to evaluate, on the one hand, the acute reproductive toxicity of TCDD on adult female pre-gestational exposed to a critical single dose of TCDD (25 μg/kg) for a week (group referred to as AFnG; adult female/non-gestation). On the other hand, the transcription, hormonal and histological effects of TCDD on the females of two generations F1 and F2, were also investigated after the exposure of pregnant females to TCDD on gestational day 13 (GD13) (group referred to as AFG; adult female/gestation). First, our data showed alternations in the ovarian expressional pattern of certain key genes involved in the detoxification of TCDD as well as in the biosynthesis of steroidal hormones. The expression of Cyp1a1 was highly induced in TCDD-AFnG group, but reduced in both F1 and F2. While the transcripts levels of Cyp11a1 and 3βhsd2 were decreased, Cyp19a1 transcripts were increased as a function of TCDD exposure. This was synchronized with a dramatic increase in the level of estradiol hormone in the females of both experimental groups. Beside a significant reduce in their size and weight, ovaries of TCDD-exposed females showed serious histological alterations marked by atrophy of the ovary, congestion in the blood vessels, necrosis in the layer of granular cells, dissolution of the oocyte and nucleus of ovarian follicles. Finally, the female fertility was dramatically affected across generations with a reduced male\female ratio. Our data indicate that the exposure of pregnant female to TCDD has serious negative effects in the female productive system across generations and suggest the use of hormonal alternation as biomarker to monitor and assess the indirect exposure of these generations to TCDD.
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Understanding the sex ratio at fertilization (primary) and birth (secondary) as well as the ratio of X and Y chromosome-bearing sperm is crucial to identifying the cause of offspring sex ratio distortion. Our findings on secondary sex ratio and the unaltered percentage of X chromosome-bearing sperm in TCDD-exposed sires is in line with the studies by Ishihara et al. [14,15]. While we did not assess primary sex ratio, Ishihara et al. reported a female predominant primary sex ratio in preimplantation embryos conceived with sperm from TCDD-exposed sires.
Many reports describe an association between preconceptional paternal exposure to environmental chemicals, including the persistent organic pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with an increased number of female offspring. We chronically treated wild-type C57BL/6 male mice with TCDD to investigate a role for the aryl hydrocarbon receptor (AHR) transcription factor. These mice had a 14 % lower male:female sex ratio than control mice, which was not observed in TCDD-treated Ahr knock out mice. AHR target genes Cyp1a1 and Ahrr were upregulated in the liver and testis of WT mice and Ahr expression was higher in the epididymis (2-fold) and liver (18-fold) than in whole testis tissue. The AHR protein was localized to round spermatids, elongating spermatids, and Leydig cells in the testis of WT mice. These studies demonstrate AHR involvement in the sex ratio distortion of TCDD-exposed males and the need for evaluating the molecular and genetic mechanism of this process.
Frequent and mild scrotal heat stress impairs embryo development, implantation and offspring sex ratio in mice
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Frequent and mild scrotal heat stress (fmSHS) often occurs in humans. The aim of this study was to determine the impact of fmSHS on natural fertility, the IVF–embryo transfer (IVF–ET) process and the offspring sex ratio.
Male mice were randomly divided into four groups: no scrotal heat stress (SHS) (controls) and those subjected to SHS at 37°C, 39°C or 41°C for 30 min once a week for 5 consecutive weeks. The testis, epididymis and sperm quality were assessed to evaluate the effects of different degrees of SHS, to establish an fmSHS model. Then, natural fertility, IVF–embryo transfer (IVF–ET) results, embryo development, offspring sex ratio and the X/Y chromosome-bearing sperm ratio were examined.
SHS at 39°C and 41°C caused mild impairment to spermatozoa, leading to a phenotype similar to oligoasthenozoospermia in humans. Given that most SHS conditions in humans are close to body temperature, SHS of 39°C was adopted to build the fmSHS model. fmSHS reduced the fertilization rate, impaired on-time development and reduced the implantation rate of the embryos in the IVF–ET process, but it did not affect the development or function of blastocysts. The fmSHS mice produced more Y chromosome-bearing spermatozoa and propagated more male offspring.
fmSHS not only reduced the fertilization ability of spermatozoa but also influenced their function beyond fertilization, in addition to changing the offspring sex ratio. These results may help to shed new light on the infertility treatment of males with scrotal heat risk and the health concerns of offspring propagated from these fathers.
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Another study reported human exposure to high levels of TCDD are associated with the birth of more girls, but only in cases of paternal exposure [9]. A similar alteration of sex ratio by TCDD has been observed using animal models [21–23]. Although dioxins are associated with the birth of fewer males, PCBs, which are acquired through diet and primarily fish consumption, are associated with an increased proportion of male births.
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2,3,7,8-Tetrachlorodibenzo-p-dioxin can alter the sex ratio of embryos with decreased viability of Y spermatozoa in mice
2018, Reproductive Toxicology
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive and developmental toxicant that can alter the sex ratio of offspring (proportion of male offspring). We hypothesized that the alteration of sex ratio is associated with sex chromosome ratio of live spermatozoa affected by exposure to TCDD. After exposure to TCDD we analyzed simultaneously sperm sex chromosome constitution and viability, and evaluated sperm sex chromosome ratio association with embryo sex ratio in mice. Short-term exposure to TCDD affects the decreased sperm motility and viability, and the increased acrosome reaction. Interestingly, Y spermatozoa survived shorter than X spermatozoa at high concentrations of TCDD. Moreover, the decreased sex ratio of embryos was associated with the short lifespan of Y spermatozoa. Our results suggest that TCDD may affect the fertility of Y spermatozoa more than X spermatozoa. Further studies are needed to compare the difference of fertilizing capability between X and Y spermatozoa by the effect of TCDD.
Pubertal exposure to bisphenol A affects the reproduction of male mice and sex ratio of offspring
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To study the effects of pubertal exposure to bisphenol A (BPA) on the reproduction of male mice in adulthood and subsequent generation mice.
Male mice aged 21 d were exposed to BPA at a dose of 50 mg/kg per day for 7 d by intraperitoneal injection. Sperm count, sperm deformity rate and testis histology were evaluated 35 d after exposure. Male fertility index and newborns were further observed by mating with the normal female mice.
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When does the sex ratio of offspring of the paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decrease: In the spermatozoa stage or at fertilization?

Abstract

Introduction

Section snippets

Chemicals

Animals and treatments

Epididymal sperm concentration and motility

Discussion

Conflicts of interest

Acknowledgments

Lancet

Lancet

Toxicology Letters

Journal of Theoretical Biology

Toxicology and Applied Pharmacology

Toxicology and Applied Pharmacology

Theriogenology

Paternal exposures and pregnancy outcome: miscarriage, stillbirth, low birth weight, preterm delivery

Paternal occupation and birth defects

Paternal cranial irradiation induces distant bystander DNA damage in the germline and leads to epigenetic alterations in the offspring

Cell Cycle