Maternal Zearalenone exposure impacted ovarian follicle formation and development of suckled offspring

Highlights

• Maternal exposure of Zearalenone through breast milk impaired folliculogenesis in offspring mice.

• Zearalenone triggered apoptosis in offspring ovaries via activation of RLRs.

• Zearalenone exposure through breast milk impaired follicular development of adolescence mice.

Abstract

Zearalenone (ZEN) is a secondary metabolite, which is mainly produced by Fusarium fungi and exists in various feeds and agricultural products. Recently, an increasing amount of data has shown that ZEN, as an estrogen-like hormone, can have harmful effects on the female reproductive system, especially on oogenesis and folliculogenesis. Breast milk is considered to be the ideal form of nutrition for infants; however, there are some records of contaminants in food, such as mycotoxins, which may be transferred from maternal blood to milk. In this study, we investigated the toxic effects of breast milk on folliculogenesis in offspring following maternal ZEN exposure. Our results showed that maternal ZEN exposure significantly inhibited the process of primordial follicle (PF) assembly and reduced the number of PFs in suckled offspring's ovaries. In addition, RNA-seq analysis showed that RIG-I-like receptor (RLRs) signaling pathways were activated after exposed to ZEN, which increased the expression levels of DNA damage (γ-H2AX, RAD51, and PARP1) and apoptosis related protein (BAX/BCL2 and Caspase-3). Finally, ZEN exposure interfered with follicular development, as evidenced by the reduced percentages of oocyte maturation and embryonic development when the offspring grew to adolescence. It is worth noting that maternal ZEN exposure disrupted the tri-methylation levels of H3K4, H3K9, and H3K27 in the offspring's oocytes. Our results indicated that maternal ZEN exposure affected ovarian development in offspring through the breast milk, which may be detrimental to their reproductive capability in adult life.

Introduction

In recent years, mycotoxins have become a serious risk factor for the health of both humans and animals (Minervini and Dell'Aquila, 2008; Zheng et al., 2019). Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin of the Fusarium fungi, is a secondary metabolite (Malekinejad et al., 2006), which is widely found in food crops, such as corn, wheat, sorghum, barley, and oats (Biehl et al., 1993; Hueza et al., 2014; Mukherjee et al., 2014; Abbasian et al., 2018; Bertero et al., 2018). Statistically, approximately 81% of food crops around the globe are polluted by mycotoxins (Appell et al., 2017). Previous research has shown that ZEN has a similar chemical structure to endogenous estrogen (17β-estradiol, E2), and can combine with cytoplasmic estrogen receptors in the breast, uterus, hypothalamus, and pituitary, to influence development of the female reproductive system (Pfeiffer et al., 2009; Rai et al., 2020; Grenier et al., 2019; Muthulakshmi et al., 2018a). Moreover, numerous studies have confirmed that the harmful toxic effects of ZEN include neurotoxicity, hepatotoxicity, immunotoxicity, and genotoxicity (Kowalska et al., 2016; Gao et al., 2018; Islam et al., 2017; Muthulakshmi et al., 2018b; Liu et al., 2018). Thus, it is important to research the toxicokinetics and potential biological threat of ZEN.

In mammals, primordial follicle (PF) formation mainly occurs during the later stage of fetal development to the early postnatal period; this is an extremely complex biological process for female animals. The PF pool is a crucial marker of female fertility, and any harmful effect at this early phase may induce dysgenesis (Ge et al., 2019; Wang et al., 2017; Kuiper-Goodman et al., 1987). A number of scientists consider that maternal hormones and especially estrogen are crucial in the promotion of PF formation in the fetus (Zhang et al., 2017; Hou et al., 2014; Lai et al., 2018). Several studies have revealed that ZEN, as a non-steroidal estrogen, has significant harmful effects on the reproductive system in many different species including humans, mice, cattle, and pigs (Malekinejad et al., 2007; Zhang et al., 2017; Silva et al., 2021; Rogowska et al., 2019; Li et al., 2019). Studies have found that ZEN exposure of pregnant mice at 12.5–17.5 days post coitum (dpc) can inhibit passage through first meiosis (K.H. Liu et al., 2017), and impair PF formation through changing the DNA methylation dynamics of the PF assembly related gene Lhx8 3′-UTR (Zhang et al., 2017). The single-cell transcriptomic profiling was used to explore the toxic effects of ZEN on PF assembly, and found it indicated that ZEN exposure can disrupt the Hippo signaling pathway and alter the developmental trajectory of both germ cells and granulosa cells (Tian et al., 2021). Furthermore, ZEN exposure has been reported to reduce the number of normal follicles, causing premature oocyte discharge in adult pigs (Zourgui et al., 2008), inhibition of oocyte survival, and increased abnormal spindle morphology in oocytes (Hassen et al., 2007). And the transcriptome analysis found that ZEN exposure of porcine granulosa cells can arrest the cell cycle and impair genomic stability in vitro (Liu et al., 2018; Li et al., 2020). In addition, recent studies have demonstrated that ZEN exposure can cause dysfunction in gut microbiome and affect the ovarian reserve (Tan et al., 2020). Although previous studies have provided abundant evidence that exposure to ZEN damages reproductive ability, the exact mechanism by which ZEN causes ovarian reserve failure remains unclear.

It is well known that breast milk is of superior nutritional value for offspring, and it is the main source of nutrition for newborn mammals. Breast milk consists of proteins, lipids, sugars, vitamins, minerals, immune factors and biologically active factors, which are critical for the development of the newborn reproductive system (Appell et al., 2017). Exposure of infants, particularly premature newborns, is extremely important because they are generally more susceptible and have a less developed immune system during the first few months of life (Committee et al., 2017). However, several mycotoxins are able to enter breast milk following maternal exposure to contaminated foodstuffs (Braun et al., 2018). ZEN is commonly found in cereals in different regions around the world; and when ingested in a mother's daily diet they can interfere with her endocrine system (Kollarova et al., 2018). All newborn mammals are dependent on breast milk as the primary food source; therefore, when ZEN is transferred into an infant through suckling, it can affect health later in life. Previous studies have mainly focused on the immune response of fetuses to ZEN in breast milk (Githang'a et al., 2019; Gao et al., 2020), while the related problems of reproductive ability have not been addressed. Therefore, we hypothesized that the exposure of lactating mothers to ZEN could affect the reproductive system of newborn mice through the maternal milk.

With the deepening of research in this area, ZEN exposure is now attracting more attention for its impact on corporeal health, especially on the reproductive system (Zhang et al., 2017; K.H. Liu et al., 2017). However, the toxic effects of ZEN via breast milk on the next generation have not been investigated. Thereby, the objective of this study was to detect damage to the reproductive system in offspring, caused by the maternal ZEN exposure of breast milk. Our study demonstrated for the first time that ZEN's effect on breast milk could impair ovarian development and early folliculogenesis in offspring.