A warning sign: the impact of environmental and food contaminants on cow reproduction

November 11, 2019

The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

Environmental and food contaminant concerns. In the last decade, major attention has been given to environmental and food contaminants, mainly the group of endocrine-disrupting compounds (EDCs). The name refers to these compounds' endocrine activity, which might have an adverse impact on health by interfering with synthesis, secretion, transport, metabolism, binding activity, or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis, reproduction, and developmental processes in both females and males.

Common sources of EDCs are natural compounds originating from plants or fungi (such as mold, clover, soybeans), and man-made chemicals that are manufactured for use as pesticides, drugs, and plasticizers, among others. Many more EDCs are manufacturing byproducts. EDCs can leach into the environment and contaminate food and water sources. Humans and animals are exposed to EDCs through consumption of food and water, direct skin contact, inhalation, and transfer across the placenta to the fetus or through lactation to infants.
A few representative EDCs have been studied in our laboratory: the plasticizer di(2-ethylhexyl) phthalate (DEHP) and its metabolite mono(2-ethylhexyl) phthalate (MEHP), and the herbicide atrazine (ATZ) and its metabolite diaminochlorotriazine (DACT). DEHP is added to polyvinyl chloride (PVC) plastics to confer softness and flexibility. DEHP does not covalently bind to the PVC polymer, and thus leaches into the environment and can be frequently found in the atmosphere, soil, sediments, and water sources. DEHP contamination of food is also possible due to its bioaccumulation in fatty foods during handling, packing and storage.

Following consumption, DEHP is metabolized in the body to MEHP, which is considered to have a more toxic effect. ATZ is a herbicide that is most extensively used to control the growth of broadleaf and grassy weeds in agricultural crops. ATZ is considered a ubiquitous environmental contaminant that is frequently detected in ground and surface water because of its mobility in soil. The main concern with ATZ is exposure via consumption of contaminated drinking water. Once ATZ enters the body, it metabolizes into a number of metabolites, the most frequently detected being DACT. 

Health and fertility concerns. EDCs are suspected of having a major impact on human health, as environmental factors are responsible for about 80% of the deadliest diseases involving the endocrine system (cancer, cardiac diseases and others). Consequently, EDC contamination of food and water poses a serious health risk.  For instance, infertility is a global public health problem, defined as a "disease of the reproductive system." One of the main causes of infertility is consumption of EDC-contaminated food and water. This risk is not limited to humans; wild and domesticated animals are also exposed to environmental and food contamination. Research in our laboratory focuses on the physiological, cellular and molecular impacts of contaminants on the female (oocyte) and male (sperm) gametes and preimplantation embryos (i.e., blastocysts), using state-of-the-art reproductive technologies (Fig. 1).



 Fig 1. In-vitro embryo production. Oocyte handling, including oocyte aspiration, collection and maturation. Sperm handling, including semen collection, swim-up and sperm capacitation. Matured oocytes were in-vitro fertilized with capacitated sperm and cultured for 8 days to allow embryonic development to the blastocyst stage.


Effect of phthalates on the oocyte. Using our in-vitro embryo-production system, we documented the oocyte's high sensitivity to DEHP and its metabolite MEHP. This was reflected by altered nuclear and cytoplasmic maturation of the oocyte, both required for the oocyte to be fertilizable. Disruptions included alterations in meiosis resumption and reorganization of cytoplasmic organelles (mitochondria, cortical granules and endoplasmic reticulum), impaired expression of genes associated with early embryonic development and pluripotency, and increased production of reactive oxygen species. Moreover, MEHP interfered with the oocyte's ability to be fertilized and develop into an embryo, reflected by a lower proportion of developing blastocysts.

Fig. 2. Simultaneous fluorimetric assessment of plasma and acrosome membrane integrity and mitochondrial membrane potential. Purple-stained sperm are dead sperm, stained both red (propidium iodide) and blue (DAPI). Blue-stained sperm indicate viable sperm. A green cap at the top of the sperm indicates acrosome-reacted sperm (FITC-PSA). Red or green sperm tail indicates high or low mitochondrial membrane potential, respectively (JC-1). 

Effects of ATZ and DACT on sperm. Recent findings from our laboratory provide evidence of adverse effects of both ATZ and DACT on sperm, expressed by a damaged sperm membrane. Staining the sperm with specific fluorescent dyes (Fig. 2) revealed that exposure to ATZ or DACT disrupts its viability, induces spontaneous acrosome reaction and impairs mitochondrial membrane potential. Moreover, both ATZ and DACT increased the proportion of sperm with fragmented DNA, reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, and reduced blastocyst-formation rate. 
The harmful effect of EDCs on the developing embryo. Interestingly, although it was the oocytes/sperm that were exposed to EDCs, alterations were further expressed at advanced embryonic developmental stages, suggesting a long-lasting effect. Blastocysts developing from either MEHP-treated oocytes or ATZ/DACT-treated sperm expressed impaired patterns of transcript abundance, indicating that these embryos are of low quality. Accordingly, microarray analysis to compare gene-expression profiles of blastocysts developed from non-treated vs. EDC-treated oocytes/sperm revealed differential expression of genes involved in a variety of biological processes, as well as intracellular pathways for transcription, metabolism, methylation, apoptosis, and others (Fig. 3).

 Fig. 3. Transcriptomic alterations in blastocysts developed from MEHP-treated oocytes (top) and ATZ-treated sperm (bottom). Presented are the alterations in biological processes in the blastocysts. 


Synopsis. The potential risk of exposure to other EDCs must be taken into consideration, at least when evaluating their effect on the reproductive system.  While the presented data are directly related to cows and limited to a few EDCs, they might serve as a toxicology model for other mammalian species and a variety of environmental and food contaminators.