Phthalate effects on early gonad development in vitro.

Increasing evidence relating to a temporal decline in human fertility has been published during the last thirty years. Several studies have linked this with environmental factors and have flagged a number of reproductive anomalies which may all share a common environmental aetiology. Observations indicative of an environmental drive include the rate of change which is not consistent with genetics along with the widespread occurrence in the human and other species. Amidst this there has been in recent years an almost epidemic increase in the use of artificial reproductive technologies which further highlights the scope of the problem, with 1 in 6 couples in the UK reported to experience sub- or infertility. Although the problem is multi-factorial, it is clear that, over a similar time frame, there has been a drastic increase in the amount and concentrations of persistent and ubiquitous chemicals released into the environment. Of key interest among this, is the gargantuan increase in the use of plastics and the simultaneous increase in plastic pollution. Coinciding with plastic use increase is the increase in plasticisers which freely leach from many plastics and are also present in a wide variety of cosmetic and household products. Several research studies have estimated phthalate exposure in the human. These studies often rely on the use of biomonitoring of phthalate metabolites in urine samples. Due to the inherent physico-chemical properties of many environmental chemicals these approaches may drastically underestimate the concentrations of these chemicals within target tissues. This is supported by the central dogma which suggests that phthalates are quickly metabolised and excreted. Recent research has suggested that the dog may be a species which, due to shared environment, may exhibit similar environmental exposures to humans. This has resulted in the dog being considered as a sentinel for the human with the added bonus of better control over lifestyle confounders that influence the human and access to reproductive tissues. In this study we report that both the ovary and testis contain (at least) three types of environmental pollutants: Di(2-ethylhexyl) phthalate (DEHP), several congeners of Polychlorinated biphenyls (PCBs) and Polybrominated diphenyl ethers (PBDEs). Of these, total PCBs and DEHP were present at higher levels in the ovary than in the testis. Since these chemicals were positioned to potentially influence gonadal function and since plastics and plastic-derived chemicals are of global concern, experiments were designed to test the hypothesis that DEHP and its metabolite, Mono (2-Ethylhexyl) phthalate (MEHP), at concentrations relevant to those measured within gonads, perturb gonad development and function, possibly through androgenic or oestrogenic mechanisms of action. To this end, a mouse ovary and testis explant culture system was optimised. The baseline for oestrogenic and androgenic effects on ovarian follicle development and health was characterised. A key observation was the enhanced sensitivity of the earliest follicle types, primordial and in particular transitional follicles, to exogenous influence. Within two ovary culture systems, one mirroring the above and one a ‘real-time’ ovarian fragment culture, ovaries were then exposed to a range of DEHP and MEHP. The concentrations used were based on those measured within dog ovaries. In the fragmented mouse ovary culture, sensitivity to phthalate was seen at the lowest concentration tested, namely 0.06X the mean dog ovarian concentration after a very short exposure period. This indicated that the whole ovary acted as some barrier to phthalate effects, as in the whole ovary culture, effects were only seen at higher concentrations. Interestingly, all follicles types, present within this culture system, were sensitive to phthalate exposure with both oocytes and granulosa cells affected. The earliest follicle stages were once again the most sensitive, with transitional follicle health negatively impacted through all cell types. Neither DEHP nor MEHP acted on ovarian follicles in a purely androgenic or oestrogenic manner, with a more complex mechanism of action suspected. To further investigate DEHP and MEHP an ‘in-house’ method to measure DEHP and MEHP concentrations in a range of biological and media samples was optimised. This study revealed significant movement and interconversion of phthalates within culture, which may explain the contradictory reports of phthalate mechanisms of action as well as the presence of DEHP within the ovary of the dog. Following central dogma, the ovary should not contain measurable concentrations of DEHP, this due to the assumption that DEHP is readily metabolised and therefore not bio accumulative. This raised considerable questions pertaining to the nature of phthalates and other environmental chemicals. Data presented in this doctoral thesis showed sensitivity of developing neonatal ovaries to exogenous androgen, oestrogen and phthalates, at relevant exposure concentrations. Furthermore, data presented here reveals the drastic and severely understudied impact of the physico-chemical properties, of enduring and omni-present environmental pollutants, on the exposure of tissues sensitive to perturbation through both endocrine and non-endocrine mechanisms.