Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†.

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7 days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF diet alone.

Endothelin 2: a key player in ovulation and fertility.

Ovulation is the fundamental biological process during which an oocyte is expelled from the ovary, and it is an essential step toward establishing a pregnancy. Understanding regulatory mechanisms governing the ovulation process is essential for diagnosing and treating causes of infertility, identifying contraceptive targets, and developing novel contraception methods. Endothelin-2 (EDN2) is a 21 amino acid-long peptide that is transiently synthesized by granulosa cells of the ovulatory follicle prior to ovulation and plays an essential role in ovulation via promoting contraction in the myofibroblast cells of the theca layer of the follicle. This review describes the organization of the endothelin system, summarizes recent findings on the expression and synthesis of the endothelin system in the ovary, illustrates the roles that EDN2 plays in regulating ovulation, and discusses EDN2 as a potential target of contraception.

Early postnatal exposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal development in pigs.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children's toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

Germline-dependent transmission of male reproductive traits induced by an endocrine disruptor, di-2-ethylhexyl phthalate, in future generations.

In males, defective reproductive traits induced by an exposure to an endocrine disruptor are transmitted to future generations via epigenetic modification of the germ cells. Interestingly, the impacted future generations display a wide range of heterogeneity in their reproductive traits. In this study, the role that the Y chromosome plays in creating such heterogeneity is explored by testing the hypothesis that the Y chromosome serves as a carrier of the exposure impact to future generations. This hypothesis implies that a male who has a Y chromosome that is from a male that was exposed to an endocrine disruptor will display a more severe reproductive phenotype than a male whose Y chromosome is from an unexposed male. To test this hypothesis, we used a mouse model in which F1 generation animals were exposed prenatally to an endocrine disruptor, di-2-ethylhexyl phthalate (DEHP), and the severity of impacted reproductive traits was compared between the F3 generation males that were descendants of F1 males (paternal lineage) and those from F1 females (maternal lineage). Pregnant dams (F0 generation) were exposed to the vehicle or 20 or 200 µg/kg/day of DEHP from gestation day 11 until birth. Paternal lineage F3 DEHP males exhibited decreased fertility, testicular steroidogenic capacity, and spermatogenesis that were more severely impaired than those of maternal lineage males. Indeed, testicular transcriptome analysis found that a number of Y chromosomal genes had altered expression patterns in the paternal lineage males. This transgenerational difference in the DEHP impact can be attributed specifically to the Y chromosome.

Generation and characterization of an estrogen receptor alpha-iCre knock-in mouse.

Two estrogen receptors, ESR1 and ESR2, are responsible for the classical actions of estrogens in mammalian species. They display different spatiotemporal expression patterns and nonoverlapping functions in various tissues and physiological conditions. In this study, a novel knock-in mouse line that expresses codon-improved Cre recombinase (iCre) under regulation of the natural Esr1 promoter (Esr1-iCre) was developed. Functional characterization of iCre expression by crossing them with reporter lines (ROSA26-lacZ or Ai9-RFP) showed that iCre is faithfully expressed in Esr1-lineage cells. This novel transgenic mouse line will be a useful animal model for lineage-tracing Esr1-expressing cells, selective gene ablation in the Esr1-lineage cells and for generating global Esr1 knockout mice.

Birth of clones of the world's first cloned dog.

Animal cloning has gained popularity as a method to produce genetically identical animals or superior animals for research or industrial uses. However, the long-standing question of whether a cloned animal undergoes an accelerated aging process is yet to be answered. As a step towards answering this question, we compared longevity and health of Snuppy, the world's first cloned dog, and its somatic cell donor, Tai, a male Afghan hound. Briefly, both Snuppy and Tai were generally healthy until both developed cancer to which they succumbed at the ages of 10 and 12 years, respectively. The longevity of both the donor and the cloned dog was close to the median lifespan of Afghan hounds which is reported to be 11.9 years. Here, we report creation of 4 clones using adipose-derived mesenchymal stem cells from Snuppy as donor cells. Clinical and molecular follow-up of these reclones over their lives will provide us with a unique opportunity to study the health and longevity of cloned animals compared with their cell donors.

Label-free, multi-scale imaging of ex-vivo mouse brain using spatial light interference microscopy.

Brain connectivity spans over broad spatial scales, from nanometers to centimeters. In order to understand the brain at multi-scale, the neural network in wide-field has been visualized in detail by taking advantage of light microscopy. However, the process of staining or addition of fluorescent tags is commonly required, and the image contrast is insufficient for delineation of cytoarchitecture. To overcome this barrier, we use spatial light interference microscopy to investigate brain structure with high-resolution, sub-nanometer pathlength sensitivity without the use of exogenous contrast agents. Combining wide-field imaging and a mosaic algorithm developed in-house, we show the detailed architecture of cells and myelin, within coronal olfactory bulb and cortical sections, and from sagittal sections of the hippocampus and cerebellum. Our technique is well suited to identify laminar characteristics of fiber tract orientation within white matter, e.g. the corpus callosum. To further improve the macro-scale contrast of anatomical structures, and to better differentiate axons and dendrites from cell bodies, we mapped the tissue in terms of its scattering property. Based on our results, we anticipate that spatial light interference microscopy can potentially provide multiscale and multicontrast perspectives of gross and microscopic brain anatomy.