Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy.

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from -10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic ß and ß' phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing.

Reproductive abnormalities in adult male mice following preimplantation exposures to estradiol or pesticide methoxychlor.

Adult females of ICR strain of mice were bred, separated into different experimental groups, and treated as follows. On Days 2-4 of pregnancy, the mice received daily subcutaneous injections of either 0.05 ml sesame oil (vehicle) or same volume of 5.0mg of purified methoxychlor (MXC) suspended in the vehicle. Another group received a single subcutaneous injection of 1.0 microg of estradiol-17beta (E) on Day 2 of pregnancy only. Male offspring were tested at 3 and 6 months of age. At 3 months, E or MXC did not alter the weights of seminal vesicles, preputial glands, or testes, although after exposure for 30 min to a female in estrus behind a partition, testosterone levels were significantly reduced in treated males in comparison to control males exposed to the same partition test. At 6 months, the preputial glands and testes weight remained unchanged, while the seminal vesicles were significantly heavier in E- and MXC-treated males. Same partition tests again revealed that in E and MXC groups, testosterone levels remained significantly lower in comparison to control males. MXC or E exposures during preimplantation appear to induce long-term effects on the sexual development in 3 and 6 month-old-males by compromising their sexual arousal and altering seminal vesicles weights in the older group.

Preimplantation exposures of murine embryos to estradiol or methoxychlor change postnatal development.

Long-term effects of in vivo exposures to proestrogen methoxychlor (MXC) or estradiol-17beta (E) were studied during early pregnancy (preimplantation) in ICR mice. Pregnant dams received either subcutaneous injections of 1 microg of E on Day 2 of pregnancy only (vaginal plug = Day 1), or 5.0mg of MXC on Days 2-4 of pregnancy in sesame oil. Pregnant control mice were treated with the vehicle only. Litter size, postnatal survival, sex ratio at birth, and anogenital distance (AGD) in offspring of both sexes were examined, as well as vaginal opening in female offspring. High mortality rate was recorded in MXC-exposed offspring due to infanticide. Exposures to either E or MXC did not change sex ratio at birth, but the litter size was smaller in the former group. On postnatal Day 21, male pups exposed to either E or MXC at preimplantation stage exhibited shorter AGD than the controls, with the change most pronounced after MXC treatments. AGD in female offspring was unaffected after MXC exposures, but E treatments produced longer AGD in the females than that recorded in the controls. Preimplantation exposures to E or MXC also accelerated sexual maturation as significantly more females exhibited precocious vaginal opening at weaning. Our study shows that exposures to MXC or E at preimplantation stages cause long term alteration of sexual development during weaning in offspring of both sexes. Also, MXC treatments retarded both growth and weight of both sexes of offspring, in comparison to controls.

Altered behaviors in male mice, male quail, and salamander larvae following early exposures to the estrogenic pesticide methoxychlor.

Numerous publications show that methoxychlor (MXC), in use today as a DDT substitute, is a reproductive toxicant; it produces deleterious effects on the structure and function of the reproductive organs in exposed species. Exposure of mice (33 mg/kg body weight) to purified (95%) MXC at the time of implantation, or injection, of 5 mg MXC into freshly laid quail eggs prior to artificial incubation, altered sexual arousal and sexual behavior in adult males of both species. When placed near a plastic partition with an estrus female behind it, the MXC-exposed male mice showed no sexual arousal, spent less time near the partition, and exhibited lower testosterone levels. Similarly, adult quail males that were exposed to MXC during incubation showed a lack of sexual interest and copulatory behavior when presented with a receptive female. Some males showed a longer latency period before mounting the female, while others did not show any sexual interest at all. Exposure of salamander embryos to purified MXC at or above 0.3 microM (0.1 mg/l) induced precocial hatching of embryos and reduced the startle response and the distance traveled in response to startle. Exposing hatched larvae to MXC for 3 days also resulted in a blunted startle response. Due to the blunted startle response and decreased avoidance travel, the exposed salamander larvae appear more susceptible to predation and these effects may contribute to amphibian population declines. The results of these studies indicate that developmental exposures to environmental chemicals with hormonal activities produce undesirable behaviors that may affect population dynamics and survivability of exposed species.