Abstract
Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.
Highlights
Chinese toads, Bufo bufo gargarizans, usually lay eggs in February every year
Hibernating toads ovulate in response to stimulation with pituitary extracts in vivo, and the artificially separated oocytes can be induced to mature by progesterone exposure in vitro; fully grown oocytes obtained with the hibernation interruption method will not undergo GVBD under progesterone stimulation [1]
With the help of oocyte cytoplasm transplantation, it was determined that LTE-oocyte cytoplasm could remedy the defective maturation competence of HTE-oocytes, but the auto-amplification capability to promote oocyte maturation had been lost in the HTE-oocytes [3]
Summary
Bufo bufo gargarizans, usually lay eggs in February every year. The small oocytes remain in the ovaries over the summer and grow to their full size during October and November, reaching a diameter of up to approximately 1.8 mm. When toads captured during November and December are maintained at 4 °C for at least one month, the oocytes achieve maturation competence. These oocytes, subjected to a low-temperature environment treatment, are hereafter referred to as LTE-oocytes (Low-temperature environment-oocytes). When the toads are raised in a 28 °C environment for 2-3 months, their oocytes lose the capacity to undergo GVBD in response to progesterone Such oocytes are hereafter referred to as HTE-oocytes (Hightemperature environment-oocytes). If the p34cdc and cyclin B1/B2 protein content in oocytes is not affected by the high-temperature (winter hibernation interruption) treatment, the question is raised about what factors in HTE-oocytes are involved in blocking GVBD in response to progesterone stimulation
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