Winter hibernation and UCHL1-p34cdc2 association in toad oocyte maturation competence

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 p34(cdc2), a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13(suc1) 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 p34(cdc2) 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 p34(cdc2) 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.

Figure 1. Tissue-specific expression of tUCHL1 in toads.

The soluble protein extracts were separated by 12% SDS-PAGE, and the protein bands were identified using mouse monoclonal antibody against tUCHL1, colored with DAB on the nitrocellulose membrane. In each lane, 20 μg of soluble protein extract from various tissues was loaded separately, except for 0.5 μg of recombinant protein as a control. The abbreviations are as follows: B: brain; Sk: skin; Sp: spermary; St: stomach; Li: liver; In: intestine; tUCHL1: Recombinant tUCHL1 expressed in E. coli (DE3), which has a molecular weight of approximately 29 kDa; O: oocyte; Ki: Kidney; Mu: muscle; Lu: lung; H: heart; M: marker. The experiment was independently repeated twice.

Figure 2. Immunological localization of tUCHL1 in toad oocytes.

A: Control for immunological detection in B with primary antibody blocking. B: Immunohistochemical detection of tUCHL1 on sections of a juvenile toad's ovary, visualized in brown using a DAB kit. C: Control for immunological detection in D and E with primary antibody blocking. D: tUCHL1 immunological detection in immature LTE-oocytes (Li) at stage IV, colored red with an AEC kit. E: tUCHL1 detection in mature oocytes (Lm) in parallel with D. F: control for immunofluorescent detection in G and H with primary antibody blocking. G: Immunofluorescence of tUCHL1 in HTE-oocytes without progesterone treatment (Hi). H: tUCHL1 immunofluorescence detection in immature LTE-oocytes (Li) in parallel with G. Yellow arrows indicate the distribution of tUCHL1. B: Western blotting analysis of tUCHL1 in different parts of toad oocytes. Lane I: 1/10 volume of the insoluble protein part derived from 40 LTE-oocytes; S: 1/10 volume of the soluble protein part derived from 40 LTE-oocytes; GV: protein extract derived from 10 nuclei isolated from LTE-oocytes; Li: soluble protein extract from 2 immature LTE-oocytes; Lm: soluble protein extract from 2 mature LTE-oocytes; Hi: soluble protein extract from 2 HTE-oocytes without progesterone treatment; Hm: soluble protein extract from 2 progesterone treated HTE-oocytes. All the bands in Panel B were visualized by chemiluminescence. Each experiment was repeated twice independently.

Figure 3. The protein quantification assays of tUCHL1, p34^cdc2 and cyclin B1 in four different oocyte protein extracts.

The protein amounts of tUCHL1 and MPF subunits (p34^cdc2 and cyclin B1) in the four types of oocyte protein extracts were evaluated in reference to beta-actin. The total soluble protein extracts of 20 µg individually from Lm, Li, Hm and Hi samples were used to detect tUCHL1, p34^cdc2 and cyclin B1 by the western Blotting method, and here, all the bands were visualized by chemiluminescence. Lm and Li indicate immature or mature LTE-oocytes; Hm and Hi indicate THE-oocytes with or without progesterone treatment. Panel A presents a typical picture of western Blotting bands among three independent experiments. Panel B shows the optical density values from three independent western Blotting results. The longitudinal axis indicates ratios between the optical density values of the denoted proteins and those of beta-actin. According to t-test analysis, only p34^cdc2 among the denoted proteins was significantly different in its relative values between Lm and Hm (p=0.027), or Li and Hi (p=0.010). A significant level less than 0.05 is marked at the tops of corresponding bars of Hm and Hi with an asterisk.

Figure 4. The comparisons of UCH activity of tUCHL1 between LTE-oocytes and HTE-oocytes.

A: ubiquitin carboxyl-terminal hydrolase (UCH) activity of four types of oocyte protein extracts. Lm: mature LTE-oocytes; Li: immature LTE-oocytes; Hm: progesterone treated HTE-oocytes; Hi: HTE-oocytes without progesterone treatment. Oocyte soluble protein extracts were prepared at the final concentration of 20 µg/ml, and the two types of recombinant proteins are both used at the concentration of 20 nmol/L. The sharp drops in the curves at the beginning were caused by the light path closure to add reaction substrate Ub-AMC. The experiment was repeated twice independently, and the recombinant tUCHL1 was applied at a relative low concentration to obtain an appropriate slope. To draw all the curves in the same diagram, their starting points were adjusted.

Figure 5. tUCHL1 binds to MPF components p34^cdc2 or cyclin B1 in different oocyte protein extracts.

Panel A: A pull-down analysis (specific adsorption) with recombinant GST-tUCHL1 and with GST as a control, which were coupled to Sepharose 4B beads, respectively, from 200 µg of Lm, Li, Hm, Hi oocyte soluble protein extracts, was performed for p34^cdc2 assay by western Blotting analysis with beta-actin detection as a pull-down sample loading control. Panel B: Western blotting analysis for cyclin B1 after pull-down analysis with GST-tUCHL1 from oocyte protein extracts of Lm and Li, with 20 µg of total protein extracts (Lm and Li) as loading control was performed. Panel C: The results of immunoprecipitation (IP) with mouse monoclonal antibodies against p34^cdc2 and cyclin B1 from 120 µg of toad oocyte protein extracts are shown. Isolated protein complexes were then detected by western blotting analysis for tUCHL1. Loading denotes the lanes with 15 µg of total protein extracts as loading control. Lm and Li indicate immature or mature LTE-oocytes; Hm and Hi indicate THE-oocytes with or without progesterone treatment, respectively.

Figure 6. Pull-down analysis with recombinant p13^suc1 for tUCHL1 isolation from oocyte protein extracts.

Panel A: After toad oocyte protein 500 µg extracts were treated with the ATP system, and affinity adsorption (pull-down) was performed with recombinant p13^suc1-argrose to isolate protein complex from the oocyte protein extracts. The isolated protein complex was used to detect tUCHL1 by western Blotting analysis. Lanes 1-4 show results of the pull-down, and lanes 5-6 contained two loading controls with 20 μg of total protein extracts. B: Affinity adsorption results without the ATP system treatment run in parallel to the lanes in panel A. Lanes 1-4 are the corresponding adsorption results, and lane 5-6 indicate two other loading controls than those used in panel A. Lm and Li indicate immature or mature LTE-oocytes; Hm and Hi indicate THE-oocytes with or without progesterone treatment, respectively.