Molecular characterization and expression of DERL1 in bovine ovarian follicles and corpora lutea

Abstract

The endoplasmic reticulum (ER) is a major site of protein synthesis and facilitates the folding and assembly of newly synthesized proteins. Misfolded proteins are retrotranslocated across the ER membrane and destroyed at the proteasome. DERL1 is an important protein involved in the retrotranslocation and degradation of a subset of misfolded proteins from the ER. We characterized a 2617 bp cDNA from bovine granulosa cells that corresponded to bovine DERL1. Two transcripts of 3 and 2.6 kb were detected by Northern blot analysis, and showed variations in expression among tissues. During follicular development, DERL1 expression was greater in day 5 dominant follicles compared to small follicles, ovulatory follicles, or corpus luteum (CL). Within the CL, DERL1 mRNA expression was intermediate in midcycle, and lowest in late cycle as compared to early in the estrous cycle. Western blot analyses demonstrated the presence of DERL1 in the bovine CL at days 5, 11, and 18 of the estrous cycle. Co-immunoprecipitation using luteal tissues showed that DERL1 interacts with class I MHC but not with VIMP or p97 ATPase. The interaction between DERL1 and MHC I suggests that, in the CL, DERL1 may regulate the integrity of MHC I molecules that are transported to the ER membrane. Furthermore, the greater expression of DERL1 mRNA is associated with the active follicular development and early luteal stages, suggesting a role of DERL1 in tissue remodeling events and maintenance of function in reproductive tissues.

Figure 1

Analysis of DERL1 mRNA expression in bovine tissues by Northern blot. Two transcripts of 3 and 2.6 kb corresponding to bovine DERL1 were observed and showed a variable pattern of expression among tissues.

Figure 2

Analysis of alternative splicing of DERL1 mRNA. The specific DERL1 PCR product corresponding to the entire open reading frame is represented by a single DERL1 PCR product; the sample without RT enzyme control shows that the specific DERL1 product amplified is not from genomic DNA.

Figure 3

Analysis of DERL1 mRNA expression during follicular development by semi-quantitative RT-PCR. The control gene was GAPDH and showed no significant differences in mRNA expression among the different groups. The DERL1 PCR signals were normalized with their corresponding GAPDH signals, and the results are presented as a relative change in ratio among groups. Different letters denote samples that differed significantly (p < 0.05) when Tukey-Kramer multiple comparison tests were performed to compare group means. Data are presented as means ± SEM. DF = dominant follicles (n = 4); OF = ovulatory follicles (n = 4); SF = 2-4 mm small follicles (n = 3); CL = corpus luteum collected on day 5 of the estrous cycle (n = 3).

Figure 4

A) RT-PCR detection of DERL1 mRNA expression in bovine corpus luteum at different stages of luteal development and B) at different hours post-PGF_2α (0, 1, 4, 8, and 12 hours). The control CL (0 h) was obtained on day 11 of the estrous cycle. Different letters denote significant differences (p < 0.05). Data are presented as means ± SEM; n = 4 animals at each day or time point.

Figure 5

Representative Western blot analyses using antibodies against DERL1 for protein expression in bovine corpus luteum. DERL1 protein was observed in bovine CL at day 5, day 11, and day 18 of the estrous cycle, with corresponding β actin blots. The experiment was repeated using CL protein lysates from four animals with similar results. The histogram in the lower panel represents the quantitative expression of DERL1 during luteal development.

Figure 6

Co-immunoprecipitation of DERL1 and bound proteins. A) Western blotting using the pelleted precipitates from immunoprecipitation. Blots were incubated, separately, with anti-DERL1 (1; C = DERL1 control), anti-MHC I (2), anti-VIMP (3), and anti-p97 (4) antibodies. B) Western blotting using the supernatant collected following co-immunoprecipitation. Blots were incubated with anti- MHC I (1), anti-VIMP (2), and anti-p97 (3) antibodies.