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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.

SUBMITTER: Ndiaye K

PROVIDER: S-EPMC2920863 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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

Ndiaye Kalidou K Lussier Jacques G JG Pate Joy L JL

Reproductive biology and endocrinology : RB&E 20100803

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 det ...[more]

PMID: 20682045

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Project description:Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn-amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn-amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn-amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

Project description:BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Project description:BACKGROUND: Estradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells. METHODS: Granulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes. RESULTS: ESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 ?M fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes. CONCLUSIONS: Our findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.

Dataset Information

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

Publications

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

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