Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The ONWARDS insulin icodec trials

A round-up of the ONWARDS phase 3 clinical trials evaluating the novel weekly insulin icodec in people with diabetes.
ADVANCED SEARCH
Log in
Top
Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2013

Open Access 01-12-2013 | Research

Molecular cloning and expression analyses of porcine MAP1LC3A in the granulosa cells of normal and miniature pig

Authors: Sang H Kim, Sue Y Hwang, Kwan S Min, Jong T Yoon

Published in: Reproductive Biology and Endocrinology | Issue 1/2013

Login to get access

Abstract

Background

The members of the microtubule-associated protein 1 light chain (MAP1LC) family, especially those of the LC3 family (MAP1LC3A, B, C), are known to induce autophagy upon localization onto the autophagosomal membrane. In this regard, LC3 can be utilized as a marker for the formation of autophagosomes during the process of autophagy. The aims of this study are to clone porcine MAP1LC3A, and analyze the pattern of its expression in the ovarian tissues of normal and miniature pig ovary in an attempt to understand the distinct mode of apoptosis between two strains.

Methods

Rapid amplification of cDNA ends (RACE) were used to obtain the 5′ and 3′ ends of the porcine MAP1LC3A full length cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, and western blot analysis were performed to examine the expression of porcine MAP1LC3A. The localization of MAP1LC3A in the ovary was determined by In situ Hybridization and Immunohistochemical staining.

Results

We cloned the full-length cDNA of porcine MAP1LC3A and identified an open reading frame of 980 bp encoding 121 amino acids. Based on its homology to known mammalian proteins (98%) this novel cDNA was designated as porcine MAP1LC3A and registered to the GenBank (Accession No. GU272221). We compared the expression of MAP1LC3A in the Graafian follicles of normal and miniature pigs by in situ hybridization at day 15 of the estrus cycle. While normal pigs showed a stronger expression of MAP1LC3A mRNA than miniature pigs in the theca cell area, the expression was lower in the granulosa cells. Immunofluorescence analysis of the MAP1LC3A fusion reporter protein showed the subcellular localization of porcine MAP1LC3A and ATG5 as a punctate pattern in the cytoplasm of porcine granulosa cells under stress conditions. In addition, the expressions of MAP1LC3A and ATG5 were higher in normal pigs than in miniature pigs both in the presence and absence of rapamycin.

Conclusions

The newly cloned porcine MAP1LC3A provides a novel autophagosomal marker in both normal and miniature pig. We demonstrated that the expression of MAP1LC3A in graafian follicle is distinct in normal and miniature pig, which may explain the unique folliculogenesis of miniature pigs.
Appendix
Available only for authorised users
Literature
1.
Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem. 1998, 67: 425-479. 10.1146/annurev.biochem.67.1.425.CrossRefPubMed
2.
Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P: Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem. 2000, 275: 992-998. 10.1074/jbc.275.2.992.CrossRefPubMed
3.
Levine B, Klionsky DJ: Development by self-digestion: Molecular mechanisms and biological functions of autophagy. De Cell. 2004, 6: 6463-6477.
4.
Tsukada M, Ohsumi Y: Isolation and characterization of autophagy defective mutants of Saccharomyces cerevisiae. FEBS Lett. 1993, 333: 169-174. 10.1016/0014-5793(93)80398-E.CrossRefPubMed
5.
Choi JY, Jo MW, Lee EY, Yoon BK, Choi DS: The role of autophagy in follicular development and atresia in rat granulosa cells. Fertil Steril. 2010, 93: 2532-2537. 10.1016/j.fertnstert.2009.11.021.CrossRefPubMed
6.
Billig H, Furuta I, Hsueh AJW: Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology. 1993, 133: 2204-2212. 10.1210/en.133.5.2204.PubMed
7.
Mizushima N: Methods for monitoring autophagy. J Biochem Cell Biol. 2004, 36: 2491-2502. 10.1016/j.biocel.2004.02.005.CrossRef
8.
Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B: Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 2005, 122: 927-939. 10.1016/j.cell.2005.07.002.CrossRefPubMed
9.
Maiuri MC, Criollo A, Tasdemir E, Vicencio JM, Tajeddine N, Hickman JA, Geneste O, Kroemer G: BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1 and Bcl-2/Bcl-X(L). Autophagy. 2007, 3: 374-376.CrossRefPubMed
10.
Maiuri MC, Toumelin GL, Criollo A, Rain JC, Gautier F, Juin P, Tasdemir E, Pierron G, Troulinaki K, Tavernarakis N, Hickman JA, Geneste O, Kroemer G: Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. EMBO J. 2007, 26: 72527-72539.CrossRef
11.
Xie Z, Klionsky DJ: Autophagosome formation: core machinery and adaptations. Nat Cell Bio. 2007, 9: 1102-1109. 10.1038/ncb1007-1102.CrossRef
12.
He H, Dang Y, Dai F, Guo Z, Wu J, She X, Pei Y, Chen Y, Ling W, Wu C, Zhao S, Liu JO, Yu L: Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B. J Biol Chem. 2003, 278: 29278-29287. 10.1074/jbc.M303800200.CrossRefPubMed
13.
Wu J, Dang Y, Su W, Liu C, Ma H, Shan Y, Pei Y, Wan B, Guo J, Yu L: Molecular cloning and characterization of rat LC3A and LC3B–two novel markers of autophagosome. Biochem Biophys Res Commun. 2006, 339 (1): 437-442. 10.1016/j.bbrc.2005.10.211.CrossRefPubMed
14.
Fengsrud M: Lunde Sneve M, Overbye A, Seglen PO: Structural aspects of mammalian autophagy. Autophagy. Edited by: Klionsky DJ. 2004, Georgetown, TX: Landes Bioscience, 11-25.
15.
16.
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T: Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol. 2001, 152: 657-667. 10.1083/jcb.152.4.657.PubMedCentralCrossRefPubMed
17.
Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73.CrossRefPubMed
18.
Duerrschmidt N, Zabirnyk O, Nowicki M, Ricken A, Hmeidan FA, Blumenauer V, Borlak J, Spanel-Borowski K: Lectin-like oxidized low-density lipoprotein receptor-1-mediated autophagy in human granulosa cells as an alternative of programmed cell death. Endocrinology. 2006, 147: 3851-3860. 10.1210/en.2006-0088.CrossRefPubMed
19.
Howard PK, Chakraborty PK, Camp JC, Stuart LD, Wildt DE: Correlates of ovary morphology, estrous behavior and eyclicity in an inbred strain of miniature swine. Anat Rec. 1982, 203: 55-65. 10.1002/ar.1092030106.CrossRefPubMed
20.
Dor FJ, Tseng YL, Cheng J, Moran K, Sanderson TM, Lancos CJ, Shimizu A, Yamade K, Awwad M, Sachs DH, Hawley RJ, Schuurman HJ, Cooper DK: Alpha 1,3-galactosyltransferase gene-knockout miniature swine produce natural cytotoxic antiGal antibodies. Transplantation. 2004, 78: 15-20.CrossRefPubMed
21.
22.
McGee EA, Hsueh AJ: Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000, 21: 200-214. 10.1210/er.21.2.200.PubMed
23.
Hughes FMJ, Gorospe WC: Biochemical identification of apoptosis (programmed cell death) in granulosa cell: evidence for a potential mechanism underlying follicular atresia. Endocrinology. 1991, 129: 2415-2422. 10.1210/endo-129-5-2415.CrossRefPubMed
24.
Byskov AGS: Atresia: Ovarian follicular development and function. Edited by: Midgley AR, Sadler WA. 1979, New York: Raven Press, 41-57.
25.
Guthrie HD, Cooper BS, Welch GR, Zakaria AD, Johnson LA: Atresia in follicles grown after ovulation in the pig: measurement of increased apoptosis in granulosa cells and reduced follicular fluid estradiol-17β. Biol Reprod. 1995, 52: 920-927. 10.1095/biolreprod52.4.920.CrossRefPubMed
26.
Guthrie HD, Grimes RW, Cooper BS, Hammond JM: Follicular atresia in pigs: measurement and physiology. J Anim Sci. 1995, 73: 2834-2844.PubMed
27.
Tilly JL: Ovarian follicular atresia: a model to study the mechanisms of physiological cell death. Endocrinol J. 1993, 1: 67-72.
Metadata
Title
Molecular cloning and expression analyses of porcine MAP1LC3A in the granulosa cells of normal and miniature pig
Authors
Sang H Kim
Sue Y Hwang
Kwan S Min
Jong T Yoon
Publication date
01-12-2013
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2013
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/1477-7827-11-8

Other articles of this Issue 1/2013

Reproductive Biology and Endocrinology 1/2013 Go to the issue

Research

RUNX2, GPX3 and PTX3 gene expression profiling in cumulus cells are reflective oocyte/embryo competence and potentially reliable predictors of embryo developmental competence in PCOS patients

Research

Differential neutrophil gene expression in early bovine pregnancy

Research

The classical progesterone receptor mediates the rapid reduction of fallopian tube ciliary beat frequency by progesterone

Research

The characteristics of oviposition and hormonal and gene regulation of ovarian follicle development in Magang geese

Research

Ovarian HMW adiponectin is associated with folliculogenesis in women with polycystic ovary syndrome

Research

Decreased myometrial p160 ROCK-1 expression in obese women at term pregnancy