Role of PKCζ signal pathway in the ovulatory process in the rat = 흰쥐 난소의 배란과정동안 PKCζ 신호전달경로의 역할

다국어 초록 (Multilingual Abstract)

LH binding to its receptor can activate several pathways, including protein kinase A/C, phospholipase A2/C, and tyrosine kinase. cAMP-PKA or PKC pathway is considered to be the most important signals in LH action. However, previously studies shown that LH treatment of granulosa cells resulted in a rapid translocation of atypical PKCζ from cytoplasm to the plasma membrane and the stimulation of atypical PKCζ activity by immunocytochemical and kinase assay. Although the presence of PKCζ in the ovary has been reported (Cutler et al., 1993), the activation of a specific PKC isoform PKCζ by LH in granulosa cells of preovulatory follicles has not been previously documented. Therefore, to understand mechanism of PKCζ during ovulatory process, the present study was designed to identify PKCζ regulated-genes in rat ovarian preovulatory granulosa cells. Preovulatory granulosa cells, obtained from ovaries of PMSG-primed immature rats, were cultured in serum-free medium in the presence of myristoylated PKCζ pseudosubstrate peptide (myr-SIYRRGARRWRKL-NH2) and a similarly sized control peptide (myr-ITRARRAPSVAN-NH2) with or without LH for 1.5 h. Total RNA extracted from cultured granulosa cells was used for ACP RT-PCR. As a result, sixteen genes including testin and aminolevulinic acid synthase1 have been identified.
Particularly, six genes (testin, glypican 4, retrovirus SC1, connective growth factor (ctgf), aminolevulinic acid synthase 1 (alas1), serum-inducible kinase(snk)) were rapidly stimulated by hCG. To determine mRNA expression and localization of these six genes, Northern blot analysis and in situ analysis was performed. Testin mRNA is rapidly and transiently stimulated in PMSG-primed rats with hCG, reaching a peak within 3 h. In situ hybridization analysis revealed that hCG treatment induced the expression of testin in granulosa cells of preovulatory follicles. Glypican-4 mRNA expression was increased from 3 h to 24 h after gonadotropin treatment in granulosa cells of preovulatory follicles. Increase in levels of ovarian retrovirus SC1 mRNA was detected at 3 and 6 h after hCG administration, and the levels rapidly declined. Furthermore, high expression of SC1 mRNA was localized to both granulosa cells of preovulatory follicles and the theca-interstitial layer at 3 h after hCG. Moderate levels of alas1 mRNA were detected in both immature untreated or PMSG-treated ovaries before hCG injection, and also rapidly and transiently stimulated alas1 mRNA expression in granulosa cells of growing follicles and preovulatory follicles, reaching a maximum stimulation 1 h after hCG treatment. Treatment of PMSG-primed rats with hCG for 1 h resulted in the rapid and transient stimulation of snk mRNA expression, reaching a maximum stimulation for 3 h after hCG treatment in granulosa cells of preovulatory follicles. The levels of snk mRNA declined by 6 h after treatment. Also, the rest of the ten genes were slowly stimulated by hCG. Northern analysis revealed that these ten gene expression was increased within 12-72 h after hCG treatment.
As the next step, I examined a role of PKCζ for LH action during ovulation in cultured rat granulosa cells of preovulatory follicles. Sixteen genes regulated by PKCζ and PKCζ signaling pathway were mostly involved in cell survival, apoptosis, and differentiation in other system. To determine whether PKCζ signaling pathway by LH in preovulatory granulosa cells plays a role of inhibition of granulosa cell apoptosis, MTT assay and DNA fragmentation assay was performed on preovulatory granulosa cells cultured under serum-free conditions for 48 h with increasing dose of RO 31-8220 (RO), myristoylated PKCζ pseudosubstrate peptide or a similarly sized control peptide with or without LH. MTT assay was shown that apoptosis of preovulatory granulosa cells is stimulated by myristoylated PKCζ pseudosubstrate peptide and 10 μM RO known to be a potent inhibitor of PKC ζ. DNA fragmentation assay showd that 10 μM RO markedly increased the ladder-pattern fragmentation of genomic DNA of cells, which was known as a characteristic feature of apoptosis suggest granulosa cell survival role for PKCζ signaling pathway in ovulation.
I then further investigated a part of the genes (testin, alas1) regulated following PKCζ activation during LH surge.
Testin is widly expressed in normal tissues and is predicted to encode a highly conserved protein of 421 amino acids containing three C-terminal LIM domains. To access a role of testin during LH-induced ovulation, I examined hormonal expression of testin in the ovary. Treatment of PMSG-primed rats with hCG resulted in the rapid and transient stimulation of testin mRNA expression, reaching a maximum stimulation 3 h after treatment. Testin mRNA was undetected in the ovaries of 26-day-old immature rats. In the ovaries obtained 48 h after PMSG treatment, testin signals were detected in atretic follicles. Interestingly, in the ovaries of PMSG-primed immature rats followed by hCG stimulation for 3 h, testin mRNA was induced in granulosa cells of mature preovulatory follicles, but not of growing follicles, as well as atretic follicles. These observation raises the possibility that testin may play a role in granulosa cell apoptosis. I have also cloned rat testin cDNA. This cDNA clone of 1397 bp is a partial representation of 2.8 kb mRNA, containing a long open reading frame (ORF) of 1260 bp. The ORF in the testin cDNA predicts a 420 amino acid polypeptide. Comparison of the amino acid sequences of human and rat testin revealed that they display 86% overall identity. The rat testin protein sequence, like the murine sequence, contains three LIM domains. Collectively, hormonal regulation of testin suggests that testin may play a role as a cell survival and apoptotic factor leading granulosa cells of ovulating follicles and small follicles to survive or death during ovulatory process.
Alas1 is perhaps best known for its role in catalyzing the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate, a universal precursor of tetrapyrrole compounds that function in a variety of reactions including the biosynthesis of heme, the transport of single electrons, and the catalysis of redox reactions. Alas1 mRNA were detected in both immature untreated or PMSG-treated theca-interstitial cells of ovaries before hCG injection, and treatment of PMSG-primed rats with hCG, however, resulted in the rapid and transient stimulation of alas1 mRNA expression, reaching a maximum stimulation 1 h after hCG treatment in theca-interstitial cells as well as granulosa cells of preovulatory follicles and some growing follicles. In addition, alas1 mRNA signals were detected in corpus luteum of ovaries stimulated with hCG for 24 h. Next, I studied a role of alas1 using 5-aminolevulinate (ALA) or hemin, inhibitor of alas1. hCG/LH-stimulated alas1 mRNA was remarkably decreased by ALA or hemin in vivo and in vitro. Because induction of alas1 induce the synthesis of several members of the CYP family, I determined whether P450scc and aromatase are regulated by alas1. Furthermore, inhibition of alas1 mRNA by ALA or hemin decreased P450scc and aromatase mRNA expression in gonadotropin-stimulated ovary and preovulatory granulosa cells. Furthermore, alas1 inhibiton by ALA or hemin had not effect on ovulation suggesting a role for steroidogenesis during ovulation.
In addition, I examined a signal pathway for LH action on testin and alas1 gene expression in cultured rat granulosa cells of preovulatory follicles. Treatment with an inhibitor of protein kinase A (PKA) did not affect LH-induced testin and alas1 levels. Interestingly, LH-induced testin and alas1 expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 μM), but not by low doses (0.1-1.0 μM), implicating the involvement of atypical PKCs. Treatment with the cell-permeable PKCζ-specific inhibitor pseudosubstrate peptide inhibited LH-induced alas1 expression, indicating the essential role of PKCζ.
In summary, the expression of PKCζ-regulated genes, testin, and alas1 is induced by LH surge and they play critical roles during LH-induced ovulation.

목차 (Table of Contents)

• ABSTRACT 1
• CHAPTER I. BACKGROUNDS 1
• 1.1 Ovarian follicle development 5
• 1.2. Ovulation 5
• 1.2.1 Characteristics of ovulation in mammals 5
• 1.2.1.1 Oocyte maturation 6
• 1.2.1.2 Cumulus expansion 9
• 1.2.1.3 Follicle rupture 11
• 1.2.1.4 Corpus luteum formation 14
• 1.2.2 Signaling pathways involved in ovulation 15
• 1.2.2.1 cAMP-PKA pathway 15
• 1.2.2.2 PKC pathway 17
• 1.2.2.3 Others 17
• 1.2.2.3.1 PLA2 pathway 18
• 1.2.2.3.2 PLD pathway 18
• 1.3 PKCζ 18
• 1.3.1 Structure of PKCζ 18
• 1.3.2 Activation mechanisms 19
• 1.3.3 Signaling 20
• 1.3.3.1 Mitogen-activated protein kinase (MAPK) cascade 20
• 1.3.3.2 From receptor signaling complexes to activation of NFκB transcriptional factor 20
• 1.3.3.3 p70S6 kinase signaling cascade 21
• 1.4 Aims of the study 22
• CHAPTER II. MATERIALS AND METHODS 23
• 2.1 Materials 23
• Hormones and reagents 23
• Animals 23
• 2.2 Methods 23
• 2.2.1 In vitro culture 23
• Preovulatory follicle culture 23
• Granulosa cell culture 24
• 2.2.2 Expression and Localization analysis 24
• ACP RT-PCR 24
• Northern blot analysis 25
• Western blot analysis 25
• In situ hybridization analysis 26
• 2.2.3 Functional analysis MTT assay 27
• DNA fragmentation assay 27
• Cloning and sequence analysis of the rat Testin cDNA 27
• Yeast two-hybrid sceens 28
• RIA 29
• Flow cytometry 29
• CHAPTER III. RESULTS 30
• 3.1 Identification of genes regulated by PKCζ and role of PKCζ in the ovulatory process in the rat 30
• 3.1.1 Gonadotropin stimulation of genes regulated by PKCζ activation 30
• 3.1.2 Death of granulosa cells by blockade of PKCζ pathway 34
• 3.2 Hormonal regulation of testin expression 36
• 3.2.1 Cloning of rat testin cDNA 36
• 3.2.2 Testin expression in gonadotropin-treated ovary in vivo 37
• 3.2.3 Signaling pathway for the LH action on testin expression in vitro cultured preovulatory granulosa cells 38
• 3.2.4 Identification of proteins interacting with testin 40
• 3.3 Expression and regulation of alas1 41
• 3.3.1 Expression of alas1 in vivo gonadotropin-treated ovary 41
• 3.3.2 Regulation and signaling pathways on LH-stimulated alas1 expression in vitro cultured preovulatory granulosa cells 43
• 3.3.3 Regulation and role of alas1 by 5-aminolevulinate or hemin in vivo gonadotropin-treated ovary 44
• CHAPTER IV. DISCUSSION 48
• REFERENCES 53
• SUMMARY INKOREAN 67