A-kinase anchoring proteins AKAP1, -4, -10 and -11 with different subcellular localizations in Sertoli cells and their roles in male fertility

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WangWenwen_2016_05_18.pdf (10.1 MB)

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2016

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DOI:
http://dx.doi.org/10.22029/jlupub-15519

Abstract

Sertoli cells in the testis regulate spermatogenesis and are involved in the molecular pathogenesis of male infertility, an increasing problem worldwide. They are controlled by testosterone and FSH signaling, which increases the intracellular cAMP-levels, leading to the activation of protein kinase A (PKA) and downstream phosphorylation events. Compartmentalization of PKA-dependent phosphorylation is controlled by a variety of distinct A-kinase anchoring proteins (AKAPs), tethering PKA by high affinity binding to a particular subcellular location. In this respect, it is of interest that rat AKAP220, partially homologous to the AKAP11 family, was described in peroxisomes of rat Sertoli cells, essential organelles for maintenance of normal lipid homeostasis and spermatogenesis regulation in this cell type. Therefore, the aims of this thesis were to analyze the effects of FSH treatment on peroxisomal gene expression, to characterize the effect of PKA-AKAP220 signaling on peroxisomes and to eventually discover new AKAP family members localized in this cell organelle or in mitochondria in Sertoli cells. Mouse TM4 Sertoli cells were used as cell culture model system to study the effects of FSH signaling on the peroxisomal and mitochondrial compartment. Since it is well established in the literature that the mitochondrial steroidogenic acute regulatory protein (StAR) is stimulated by LH-receptor/PKA-AKAP-signaling in testicular Leydig cells, the Star mRNA expression was assessed in TM4 Sertoli cells. Indeed, the Star gene was expressed in TM4 Sertoli cells and its expression level was also FSH-regulated. With optimized cell culture and FSH-treatment conditions, the gene expression for a variety of peroxisomal biogenesis and metabolic proteins was analyzed. FSH-treatment induced the expression of Pex11alpha, Pex13, Cat, Acox2, and Abcd2 mRNAs, encoding proteins involved in proliferation, matrix protein import, ROS and lipid metabolism in peroxisomes, demonstrating that the organelle is influenced by FSH signaling. To verify the peroxisomal localization of AKAP220 (=AKAP11) and to study its effects on FSH-PKA-mediated spermatogenesis regulation, this protein was localized with a commercial antibody in different subcellular fractions and highly purified peroxisomes of TM4 cells. However, our follow-up experiments with Western blots, bioinformatic evolutionary tree analysis for conserved PTS1 signals in AKAPs, and sequence alignments between Akap11 cDNA and genome sequences revealed that the reported cloned rat Akap220 cDNA as well as the subsequent results of AKAP220 subcellular localization were artifacts. Furthermore, during the series of experiments on rat Akap11 mRNAs in our laboratory, total RNA from a variety of rat tissues was isolated and the tissue-specific Akap11 gene expression was analyzed with specific sets of primer pairs, which revealed the highest Akap11 mRNA levels in the testis, pituitary, total brain and spleen and intermediate levels in liver, lung, heart and skeletal muscle. The same primers were used for subcloning parts of Akap11 in plasmid vectors for in vitro transcription of DIG-labelled cRNA-probes for Akap11 as well as corresponding mRNA controls. A non-radioactive Northern blot method was established, which can be used for future experiments on alternative splicing of Akap mRNAs.

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