Influence of peroxisomes on development, maturation and adult functions of the testis
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Peroxisomes are ubiquitous cell organelles with heterogeneous enzyme composition andfunctions, depending on the metabolic needs of different cell types, tissues and organ systems.However, until the beginning of the experimental work of this thesis hardly any knowledge wasavailable on the metabolic functions of these cell organelles in the testis, despite the well-knownsevere pathological alterations in this organ in patients suffering from peroxisomal diseases. Malechildren with Zellweger syndrome, the most severe form of a peroxisomal biogenesis disorder,exhibit cryptorchism and patients with X-linked adrenoleukodystrophy (X-ALD), a single enzymedefect of a peroxisomal ABC transporter for lipids, show an impairment of testicular functions in80% of the cases.Therefore, the main goals of this thesis were to characterize the peroxisomal distribution andenzyme composition in distinct cell types of normal mouse and human testis and to analyse themolecular consequences of peroxisome deficiency and its influence on male fertility. To get acomplete overview on the peroxisomal compartment and its enzyme composition in the testis innormal mice and on the alterations in scsPex13 knockout mice, routine histological analyses withHE-stained material, Oil red O staining on frozen sections, TUNEL, immunofluorescence andimmunohistochemistry with a variety antibodies against peroxisomal and cell marker proteins,electron microscopy, laser-assisted microdissection (LAMD), isolation of different testicular celltypes and primary cell culture, cell and tissue homogenisation, subcellular fractionation andorganelle isolation, Western blot analysis, lipid extraction, fatty acid and steroid (androgen)analyses by GC-MS, genomic DNA isolation, PCR genotyping, total RNA isolation, RT-PCRanalyses and RNAi on primary Sertoli cells were used.The results of this thesis indicate that peroxisomes in distinct cell types of the testis exhibitspecific enzyme composition and serve divers functions already under normal physiologicalconditions. Despite older reports from the literature on the exclusive presence of these organellesin Leydig cells, in this study peroxisomes were identified with new specific markers in all testicularcell types, including developing germ cells (except for mature spermatozoa). Peroxisomes altertheir matrix protein composition, aggregate in clusters during spermiogenesis in late stages ofelongated spermatids (step 14 to 16) and are transported into the cytoplasmic droplets andresidual bodies, which are subsequently phagocytosed by Sertoli cells. The highest enrichment ofperoxisomal lipid transporters (ABCD1 and ABCD3) as well as of ACOX2, the key regulatoryenzyme of the b-oxidation pathway 2 for side-chain oxidation of cholesterol and medium levels ofcatalase were found in Sertoli cells, suggesting peroxisomes as protectors against lipid toxicityand oxidative stress to prevent germ cell apoptosis. Leydig cells were selectively enriched incatalase and ABCD2, indicating that peroxisomes are important also in this cell type for theprotection of steroidogenesis against ROS and for lipid homeostasis. Comparativeimmunofluorescence analysis revealed that distribution, abundance and enyzme composition ofperoxisomes were conserved between mouse and man.To study the molecular consequences of peroxisomal dysfunction on male fertility, a Sertoli cellspecificknockout mouse was generated by AMH-cre-mediated excision of exon 2 of the Pex13gene, encoding a protein of the docking complex on the peroxisomal membrane involved inmatrix protein import into the organelle. Due to the absence of PEX13 protein and deficient matrixprotein import, all matrix proteins, such as catalase and thiolase, were mislocalized into thecytoplasm in Sertoli cells. Few peroxisomal membrane ghosts, positive for PEX14 were stillpresent in this cell type surrounding large lipid droplets. Within 130 days of postnataldevelopment, the scsPex13KO animals developed a complete Sertoli cell only syndrome. TUNELpreparations of paraffin sections revealed that germ cell apoptosis occurred between 90 and 130days of postnatal development. A strong increase of neutral lipids, such as triglycerides andcholesteryl esters was observed in Sertoli cells of the seminiferous tubules of P130 scsPex13KOanimals. The accumulation of peroxisome-metabolised fatty acids (VLCFA, pristanic and phytanicacid) in frozen testis samples suggested the deficiency of peroxisomal a- and b-oxidation. Steroidmeasurements revealed normal testosterone levels, but a strong accumulation ofdehydroepiandrosterone (DHEA) and a decrease of all other androgen precursors, suggesting ablock of the D5 pathway in these animals. The expression of mRNAs for the FSH- and LHreceptorswas significantly increased and the ones for transcription factors (Sf1, Gata4) andenzymes for steroidogenesis (Star, Cyp450scc, Cyp450c17, 3b-Hds) were elevated in Sertolicells as well as in Leydig cells of scsPex13KO mice. Specific Sertoli cell markers were alsoenhanced (Inha, Trf, Sgp2). Strongly proliferated Leydig cells seemed to dramatically upregulatetheir peroxisomal compartment and exhibited a strong increase of peroxisomal ABCDtransporters.Ultrastructural analysis of different subcellular compartments revealed mitochondriawith rearrangement of their cristae and giant whorl-like structures of SER in Leydig cells.Mitochondria in Sertoli cells were proliferated and exhibited extremely high levels of SOD2.Strong ROS production and a similar increase in mitochondrial SOD2 were detected in primarySertoli cells with a siRNA-mediated knockdown of the Pex13 gene. Peroxisomal dysfunction inSertoli cells led to an induction of constitutive and inducible cyclooxygenases (COX1, COX2),production of pro-inflammatory cytokines (Il1a, Il6, Tnfa, Mif) and local testicular inflammation,showing infiltration of macrophages in the interstitial space of the testis in scsPex13KO animals.In conclusion, peroxisomes in Sertoli cells are essential organelles in the testis for male fertilitywhich (1) regulate lipid homeostasis in the seminiferous tubules, (2) protect spermatogenesisagainst oxidative stress due to ROS increase, (3) are involved in androgen precursor synthesisand keep androgens in balance, and (4) protect against inflammation through the degradation ofbioactive lipid mediators (e.g. arachidonic acid or eicosanoids).Since peroxisomal enzyme composition is similar between mice and humans, scsPex13 knockoutmice provide an excellent model system to study human infertility due to peroxisomal deficiency.Future studies on tissue samples from infertile patients are needed to clarify in which extentperoxisomes are involved in the pathogenesis of idiopathic infertility.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler