Sena E AYDOS, Yunus YÜKSELTEN, Merve Gulsen BAL, Isıl YUKSELEN, Kaan AYDOS, Asuman SUNGUROĞLU

Azoospermi etyolojisinde sertoli hücresi enerji metabolizması bozuklukları

Amaç: Nonobstrüktif NOA ve obstrüktif OA azoospermili bireylerin Sertoli hücrelerinde spermatozoanın başlıca kullandığı enerji kaynağı olan laktatın sentezlenmesinde farklılık olabileceği hipotezinden yola çıkarak, PKM1, PKM2 ve LDHB enzimlerinin ifadelerinin, mitokondri sayısının hasta ve kontrol grubunun Sertoli hücrelerinde karşılaştırmalı olarak belirlenmesi ve metabolizmanın fertilite üzerine olası etkilerinin araştırılması amaçlandı. Gereç ve Yöntem: Bu çalışmada NOA ve OA’lı bireylerin testis dokularından Sertoli hücreleri izole edildi. Sertoli hücrelerinde enerji metabolizmasında görevli PKM1,PKM2 ve LDHB genlerinin ifadesi RT-PCR analizi ile ölçüldü. OA ve NOA’lı Sertoli hücrelerinde mitokondri boyaması Mitotraker Red ile gerçekleştirildi. Boyanan Mitokondri yüzdeleri flow sitometi analizi ile tespit edildi. Bulgular: Real Time PCR analiz sonuçlarına göre PKM1, PKM2, LDHB gen ifadeleri OA’lı gruba göre sırasıyla 198,01-, 162,01-, 50.79- kat düşük olduğu hesaplandı. Mitotraker boyama sonuçlarına göre OA’lı bireylerin Sertoli hücreleri NOA’lı bireylerin Sertoli hücreleri ile karşılaştırıldığında, NOA’lı grupta %34,6 boyanma mevcutken OA’lı grupta %52,4 boyanma tespit edildi. Sonuç: Hastalarımızın mitokondri sayısında azalma olmasına rağmen glikolitik yolak enzimlerinde de kontrole göre önemli bir azalma saptanması, Sertoli hücrelerindeki metabolik disfonksiyonun sperm hücre gelişiminde, dolayısıyla infertilitede önemli rolü olduğunu desteklemektedir.

Anahtar Kelimeler:

Sertoli hücresi, enerji metabolizması, erkek infertilitesi, azoospermi

Energy metabolism disorders of sertoli cells in azoospermia etiology

Objective: Based on the hypothesis, there may be difference in synthesis of lactose which is the main energy source used by spermatozoa in Sertoli cells between nonobstructive azoospermia NOA and obstructive azoospermia OA males, we aimed to analyze PKM1, PKM2 and LDHB gene expression levels and mitochondria numbers in the patient and control groups’ Sertoli cells comparatively. Material and Methods: In this study, Sertoli cells were isolated from NOA and OA patients’ testis tissues. PKM1, PKM2 and LDHB genes expression levels were measured by RT-PCR. Mitochondria were stained by Mitotracker red. Stained mitochondria percentage were determined by flow cytometry analysis. Results: PKM1, PKM2, LDHB genes expression levels were observed 198,01-, 162,01-, 50.79- fold lower in NOA group than OA group, respectively. According to the results of mitotracker stained test, 34,6% mitochondria were stained in NOA group, 52,4% mitochondria were stained in OA group. Conclusion: Despite the reduction in the mitochondria numbers of our patients, significant decrease in gene expression levels of the enzyme in the glycolytic pathway compared to the control was observed. This result showed that there was metabolic dysfunction in the development of sperm cells, thus playing an important role in infertility

Keywords:

Sertoli cells, energy metabolism, male infertility, azoospermia,

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Grisworld MD. The central role of Sertoli cells in sperma- togenesis. Semin Cell Dev Biol 1998; 9:411–416.
Alves MG, Rato L, Carvalho RA et al. Hormonal control of Sertoli cell metabolism regulates spermatogenesis. Cell Mol Life Sci 2013;70:777–793.
Benahmed M. Growth factors and cytokines in the testis. In:Comhaire FH, ed. Male Infertility. Chapman and Hall Medical 1996 ; 324–336.
Dorrington JH, Kahn SA. Steroid production, metabolism, and release by Sertoli cells. In: Russell LD, Griswold MD, eds. The Sertoli Cell. Clearwater, FL: Cache River Press 1993; 537–549.
Mruk DD, Cheng CY. Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Mo- vement in the Seminiferous Epithelium during Spermato- genesis. Endocrine Reviews 2004; 25:747–806.
Hunter D, Anand-Ivell A, Danner S, Ivell R. Models of in vitro spermatogenesis. Landes Bioscience 2012; 2: 1 – 12.
Chehtane M, Khaled AR. Interleukin-7 mediates glucose utilization in lymphocytes through transcriptional regula- tion of the hexokinase II gene.American Journal of Physio- logy - Cell Physiology 2010; 298:1560-1571.
Martins AD, Alves MG, Simoes VL et al. Control of Ser- toli cell metabolism by sex steroid hormones is mediated through modulation in glycolysis-related transporters and enzymes. Cell Tissue Res 2013;354:861–868.
Heinrichs M, Jacobasch G, Scheiner-Bobis K et al. Human erythrocyte pyruvate kinase (L/R-piruvat kinaz): produc- tion and characterization of a monoclonal antibody. Bio- med. Biochim Acta 1987; 46: 223-228.
Mazurek S, Grimm H, Oehmke M et al. Tumor M2-Piruvat Kinaz and glutaminolytic enzymes in the metabolic shift of tumor cells. Anticancer Res 2000; 20: 5151- 5154.
Hawtrey C, Goldberg E. Differential Synthesis of LDH In Mouse Testes. Annals of the New York Academy of Scien- ces 1968; 151: 611–615.
Mita M, Hall PF. Metabolism of round spermatid from rats: Lactate as the preferred substrate. Biol. Reprod 1982; 26:445-455.
Bajpai M, Gupta G, Setty BS. Changes in carbohydrate me- tabolism of testicular germ cells during meiosis in the rat. Eur J Endocrinol 1998; 138:322-7.
Grootegoed JA , Den Boer PJ. Energy metabolism in sper- matids: a review. In Cellular and Molecular Events in Sper- miogenesis. Cambridge University Press 1989;193–215.
Urner F, Sakkas D. A possible role for the pentose phospha- te pathway of spermatozoa in gamete fusion in the mouse. Biol Reprod 1999; 60:733-9.
Gupta GS. LDH-C4: A Unique Target of Mammalia Sper- matozoa. Clin Rev Biochem Molec Biol 1999; 34:361-385.
Cahn RD, Zwilling E, Kaplan NO, Levine L. Nature and Development of Lactic Dehydrogenases. Science 1962;136:962-969.
Markert CL, Holmes RS. Lactate dehydrogenase isozymes of the flatfish, Pleuronectiformes: kinetic, molecular and immunochemical analysis. J. exp. Zool 1969; 171: 85-104.
Burgos C, Maldonado C, Gerez de Burgos NM, Aoki A, Blanco A. Intracellular localization of the testicular and sperm-specific lactate dehydrogenase isozyme C4 in mice. Biol Reprod 1995;53:84–92.
Ramalho-Santos J, Amaral A, Sousa AP et al. Probing the Structure and Function of Mammalian Sperm using Opti- cal and Fluorescence Microscopy. In Modern Research and Educational Topics in Microscopy 2007:1;394-402.
Aitken J, Auger J, Baker HWG et al. WHO laboratory ma- nual for the examination and processing of human semen - 5th ed: World Health Organization 2010.
Koppenol WH, Bounds PL, Dang CV. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer 2011;11:325-337.
Anway MD, Folmer J, Wright WW, Zirkin BR .Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of sertoli cell function. Biology of Reproduction 2003;68 : 996-1002.
Alcivar AA. DNA methylation and expression of the genes coding for lactate dehydrogenases A and C during rodent spermatogenesis. Biol. Reprod 1991; 44:527–535.
Jen J, Deschepper CF, Shackleford GM, Lee CYG. Stage- specific expression of the lactate dehydrogenase-X gene in adult and developing mouse testes. Mol. Reprod. Dev 1990; 25: 14–21 .
Thomas K, Del Mazo J, Eversole P, Bellve A, Hiraoka Y, De- velopmental regulation of expression of the lactate dehy- drogenase (LDH) multigene family during mouse sperma- togenesis. Development 1990; 109, 483–493.
Rato L, Alves MG, Socorro S et al. Metabolic regulation is important for spermatogenesis, Nature Reviews Urology 2012; 9:6, 330–338.
Boussouar F, Rene EG, Jingwei J, Mohamed B. Tumor nec- rosis factor-a stimulates lactate dehydrogenase A expressi- on in porcine cultured Sertoli cells: mechanisms of action. Endocrinology 1999;140: 3054–3062.
Nehar D, Mauduit C, Boussouar F , Mohamed B. Tumor necrosis factor-a-stimulated lactate production is linked to lactate dehydrogenase A expression and activity increase in porcine cultured Sertoli cells. Endocrinology 1997: 138; 1964–1971.
Nehar D, Mauduit C, Boussouar F , Mohamed B. Interleu- kin 1a stimulates lactate dehydrogenase A expression and lactate production in cultured porcine Sertoli cells. Biol. Reprod 1998; 59, 1425–1432.
Riera MF, Meroni SB, Schteingart HF, Pellizzari EH, Ci- gorraga SB. Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells. J. Endocrinol 2002 ;173, 335–343.
Grataroli R, Boussouar F , Mohamed B. Role of sphingo- sine in the tumor necrosis factor a stimulatory effect on lactate dehydrogenase A expression and activity in porcine Sertoli cells. Biol. Reprod 2000 ; 63: 1473–1481.
Rhoden EL, Morgentaler A. Treatment of testosterone-in- duced gynecomastia with the aromatase inhibitor, anastro- zole. Int J Impot Res 2004;16:95-7.
Otasevic V, Korac A, Vucetic M et al. Is manganese (II) pentaazamacrocyclic superoxide dismutase mimic benefi- cial for human sperm mitochondria function and motility? Antioxid Redox Signal 2013;18:170–178.
Veitinger S, Veitinger T, Cainarca S, et al. Purinergic signal- ling mobilizes mitochondrial Ca2+ in mouse Sertoli cells. The Journal of Physiology 2011;589:5033-5055.