Karakterizacija i putevi sinhronizacije perifernog biološkog časovnika i steroidogeneze u Lajdigovim ćelijama pacova

Abstract

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), gena uključenih u metabolizam holesterola (Lipe, Soat2, Scarb1) i steroidogenih gena, (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4)...

Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4). Abolishment of melatonin, a main cue that spread information of light regime via cAMP signaling, stimulated expression of clock (Bmal1/BMAL1, Per1/2) and steroidogenic (Star/StAR, Hsd3b/HSD3B) elements...