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  • Flutamide Alters the Expression of Chemerin, Apelin, and Vaspin and Their Respective Receptors in the Testes of Adult Rats.

Flutamide Alters the Expression of Chemerin, Apelin, and Vaspin and Their Respective Receptors in the Testes of Adult Rats.

International journal of molecular sciences (2020-06-26)
Malgorzata Brzoskwinia, Laura Pardyak, Agnieszka Rak, Alicja Kaminska, Anna Hejmej, Sylwia Marek, Malgorzata Kotula-Balak, Barbara Bilinska
ABSTRACT

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-CCRL2 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-74, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Flutamide
Sigma-Aldrich
p-Xylene-bis(N-pyridinium bromide), ≥95% (TLC)
Sigma-Aldrich
3,3′-Diaminobenzidine tetrahydrochloride hydrate, ≥96%