Evaluation of the MA-10 cell line as a model of insl3 regulation and Leydig cell function
Introduction
Cryptorchidism is a developmental disorder characterized by failure of one or both testes to descend to an extra-abdominal location. Cryptorchidism can be detrimental to fertility in males and can lead to complications in normal castration procedures in domesticated animals, requiring more extensive surgical intervention than routine castration. Insulin-like peptide 3 (INSL3), a peptide hormone produced by the Leydig cells of the testes, is an important mediator of testicular descent as evidenced by functional studies in mice (Zimmermann et al., 1999; Adham et al., 2002). Though the central role of INSL3 in normal sexual development is acknowledged, the factors that regulate INSL3 production have yet to be comprehensively defined.
Secretion of INSL3 is a marker of Leydig cell differentiation and maturation rather than acute regulation (reviewed by Ivell et al., 2014). Evidence from clinical observations and intervention also indicates a strong correlation between LH and INSL3 even over relatively short-term treatment periods, possibly indicating a more direct relationship between gonadotropins and INSL3 (Trabado et al., 2014). Whether directly or indirectly, LH is widely considered as the secreteagogue for INSL3 though the mechanisms connecting LHR activation to insl3 activation are not well understood. Activation of the cAMP/PKA pathway has been implicated, though LHR activation has also been associated with several other signal transduction pathways (Kühn and Gudermann, 1999; Salvador et al., 2002; Robert et al., 2006; Evaul and Hammes, 2008). Several transcription factors have been implicated in regulating the insl3 promoter in vitro, including Nur77, KLF6, SF1 and COUP-TFII, though the complete mechanism of regulation of insl3 mRNA production in Leydig cells remains unclear (Zimmermann et al., 1998; Martin and Tremblay, 2005; Robert et al., 2006; Mendoza-Villarroel et al., 2014; Tremblay et al., 2016).
The murine MA-10 Leydig cell line is a widely used cell model for study of Leydig cell function in mammals including most studies of insl3 regulation (Ascoli, 1981). In the present study, there was direct testing of the hypothesis that LH functions through the cAMP/PKA pathway to increase the abundance of insl3 mRNA in MA-10 cells.
Section snippets
Materials and methods
All reagents were purchased from Sigma Aldrich, unless otherwise indicated.
Results
Treatment with 100 ng/ml hCG did not have an effect on insl3 mRNA abundance at 1, 2, or 3 h (Fig. 1A). The capacity of MA-10 cells to respond to gonadotropin stimulation was confirmed by measuring nur77 mRNA abundance in hCG-treated samples. There was a significant but transient increase in nur77 mRNA abundance at 1 h of treatment, while at 2 and 3 h, nur77 mRNA abundance was less than that of the control (Fig. 1B).
To determine which signal transduction pathway the LHR uses to regulate nur77
Discussion
Though there is evidence in the literature implicating LH as a direct stimulus for insl3 gene transcription stimulation, treatment with hCG did not affect insl3 mRNA abundance in the MA-10 cell line. These data could indicate either a lack of capacity of the MA-10 cell to respond to gonadotropin stimulation or that there are other means by which the insl3 gene is regulated. The robust increase in nur77 mRNA abundance in response to hCG indicates that these cells are indeed capable of responding
Conclusions
The MA-10 cells are functionally capable of responding to gonadotropin stimulation and have been considered to be a useful model to study regulation of the insl3 gene. Results from the present study indicate that while the MA-10 cell line is responsive to gonadotropin stimulation through nur77 and cyp17 stimulation of gene transcription, that response does not include acute regulation of insl3 mRNA abundance. Data from the present study are inconsistent with previously published data in terms
Funding
This research was not conducted as result of funding from any specific grant agencies in the public, commercial, or not-for-profit sectors.
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