Expression patterns of C1ql4 and its cell-adhesion GPCR Bai3 in the murine testis and functional roles in steroidogenesis

C1q-like 4 (C1QL4), a novel member of the C1q- and TNF-related protein family, was found to be highly expressed in rodent and human testis. However, the localization, developmental, and hormonally regulated expression and biologic function of C1ql4 in the testis have not been investigated. Here, we demonstrated that C1ql4 mRNA and protein levels in murine testes gradually increased from the postnatal period to the adult stage and were up-regulated by LH in vivo. In situ hybridization demonstrated that the distribution and expression levels of C1ql4 mRNA varied at different developmental stages, although C1ql4 mRNA was detected in the seminiferous tubule and interstitial Leydig cells. Recombinant C1QL4 did not affect cell proliferation but did increase testosterone production in TM3 Leydig cells, as well as in cultured seminiferous tubules. C1QL4-induced testosterone secretion in Leydig cells was accompanied by increased expression of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes. During this process, the c-Raf/extracellular signal-regulated protein kinase kinases 1 and 2/ERK1/2/mitogen- and stress-activated protein kinase-1 and cAMP/PKA/cAMP-responsive element binding protein signaling cascades were activated by C1QL4. The cell-adhesion GPCR brain-specific angiogenesis inhibitor 3 (BAI3), a putative receptor of C1QL4, was detected in the seminiferous tubule and interstitial Leydig cells during testicular development. Knockdown of Bai3 expression in Leydig cells led to a reduction in Star expression, accompanied by increases in phosphorylation of ERK1/2 and intercellular cAMP levels. However, C1QL4-induced StAR expression was not completely suppressed in the Bai3-deficient Leydig cells, and phosphorylation of ERK1/2 and intercellular cAMP levels were not significantly changed before and after C1QL4 stimulation. Our results suggested that although BAI3 played a role in C1QL4-induced steroidogenesis, there was an unidentified receptor that mediated C1QL4-activated testosterone secretion in Leydig cells through phosphorylation of ERK1/2 and up-regulation of intracellular cAMP levels. Taken together, our results showed, for the first time to our knowledge, that C1QL4 served as a novel acute regulator of testosterone secretion, and BAI3 functioned as a new receptor that is involved in steroidogenesis in Leydig cells. BAI3-independent ERK1/2 activation and cAMP activation mediated C1QL4-induced testosterone secretion. This study expanded the reproductive roles and mechanisms of C1QL4 and BAI3 signaling pathways.