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.-Tan, A., Ke, S., Chen, Y., Chen, L., Lu, X., Ding, F., Yang, L., Tang, Y., Yu, Y. Expression patterns of C1ql4 and its cell-adhesion GPCR Bai3 in the murine testis and functional roles in steroidogenesis.

C1QTNF3 in the murine ovary and its function in folliculogenesis.

C1q/tumor necrosis factor-related protein 3 (C1QTNF3) is a novel adipokine with modulating effects on metabolism, inflammation and the cardiovascular system. C1QTNF3 expression levels in the sera and omental adipose tissues of women with PCOS are low compared to control subjects. However, the expression and function of C1QTNF3 in the ovary has not previously been examined. Here, we assessed the expression patterns of C1qtnf3 in the ovary and explored its role in folliculogenesis. The C1qtnf3 transcript abundance was higher in large follicles than in small follicles and was under the influence of gonadotropin. C1QTNF3 was detected mainly in the granulosa cells and oocytes of growing follicles and modestly in the granulosa cells of atretic follicles and in luteal cells. Excess androgen significantly decreased C1QTNF3 expression in the ovaries in vivo and in granulosa cells in vitro Recombinant C1QTNF3 protein accelerated the weight gain of ovarian explants and the growth of preantral follicles induced by follicle stimulating hormone (FSH) in vitro The stimulatory effect of C1QTNF3 on ovarian growth was accompanied by the initiation of AKT, mTOR, p70S6K and 4EBP1 phosphorylation, an increase in CCND2 expression and a reduction in cleaved CASP3 levels. Moreover, the addition of C1QTNF3 accelerated proliferation and reduced activated CASP3/7 activity in granulosa cells. In vivo, the ovarian intrabursal administration of the C1QTNF3 antibody delayed gonadotropin-induced antral follicle development. Taken together, our data demonstrate that C1QTNF3 is an intraovarian factor that promotes follicle growth by accelerating proliferation, decelerating apoptosis and promoting AKT/mTOR phosphorylation.

Peri-ovarian adipose tissue contributes to intraovarian control during folliculogenesis in mice.

Peri-ovarian adipose tissue (POAT) is a kind of intra-abdominal white adipose tissue that is present surrounding the ovaries in rodents. Recent studies demonstrated that POAT-deficient mice displayed a phenotype of delayed antral follicular development, for which decreases in serum estrogen, serum FSH and FSHR levels were responsible. However, folliculogenesis is regulated by endocrine signals and also modulated by a number of locally produced intraovarian factors whose acts are both autocrine and paracrine. Here, we used a model of surgical removal of POAT unilaterally and contralateral ovaries as controls, as both were under the same endocrine control, to assess the paracrine effect of the POAT on folliculogenesis. Surgical removal of unilateral POAT resulted in delayed antral follicular development and the increased number of atretic follicles, accompanied by decreased levels of intraovarian adipokines and growth factors, lipid accumulation and steroidogenic enzyme expression. POAT-deficient ovaries displayed compensatory increased expressions of intraovarian genes, such as Vegf and Adpn for angiogenesis, Acc, Fasn, and Gapdh involved in lipogenesis and Fshr in response to FSH stimulation. Furthermore, we demonstrated that removal of POAT promoted follicular apoptosis, caused retention of cytoplasmic YAP and inhibited PTEN-AKT-mTOR activation. These alterations were observed only in the POAT-deficient ovaries but not in the contralateral ovaries (with POAT), which suggests that a paracrine interaction between POAT and ovaries is important for normal folliculogenesis.

C1ql1/Ctrp14 and C1ql4/Ctrp11 promote angiogenesis of endothelial cells through activation of ERK1/2 signal pathway.

C1ql-like (C1QL)-1 and -4 proteins are encoded by homologous genes that are highly expressed in brain and adipose tissues. However, functional properties of C1QL proteins outside of the brain and adipocytes remain unknown. Here, we report that the globular domain of C1ql1/Ctrp14 and C1ql4/Ctrp11 proteins directly stimulate the angiogenesis of endothelial cells. In this study, soluble C1ql1/CTRP14 and C1ql4/Ctrp11 proteins, produced in prokaryote expression system, are co-cultured with human umbilical vein endothelium cells (HUVECs), which phenotype is identified with von Willebrand factor antibody. C1ql1/Ctrp14 and C1ql4/Ctrp11 promote the migration and capillary tube formation of HUVECs in a dose-dependent manner. During this process, phosphorylation of c-Raf, MEK1/2, ERK1/2, and p90RSK are activated by C1ql1/Ctrp14 and C1ql4/Ctrp11. MEK1/2 inhibitor, U0126, blocks C1ql1/Ctrp14-, and C1ql4/Ctrp11-induced capillary tube formation and cell migration. Moreover, the immunoreactivity of the receptor of C1QL1-C1QL4, brain-specific angiogenesis inhibitor 3 (BAI3), is detected in HUVECs, suggesting that BAI3 may mediate C1QL1/CTRP14- and C1QL4/CTRP11-induced angiogenesis. Meanwhile, C1ql1/Ctrp14 and C1ql4/Ctrp11 exposure also causes a stimulatory response of angiogenesis in chick yolk sac membrane. These data demonstrate that C1ql1/Ctrp14 and C1ql4/Ctrp11 stimulate the new blood vessel growth by activation of ERK1/2 signal pathway. The proangiogenic activity of C1ql1/Ctrp14 and C1ql4/Ctrp11 provides novel insights into the new opportunities for therapeutic intervention by targeting C1QLs in tumorigenesis, tissue regeneration, and recovery of ischemic heart disease.