Suppressive action of pituitary adenylate cyclase activating polypeptide (PACAP) on proliferation of immature mouse Leydig cell line TM3 cells

Abstract

We aimed in the current study to understand the participation of PACAP in stage-specific Leydig and Sertoli cell functions. For this purpose, clonal cell lines TM3 (Leydig) and TM4 (Sertoli) cells, derived from the testis of immature BALB/c mice, were used. PACAP-specific receptors were detected in TM3 cells, but not in TM4 cells, which were characterized as PAC1 (type I) receptors. Stimulation of cAMP accumulation and testosterone secretion were observed in TM3 cells during 1~2 h treatment with PACAP38 (10 −10~1 0 −7 M) or PACAP27 (10 −11~1 0 −7 M). After around 10 h treatment with 10 −11~1 0 −7 M PACAP38 or PACAP27, proliferation of TM3 cells was suppressed in time-and dose-dependent manners, which was confirmed by real-time cell electronic sensing (RT-CES) system and phase-contrast microscopy. At 6 h after the addition of PACAP38, the percent cell population in G 2 /M phases increased significantly, while that in S phase showed significant decrease with little change in G 0 /G 1 phases. The results revealed that PACAP exerts, in addition to early stimulatory effect on cAMP formation-steroidogenesis, sustained suppressive effect on cell proliferation in TM3 cells by controlling progression of the cell cycle. The suppressive action of PACAP on proliferation in TM3 cells supports the stage-specific participation of the peptide in differentiation of immature mouse Leydig cells.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalamic tissues for its ability to stimulate adenylate cyclase (24). On the basis of structure similarity, the peptide is known to belong to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) family (24). Two biologically active forms with 38 (PACAP38) and 27 (PACAP27) amino acid residues have been identified (25). PACAP27 corresponds to the first 27 residues of PACAP38 and exhibits 68% sequence homology with VIP (25).Highly abundant PACAP was first demonstrated in the rat brain and testis by radioimmunoassay and the total PACAP immunoreactivity (PACAP-IR) in both testes was found to be almost two times greater than that in the whole brain, which exceeded the concentrations of any other peptides found in the testis (3). Subsequent several studies have confirmed the localization of PACAP-IR (11,15,28,34) and PACAP mRNA (11,14,16,28) in the testis of the rat in various stage of age. On the other hand, two high affinity binding sites for PACAP have been demonstrated (9), and in the adult rat testis, PACAP27 binding sites were localized in germinal cells (29).

Huaier Extract Inhibits Proliferation and Promotes Apoptosis via the MAPK Pathway in Human Osteosarcoma Cells

“…Flow cytometry was performed to detect the cell cycle of MG-63 and MNNG/HOS cells under treatment with Huaier. In contrast to results obtained by other study(Matsumoto et al 2008;Yan et al 2015), Huaier increased the percentage of OS cells in the G0/G1 phase, while decreasing the cell percentage in the G2/M phase. Similar medicine-induced of G0/G1 arrest has also been detected in other cell type, such as human breast cancer cells(Zhou et al 2018), and rheumatoid arthritis broblast-like synoviocytes(Zhang et al 2018a).…”

Effects of the Neuropeptides Pituitary Adenylate Cyclase Activating Polypeptide and Vasoactive Intestinal Peptide in Male Fertility

“…More specifically, through engaging with type I PACAP receptors in fetal rats, the results suggested that PACAP is a highly potent regulator of fetal testicular steroidogenesis [50]. PACAP involvement in the differentiation of immature mouse Leydig cells has been shown by exerting early stimulatory effect on cAMP formation-steroidogenesis and controlling the course of the cell cycle, inducing a prolonged suppressive effect on cell proliferation in TM3 cells [51]. Through studying PACAP-deficient mice, down-regulating testosterone production prevents the testicles from aging as a result of apoptosis and lowers the generation of reactive oxygen species [20].…”

Seasonal changes in the expression of PACAP, VPAC1, VPAC2, PAC1 and testicular activity in the testis of the muskrat (<em>Ondatra zibethicus</em>)

“…also confirmed the existence of PACAP specific receptor PAC1 in the Leydig cells through which the PACAP could regulate the development of immature Leydig cells. 29 Moreover, Shivers et al . and Li et al .…”