Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty-related QTL region down to a 1.7 Mb region on chromosome X in female mice and inferred miR-505-3p as the functional gene. We conducted ectopic expression of miR-505-3p in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of miR-505-3p on female puberty was evaluated by the measurement of pubertal/reproduction events and histological analysis. The results showed that female mice with overexpression of miR-505-3p in the hypothalamus manifested later puberty onset timing both in vaginal opening and ovary maturation, followed by weaker fertility lying in the longer interval time between mating and delivery, higher abortion rate and smaller litter size. We also constructed miR-505-3p-knockout mice by CRISPR/Cas9 technology. miR-505-3p-knockout female mice showed earlier vaginal opening timing, higher serum gonadotrophin and higher expression of puberty-related gene in the hypothalamus than their WT littermates. Srsf1 proved to be the target gene of miR-505-3p that played the major role in this process. The results of RNA immunoprecipitation sequencing showed that SRSF1 (or SF2), the protein product of Srsf1 gene, mainly bound to ribosome protein (RP) mRNAs in GT1-7 cells. The collective evidence implied that miR-505-3p/SRSF1/RP could play a role in the sexual maturation regulation of mammals.
Yuxun Zhou, Li Tong, Maochun Wang, Xueying Chang, Sijia Wang, Kai Li and Junhua Xiao
Kai-Chun Cheng, Ying-Xiao Li, Akihiro Asakawa, Miharu Ushikai, Ikuo Kato, Yuki Sato, Juei-Tang Cheng and Akio Inui
We aimed to characterize the effects of preptin on insulin secretion at the single-cell level, as well as the mechanisms underlying these changes, with respect to regulation by intracellular Ca2+ [Ca2+]i mobilization. This study assessed the effect of preptin on insulin secretion and investigated the link between preptin and the phospholipase C (PLC)/protein kinase C (PKC) pathway at the cellular level using fura-2 pentakis(acetoxymethyl) ester-loaded insulin-producing cells (Min 6 cells). Our results demonstrate that preptin promotes insulin secretion in a concentration-dependent manner. Using a PLC inhibitor (chelerythrine) or a PKC inhibitor (U73122) resulted in a concentration-dependent decrease in insulin secretion. Also, preptin mixed with IGF2 receptor (IGF2R) antibodies suppressed insulin secretion in a dose-dependent manner, which indicates that activation of IGF2R is mediated probably because preptin is a type of proIGF2. In addition, preptin stimulated insulin secretion to a similar level as did glibenclamide. The activation of PKC/PLC by preptin stimulation is highly relevant to the potential mechanisms for increase in insulin secretion. Our results provide new insight into the insulin secretion of preptin, a secreted proIGF2-derived peptide that can induce greater efficacy of signal transduction resulting from PLC and PKC activation through the IGF2R.
Jun-Ping Wen, Chune Liu, Wen-Kai Bi, Ya-Ting Hu, Qingshi Chen, Huibing Huang, Ji-Xing Liang, Lian-Tao Li, Li-Xiang Lin and Gang Chen
Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60–70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Ki ss 1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.
Xiaoning Li, Junhua Xiao, Yating Fan, Kan Yang, Kai Li, Xin Wang, Yanhua Lu and Yuxun Zhou
Gonadotropin-releasing hormone (GnRH) is the ultimate signal by which the neuroendocrine system controls the puberty onset and fertility in mammals. The pulsatile release of GnRH is regulated by numerous extracellular and intracellular factors, including miRNAs. Here, we report a novel regulation mechanism mediated by miR-29 family. We found that the absence of miR-29s resulted in elevated expression of Gnrh1 in GT1-7 cells. Through in silico and wet analysis, we identified Tbx21, a target gene of miR-29, as the main effector. As a transcription activator, TBX21 stimulates the expression of Gnrh1 directly by binding to its promoter region, and indirectly by activating the expression of Dlx1, another transcription activator of Gnrh1. Stereotactic brain infusion of miR-29 inhibitor into the hypothalamus caused earlier puberty onset in prepubertal female mice than that of intact controls. The female mice with ectopic expression of Tbx21 in the hypothalamus were affected in both puberty onset and fertility, as they had higher level of serum LH and FSH, larger litter size but steeper decline of fertility compared with those of controls. Our results revealed that miR-29-3p and its target Tbx21 played a role in regulating the mammalian puberty onset and reproduction by modulating the Gnrh1 expression.