Casein kinase 1α regulates murine spermatogenesis via p53-Sox3 signaling.

Casein kinase 1α (CK1α), acting as one member of the ß-catenin degradation complex, negatively regulates the Wnt/ß-catenin signaling pathway. CK1α knockout usually causes both Wnt/ß-catenin and p53 activation. Our results demonstrated that conditional disruption of CK1α in spermatogonia impaired spermatogenesis and resulted in male mouse infertility. The progenitor cell population was dramatically decreased in CK1α conditional knockout (cKO) mice, while the proliferation of spermatogonial stem cells (SSCs) was not affected. Furthermore, our molecular analyses identified that CK1α loss was accompanied by nuclear stability of p53 protein in mouse spermatogonia, and dual-luciferase reporter and chromatin immunoprecipitation assays revealed that p53 directly targeted the Sox3 gene. In addition, the p53 inhibitor pifithrin α (PFTα) partially rescued the phenotype observed in cKO mice. Collectively, our data suggest that CK1α regulates spermatogenesis and male fertility through p53-Sox3 signaling, and they deepen our understanding of the regulatory mechanism underlying the male reproductive system.

RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells.

Background: The v-raf-leukemia viral oncogene 1 (RAF1) plays an essential physiological role in reproduction and development through the mediation of steroid hormone synthesis. Follicle-stimulating hormone (FSH) signaling pathway was not involved in the majority of RAF1 studies, whether RAF1 takes part in the signaling events of gonadotropic hormones such as FSH in ovarian tissue is unknown. Methods: The process is blocked by treating granulosa cells (GCs) with the RAF1 inhibitor, RAF709. Inhibition of RAF1 activity by RAF709 decreased extracellular regulated protein kinases (ERK) phosphorylation and suppressed the expression of the cytochrome P450 subfamily 19 member 1 (CYP19A1), which is a major rate-limiting enzyme that participates in the last step of E2 biosynthesis. Results: We found that RAF1, acting as a downstream molecule, mediates FSH signalling to stimulate estradiol (E2) synthesis and secretion in mouse ovarian GCs. Gene expression of RAF1 was induced by FSH and the secretion of E2 increased into the bloodstream of mice and into the supernatant of primary GCs. Our in vitro and in vivo studies clearly illustrate RAF1 plays an important medium adjusting role in the FSH signaling pathway, and RAF1 acting as a downstream molecule to trigger ERK phosphorylation to stimulate GC E2 synthesis and secretion. Conclusions: RAF1 plays a pivotal mediating role in the FSH signaling pathway by inducing the phosphorylation of ERK and promoting E2 synthesis.

miR-375 acts as a novel factor modulating pituitary prolactin synthesis through Rasd1 and Esr1.

Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PRL synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7 and 60.4%, respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and Western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9 and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using the dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.

GRP94 regulates M1 macrophage polarization and insulin resistance.

Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.

Amitriptyline plays important roles in modifying the ovarian morphology and improving its functions in rats with estradiol valerate-induced polycystic ovary.

Previous studies demonstrated that depression is more prevalent in women with polycystic ovary syndrome (PCOS). In this study, we aimed to determine whether amitriptyline (AMT), an antidepressant drug, plays a role in preventing PCOS. The results showed that AMT modified ovarian morphology improved the ovarian functions and estrus cycle in estradiol valerate (EV)-induced polycystic ovary (PCO). AMT restored the levels of estradiol (E2), testosterone (T) and progesterone (P4) to normal, and elevated the level of luteinizing hormone (LH) in EV-induced PCO. No significant changes in follicle stimulating hormone (FSH) levels were observed in rats with EV or AMT treatment. The restoration of norepinephrine (NE) level was detected in rats with EV-induced PCO. AMT also altered the expression levels of steroidogenesis genes and beta2-adrenoceptor (beta2-AR) in EV-induced PCO. Our data revealed that AMT improves the ovarian morphology and modifies ovarian expression of beta2-AR and steroidogenesis genes in rats with EV-induced rat PCO. Our data provide support for the hypothesis that AMT is considered as a candidate drug for preventing and treating PCOS along with depression.

Loss of smooth muscle myosin heavy chain results in the bladder and stomach developing lesion during foetal development in mice.

Smooth muscle myosin heavy chain (SM-MHC) is exclusively expresses in smooth muscle, which takes part in smooth muscle cell contraction. Here, we used an insertional mutation mouse whose heavy polypeptide 11 (Myh11) gene has been disrupted and no SM-MHC protein has been detected. Compared to the wild-type and SM-MHC+/- mice, the SM-MHC-/- neonates had large round bellies, thin-walled giant bladders, and large stomachs with huge gas bubbles. Most of it died within 10 h and the rest within 20 h after birth. Further analysis of the developing foetuses from 16.5 days postcoitum (dpc) stage to newborn showed no significant (P<0.05) difference in the ratio of Mendelian inheritance and average body weight among SM-MHC+/+ , SM-MHC+/- and SM-MHC-/- mice, whereas the abnormal exterior appearance was observed in each SM-MHC-/- bladders from 16.5 dpc. Histological analysis showed no difference in stomach tissues but evidently thin-walled smooth muscle layer and a giant cavity in bladders of SM-MHC-/- foetuses at various stages from 15.5 dpc to newborn. The results indicated that the defect of SM-MHC lead to the bladder developing lesions initially at 15.5 dpc stage in mouse and also implied that the SM-MHC loss might result in the gas bubbles in stomach. The study should facilitate further detailed analyses of the potential role of SM-MHC in bladder and stomach development.

MiR-7 Mediates the Zearalenone Signaling Pathway Regulating FSH Synthesis and Secretion by Targeting FOS in Female Pigs.

Zearalenone (ZEA) acts as an environmental endocrine disruptor (EED) to cause health detriments. miRNAs were reported to influence the synthesis and secretion of pituitary hormones. However, the interactions between ZEA and miRNAs and related mechanisms remain unclear. The aims of this study were to determine whether and how miR-7 affects animal reproduction by its interactions with ZEA in the pig pituitary, which is sensitive to ZEA and has been used as an important animal model in medical research. Expressions of miRNA were detected by real-time PCR, in situ hybridization, and immunohistochemistry. The effects of ZEA, miR-7, and their interactions in the pituitary gland were identified by using an ovariectomized pig model, transfecting miR-7 mimics and inhibitor, radioimmunoassay, luciferase reporter assay, and Western blotting. The ZEA dosage was 7.5 mg/kg body weight in vivo and 1 µM in vitro. Our results demonstrate miR-7 acts to regulate gonadotropin synthesis and secretion. Furthermore, we found that ZEA leads to reproductive defects by enhancing miR-7 expression, which subsequently inhibits FSH synthesis and secretion. In vitro and in vivo experiments revealed that the effects of ZEA rely on G protein-coupled estrogen receptor 1, and miR-7 functions by mediating ZEA signaling pathway and targeting the Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS) gene. These findings show that miRNAs are key intrinsic factors regulating pituitary gonadotropins by mediating EED signaling in pituitary glands, and the actions of miRNAs and EEDs should be seriously considered in related studies about medical practice and animal production.

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

Molecular mechanisms of bladder outlet obstruction in transgenic male mice overexpressing aromatase (Cyp19a1).

We investigated the etiology and molecular mechanisms of bladder outlet obstruction (BOO). Transgenic (Tg) male mice overexpressing aromatase (Cyp19a1) under the ubiquitin C promoter in the estrogen-susceptible C57Bl/6J genetic background (AROM+/6J) developed inguinal hernia by 2 months and severe BOO by 9 to 10 months, with 100% penetrance. These mice gradually developed uremia, renal failure, renal retention, and finally died. The BOO bladders were threefold larger than in age-matched wild-type (WT) males and were filled with urine on necropsy. Hypotrophic smooth muscle cells formed the thin detrusor urinae muscle, and collagen III accumulation contributed to the reduced compliance of the bladder. p-AKT and ERα expression were up-regulated and Pten expression was down-regulated in the BOO bladder urothelium. Expression of only ERα in the intradetrusor fibroblasts suggests a specific role of this estrogen receptor form in urothelial proliferation. Inactivation of Pten, which in turn activated the p-AKT pathway, was strictly related to the activation of the ERα pathway in the BOO bladders. Human relevance for these findings was provided by increased expression of p-AKT, PCNA, and ERα and decreased expression of PTEN in severe human BOO samples, compared with subnormal to mild samples. These findings clarify the involvement of estrogen excess and/or imbalance of the androgen/estrogen ratio in the molecular pathogenetic mechanisms of BOO and provide a novel lead into potential treatment strategies for BOO.

Homologous illegitimate random integration of foreign DNA into the X chromosome of a transgenic mouse line.

BACKGROUND: It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integration. RESULTS: We confirmed that one fad2 line had a sole integration site on the X chromosome according to the inheritance patterns. Mapping of insertion sequences with thermal asymmetric interlaced and conventional PCR revealed that the foreign DNA was inserted into the XC1 region of the X chromosome by a homologous illegitimate replacement of an entire 45,556-bp endogenous genomic region, including the ovarian granulosa cell tumourigenesis-4 allele. For 5' and 3' junction sequences, there were very short (3-7 bp) common sequences in the AT-rich domains, which may mediate the recognition of the homologous arms between the transgene and the host genome. In addition, analysis of gene transcription indicated that the transgene was expressed in all tested fad2 tissues and that its transcription level in homozygous female tissues was about twice as high as in the heterozygous female (p < 0.05). CONCLUSIONS: Taken together, the results indicated that the foreign fad2 behaved like an X-linked gene and that foreign DNA molecules were inserted into the eukaryotic genome through a homologous illegitimate random integration.

[Molecular cloning, expression mutation of myostatin and study on biochemical activity of its C-terminal peptide].

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Mutation of myostatin activity leads to increases muscle growth and carcass lean yield. The bovine myostatin mutation cDNA was amplified by polymerase chain reaction, and then sub-cloned into the expression vector pET-30a( + ) to form the expression plasmid pET30a (+)-action/ Myostatin. The recombinant plasmid was transformed into E. coli BL21. The overexpression product of pET30a (+)-action/ Myostatin was been showed in vitro. Sheep skeletal muscle cell were cultured with the purified myostatin mutation C-terminal peptide. The results of this study suggest that had a powerful activity to stimulate the hyperplasia and proliferation of sheep muscle cells and shows high biochemical activity.