A combination containing natural extracts of clove, Sophora flower bud, and yam improves fertility in aged female mice via multiple mechanisms.

Introduction: With society development, the age at which women choose to have children has been gradually delayed. To improve the reduced fertility in women at advanced maternal age, we developed a combination containing natural extracts from clove, Sophora flower bud and Chinese yam with a mass ratio 15:6:10 and named it as DACHAO. Methods and Results: We then gavage DACHAO at a dose of 310 mg/kg BW to female mice at 10 month of age and investigated its effects on ovarian functions. Using MitoTracker probes, ROS, and JC-1 staining, we found that DACHAO treatment improved mitochondria functions in oocytes from aged mice. We also observed increased blastocyst formation when mature oocytes from control and DACHAO treated mice were for IVF and in vitro embryo culture. Cell counting and TUNEL assay further revealed increased cell numbers and decreased apoptosis in blastocysts of DACHAO group. After control or DACHAO treated mice being mated with fertile male mice, fertility test revealed a greater first litter size in the DACHAO group. Further studies demonstrated that DACHAO treatment could alleviate the retarded ovarian function in aged mice via changes in serum hormone levels, over-expression of antioxidant factors, under-expression of inflammation-related factors, and reduced apoptosis in the ovaries. Discussion: Thus, the new combination DACHAO will be a good choice in clinic to improve ovarian functions for women at advanced maternal age.

Macrophage-derived extracellular vesicles regulate follicular activation and improve ovarian function in old mice by modulating local environment.

In mammals, ovarian function is dependent on the primordial follicle pool and the rate of primordial follicle activation determines a female's reproductive lifespan. Ovarian ageing is characterised by chronic low-grade inflammation with accelerated depletion of primordial follicles and deterioration of oocyte quality. Macrophages (Mφs) play critical roles in multiple aspects of ovarian functions; however, it remains unclear whether Mφs modulate the primordial follicle pool and what is their role in ovarian ageing. Here, by using super- or naturally ovulated mouse models, we demonstrated for the first time that ovulation-induced local inflammation acted as the driver for selective activation of surrounding primordial follicles in each estrous cycle. This finding was related to infiltrating Mφs in ovulatory follicles and the dynamic changes of the two polarised Mφs, M1 and M2 Mφs, during the process. Further studies on newborn ovaries cocultured with different subtypes of Mφs demonstrated the stimulatory effect of M1 Mφs on primordial follicles, whereas M2 Mφs maintained follicles in a dormant state. The underlying mechanism was associated with the differential regulation of the Phosphatidylinositol 3-kinase/Mechanistic target of rapamycin (PI3K/mTOR) signaling pathway through secreted extracellular vesicles (EVs) and the containing specific miRNAs miR-107 (M1 Mφs) and miR-99a-5p (M2 Mφs). In aged mice, the intravenous injection of M2-EVs improved ovarian function and ameliorated the inflammatory microenvironment within the ovary. Thus, based on the anti-ageing effects of M2 Mφs in old mice, M2-EVs may represent a new approach to improve inflammation-related infertility in women.

RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging.

There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what's the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7Q67L or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality.Abbreviations: ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.

HucMSC-Derived Exosomes Mitigate the Age-Related Retardation of Fertility in Female Mice.

In mammals, resting primordial follicles serve as the ovarian reserve. The decline in ovarian function with aging is characterized by a gradual decrease in both the quantity and quality of the oocytes residing within the primordial follicles. Many reports show that mesenchymal stem cells have the ability to recover ovarian function in premature ovarian insufficiency (POI) or natural aging animal models; however, the underlying mechanism remains unclear. In this study, using exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-exos), we found the specific accumulation of exosomes in primordial oocytes. The stimulating effects of exosomes on primordial follicles were manifested as the activation of the oocyte phosphatidylinositol 3-kinase (PI3K)/mTOR signaling pathway and the acceleration of follicular development after kidney capsule transplantation. Further analysis revealed the stimulatory effects of HucMSC-exos on primordial follicles were through carrying functional microRNAs, such as miR-146a-5p or miR-21-5p. In aged female mice, the intrabursal injection of HucMSC-exos demonstrated the recovery of decreased fertility with increased oocyte production and improved oocyte quality. Although assisted reproductive technologies have been widely used to treat infertility, their overall success rates remain low, especially for women in advanced maternal age. We propose HucMSC-exos as a new approach to mitigate the age-related retardation of fertility in women.

Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis.

Liver serine/threonine kinase B1 (LKB1) is a tumor suppressor associated with the pathogenesis of Peutz-Jeghers syndrome. Affected males are at increased risk of developing Sertoli cell tumors and display defective spermatogenesis. Male mice lacking the short isoform (Lkb1S) of Lkb1 were sterile and exhibited abnormal spermiogenesis. In addition to the short isoform, the long isoform of Lkb1 (Lkb1L) is also expressed in testis; however, the requirement of the long isoform for fertility and the functional difference between the isoforms remain unknown. Herein, different from the spermiation failure reported in Lkb1S knockout mice, conditional deletion (cKO) of both isoforms of Lkb1 in germ cells resulted in male sterility stemming from defects in acrosome formation, as well as nuclear elongation and condensation during spermatid differentiation. Additionally, cKO mice showed a progressive germ cell loss that was never reported in mice with Lkb1S deletion. Further experiments revealed that the defect resulted from the failure of spermatogonial stem/progenitor cells (SPCs) maintenance. Although increased mTORC1 activity in postnatal cKO testes was consistent with a tendency toward germline stem cell differentiation, in vivo inhibition of the pathway by rapamycin treatment failed to rescue the phenotype. Concurrently, we detected a significant reduction of mitochondrial activity in Lkb1deficient SPCs. The results suggest that the regulation of LKB1 on SPCs' maintenance is associated with mitochondrial functions but not through the mTOR signaling pathway. In summary, our study supports different roles of Lkb1 isoforms in spermatogenesis with Lkb1L directing SPCs maintenance, and Lkb1L and Lkb1S coordinately regulating spermatid differentiation.

Restricting the induction of NGF in ovarian stroma engenders selective follicular activation through the mTOR signaling pathway.

In mammalian ovaries, primordial follicles remain in a quiescent state until activation by the surrounding microenvironment. Ovarian intervention, for example, ovarian cystectomy, ovarian wedge resection or laser drilling therapies for polycystic ovarian syndrome, has long been reported to change follicular development by an unknown mechanism(s). Herein, we established a murine model with partial ovarian resection of one ovary unilaterally, with the contralateral ovary undamaged. We found the injury accelerated follicular activation and development through the mTORC1 signaling pathway. Moreover, the stimulation of primordial follicles was restricted near the incision site where the mTORC1 pathway showed sequential activation beginning at the interstitial cells and proceeding to the primordial follicles. Total and polysome-associated RNA-seq revealed the increase of the nerve growth factor (NGF) family member, in both two fractions and immunostaining showed the restricted induction of NGF near the incision site. In cultured newborn ovaries, NGF demonstrated increase of follicular activation, and moreover, the NGF inhibitor K252a effectively blocked activation of primordial follicles stimulated by the surgery. We liken ovulation in mammals to minor tissue trauma, which happens naturally and cyclically in the body. As the increase in NGF accompanied the accumulation of activated primordial follicles after ovulation, our study may represent a common mechanism for selective follicular activation induced by a localized increase in NGF in interstitial cells and mediated via the mTOR signaling pathway. In addition, the NGF inhibitor K252a and the mTOR inhibitor rapamycin constitute good candidates for protecting follicular reserve against over exhaustion after ovarian surgery.

RNA purification by preparative polyacrylamide gel electrophoresis.

Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes.

Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

B and T lymphocyte attenuator interacts with CD3zeta and inhibits tyrosine phosphorylation of TCRzeta complex during T-cell activation.

B and T lymphocyte attenuator (BTLA) is an important negative regulator of T-cell activation. T-cell activation involves partitioning of receptors into discrete membrane compartments known as lipid rafts and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Here we show that after T-cell stimulation, BTLA co-clusters with the CD3zeta and is then involved in IS, as determined by a two-photon microscope. BTLA can interact with the phosphorylated form of T-cell receptor (TCR) within the lipid raft, which is associated with the T-cell signaling complex. Coligation of BTLA with the TCR significantly decreased the amount of phosphorylated TCR-related signal accumulation in the lipid raft during T-cell activation. These results suggest that BTLA functions to regulate T-cell signaling by controlling the phosphorylated form of TCRzeta accumulation in the lipid raft.

The influence of cyclosporin A on lymphocyte attenuator expression.

B and T lymphocyte attenuator (BTLA), a recently identified immune inhibitory receptor, has been demonstrated to have the ability to maintain self-tolerance and transplant-tolerance in mice. However, little is known about the effects of immunosuppressive drugs on the expression of BTLA. In the present study, we observed that the immunosuppressive drug cyclosporin A (CsA) could significantly reduce BTLA but not CD25 and CD69 expression on CD4+ T cells during activation in vitro, while rapamycin (RPM) had little effect on it. Exogenous interleukin-2 (IL-2) failed to reverse the inhibitory effect that CsA had on BTLA expression. Furthermore, phorbol 12-myristate 13-acetate (PMA) or ionomycin alone could efficiently induce BTLA protein expression on CD4+ and CD8+ T cells, while CsA significantly suppressed BTLA expression in this system. The present data indicate that the regulation of BTLA expression on CD4+ T cells does not depend on IL-2 and T cell activation but depends on calcineurin-dependent and calcineurin-independent pathways. The observation that CsA significantly inhibits BTLA expression on CD4+ T cells during activation, suggests that CsA might block the immune tolerance induced by BTLA and potentially increase the susceptibility to autoimmune diseases and graft rejection.

Inhibition of cell growth and induction of G1-phase cell cycle arrest in hepatoma cells by steroid extract from Meretrix meretrix.

In this study, we report that the steroid extract 5alpha, 8alpha-epidioxycholest-6-ene-3beta-ol (MME) from Meretrix meretrix has the ability to inhibit growth of hepatoma cells and to induce G1-phase cell cycle arrest in two human hepatoma cell lines, HepG2 and Hep3B. HepG2 cells were more sensitive than Hep3B to MME. The extract markedly up-regulated the expression of p53 and p21WAF1/CIP1 in HepG2, suggesting that MME-induced G1 phase cell cycle arrest in HepG2 might be p53-dependent. Therefore, the up-regulation of p27KIP1and p16INK4A in both cell lines indicates that a p53-independent pathway might be involved in the mechanism of MME inducing cell cycle arrest. In conclusion, MME induces G1 phase cell cycle arrest via both p53-dependent and p53-independent pathways.

BTLA, a new inhibitory B7 family receptor with a TNFR family ligand.

B and T lymphocyte attenuator (BTLA), identified as an immune inhibitory receptor recently, plays widespread roles on T and B cells. Emerging evidence has generated plentiful information on the mechanisms which BTLA mediates negative regulation in immune responses and involves in a variety of physiological and pathological processes. The exploration of the biological mechanisms and regulation of BTLA will open possibilities on novel therapeutic strategies in immune-related diseases.

Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein.

Fusion of paramyxovirus to the cell involves receptor binding of the HN glycoprotein and a number of conformational changes of F glycoprotein. The F protein is expressed as a homotrimer on the virus surface. In the present model, there are at least three conformations of F protein, i.e. native form, pre-hairpin intermediate and the post-fusion state. In the post-fusion state, the two highly conserved heptad repeat (HR) regions of F protein form a stable 6-helix coiled-coil bundle. However, no crystal structure is known for this state for the Newcastle disease virus, although the crystal structure of the F protein native form has been solved recently. Here we deployed an Escherichia coli in vitro expression system to engineer this 6-helix bundle by fusion of either the two HR regions (HR1, linker and HR2) or linking the 6-helix [3 x (HR1, linker and HR2)] together as a single chain. Subsequently, both of them form a stable 6-helix bundle in vitro judging by gel filtration and chemical cross-linking and the proteins show salient features of an alpha-helix structure. Crystals diffracting X-rays have been obtained from both protein preparations and the structure determination is under way. This method could be used for crystallization of the post-fusion state HR structures of other viruses.

Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion protein.

Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers [(HR1-HR2)(3)] after the fusion between viral and cellular membranes. In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli. Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method. A complete data set was collected in-house to 1.9 A resolution from a single crystal. The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058 A. To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8 A using synchrotron radiation.

Fruit bats as a natural reservoir of zoonotic viruses.

As a natural reservoir of manifold zoonotic viruses, fruit bats have been involved in at least three emerging zoonoses in recent years. This paper aims to introduce the epidemiological characteristics of these diseases emerged in the Australasian region between 1994 and 1999, transmission pathways of the newly discovered viruses and the relationship between the changed entironment of fruit bats and occurrences of these emerging diseases and provide a clue for the epidemiological investigations of SARS.