Obesity-induced inflammatory miR-133a mediates apoptosis of granulosa cells and causes abnormal folliculogenesis.

Obesity has been reported to promote disordered folliculogenesis, but the exact molecular mechanisms are still not fully understood. In this study, we find that miR-133a is involved in obesity-induced follicular development disorder. After feeding with a high-fat diet (HFD) and fructose water for nine weeks, the mouse body weight is significantly increased, accompanied by an inflammatory state and increased expression of miR-133a in the adipose tissues and ovaries as well as accelerated follicle depletion. Although miR-133a is increased in the fat and ovaries of HFD mice, the increased miR-133a in the HFD ovaries is not derived from exosome transferred from obese adipose tissues but is synthesized by ovarian follicular cells in response to HFD-induced inflammation. In vivo experiments show that intrabursal injection of miR-133a agomir induces a decrease in primordial follicles and an increase in antral follicles and atretic follicles, which is similar to HFD-induced abnormal folliculogenesis. Overexpression of miR-133a modestly promotes granulosa cell apoptosis by balancing the expression of anti-apoptotic proteins such as C1QL1 and XIAP and pro-apoptotic proteins such as PTEN. Overall, this study reveals the function of miR-133a in obesity-induced ovarian folliculogenesis dysfunction and sheds light on the etiology of female reproductive disorders.

Echocardiographic assessment of Xenopus tropicalis heart regeneration.

BACKGROUND: Recently, it was reported that the adult X. tropicalis heart can regenerate in a nearly scar-free manner after injury via apical resection. Thus, a cardiac regeneration model in adult X. tropicalis provides a powerful tool for recapitulating a perfect regeneration phenomenon, elucidating the underlying molecular mechanisms of cardiac regeneration in an adult heart, and developing an interventional strategy for the improvement in the regeneration of an adult heart, which may be more applicable in mammals than in species with a lower degree of evolution. However, a noninvasive and rapid real-time method that can observe and measure the long-term dynamic change in the regenerated heart in living organisms to monitor and assess the regeneration and repair status in this model has not yet been established. RESULTS: In the present study, the methodology of echocardiographic assessment to characterize the morphology, anatomic structure and cardiac function of injured X. tropicalis hearts established by apex resection was established. The findings of this study demonstrated for the first time that small animal echocardiographic analysis can be used to assess the regeneration of X. tropicalis damaged heart in a scar-free perfect regeneration or nonperfect regeneration with adhesion manner via recovery of morphology and cardiac function. CONCLUSIONS: Small animal echocardiography is a reliable, noninvasive and rapid real-time method for observing and assessing the long-term dynamic changes in the regeneration of injured X. tropicalis hearts.

Deficiency of C1QL1 Reduced Murine Ovarian Follicle Reserve Through Intraovarian and Endocrine Control.

Ovarian aging is associated with depletion of the ovarian follicle reserve, which is the key determinant of fertility potential in females. In this study, we found that the small, secreted protein complement 1Q-like (C1QL1) is involved in the regulation of follicle depletion through intraovarian and endocrine control in a multidimensional collaborative manner. C1ql1 was detected to be conserved in the ovary and showed high transcript levels during folliculogenesis. Blockade of C1QL1 by IP and ovarian intrabursal injection of C1QL1 antiserum into prepubertal mice impaired folliculogenesis accompanied by reductions in body weight, fat mass, and intraovarian lipid accumulation. An elevation of circulating estradiol levels, reduction of hypothalamic KISS1 and GnRH expression, and a decrease in serum FSH levels were found in C1QL1-deficient mice. In C1QL1-deficient ovaries, many primordial follicles were recruited and developed into medium follicles but underwent atresia at the large follicle stages, which resulted in depletion of follicle reserve. Depletion of C1QL1 alleviated the inhibitory effect of C1QL1 on granulosa cell apoptosis and the stimulatory effect of C1QL1 on granulosa cell autophagy, which resulted in accumulation in the preantral and early antral follicles and an increase in the atretic follicles. The abnormal profile of endocrine hormones accelerated the intraovarian effect of C1QL1 deficiency and further led to depletion of ovarian reserve. Altogether, this study revealed the expression patterns and the mechanism of action of C1QL1 during folliculogenesis and demonstrated that deficiency of C1QL1 caused ovarian follicular depletion.

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.

[Effect of globular adiponectin on proliferation, migration and tube formation of ovarian microvascular endothelial cells].

OBJECTIVE: To analyze the effect of globular adiponectin on angiogenesis of ovarian microvascular endothelial cells (OMECs). METHODS: Mouse OMECs were isolated and purified by density gradient centrifugation with Percoll and identified by immunofluorescence analysis of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and endothelial cell marker von Willebrand factor (vWF). The capillary-like tube formation of OMECs was determined by vascular endothelial growth factor A (VEGFA) treatment in Matrigel matrix. OMECs treated with recombinant globular adiponectin protein were examined for cell proliferation with MTS assay and cell migration with scratch wound healing assay, and capillary-like tube formation was tested in Matrigel matrix. Western blotting was performed to detect the effect of globular adiponectin on AMPK phosphorylation. RESULTS: The signals of LHR and vWF, but not that of FSHR, were detected in the isolated cells. VEGFA treatment of the cells induced capillary-like tube formation, indicating their properties of ovarian-specific endothelial cells. Treatment with 1 and 3 µg/mL of recombinant globular adiponectin significantly increased the number of OMECs by (158.72∓14.50) % and (186.50∓4.20)% (P<0.01) and resulted in scratch wound closure rates of (49.43∓3.43)% (P<0.05) and (69.67∓1.2) % (P<0.01) respectively. The cells treated with 3 µg/mL globular adiponectin formed a capillary-tube length 6.63∓0.66 folds greater than that formed by the control cells (P<0.01). Treatment of the cells with 3 µg/mL globular adiponectin for 15 and 30 min resulted in pAMPK/AMPK ratios of 0.86∓0.08 and 0.66∓0.13, respectively significantly higher than that in the control cells (0.13∓0.12, P<0.01). Compound C obviously suppressed the tube formation and AMPK phosphorylation induced by globular adiponectin. CONCLUSION: Globular adiponectin promotes angiogenesis of OMECs through activation of the AMPK signal pathway.

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.

[Effect of mesenchymal stem cell transplantation on immunological injury of the ovary in mice].

OBJECTIVE: To investigate the effect of mesenchymal stem cell (MSC) transplantation in repairing ovarian injury in mice sensitized with porcine ovarian proteins. METHODS: Wild-type female mice with ICR background (6-8 weeks old) were divided randomly into groups A, B and C (n=12). In groups B and C, the mice were treated with the total protein extract from porcine ovary to induce immunological injury of the ovary, while those in group A received no treatment. MSCs-derived from GFP transgenic mice were transplanted into the mice of group C, and equal volume of PBS was injected intraperitoneally in mice of the other two groups. PCR was used to detect GFP gene in the genomic DNA of the ovaries to assess MSCs homing in the ovary, and the reparative effect of MSCs on ovarian injury was evaluated using HE staining and TUNEL analysis. RESULTS: After transplantation, the MSCs could reach the injured ovaries to promote the repair of the ovarian injury, resulting also in reduced apoptosis of the granulosa cells (GCs) in the injured ovaries. CONCLUSION: MSCs transplantation can promote the recovery of the immunological injury of the ovary in mice, the mechanism of which may involve reduced apoptosis of the GCs.

[Secretory expression of recombinant porcine zona pellucida glycoprotein-3alpha (rpZP3alpha) in Pichia pastoris].

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.

[Comparison of three methods for evaluating acrosome reaction in human spermatozoa].

OBJECTIVE: To find a convenient and exact method for evaluating acrosome reaction in human spermatozoa. METHODS: The semen of the normal male was mixed and then divided into 6 groups. Coomassie brilliant blue (CBB) staining, chlortetracycline (CTC) fluorescence staining and acid phosphatase (ACP) detection were used for morphological observation and data analysis of the acrosome status of the human sperm treated with or without progesterone. RESULTS: There were obvious morphological differences between the acrosome-reaction and acrosome-intact spermatozoa in CBB staining and CTC fluorescence staining, and significant differences were observed between the experimental and control spermatozoa by the three methods (P < 0.05). CONCLUSION: All the three methods can be used to assess acrosome reaction in human spermatozoa, but Coomassie brilliant blue (CBB) staining is much more convenient and stable.

Preparation of chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3alpha and its expression in mouse.

In the present study, the porcine zona pellucida (ZP)-3alpha eukaryotic expression vector pVAX1-pZP3alpha was constructed by genetic recombinant technology, then the recombinant plasmid was encapsulated in nanoparticles with chitosan, and the imaging of chitosan/pVAX1-pZP3alpha nanoparticles by Atomic Force Microscope (AFM) was processed. Feeding mouse with those microencapsulation by gastric larvae, and after 5 days, detecting its expression in mouse intestine by RT-PCR and indirect immunofluorescence (IIF). Results show that the porcine ZP-3alpha eukaryotic expression vector pVAX1-pZP3alpha had been constructed correctly, and the chitosan-DNA expressing ZP microencapsulation was prepared successfully. After 5 days of feeding mouse, the transcription and expression of those DNA vaccines were found in mouse alvine chorion. The preparation of chitosan/pVAX1-pZP3alpha plasmid DNA nanoparticles and its expression in mice will help to investigate the feasibility of ZP DNA vaccine to induce oviduct local mucosal immunity against ZP to block the fertilization without causing ovarian dysfunction, which will provide new ideas and ways for research and exploiting more effective, more convenient oral contraceptive vaccines.

[Expression of recombinant human zona pellucida-3 protein (rhZP3) in Pichia pastoris].

Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.