Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

齐口裂腹鱼zona pellucida3的克隆表达及其蛋白活性研究

为了研究齐口裂腹鱼Schizothorax prenanti的低温适应性,根据齐口裂腹鱼卵巢转录组测序结果设计引物,采用RT-PCR法克隆得到齐口裂腹鱼zona pellucida 3(Schizothorax prenanti zona pellucida 3,sp-zp3)基因的编码区,将目的基因重组到表达载体,并将质粒转染至中国仓鼠的卵巢细胞(CHO-K1)中进行表达,分离纯化得到ZP3蛋白后,对其冰晶结合活性进行初步测定。结果表明:齐口裂腹鱼zp3基因在中国仓鼠卵巢细胞中高效表达,并纯化得到重组蛋白SP-ZP3,该蛋白具有一定的冰晶结合活性。研究表明,一些鱼类ZP蛋白可能具有冰晶结合活性,当选择压力存在时这些ZP蛋白将进化出具有冷适应性的功能。

Acrosome reaction： relevance of zona pellucida glycoproteins

During mammalian fertilisation, the zona pellucida （ZP） matrix surrounding the oocyte is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction （AR） in the ZP-bound spermatozoon. The AR is crucial for the penetration of the ZP matrix by spermatozoa. The ZP matrix in mice is composed of three glycoproteins designated ZP1, ZP2 and ZP3, whereas in humans, it is composed of four （ZP1, ZP2, ZP3 and ZP4）. ZP3 acts as the putative primary sperm receptor and is responsible for AR induction in mice, whereas in humans （in addition to ZP3）, ZP1 and ZP4 also induce the AR. The ability of ZP3 to induce the AR resides in its C-terminal fragment. O-linked glycans are critical for the murine ZP3-mediated AR. However, N-linked glycans of human ZP1, ZP3 and ZP4 have important roles in the induction of the AR. Studies with pharmacological inhibitors showed that the ZP3-induced AR involves the activation of the Gi-coupled receptor pathway, whereas ZP1- and ZP4-mediated ARs are independent of this pathway. The ZP3-induced AR involves the activation of T-type voltage-operated calcium channels （VOCCs）, whereas ZP1- and ZP4-induced ARs involve both T- and L-type VOCCs. To conclude, in mice, ZP3 is primarily responsible for the binding of capacitated spermatozoa to the ZP matrix and induction of the AR, whereas in humans （in addition to ZP3）, ZP1 and ZP4 also participate in these stages of fertilisation.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Cellular mechanisms regulating sperm-zona pellucida interaction

For mammalian spermatozoa to exhibit the ability to bind the zona pellucida （ZP） they must undergo three distinct phases of maturation, namely, spermatogenesis （testis）, epididymal maturation （epididymis） and capacitation （female reproductive tract）. An impressive array of spermatozoa surface remodeling events accompany these phases of maturation and appear critical for recognition and adhesion of the outer vestments of the oocyte, a structure known as the ZP. It is becoming increasingly apparent that species-specific zona adhesion is not mediated by a single receptor. Instead, compelling evidence now points toward models implicating a multiplicity of receptor-ligand interactions. This notion is in keeping with emerging research that has shown that there is a dynamic aggregation of proteins believed to be important in sperm-ZP recognition to the regions of sperm that mediate this binding event. Such remodeling may in turn facilitate the assembly of a multimeric zona recognition complex （MZRC）. Though formation of MZRCs raises questions regarding the nature of the block to polyspermy, formation and assembly of such a structure would no doubt explain the strenuous maturation process that sperm endure on their sojourn to functional maturity.

Optimization of ionic liquid-based microwave-assisted extraction of polyphenolic content from Peperomia pellucida (L) kunth using response surface methodology

Objective: To optimize the ionic liquid based microwave-assisted extraction (IL-MAE) of polyphenolic content from Peperomia pellucida (L) Kunth. Methods: The IL-MAE factors as experimental design parameters, including microwave power, extraction time, ionic liquid concentration, and liquid–solid ratio had been involved. Response surface methodology and Box–Behnken design were used to obtain predictive model (multivariate quadratic regression equation) and optimization of the extraction process. The response surface was analyzed by using the yields of total polyphenolic content as response value. Results: Based on the obtained results the optimum extraction condition, including microwave power of 30% Watts, extraction time of 18.5 min, the ionic liquid concen-tration of 0.79 mol/L, and the liquid–solid ratio of 10.72 mL/g 1-Buthyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) as a solvent was selected. The regression model was obtained to predicts the yields from Peperomia pellucida:Y = 30.250 – 1.356X1 + 2.655X2 + 2.252X3 – 0.565X4 + 0.990 X1X3 – 8.172 X1X4 – 3.439 X3X4 – 4.178 X12 – 3.210 X32 – 6.786 X42 – 7.290 X12X3 + 5.575 X1X32 – 4.843 X32X4 with R2 = 0.82519. Scale-up confirmation test was obtained the maximum yields of total polyphenolics content with the amount of 31.1725μg GAE/g. Conclusions: The IL-MAE method produced a higher extraction polyphenolic and performed rapidly, easily and efficiently.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

Relationship between seminal plasma zinc concentration and spermatozoa-zona pellucida binding and the ZP-induced acrosome reaction in subfertile men

The aim of this study was to determine the relationship between seminal zinc concentration and spermatozoazona pellucida （ZP） binding and the ZP-induced acrosome reaction （ZPIAR） in subfertile men. Semen analyses and seminal zinc concentration assessments were carried out according to the World Health Organization manual for 458 subfertile men. A spermatozoa-ZP interaction test was carried out by incubating 2 × 10^6 motile spermatozoa with a group of four unfertilized oocytes obtained from a clinical in vitro fertilization programme. After 2 h of incubation, the number of spermatozoa bound per ZP and the ZPIAR of ZP-bound spermatozoa were examined. The effect of adding 0.5 mmol L^-1 zinc to the media on the ZPIAR of spermatozoa from normozoospermic men was also tested in vitro. Seminal zinc concentration positively correlated with sperm count and duration of abstinence, but negatively correlated with semen volume. On analysis of data from all participants, both spermatozoa-ZP binding and the ZPI- AR were significantly correlated with sperm motility and normal morphology, but not with seminal zinc concentration. However, in men with normozoospermic semen, the seminal zinc concentration was significantly higher in men with defective ZPIAR （ 〈 16%） than in those with normal ZPIAR （ ≥ 16% ） （P 〈 0.01）. The addition of 0.5 mmol L^-1 zinc to the culture media had no effect on spermatozoa-ZP binding, but significantly reduced the ZPIAR in vitro （P 〈 0. 001）. In conclusion, seminal zinc concentration is correlated with sperm count and the duration of abstinence in subfertile men. In men with normozoospermic semen, high seminal zinc concentration may have an adverse effect on the ZPIAR.

Effects of MⅡ stage oocytes zona pellucida birefringence on pregnancy outcome

Objective:To explore the effects of different MⅡstage oocytes zona pellucida birefringence on pregnancy outcome.Methods:A total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively,and randomly divided into the high zona birefringence(HZB)/HZB group.HZB/low zona birefringeuce(LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence.Intracytoplasmic sperm injection outcome was analyzed and compared.Results:The proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups(HZB/HZB group】HZB/ LZB group】LZB/LZB group)(P【0.05).But there was no significantly different between the number of oocytes and fertilization rate of these groups(P】0.05).Logistic regression analysis showed that factors allecl MⅡstage oocytes zona pellucida birefringence were age.basal FSH level and the LH level on the day of HCG injection.Age and KSH levels were negatively correlated with the single oocyte zona pellucida birefringence:While the LH level on the day of hCC injection was positively correlated with the single oocylc zona pellucida birefringence.Conclusions:The primary influence factors on MⅡstage oocytes zona pellucida are age.basal FSH level and the LH level on the day of hCG injection.The birefringence value of zona pellucida can affect the pregnancy outcome.

Effect of genistein on acrosome reaction and zona pellucida binding independent of protein tyrosine kinase inhibition in bull

Aim： To investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa. Methods： The effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin （FITC-PSA）/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline （CTC） assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa. Results： Immunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively. Conclusion： Our results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

A new angiotensin-converting enzyme inhibitor from Peperomia pellucida(L.) Kunth

Objective:To isolate,identify,and evaluate a new angiotensin-converting enzyme inhibitor from Peperomia pellucida(L.)Kunth herbs.Methods:A dried sample of Peperomia pellucida herb was successively macerated with n-hexane and ethyl acetate.The ethyl acetate extract solution was evaporated to obtain the crude extract.Vacuum liquid column chromatography and thin layer chromatography were performed to obtain two pure compounds.Then,both compounds were elucidated and identified using the spectroscopic method.Angiotensin-converting enzyme inhibitory activity studies of both compounds were determined using angiotensin-converting enzyme kit WST-1 with spectrophotometer microplate reader 96-well at 450 nm wavelength.Results:Two bioactive compounds were successfully isolated from Peperomia pellucida herb,including a new compound of 2,3,5-trimethoxy-9-(12,14,15-trimethoxybenzyl)-1 H-indene and pellucidin A.Both compounds demonstrated angiotensin-converting enzyme inhibitory activity,with IC50 values of 72 μM(27.95 μg/mL)and 1 1μM(4.4 μg/mL),respectively.Conclusions:In the present study,two active angiotensin-converting enzyme inhibitors were successfully isolated and purified from Peperomia pellucida which is used as an antihypertensive in traditional medicine,and support its use as an angiotensin-converting enzyme-inhibiting drug.

Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies

The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.

Analysis of anti-zona pellucida antibody and tumor necrosis factor-α,γ-interferon and interleukin-2 in sera from patients with premature ovarian failure

Objective:To investigate the role and the clinical significance of anti-zona pellucidaantibody (AzpAb) and tumor necrosis factor-α(TNF-α),γ-interferon(IFN-γ) and inter-leukin-2 (IL-2) in sera from patients with premature ovarian failure (POF).Methods: The AzpAb in the serum of POF patient was analyzed by means ofELISA. The levels of TNF-α, IL-2 and IFN-γ in the serum were determined by meansof radioimmunoassay (RIA).Results:The level of serum AzpAb in the POF patients was significantly higher thanthat of the normal controls(P＜0.001). The levels of TNF-α and IL-2 were significantlyreduced (P＜0. 001), and the level of IFN-γ was significantly elevated (P＜0.01). Thelevels of above three cytokines in AzpAb positive group were significantly higher thanthose of the negative group in POF patients.Conclusion: This study suggested that AzpAb, TNF-α, IFN-γ and IL-2 might playimportant roles in the pathogenesis of autoimmune POF.

In Vitro Fertilization Outcomes Following Assisted Hatching of Embryos with Thick Zona Pellucida—A Prospective Randomized Study

Purpose: Impaired hatching is associated with implantation failure following in vitro fertilization (IVF). Thickening or hardening of the zona pellucida (ZP) has been proposed as a factor in this impairment. We examined whether selective assisted hatching (AH) is beneficial with embryos having a thick ZP. Methods: This prospective, randomized controlled study was performed in the IVF unit of an obstetrics and gynecology department in a university-affiliated hospital. Only patients undergoing IVF and having a ZP thickness of ≥17 μm measured in all their embryos were included. In the intervention group, AH was applied to all embryos, before their transfer. In the control group, embryos were transferred without AH. Implantation, clinical pregnancy and live birth rates were the study endpoints. Results: Both study arms were comparable in most baseline parameters. The two groups did not differ in implantation rates (14.1% control vs. 8.92% intervention, odds ratio (OR) = 0.5974, 95% confidence interval (CI) 0.325 - 1.1), clinical pregnancy rates (36.7% vs. 25.8%, OR = 0.6025, 95% CI 0.274 - 1.325), or live birth rates (25% vs. 18.9%, OR = 0.7021, 95% CI 0.291 - 1.691). Conclusions: Selecting embryos for AH by their ZP thickness as a sole parameter was not found to be beneficial and to improve IVF outcome.

Cryopreservation of a Small Number of Human Sperm within Empty Zona Pellucidae

Objective To investigate the empty zona pellucida for use in the cryopreservation of human sperm. Materials & Methods Human and hamster zona pellucidae were evacuated and injected with testicular, epididymal and ejaculated sperm. The zona pellucidae with sperm were cryopreserved. Results After thawing, zona pellucidae were easily found, and sperm inside zona pellucidae were also easily observed. There were no differences in post-thaw motility and vitality between ejaculated and epididymal sperm groups (P>0.05), but these two parameters were lowered in testicular sperm group compared to both ejaculated and epididymal sperm (P<0.01). No significant difference was observed in post-thaw motilities among 6%, 7.5%, 9% glycerol concentrations (P>0.05). In addition, obvious differences in post-thaw motilities were not found between human and hamster empty zona pellucidae (P>0.05). Conclusion An evacuated zona pellucida is an ideal vehicle for the cryopreservation of a small number of human sperm.

Expression of Nonfusion Extracellular Porcine Zona Pellucida Protein 3β in E.coli

Objeetive To obtain the recombinant non fusion extracellular porcine zona pellucida protein 3β （pZP3β） in E.coli Methods By modificated the transition initiation region （TIR） in primers, synthetic nucleotide was gained by PCR. Such gene was cloned into pET-3c vector and transformed into E.coli BL21（DE3）pLysS. Results The recombi.ant nonfusion extracelhtlar pZP3β was expressed in E.coli to 10% of total cellular proteins, and identified by the Western blot method. Conclusion Modification of nucleotide without changing amino acid sequences is an effective means to increase non fusion expression rate of recombinant proteins, such as pZP3β in E. coll.

Specific Binding of 17D_3 Anti-pZP mAb to Porcine and Human Zona Pellucida but not to Other Tissue Antigens Identified by ABC Immunohistochemistry

Objective To characterize the binding effects of 17D3 anti-pZP monoclonal antibody （mAb） on porcine and human ZP,, ovary and other important tissues Methods The 17D3 anti-pZP mAb was produced by immunizing mouse with porcine zona pellucida and hybridoma and monoclonal antibody preparation. An ABC immunohistochemistry was used to evaluate reaction of mAb 17D3pZP to porcine and human ZP antigens as well as important human tissue antigens. Results mAb 17D3pZP specifically bound to porcine and human ZP antigen, but not to other cells of ovaries and other important human tissue antigens. Conclusion 17D3 anti-pZP mAb can recognize porcine and human ZP antigen without cross-reaction with other human tissue antigens including ovary, so it may further help us to abstract and purify corresponding target antigen and settle basis for producing human ZP contraceptive vaccine.

A method for mouse oocyte enucleation assisted with zona pellucida dilating

Objective: To study the reliability of a new enucleation method, zona pellucida Dilating (ZPD) assisted enucleation method, for mouse oocyte enucleation.Methods: In ZPD enucleation method, the zona pellucida was dilating with the help of outside pression in enucleation needle after breakthrough by needle tip and the nucleus was aspirated by the needle entering the perivitelline space for enucleation. This method was employed and was compared with three other methods in this experiment. The efficiency of enucleation of mouse oocyte was examed.Result: ZPD assisted enucleation method is better than three others at the survival rate after enucleation and the simplicity of micromanipulation. The rate of forming pronucleus of reconstructed embryo from enucleated oocyte by ZPD methods is as high as 62.8 % and the reconstructed embryo at 4-cell stage was obtained.Conclusion: ZPD assisted method is a highly efficient and simple enucleation method with the advantage of saving manipulating time and it does less damage to the oocyte.

High-level Expression and Purification of Human Zona Pellucida huZP3a^(22-176) and huZP3b^(177-348) Peptides in Escherichia coli

Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

Background： In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up（SU） or swim up ＋ zona pellucida（SU ＋ ZP） binding.Results： Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation（precense of one PN）. Treatments showed similar results（54, 47, 42 %, respectively） but statistically differents（P 0.05）.Conclusions： Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU ＋ ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU ＋ ZP were significantly different than frozen-thawed sperm,but between sperm treatments no significant differences were obtained.