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1.
Acta Biochim Biophys Sin (Shanghai) ; 51(8): 845-855, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31287492

RESUMO

Autoimmune ovarian disease (AOD) is considered to be a major cause of premature ovarian failure (POF). The immunomodulatory properties of human amniotic epithelial cells (hAECs) have been studied in many disease models. We previously reported that hAECs restored ovarian function in chemotherapy-induced POF mice, but the immunomodulatory mechanism of hAECs is still unclear. To investigate the effect of hAECs on recipient mice, especially on regulatory Treg cells, hAECs and hAEC-conditioned medium (hAEC-CM) were intravenously injected into AOD mice immunized with zona pellucida protein 3 peptides (pZP3). Ovarian function was evaluated through estrous cycle, hormone secretion, follicle development, and cell apoptosis analysis. Immune cells including CD3, CD4, CD8 and Treg cells in the spleens were tested by flow cytometry. To elucidate the effect of hAEC-CM on macrophage function, inflammation model in vitro was established in RAW264.7 cells induced by lipopolysaccharide (LPS). hAECs and hAEC-CM regulated estrous cycles, promoted follicle development, ameliorated cell apoptosis and fibrosis in ovaries of AOD mice. In addition, hAECs significantly reversed the decrease of pZP3-induced Treg cells in the spleens. In vitro, hAEC-CM significantly inhibited the inflammatory reaction induced by LPS in RAW264.7 cells via up-regulating the expression of M2 macrophage genes. Further study demonstrated that hAEC-secreted transforming growth factor-beta and macrophage inhibitory factor played important roles in the macrophage polarization and migration under inflammatory stimulation. Taken together, hAECs restored ovarian function by up-regulating Treg cells in the spleens and reduced the inflammatory reaction via modulating the activated macrophage function in a paracrine manner in the ovaries of AOD mice.


Assuntos
Âmnio/citologia , Doenças Autoimunes/terapia , Células Epiteliais/citologia , Doenças Ovarianas/terapia , Insuficiência Ovariana Primária/terapia , Animais , Apoptose , Movimento Celular , Meios de Cultivo Condicionados/química , Modelos Animais de Doenças , Feminino , Células da Granulosa/citologia , Humanos , Imuno-Histoquímica , Inflamação , Oxirredutases Intramoleculares/metabolismo , Lipopolissacarídeos/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Baço/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo
2.
Nat Commun ; 10(1): 3086, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300655

RESUMO

Mammalian fertilisation begins when sperm interacts with the egg zona pellucida (ZP), whose ZP1 subunit is important for fertility by covalently cross-linking ZP filaments into a three-dimensional matrix. Like ZP4, a structurally-related component absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Here we report a characterisation of ZP1 proteins carrying mutations from infertile patients, which suggests that, in human, filament cross-linking by ZP1 is crucial to form a stable ZP. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not in human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception.


Assuntos
Infertilidade Feminina/genética , Domínios Proteicos/fisiologia , Glicoproteínas da Zona Pelúcida/metabolismo , Zona Pelúcida/metabolismo , Animais , Galinhas , Cristalografia por Raios X , Feminino , Fertilização/fisiologia , Mutação da Fase de Leitura , Humanos , Simulação de Dinâmica Molecular , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Alinhamento de Sequência , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/isolamento & purificação , Glicoproteínas da Zona Pelúcida/ultraestrutura
3.
Microb Pathog ; 134: 103572, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31163251

RESUMO

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.


Assuntos
Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/prevenção & controle , Adenoviridae/genética , Vacinas , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/imunologia , Sequência de Aminoácidos , Animais , Anticoncepção Imunológica , Modelos Animais de Doenças , Feminino , Fertilidade/imunologia , Imunização , Masculino , Glicoproteínas de Membrana/genética , Folículo Ovariano/patologia , Ovário/patologia , Óvulo , Plasmídeos , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Interações Espermatozoide-Óvulo , Espermatozoides , Células Vero
4.
Cell Biol Int ; 43(8): 899-909, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31081266

RESUMO

Previous studies have shown that the ovarian failure in autoimmune-induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta-derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol-requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X-box binding protein 1 (XBP1), up-regulated 78 kDa glucose-regulated protein (GRP78) and caspase-12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis-induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation-mediating ovarian function recovery.


Assuntos
Apoptose/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Placenta/citologia , Insuficiência Ovariana Primária , Animais , Caspase 12/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Feminino , Células da Granulosa/citologia , Proteínas de Choque Térmico/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ovário/metabolismo , Gravidez , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/terapia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo
5.
Nat Commun ; 10(1): 2271, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118423

RESUMO

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacinmCherry as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/metabolismo , Animais , Antígenos/metabolismo , Membrana Celular/metabolismo , Proteínas do Ovo/metabolismo , Feminino , Microscopia Intravital , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Glicoproteínas da Zona Pelúcida/metabolismo
6.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845640

RESUMO

The in vitro maturation of oocytes is frequently used as an assisted reproductive technique (ART), and has been successfully established in humans and rodents. To overcome the limitations of ART, novel procedures for the in vitro maturation of early follicles are emerging. During the follicle isolation procedure, the unintended rupture of each follicle leads to a release of extra oocytes. Such oocytes, which are obtained during follicle isolation from marmosets, can be used for early maturation studies. Marmoset (Callithrix jacchus), which is classified as a new-world monkey, is a novel model that has been employed in reproductive biomedical research, as its reproductive physiology is similar to that of humans in several aspects. The ovaries of female marmosets were collected, and the excess oocytes present during follicle isolation were retrieved without pre-gonadotropin induction. Each oocyte was matured in vitro for 48 h in the presence of various concentrations of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF), and the maturity of oocytes and optimal maturation conditions were evaluated. Each oocyte was individually reverse-transcribed, and the expression of mRNAs and microRNAs (miRs) were analyzed. Concentrations of hCG significantly affected the maturation rate of oocytes [the number of metaphase II (MII) oocytes]. The expression of BMP15 and ZP1 was highest when the oocytes were matured using 100 IU/L of hCG without pre-treatment with gonadotropins, and that of Cja-mir-27a was highest when cultured with follicle stimulating hormone. These results suggest that these up-regulated miRs affect the maturation of oocytes. Interactions with other protein networks were analyzed, and a strong association of BMP15 and ZP1 with sperm binding receptor (ACR), anti-Müllerian hormone (AMH), and AMH receptor was demonstrated, which is related to the proliferation of granulosa cells. Collectively, on the basis of these results, the authors propose optimal maturation conditions of excess oocytes of marmoset without in vivo gonadotropin treatment, and demonstrated the roles of miRs in early oocyte maturation at the single-cell level in marmosets.


Assuntos
Gonadotropina Coriônica/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Perfilação da Expressão Gênica/métodos , Oócitos/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 15/genética , Callithrix , Feminino , Fertilização In Vitro , Hormônio Foliculoestimulante , Perfilação da Expressão Gênica/veterinária , Humanos , Técnicas de Maturação in Vitro de Oócitos , MicroRNAs/genética , Modelos Animais , Oócitos/metabolismo , Oogênese , Glicoproteínas da Zona Pelúcida/genética
7.
J Assist Reprod Genet ; 36(4): 741-747, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30778819

RESUMO

PURPOSE: Empty follicle syndrome (EFS) is a complex reproductive disorder characterized by the repeated failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization (IVF). In addition to some cases caused by iatrogenic problems and known genetic factors, there are still many unexplained aspects of EFS. Here, we aimed to assess the clinical and genetic characteristics of two EFS patients. METHODS: We have characterized two primary infertility patients with EFS in a nonconsanguineous family from China. Both the patients presented similar clinical phenotypes, that is a few granulosa cells but no oocytes could be retrieved during repeated cycles with normal follicular development, E2 levels, and bioavailable hCG plasma levels. Abnormal oocytes were obtained once or twice between multiple IVF cycles. We performed Sanger sequencing of the LHCGR and ZP1~ZP4 genes in the patients, and further bioinformatics analysis was performed to identify pathogenic elements in the genes. RESULTS: A novel mutation, c.181C>T (p.Arg61Cys), and a known mutation, c.1169_1176delTTTTCCCA (p.Ile390Thrfs*16), in the ZP1 gene were both identified in patient 2, but no mutations were identified in patient 1. The novel mutation inherited from her mother was absent in the control cohort and the ExAc database. The arginine residue is conserved at this position, and its replacement by cysteine was predicted to be deleterious. In another allele, a paternal frameshift mutation was predicted to introduce premature stop codons, resulting in the deletion of 234 amino acids from the C-terminus of the ZP1 protein. CONCLUSIONS: Our findings presented compound heterozygous mutations in ZP1 associated with EFS and abnormal oocytes and provided further new evidence for the genetic basis of EFS and support for the genetic diagnosis of infertile individuals.


Assuntos
Predisposição Genética para Doença , Infertilidade Feminina/genética , Doenças Ovarianas/genética , Glicoproteínas da Zona Pelúcida/genética , Adulto , China/epidemiologia , Feminino , Humanos , Infertilidade Feminina/patologia , Mutação , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , Doenças Ovarianas/patologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/patologia , Ovulação/genética , Indução da Ovulação/métodos , Fenótipo , Zona Pelúcida/patologia
8.
Hum Genet ; 138(4): 327-337, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30810869

RESUMO

The human zona pellucida (ZP) is an extracellular glycoprotein matrix composed of ZP1, ZP2, ZP3, and ZP4 surrounding the oocyte, and it plays an important role in sperm-egg interactions during fertilization. Structural and functional changes in the ZP can influence the process of fertilization and lead to female infertility. Previous studies have identified mutations in ZP1, ZP2, and ZP3 that lead to female infertility caused by oocyte degeneration, empty follicle syndrome, or in vitro fertilization failure. Here we describe seven patients from six independent families who had several abnormal oocytes or suffered from empty follicle syndrome, similar to the previously reported phenotypes. By whole-exome sequencing and Sanger sequencing, we identified several novel mutations in these patients. These included three homozygous mutations in ZP1 (c.1708G > A, p.Val570Met; c.1228C > T, p.Arg410Trp; c.507del, p.His170Ilefs*52), two mutations in a compound heterozygous state in ZP1 (c.1430 + 1G > T, p.Cys478X and c.1775-8T > C, p.Asp592Glyfs*29), a homozygous mutation in ZP2 (c.1115G > C, p.Cys372Ser), and a heterozygous mutation in ZP3 (c.763C > G, p.Arg255Gly). In addition, studies in CHO cells showed that the mutations in ZP1, ZP2, and ZP3 might affect the corresponding protein expression, secretion, and interaction, thus providing a mechanistic explanation for the phenotypes. Our study expands the spectrum of ZP gene mutations and phenotypes, and provides a further understanding of the pathogenic mechanism of ZP gene mutations in vitro.


Assuntos
Infertilidade Feminina/genética , Mutação , Glicoproteínas da Zona Pelúcida/genética , Zona Pelúcida/patologia , Adulto , Animais , Células CHO , Consanguinidade , Cricetinae , Cricetulus , Análise Mutacional de DNA , Feminino , Humanos , Infertilidade Feminina/patologia , Masculino , Folículo Ovariano/anormalidades , Folículo Ovariano/patologia , Linhagem , Síndrome , Zona Pelúcida/metabolismo
9.
Mutat Res ; 813: 46-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30611916

RESUMO

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35-50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.


Assuntos
Endometriose/genética , Mutação , Doenças Ovarianas/genética , Glicoproteínas da Zona Pelúcida/genética , Adulto , China , Grupos Étnicos , Feminino , Humanos , Adulto Jovem
10.
Genet Med ; 21(2): 431-440, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29895852

RESUMO

PURPOSE: The oocyte-borne genetic causes leading to fertilization failure are largely unknown. We aimed to identify novel human pathogenic variants (PV) and genes causing fertilization failure. METHODS: We performed exome sequencing for a consanguineous family with a recessive inheritance pattern of female infertility characterized by oocytes with a thin zona pellucida (ZP) and fertilization failure in routine in vitro fertilization. Subsequent PV screening of ZP2 was performed in additional eight unrelated infertile women whose oocytes exhibited abnormal ZP and similar fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type and mutant proteins were carried out in CHO-K1 cells. RESULTS: Two homozygous s PV (c.1695-2A>G, and c.1691_1694dup (p.C566Wfs*5), respectively) of ZP2 were identified in the affected women from two unrelated consanguineous families. All oocytes carrying PV were surrounded by a thin ZP that was defective for sperm-binding. Immunostaining indicated a lack of ZP2 protein in the thin ZP. Studies in CHO cells showed that both PV resulted in a truncated ZP2 protein, which might be intracellularly sequestered and prematurely interacted with other ZP proteins. CONCLUSION: We identified loss-of-function PV of ZP2 causing a structurally abnormal and dysfunctional ZP, resulting in fertilization failure and female infertility.


Assuntos
Fertilização In Vitro , Infertilidade Feminina/genética , Glicoproteínas da Zona Pelúcida/genética , Adulto , Animais , Células CHO , Cricetulus , Feminino , Humanos , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Mutação , Gravidez , Análise de Sequência de DNA , Falha de Tratamento , Zona Pelúcida/ultraestrutura , Glicoproteínas da Zona Pelúcida/metabolismo
11.
Cell Prolif ; 52(1): e12530, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30334302

RESUMO

OBJECTIVES: In vitro differentiation of oocytes from female germline stem cells (FGSCs) has exciting potential applications for reproductive medicine. Some researchers have attempted to reveal the in vitro differentiation capacity of FGSCs. However, no systematic comparative study of in vitro differentiation conditions has been performed for murine FGSCs (mFGSCs). MATERIALS AND METHODS: mFGSCs line was cultured under five different conditions for in vitro differentiation. RT-PCR was performed to detect the expression of Oct4, Fragilis, Blimp1, Mvh, Scp3 and Zp3. Immunofluorescence was carried out to test the expression of Mvh, Fragilis and Zp3. Two-photon laser-scanning microscope was used to analyze nucleus-plasma ratio, and the proportion of chromatin of GV oocytes differentiated from mFGSCs in vitro (IVD-GVO), GV oocytes from in vivo (GVO) and mFGSCs. RESULTS: RT-PCR and immunofluorescence showed that mFGSC line expressed germ cell-specific markers, but not a meiosis-specific marker. By evaluating five different in vitro differentiation conditions, condition 5, which included a hanging drop procedure and co-culture of mFGSCs with granulosa cells, was shown to be optimal. mFGSCs could be successfully differentiated into germinal vesicle (GV) -stage oocytes under this condition. 3D observation revealed that both the nucleus-plasma ratio and proportion of chromatin were not significantly different between IVD-GVO and GVO. CONCLUSION: We evaluated five in vitro differentiation conditions for mFGSCs and successfully differentiate mFGSCs into GV-stage oocytes using a three-step differentiation process.


Assuntos
Células da Granulosa/citologia , Oócitos/citologia , Oogênese/fisiologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , RNA Helicases DEAD-box/biossíntese , Proteínas de Ligação a DNA , Feminino , Proteínas de Membrana/biossíntese , Camundongos , Proteínas Nucleares/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 1 de Ligação ao Domínio I Regulador Positivo/biossíntese , Glicoproteínas da Zona Pelúcida/biossíntese
12.
Tissue Cell ; 55: 91-106, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30503065

RESUMO

The human oocyte zona pellucida (ZP) is made of four glycoproteins, ZP1-ZP4. Recently, the prostate adenocarcinoma and prostate cancer PC3 cell-line were shown to express the human oocyte ZP3 glycoprotein, which was evaluated in a single report subject to patent. To further clarify whether oocyte zona pellucida glycoproteins are expressed in prostate cancer tissue and PC3-cells, in this report we evaluated protein expression of the four ZP glycoproteins in normal prostate tissue, prostate adenocarcinoma tissue and PC3-cells, and performed quantitative mRNA expression of the four ZP glycoproteins in the PC3 cell-line. Furthermore, as PC3-cells have not yet been studied in detail regarding their ultrastructural characteristics, in the present report we bring forward the detailed ultrastructure of PC3-cells. PC3-cells were divided into pavement and aggregated cells. We observed new ultrastructural features in pavement and aggregated cells, with the later exhibiting two different cell types. In prostate carcinoma tissue and PC3-cells we found protein expression of the four oocyte glycoproteins, ZP1, ZP2, ZP3 and ZP4. In addition, mRNA expression studies revealed expression of ZP1, ZP3 and ZP4 glycoproteins, but not of ZP2. Interestingly, the ZP1 mRNA product exhibited intron retention.


Assuntos
Células PC-3/citologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Glicoproteínas da Zona Pelúcida/metabolismo , Agregação Celular/fisiologia , Humanos , Masculino , Oócitos/metabolismo , Próstata/metabolismo , Próstata/patologia , Processamento de Proteína Pós-Traducional/fisiologia
13.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 35(5): 737-740, 2018 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-30298508

RESUMO

OBJECTIVE: To assess the association of single nucleotide polymorphisms (SNP) rs547984, rs540782, rs693421 and rs2499601 of Zona Pellucida Glycoprotein 4 (ZP4) gene with primary open-angle glaucoma (POAG) among ethnic Han Chinese from Sichuan Province. METHODS: A dye terminator-based SNaPshot method was used to genotype 336 patients with POAG and 768 healthy controls. RESULTS: No significant difference was detected in allelic frequencies of rs547984, rs540782, rs693421 and rs2499601 between the two groups (P>0.05). Haplotypic analysis showed a significant difference in G-G-A-G haplotype formed by the 4 SNPs between the POAG and the control groups (P<0.05). CONCLUSION: ZP4 gene SNPs rs547984, rs540782, rs693421, rs2499601 are not associated with POAG among ethnic Hans from Sichuan.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Glaucoma de Ângulo Aberto/genética , Polimorfismo de Nucleotídeo Único , Glicoproteínas da Zona Pelúcida/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático/etnologia , China/etnologia , Feminino , Glaucoma de Ângulo Aberto/etnologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
14.
World J Microbiol Biotechnol ; 34(11): 159, 2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341457

RESUMO

The zona pellucida-3 (ZP3) protein plays a pivotal role in oocyte and gamete development. We aimed to produce a recombinant ZP3 peptide using the Escherichia coli secretory system and apply it to a protein chip for detecting anti-ZP3 antibodies. The ZP3 gene was cloned into the pHOA downstream of the phoA promoter and transformed into E. coli YK537. Recombinant ZP3 was secretory expressed by decreasing the inorganic phosphate concentration. Then, rZP3 was purified and coated onto a protein chip, which was used to detect AZP3A in serum samples from 63 infertile patients. The area under the receiver operating characteristic curve was 0.934. The results, in terms of AZP3A detection, of the rZP3-coated protein chip were consistent with those of the ELISA kit. Therefore, our protein chip assay has potential for diagnosis of infertility due to AZP3A, and represents a less costly and simpler assay for clinical and research applications.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/metabolismo , Fosfatase Alcalina , Anticorpos/análise , Antígenos/genética , Antígenos/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Humanos , Infertilidade , Análise Serial de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Glicoproteínas da Zona Pelúcida/imunologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-30189258

RESUMO

Estrogen could lead to abnormal modulation or disruption of physical development, reproduction and sexual behavior in aquatic wildlife, especially in fish. Information on the toxicity of estrogens to native species in that can be used in site-specific risk assessments is scarce. In the present study, one zona pellucida 3 (ZP3) homologue termed CaZP3 was firstly identified from topmouth culter Culter alburnus, following its structural characteristics, tissue distribution and transcriptional modulation to 17α-ethinylestradiol (EE2) exposure were investigated. Meanwhile, vitellogenin (VTG) gene was employed to provide a comparison of the reactive ability to EE2 induction. The CaZP3 characterized with analogical functional domains such as ZP domain, SP, IHP, EHP, 12 cysteine residues, one N-linked glycosylation site and two conserved O-linked glycosylation sites and equal number of eight exons and seven introns with ZP3 counterparts of higher species. CaZP3 mRNA predominantly expressed in ovary, besides, highly expressed in female heart and male muscle and relatively high expressed in testis. CaZP3 has the lower reactive ability to EE2 induction in comparison with VTG, however, CaZP3 transcripts were significantly induced in gonads of both male and female culter by EE2 and could be used as an alternative biomarker to monitor EE2 activity. The present results supplement the database for toxicity of EE2, especially for fish species endemic to China and provide some useful information for the monitoring of EE2 activity in aquatic environment.


Assuntos
Cyprinidae/fisiologia , Disruptores Endócrinos/toxicidade , Etinilestradiol/toxicidade , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Glicoproteínas da Zona Pelúcida/metabolismo , Sequência de Aminoácidos , Animais , Aquicultura , Sequência Conservada , Cyprinidae/crescimento & desenvolvimento , Estrogênios/toxicidade , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Distribuição Aleatória , Alinhamento de Sequência , Caracteres Sexuais , Vitelogeninas/genética , Vitelogeninas/metabolismo , Glicoproteínas da Zona Pelúcida/química , Glicoproteínas da Zona Pelúcida/genética
16.
Dev Cell ; 46(5): 627-640.e5, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30122633

RESUMO

The zona pellucida surrounding ovulated eggs regulates monospermic fertilization necessary for successful development. Using mouse transgenesis, we document that the N terminus of ZP2 is sufficient for sperm binding to the zona matrix and for in vivo fertility. Sperm binding is independent of ZP2 glycans and does not occur after complete cleavage of ZP2 by ovastacin, a zinc metalloendopeptidase stored in egg cortical granules. Immediately following fertilization, a rapid block to sperm penetration of the zona pellucida is established that precedes ZP2 cleavage but requires ovastacin enzymatic activity. This block to penetration is associated with release of zinc from cortical granules coincident with exocytosis. High levels of zinc affect forward motility of sperm to prevent their passage through the zona matrix. This transient, post-fertilization block to sperm penetration provides a temporal window to complete the cleavage of ZP2, which prevents sperm binding to ensure monospermy.


Assuntos
Polissacarídeos/farmacologia , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zinco/metabolismo , Glicoproteínas da Zona Pelúcida/metabolismo , Zona Pelúcida/metabolismo , Animais , Comunicação Celular , Exocitose , Feminino , Fertilização , Masculino , Camundongos , Camundongos Transgênicos , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Zona Pelúcida/efeitos dos fármacos , Glicoproteínas da Zona Pelúcida/genética
17.
Stem Cell Rev Rep ; 14(5): 715-721, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29876729

RESUMO

Data from the literature show that there are different populations of stem cells present in human adult ovaries, including small stem cells resembling very small embryonic-like stem cells (VSELs). These small ovarian stem cells with diameters of up to 5 µm are present in the ovarian surface epithelium and can grow into bigger, primitive oocyte-like cells that express several markers of a germinal lineage and exhibit pluripotency but not the zona pellucida structure when cultured in vitro. In this report, we present the results of the functional testing of such primitive oocyte-like cells from one patient with premature ovarian failure after insemination with her partners' sperm. Knowing that even immature oocytes collected in an in vitro fertilization program cannot be fertilized naturally, we were only interested in determining whether and how these cells react to added sperm and whether spermatozoa somehow "recognize" them. Interestingly, the primitive oocyte-like cells quickly released a zona pellucida-like structure in the presence of sperm. Two different populations of cells were distinguished, those with a thick and those with a thin zona pellucida-like structure. The primitive oocyte-like cells with a released zona pellucida-like structure expressed the pluripotency-related gene OCT4A (POU5F1) and zona pellucida-related gene ZP3, similar to oocytes obtained from in vitro fertilization but not somatic chondrocytes. In a small proportion of these cells, a single-spermatozoon was observed inside the cytoplasm, but no signs of fertilization were found. These observations may suggest a primitive "cortical reaction". Our data further confirm the presence of germinal stem cells in the ovarian surface epithelium cell culture.


Assuntos
Linhagem da Célula/genética , Fator 3 de Transcrição de Octâmero/genética , Oócitos/crescimento & desenvolvimento , Glicoproteínas da Zona Pelúcida/genética , Adulto , Técnicas de Cultura de Células , Diferenciação Celular/genética , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Oócitos/citologia , Ovário/citologia , Ovário/crescimento & desenvolvimento , Espermatozoides/citologia , Espermatozoides/crescimento & desenvolvimento , Células-Tronco/citologia , Células-Tronco/metabolismo , Zona Pelúcida/metabolismo
18.
Curr Top Dev Biol ; 130: 307-329, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29853181

RESUMO

Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.


Assuntos
Galinhas , Proteínas do Ovo/fisiologia , Glicoproteínas da Zona Pelúcida/fisiologia , Sequência de Aminoácidos , Animais , Embrião de Galinha , Galinhas/metabolismo , Galinhas/fisiologia , Proteínas do Ovo/química , Feminino , Masculino , Modelos Biológicos , Óvulo/química , Conformação Proteica , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/química , Zona Pelúcida/fisiologia , Glicoproteínas da Zona Pelúcida/química
19.
Curr Top Dev Biol ; 130: 331-356, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29853182

RESUMO

All mammalian eggs are surrounded by a highly specialized extracellular matrix (ECM), called the zona pellucida (ZP), that functions before, during, and after fertilization. Unlike somatic cell ECM the mouse ZP is composed of three different proteins, ZP1-3, that are synthesized and secreted by growing oocytes and assembled into long interconnected fibrils. ECM or vitelline envelope (VE) that surrounds fish, reptilian, amphibian, and avian eggs also consists of a limited number of proteins all closely related to ZP1-3. Messenger RNAs encoding ZP1-3 are expressed only by growing oocytes at very high levels from single-copy genes present on different chromosomes. Processing at the amino- and carboxy-termini of nascent ZP1-3 permits secretion of mature proteins into the extracellular space and assembly into fibrils and matrix. Structural features of nascent ZP proteins prevent assembly within secretory vesicles of growing oocytes. Homozygous knockout female mice that fail to synthesize either ZP2 or ZP3 are unable to construct a ZP, ovulate few if any eggs, and are infertile. ZP1-3 have a common structural feature, the ZP domain (ZPD), that has been conserved through 600 million years of evolution and is essential for ZP protein assembly into fibrils. The ZPD consists of two subdomains, each with four conserved cysteine residues present as two intramolecular disulfides, and resembles an immunoglobulin (Ig) domain found in a wide variety of proteins that have diverse functions, from receptors to mechanical transducers. ZP2 and ZP3 function as receptors for acrosome-reacted and acrosome-intact sperm, respectively, during fertilization of ovulated eggs, but are inactivated as sperm receptors as a result of fertilization.


Assuntos
Camundongos , Óvulo , Zona Pelúcida/fisiologia , Animais , Feminino , Fertilização/fisiologia , Óvulo/química , Óvulo/fisiologia , Domínios Proteicos , Multimerização Proteica/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/química , Zona Pelúcida/metabolismo , Glicoproteínas da Zona Pelúcida/química , Glicoproteínas da Zona Pelúcida/metabolismo
20.
Curr Top Dev Biol ; 130: 357-377, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29853183

RESUMO

Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT).


Assuntos
Embrião de Mamíferos/química , Marsupiais/embriologia , Monotremados/embriologia , Glicoproteínas da Zona Pelúcida/fisiologia , Zigoto/metabolismo , Animais , Proteínas do Ovo/química , Proteínas do Ovo/fisiologia , Embrião de Mamíferos/metabolismo , Óvulo/química , Óvulo/metabolismo , Zona Pelúcida/química , Zona Pelúcida/fisiologia , Glicoproteínas da Zona Pelúcida/química , Zigoto/química
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