RESUMEN
Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.
Asunto(s)
Capacitación Espermática , Espermatozoides , Animales , Capacitación Espermática/efectos de los fármacos , Masculino , Caballos/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Calcio/metabolismo , Zona Pelúcida/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , FosforilaciónRESUMEN
This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of É-aminocaproic acid (É-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with É-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.
Asunto(s)
Células del Cúmulo/metabolismo , Fibrinolisina/farmacología , Fibrinolíticos/farmacología , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/metabolismo , Oogénesis/efectos de los fármacos , Plasminógeno/farmacología , Ácido Aminocaproico/farmacología , Animales , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Bovinos , Medios de Cultivo , Células del Cúmulo/efectos de los fármacos , Técnicas de Cultivo de Embriones/métodos , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro/métodos , Fibrinolisina/antagonistas & inhibidores , Oocitos/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
OBJECTIVE: Isotretinoin is used in acne vulgaris treatment for more than 20 years. Isotretinoin has serious side effects on many organs, but there are no comprehensive studies investigating its possible toxic effects on reproductive organs. Thus, we aimed to investigate the possible toxic effects of isotretinoin administration on oocyte maturation in female rat gonads in this study. METHODS: Thirty-two adolescent female rats (Wistar Albino, 220 ± 35 g) were randomly divided into 4 groups with 8 subjects in each group: group 1, group 2, group 3, and group 4. Different doses of isotretinoin which was dissolved in sesame oil were given to rats by gavage: 7.5 mg/kg/day in group 3 and 15 mg/kg/day in group 4. The rats in group 2 received sesame oil by gavage. To create gavage stress, only gavage was administered to the rats in group 1. The gavages for each group continued once a day and at a certain time for 30 days. To determine the effect of isotretinoin on oocyte maturation, the periodic acid-Schiff reaction was performed for histochemical and histomorphometric evaluation of the zona pellucida, and staining of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) was performed for immunohistochemical analysis. RESULTS: When the thickness of the zona pellucida was evaluated, a statistically significant difference was found between group 1 and experimental groups (group 3 and group 4). In the experimental groups, it was determined that the thickness of the zona pellucida was decreased depending on the increase in dose. GDF-9 and BMP-15 expressions in oocytes of primordial and primary follicles decreased significantly in the experimental groups compared to group 1 and group 2. However, the expression of GDF-9 and BMP-15 in oocytes of secondary follicles was not significantly different between group 1 and group 2 and the experimental groups. CONCLUSIONS: In our study, we showed toxic effect of isotretinoin on oocyte maturation in female rats.
Asunto(s)
Acné Vulgar/tratamiento farmacológico , Fármacos Dermatológicos/efectos adversos , Isotretinoína/efectos adversos , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Acné Vulgar/fisiopatología , Animales , Proteína Morfogenética Ósea 15/metabolismo , Femenino , Factor 9 de Diferenciación de Crecimiento/metabolismo , Oocitos/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ratas , Ratas Wistar , Zona Pelúcida/efectos de los fármacosRESUMEN
DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.
Asunto(s)
Daño del ADN , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Mutágenos/toxicidad , Zona Pelúcida/efectos de los fármacos , Animales , Ensayo Cometa , Embrión de Mamíferos/patología , Desarrollo Embrionario/genética , Femenino , Masculino , Exposición Materna , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo , Zona Pelúcida/patologíaRESUMEN
The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.
Asunto(s)
Acetilglucosamina/farmacología , Fertilización/fisiología , Ácido Glucurónico/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/ultraestructura , Sus scrofa/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Femenino , Glutatión/análisis , Glutatión Peroxidasa/metabolismo , Ácido Hialurónico/análisis , Oocitos/efectos de los fármacos , Oocitos/fisiología , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/ultraestructuraRESUMEN
The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Asunto(s)
Reacción Acrosómica/fisiología , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Progesterona/farmacología , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Reacción Acrosómica/efectos de los fármacos , Calcimicina/farmacología , Calcio/farmacología , Ionóforos de Calcio/farmacología , Sistemas de Liberación de Medicamentos , Humanos , Masculino , Espermatozoides/efectos de los fármacos , Zona Pelúcida/efectos de los fármacosRESUMEN
We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.
Asunto(s)
Anisoles/farmacología , Antioxidantes/metabolismo , Blastocisto/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Derivados de Alilbenceno , Animales , Blastocisto/citología , Blastocisto/metabolismo , Bovinos , Medios de Cultivo/farmacología , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.
Asunto(s)
Calgranulina B/metabolismo , Epitelio/metabolismo , Oviductos/metabolismo , Capacitación Espermática , Interacciones Espermatozoide-Óvulo , Reacción Acrosómica/efectos de los fármacos , Adulto , Animales , Sitios de Unión , Epitelio/efectos de los fármacos , Femenino , Humanos , Masculino , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oviductos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Semen/efectos de los fármacos , Semen/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
Brefeldin A (BFA) is a lactone antibiotic synthesized from palmitic acid by several fungi that could block anterograde transport of proteins from endoplasmic reticulum to Golgi apparatus by reversible disruption of the Golgi complex. Previous investigations have shown that BFA induces the apoptosis of cancer cells in mitosis and impairs asymmetric spindle positioning in meiosis. Here, we document that exposure to BFA in porcine oocytes compromises the meiotic maturation via disrupting both nuclear and cytoplasmic maturation. We found that BFA exposure collapsed the cytoskeleton assembly by showing the aberrant spindle organization with misaligned chromosomes and defective actin dynamics. Furthermore, the distribution of both mitochondria and cortical granules (CGs), two important indexes of cytoplasmic maturation of oocytes, was disturbed following BFA exposure. We finally validated that the localization of ovastacin, a component of CGs that is essential for the postfertilization removal of sperm-binding sites in the zona pellucida, was also perturbed in BFA-exposed oocytes, which might weaken their fertilization capacity. Collectively, these findings indicate that Golgi-mediated protein transport is indispensable for the porcine oocyte meiotic maturation.
Asunto(s)
Brefeldino A/farmacología , Meiosis/efectos de los fármacos , Oocitos/efectos de los fármacos , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Cromosomas/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Femenino , Fertilización/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitosis/efectos de los fármacos , Oocitos/metabolismo , Oogénesis/efectos de los fármacos , Porcinos , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.
Asunto(s)
Oocitos/metabolismo , Interacciones Espermatozoide-Óvulo , Zinc/metabolismo , Animales , Calcio/metabolismo , Bovinos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , Oocitos/fisiología , Espectrometría por Rayos X/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Zona Pelúcida/efectos de los fármacosRESUMEN
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.
Asunto(s)
Polisacáridos/farmacología , Interacciones Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zinc/metabolismo , Glicoproteínas de la Zona Pelúcida/metabolismo , Zona Pelúcida/metabolismo , Animales , Comunicación Celular , Exocitosis , Femenino , Fertilización , Masculino , Ratones , Ratones Transgénicos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , Glicoproteínas de la Zona Pelúcida/genéticaRESUMEN
Reproductive dysfunction associated with obesity is increasing among women of reproductive age, including infertility and increasing risk of miscarriage. In females, reproductive disorders are linked to declining quality of oocytes. Using a model of diet-induced obesity, we have investigated the possible effects of obesity on oocyte quality, including metabolism, lipid accumulation, ROS levels, meiosis and changes in spindle structure in Metaphase II. Our study showed that obesity induced by a high fat diet can impair oocyte meiosis, destroy spindle assembly, and promote oxidative stress and abnormal mitochondrial distribution. With the addition of resveratrol, the negative impact of diet-induced obesity on the quality of oocytes was alleviated to some extent. In addition, we found that obesity causes mouse oocytes to soften, and resveratrol can restore the zona pellucida of oocytes to the same state as the control group. In conclusion, resveratrol can reverse the adverse effects of obesity on oocytes, which is beneficial for subsequent embryonic development.
Asunto(s)
Obesidad/tratamiento farmacológico , Oocitos/efectos de los fármacos , Estilbenos/administración & dosificación , Zona Pelúcida/efectos de los fármacos , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Humanos , Meiosis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Obesidad/fisiopatología , Oocitos/citología , Oocitos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Resveratrol , Zona Pelúcida/metabolismoRESUMEN
OBJECTIVE: To compare the influences of controlled ovarian hyperstimulation (COH) drugs using recombinant follicular stimulating hormone (rFSH) versus human menopausal gonadotropins (hMG) on morphometry and morphology of MII oocytes in ICSI cycles. MATERIALS AND METHODS: In this prospective study, 363 MII oocytes from 50 ICSI cycles with male factor infertility were evaluated. The patients were divided into two groups according to the protocols of COH: I- rFSH and II- hMG. The immature oocytes were excluded from the study. All oocytes were categorized into four morphological groups of normal, and those with single, double, or multiple defects. The inclusive morphometrical criteria were: areas and diameters of oocyte, ooplasm, and zona pellucida (ZP). Also, circumferences of oocyte and ooplasm were assessed. RESULTS: The ZP area and ooplasm diameter for both normal and abnormal oocytes were significantly higher in group I (P: .05; P: .028, respectively) compared to group II (P: .023; P: .003, respectively). In abnormal oocytes, ooplasm diameter was higher in group I compared to group II. Furthermore, ooplasm area for abnormal oocytes was significantly higher in group I compared to group II. There was an increasing trend for number of mature oocytes, in abnormal oocytes, for group I (5.53 ± 3.1) in comparison with group II (4.4 ± 2.97; P = .25). The rate of oocytes with normal morphology was significantly higher in hMG, when compared to rFSH groups. CONCLUSION: Morphometrical parameters were increased in rFSH group, but the normal morphology of oocytes were significantly enhanced in hMG group. Treatment with proper dosage of ovulation induction drugs may enhance the number of normal sized oocytes.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Menotropinas/farmacología , Oocitos/efectos de los fármacos , Oocitos/ultraestructura , Inducción de la Ovulación/métodos , Inyecciones de Esperma Intracitoplasmáticas , Tamaño de la Célula , Femenino , Hormona Folículo Estimulante/administración & dosificación , Humanos , Infertilidad Masculina/terapia , Masculino , Menotropinas/administración & dosificación , Estudios Prospectivos , Proteínas Recombinantes , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/ultraestructuraRESUMEN
Complex N-glycans of glycoproteins of the zona pellucida (ZP) of human oocytes have been implicated in the binding of spermatozoa. The termini of these unusual bi-, tri-, and tetra-antennary N-glycans consist of the tetrasaccharide sialyl-Lewisx (SLex ), which was previously identified as the minimal epitope for sperm binding. We describe here the chemoenzymatic synthesis of highly complex triantennary N-glycans derived from ZP carrying SLex moieties at the C-2 and C-2' arm and a sialyl-Lewisx -Lewisx (SLex -Lex ) residue at the C-6 antenna and two closely related analogues. The compounds were examined for their ability to inhibit the interaction of human sperm to ZP. It was found that the SLex -Lex moiety is critical for inhibitory activity, whereas the other SLex moieties exerted minimal effect. Further studies with SLex -Lex and SLex showed that the extended structure is the more potent inhibitor. In addition, trivalent SLex -Lex and SLex were prepared which showed greater inhibitory activity compared to their monovalent counterparts. Our studies show that although SLex can inhibit the binding of spermatozoa, presenting this epitope in the context of a complex N-glycan results in a loss of inhibitory potential, and in this context only SLex -Lex can make productive interactions. It is not the multivalent display of SLex on a multi-antennary glycan but the presentation of multiple SLex -Lex on the various glycosylation sites of ZP that accounts for high avidity binding.
Asunto(s)
Enzimas/metabolismo , Oocitos/metabolismo , Polisacáridos/síntesis química , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Antígeno CA-19-9 , Catálisis , Femenino , Glicosilación , Humanos , Masculino , Oligosacáridos/química , Polisacáridos/química , Polisacáridos/farmacología , Unión Proteica , Antígeno Sialil Lewis X , Espermatozoides/metabolismo , Estereoisomerismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
BACKGROUND: Retinoic acid (RA) signaling has been identified as a key driver in male and female gamete development. The presence of RA is a critical step in the initiation of meiosis and is required for the production of competent oocytes from primordial germ cells. Meiosis has been identified as a difficult biological process to recapitulate in vitro, when differentiating stem cells to germ cells. We have previously shown that primordial germ cell-like cells, and more advanced oocyte-like cells (OLCs), can be formed by differentiating mouse skin-derived stem cells. However, the OLCs remain unable to function due to what appears to be failure of meiotic initiation. The aim of this study was to determine the effect of RA treatment, during stem cell differentiation to germ cells, particularly on the initiation of meiosis. RESULTS: Using qPCR we found significant increases in the meiosis markers Stra8 and Sycp3 and a significant reduction in the meiosis inhibitor Nanos2, in the differentiating populations. Furthermore, OLCs from the RA treated group, expressed significantly more of the meiosis regulatory gene Marf1 and the oocyte marker Oct4. At the protein level RA treatment was found to increase the expression of the gap junction protein CX43 and the pluripotency marker OCT4. Moreover, the expression of SYCP3 was significantly upregulated and the localization pattern better matched that of control fetal ovarian cells. RA treatment also improved the structural integrity of the OLCs produced by initiating the expression of all three zona pellucida transcripts (Zp1-3) and improving ZP3 expression levels and localization. Finally, the addition of RA during differentiation led to an almost two-fold increase in the number of OLCs recovered and increased their in vitro growth. CONCLUSION: RA is a key driver in the formation of functioning gametes and its addition during stem cell to germ cell differentiation improves OLCs entry into meiosis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Piel/citología , Células Madre/citología , Células Madre/efectos de los fármacos , Tretinoina/farmacología , Animales , Biomarcadores , Conexina 43/genética , Conexina 43/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Germinativas/metabolismo , Meiosis/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Transporte de Proteínas , Células Madre/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Camélidos del Nuevo Mundo/fisiología , Motilidad Espermática/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , Animales , Bovinos , Medios de Cultivo , Femenino , Sangre Fetal , Masculino , Suero , Capacitación Espermática/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , EspermatozoidesRESUMEN
The purpose of this study was to determine the incidence of oocytes with severe ovoid zona pellucida (ZP), investigate the development potential of their sibling oocytes and the clinical outcomes from affected cycles. The data were collected from our medical records. Cycles having at least one oocyte with severe ovoid ZP were defined as the 'severe ovoid group', cycles having at least one oocyte with mild ovoid ZP were defined as the 'mild ovoid group', whereas cycles without oocytes with ovoid ZPs were defined as the 'control group' (n = 150 for each group). The results showed that sibling embryos in the 'severe ovoid group' were characterized by delayed development and lower available embryo rate. The implantation, clinical pregnancy and live birth rates in this group were also significantly lower than that in the other two groups. There were five cycles in which only one embryo with severe ovoid ZP was transferred and two healthy babies were born. The mild ovoid group showed comparable embryo development and clinical outcomes compared with the control group. This study suggests that cycles containing oocytes with severe ovoid ZPs had delayed embryo development, lower available embryo rate, compromised implantation, clinical pregnancy and live birth rates.
Asunto(s)
Ectogénesis/efectos de los fármacos , Fármacos para la Fertilidad Femenina/efectos adversos , Fertilización In Vitro , Infertilidad Femenina/terapia , Recuperación del Oocito/efectos adversos , Inducción de la Ovulación/efectos adversos , Zona Pelúcida/efectos de los fármacos , Adulto , Tasa de Natalidad , China/epidemiología , Estudios de Cohortes , Composición Familiar , Femenino , Hospitales Especializados , Humanos , Infertilidad Femenina/patología , Infertilidad Femenina/fisiopatología , Infertilidad Masculina , Masculino , Registros Médicos , Embarazo , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Inyecciones de Esperma Intracitoplasmáticas , Zona Pelúcida/patologíaRESUMEN
Polo-like kinaseâ 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Oocitos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirroles/farmacología , Animales , Compuestos de Azabiciclo/farmacología , Permeabilidad de la Membrana Celular , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Oligopéptidos/farmacología , Organofosfatos/síntesis química , Organofosfatos/farmacología , Dominios Proteicos , Pirroles/síntesis química , Pirroles/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/fisiología , Porcinos , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/fisiología , Quinasa Tipo Polo 1RESUMEN
STUDY QUESTION: What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes? SUMMARY ANSWER: Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno. WHAT IS KNOWN ALREADY: Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice. STUDY DESIGN, SIZE, DURATION: Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the actin cap which promotes a cortical granule (CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.
Asunto(s)
Fertilización/efectos de los fármacos , Melatonina/farmacología , Metaloproteasas/metabolismo , Oocitos/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Animales , Femenino , Fertilización/fisiología , Masculino , Ratones , Oocitos/metabolismo , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
STUDY QUESTION: Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? SUMMARY ANSWER: Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. WHAT IS KNOWN ALREADY: Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. STUDY DESIGN, SIZE, DURATION: We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). LIMITATIONS, REASONS FOR CAUTION: Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity. LARGE SCALE DATA: None. STUDY FUNDING/COMPETING INTERESTS: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture.