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Purpose: In humans, catecholamines (including dopamine) have been identified in semen and fallopian tubes, while dopamine D2 receptors (D2DR) are found in the sperm midpiece region. How dopamine dose affects human sperm function and whether dopamine treatment is useful in assisted reproductive technology is unclear. Methods: Sperm samples were obtained from patients with normal semen parameters undergoing fertility treatment. We investigated the effects of dopamine treatment on tyrosine phosphorylation and sperm motility. Sperm motility was analyzed using the computer-assisted sperm analysis (CASA) system. Results: This study revealed that various dopamine concentrations (0.1-100 µM) did not increase sperm tyrosine phosphorylation. Progressive motility increased substantially when treated with high concentrations of dopamine (10 and 100 µM) and was blocked by raclopride (a D2DR antagonist). After 24-h sperm culture, the addition of 10 µM dopamine significantly increased curvilinear velocity and amplitude of lateral head displacement, which are indicators of hyperactivation. Conclusion: Dopamine did not affect tyrosine phosphorylation, but increased sperm motility. High concentrations of dopamine were more effective to accelerate sperm motility in cases where sperm motile capacity was low.
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The structure of the brain is dramatically altered during the critical period. Physiological substances (neurotransmitters, hormones, etc.) in the body fluctuate significantly before and after sexual maturation. Therefore, the effect of chemical exposure on the central nervous system often differs depending on the developmental stage and sex. We aimed to compare the behavioural effects that emerged from the administration of chemicals to mice of different life stages (immature or mature) and different sex (male or female). We administered mice with domoic acid (DA), a marine poison, and ibotenic acid (IA), found in poisonous mushrooms. These excitatory amino acids act as agonists for glutamate and are potent neurotoxins. Interestingly, the behavioural effects of these chemicals were completely different. Following DA administration, we observed memory deficits only in groups of male mice treated at maturity. Following IA administration, we observed deviations in emotional behaviour in groups of male mice treated at both immaturity and maturity. In contrast, few characteristic changes were detected in all groups of females. Our results support the theory that the behavioural effects of chemical administration vary considerably with developmental stages and sex. In conclusion, our findings promote better understanding of individual differences in excitatory chemical-induced neurotoxicity and provide evidence for future risk strategies and treatments.
Assuntos
Comportamento Animal/efeitos dos fármacos , Ácido Ibotênico/toxicidade , Ácido Caínico/análogos & derivados , Administração Oral , Animais , Comportamento Animal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Ácido Ibotênico/administração & dosagem , Ácido Caínico/administração & dosagem , Ácido Caínico/toxicidade , Masculino , Toxinas Marinhas/administração & dosagem , Toxinas Marinhas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neurotoxinas/administração & dosagem , Neurotoxinas/toxicidade , Fatores Sexuais , Maturidade Sexual/fisiologiaRESUMO
The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.
Assuntos
Acetiltransferases/metabolismo , Genitália Masculina/metabolismo , Proteínas dos Microtúbulos/metabolismo , Animais , Epididimo/metabolismo , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Testículo/metabolismo , Distribuição TecidualRESUMO
Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 µm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 µm) as compared to those from the control group (178.9 ± 9.0 µm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.
Assuntos
Blastocisto/metabolismo , Proteína 9 Associada à CRISPR/genética , Desenvolvimento Embrionário/fisiologia , Exodesoxirribonucleases/genética , Edição de Genes/métodos , Mosaicismo , Fosfoproteínas/genética , Zigoto/metabolismo , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Mutação , SuínosRESUMO
In mammals, ejaculated sperm acquire their fertilizing ability during migration through the female reproductive tract, which secretes several factors that contribute to sperm capacitation. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter in the central nervous system, but additionally enhances the sperm acrosome reaction in the rat and cow. However, the detailed effects of GABA concentration on sperm function remain unclear. In this study, we detected the presence of the GABA type A receptor (GABA A) in mouse epididymal sperm by western blot analysis and in the sperm acrosome by immunocytochemistry. We also investigated the effects of GABA on sperm fertilizing ability. We found that GABA facilitated the tyrosine phosphorylation of sperm proteins, which is an index of sperm capacitation. GABA also promoted the acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. We then found that the effective GABA concentration for the acrosome reaction corresponds to sperm concentration, but we did not detect any marked effect of GABA on sperm motility using a computer-assisted sperm analysis system. Using immunohistochemistry, we also detected GABA expression in the epithelia of the mouse uterus and oviduct. Furthermore, we found that the mRNA levels of glutamate decarboxylase (Gad), which generates GABA from L-glutamate, were higher in the oviduct than in the uterus, and that Gad mRNA levels were higher at estrus than at the diestrus stage. These results indicate that the GABA concentration can act as a modulator of the acrosome reaction and sperm capacitation in the female reproductive tract.
Assuntos
Capacitação Espermática/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia , Reação Acrossômica/efeitos dos fármacos , Animais , Feminino , Fertilização/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise do Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
Previously, we reported that neurotensin (NT), which is expressed in the uterus and oviduct, enhanced bovine sperm capacitation and acrosome reactions. As NT mRNA expression in bovine oviducts increases dramatically in the follicular phase, we hypothesized that NT modulates fertilization and subsequent conception in cattle. The objective of this study was to evaluate the effect of NT on embryo development and blastocyst quality. The rate of embryo cleavage was significantly increased by the addition of NT to the fertilization medium. Furthermore, the total number of cells and numbers of cells in the inner cell mass of blastocysts were significantly increased by NT during in vitro fertilization (IVF). These results suggested that NT enhanced the efficiency of early bovine embryo development and blastocyst quality. The expression of NT receptors (NTRs) in sperm, testes, oocytes, and cumulus cells was evaluated to determine whether NT acted via NTRs in sperm alone or in both male and female reproductive cells during IVF. Immunocytochemistry and reverse transcription polymerase chain reaction revealed that NTR1 and NTR2 were expressed in sperm and testes, but not in oocytes and cumulus cells. We propose that NT selectively acts upon sperm via NTR1 and NTR2 during IVF to improve the cleavage rate and quality of blastocysts, which are important determinants of sperm quality for successful conception. This research supports our hypothesis that NT acts as a key modulator of fertilization and conception in cattle. Further studies are necessary to apply our findings to the industrial framework of bovine reproduction.
Assuntos
Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Fertilização in vitro , Neurotensina/farmacologia , Receptores de Neurotensina/fisiologia , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/genética , Animais , Blastocisto/fisiologia , Bovinos/embriologia , Células Cultivadas , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Fertilização/efeitos dos fármacos , Fertilização/genética , Fertilização in vitro/veterinária , Masculino , Neurotensina/metabolismo , Neurotensina/fisiologia , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/genética , Espermatozoides/fisiologiaRESUMO
PURPOSE: As disturbed mitochondrial distribution is thought to be a cause of the aging of oocytes, it was investigated whether oxidizing agents exert harmful effects on nuclear maturation and mitochondrial cluster formation in murine oocytes and whether antioxidants could rescue such harmful effects in vitro. METHODS: Oocytes were obtained from female Institute of Cancer Research mice 48 h after an intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin. The oocytes were cultured with potassium bromate, an oxidizing agent, in the presence or absence of the antioxidant, resveratrol. After 12 h, the nuclear phases and mitochondrial distribution were observed. RESULTS: Significantly decreased rates of metaphase II (MII) oocytes were observed with 750 µM and 1000 µM of potassium bromate, while a significant increase in abnormal mitochondrial clusters was induced at 500 µM, 750 µM, and 1,000 µM. The addition of 10 µM or 20 µM resveratrol improved both MII maturity and the cluster formation rates in the presence of potassium bromate. CONCLUSIONS: The addition of potassium bromate reduced MII maturity rates and induced abnormal mitochondrial cluster formation. This effect was alleviated by the antioxidant, resveratrol. The in vitro model used herein is useful for investigating the functions of antioxidants in the aging of oocytes.
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Sperm sorting by flow cytometry is a useful technology in the bovine industry, but the conception rates after artificial insemination using sex-sorted sperm are lower than when using the un-sorted sperm. In this study, we have investigated the causes for these low conception rates. We have focused on changes caused by flow cytometry to the glycocalyx, which forms the outermost surface of the sperm membrane. We have also evaluated the effects of capacitation on the glycocalyx since capacitation involves a redistribution of the sperm membrane that is vital for successful fertilization and conception. Lectin histochemistry was used to visualize the structure of the sperm glycocalyx. Lectin-staining sites were examined in non-treated sperm, sex-sorted sperm, and capacitated sperm. We have detected six different staining patterns related to different labeling regions of the sperm. Phaseolus vulgaris-erythroagglutinin (PHA-E) lectin-staining patterns of non-treated sperm were very different from those observed for sex-sorted sperm or capacitated sperm, suggesting that both, sex sorting by flow cytometry and the capacitation process affected the glycocalyx structures in the sperm. In addition, the total tyrosine-phosphorylation level in sex-sorted sperm was significantly higher than that in the non-treated sperm. Therefore, we concluded that the unexpected capacitation of bovine sperm during flow cytometry is associated with changes in the glycocalyx. Since premature capacitation leads to low conception rates, this unexpected capacitation could be a cause of low conception rates after artificial insemination using sex-sorted sperm.
Assuntos
Citometria de Fluxo , Congelamento , Glicocálix/química , Capacitação Espermática/fisiologia , Espermatozoides/citologia , Espermatozoides/ultraestrutura , Animais , Bovinos , Citometria de Fluxo/métodos , Glicocálix/metabolismo , Glicocálix/ultraestrutura , Lectinas/metabolismo , Masculino , Análise do Sêmen , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Pré-Seleção do Sexo/métodos , Espermatozoides/química , Espermatozoides/metabolismo , Distribuição TecidualRESUMO
Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice. In this study, we confirmed the expression of the NT receptor (NTR1) in the bovine sperm neck region and the secretion of NT in the bovine uterus and oviduct. The similar expression patterns of NT and NTR1 suggests a conserved mechanism of sperm functional regulation between mouse and cattle. Thus, we examined the effects of exogenous NT on the bovine sperm functions. First, we showed that NT induced sperm protein tyrosine phosphorylation in a dose-dependent manner, suggesting that NT enhances sperm capacitation. Second, we showed that NT induced acrosome reactions of capacitated sperm in a dose-dependent manner, suggesting that NT facilitates acrosome reaction. Finally, we used a computer-aided sperm analysis system to show that NT did not have a great effect on sperm motility. These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tracts in cattle, highlighting the importance of NT-mediated signaling to regulate sperm functions.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Neurotensina/farmacologia , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/fisiologia , Animais , Bovinos , Relação Dose-Resposta a Droga , Feminino , Masculino , Neurotensina/metabolismo , Oviductos/metabolismo , Fosforilação , Receptores de Neurotensina/metabolismo , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Útero/metabolismoRESUMO
Many studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus-oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.
Assuntos
Conexina 43/metabolismo , Células do Cúmulo/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/metabolismo , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Imuno-Histoquímica , Suínos , Fatores de TempoRESUMO
Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 µg/ml) for 18 h. At concentrations of 50 and 100 µg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 µg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 µg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 µg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.
Assuntos
Compostos Benzidrílicos/farmacologia , Oócitos/efeitos dos fármacos , Fenóis/farmacologia , Corpos Polares/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Disruptores Endócrinos/farmacologia , Estrogênios não Esteroides/farmacologia , Feminino , Proteínas Mad2/metabolismo , Camundongos Endogâmicos ICR , Microscopia Confocal , Oócitos/citologia , Oócitos/metabolismo , Corpos Polares/metabolismo , Fuso Acromático/metabolismo , Fatores de Tempo , Tubulina (Proteína)/metabolismoRESUMO
During mammalian spermatogenesis, spermatogenic cells undergo mitotic division and are subsequently divided into haploid spermatids by meiotic division, but the dynamics of sex chromosomes during spermatogenesis are unclear in vivo. To gain insight into the distribution of sex chromosomes in the testis, we examined the localization of sex chromosomes before and after meiosis in mouse testis sections. Here, we developed a method of fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes to obtain their positional information in histological testis sections. FISH analysis revealed the sex chromosomal position during spermatogenesis in each stage of seminiferous epithelia and in each spermatogenic cell. In the spermatogonia and leptotene spermatocytes, sex chromosomes were distantly positioned in the cell. In the zygotene and pachytene spermatocytes at prophase I, X and Y chromosomes had a random distribution. After meiosis, the X and Y spermatids were random in every seminiferous epithelium. We also detected aneuploidy of sex chromosomes in spermatogenic cells using our developed FISH analysis. Our results provide further insight into the distribution of sex chromosomes during spermatogenesis, which could help to elucidate a specific difference between X and Y spermatids and sex chromosome-specific behavior.
Assuntos
Posicionamento Cromossômico , Meiose , Epitélio Seminífero/metabolismo , Espermatogênese , Cromossomo X/metabolismo , Cromossomo Y/metabolismo , Aneuploidia , Animais , Endopeptidase K/metabolismo , Hibridização in Situ Fluorescente , Masculino , Prófase Meiótica I , Camundongos Endogâmicos C57BL , Microscopia Confocal , Estágio Paquíteno , Proteólise , Epitélio Seminífero/citologia , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/citologia , Testículo/metabolismoRESUMO
Neurotensin (NT) has multiple functions, ranging from acting as a neurotransmitter to regulating intestinal movement. However, its function in reproductive physiology is unknown. Here, we confirmed the expression and localization of NT receptors (NTR1) in mouse epididymal spermatozoa and investigated the effect of NT on sperm function. Sperm protein tyrosine phosphorylation, one of the indices of sperm capacitation, was facilitated dose-dependently by NT administration. In addition, the acrosome reaction was promoted in capacitated spermatozoa, and addition of a selective antagonist of NTR1 and NTR2 blocked the induction. Furthermore, intracellular calcium mobilization by NT addition was observed. This showed that NT was an accelerator of sperm function via its functional receptors. The presence of NT was confirmed by immunohistochemistry and its localization was observed in epithelia of the uterus and oviduct isthmus and ampulla, which correspond to the fertilization route of spermatozoa. The NT mRNA level in ovulated cumulus cell was remarkably increased by treatment with human chorionic gonadotropin (hCG). Using an in vitro maturation model, we analyzed the effects of FSH, epidermal growth factor (EGF), estradiol, and progesterone in NT production in cumulus cells. We found that FSH and EGF upregulated NT release and mRNA expression. Both FSH- and EGF-induced upregulation were inhibited by U0126, an MAPK kinase inhibitor, indicating that FSH and EGF regulate NT expression via a MAPK-dependent pathway. This evidence suggests that NT can act as a promoter of sperm capacitation and the acrosome reaction in the female reproductive tract.
Assuntos
Reação Acrossômica/fisiologia , Neurotensina/farmacologia , Receptores de Neurotensina/metabolismo , Capacitação Espermática/efeitos dos fármacos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Tubas Uterinas/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Neurotensina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Neurotensina/genética , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Útero/metabolismoRESUMO
The core histone is composed of four proteins (H2A, H2B, H3 and H4). Investigation of the modification patterns of histones is critical to understanding their roles in biological processes. Although histone modification is observed in multiple cells and tissues, little is known about its function in spermatogenesis. We focused on the modification patterns of histone H4 during murine spermatogenesis. We demonstrated that the individual N-terminal sites of H4 show different modification patterns during the differentiation of male germ cells. The methylation pattern varied depending on the residues that were mono-, di-, or tri-methylated. All the H4 modifications were high during the meiotic prophase, suggesting that histone H4 modification plays an important role during this stage of spermatogenesis. Elongating spermatids showed increased acetylation of histone H4, which may be associated with a histone-to-protamine substitution. Our results provide further insight into the specific relationship between histone H4 modification and gene expression during spermatogenesis, which could help to elucidate the epigenetic disorders underlying male infertility.
Assuntos
Histonas/metabolismo , Espermatogênese , Acetilação , Animais , Arginina/metabolismo , Histonas/química , Imuno-Histoquímica , Lisina/metabolismo , Masculino , Meiose , Metilação , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Microtubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus-oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.
Assuntos
Western Blotting/métodos , Técnicas de Maturação in Vitro de Oócitos/métodos , Proteínas Associadas aos Microtúbulos/análise , Oócitos/fisiologia , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo , Feminino , Leucina/análogos & derivados , Leucina/farmacologia , Oócitos/efeitos dos fármacos , Pepstatinas/farmacologia , SuínosRESUMO
Mammalian target of rapamycin (mTOR), a Ser/Thr protein kinase, is the catalytic component of two distinct signaling complexes, mTOR-raptor complex (mTORC1) and mTOR-rictor complex (mTORC2). Recently, studies have demonstrated mitosis-specific roles for mTORC1, but the functions and expression dynamics of mTOR complexes during meiotic maturation remain unclear. In the present study, to evaluate the roles of respective mTOR complexes in maternal meiosis and compare them with those in mitosis, we sought to elucidate the spatiotemporal immunolocalization of mTOR, the kinase-active Ser2448- and Ser2481-phosphorylated mTOR, and raptor and rictor during cumulus-cell mitosis and oocyte meiotic maturation in mice. mTOR principally accumulated around the chromosomes and on the spindle. Phosphorylated mTOR (Ser2448 and Ser2481) exhibited elevated fluorescence intensities in the cytoplasm and punctate localization adjacent to the chromosomes, on the spindle poles, and on the midbody during mitotic and meiotic maturation, suggesting functional homology of mTOR between the two cell division systems, despite their mechanistically distinctive spindles. Raptor colocalized with mTOR during both types of cell division, indicating that mTORC1 is predominantly associated with these events. Mitotic rictor uniformly distributed through the cytoplasm, and meiotic rictor localized around the spindle poles of metaphase-I oocytes, suggesting functional divergence of mTORC2 between mitosis and female meiosis. Based on the general function of mTORC2 in the organization of the actin cytoskeleton, we propose that mTORC1 controls spindle function during mitosis and meiosis, while mTORC2 contributes to actin-dependent asymmetric division during meiotic maturation in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Células do Cúmulo/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Cromossomos de Mamíferos/metabolismo , Células do Cúmulo/citologia , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos ICR , Complexos Multiproteicos/metabolismo , Oócitos/citologia , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Fuso Acromático/metabolismoRESUMO
The widely used porcine artificial insemination procedure involves the use of liquid-stored semen because it is difficult to control the quality of frozen-thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma-aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABAA receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABAA receptor antagonist. GABA binds to GABAA receptors, resulting in chloride ion influx. We found that treatment with 1 µM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function.
Assuntos
Reação Acrossômica , Motilidade dos Espermatozoides , Acrossomo/fisiologia , Animais , Cloretos/farmacologia , Masculino , Camundongos , Espermatozoides/fisiologia , Suínos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.
Assuntos
Adrenomedulina/metabolismo , Células do Cúmulo/metabolismo , Oócitos/metabolismo , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/farmacologia , Animais , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Oócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Adrenomedulina/genéticaRESUMO
The nicotinic acetylcholine receptor (nAChR) is one of the receptors of acetylcholine (ACh), and nicotine (NIC) acts as an agonist of this receptor. Among the nAChR subunits, we found that the ε subunit (AChRe) had approximately 10 to 1000 times higher level of mRNA expression in mouse testes than the other subunits. In this study, we aimed to elucidate the expression and localization of AChRe in the testes and spermatozoa of mice and clarify the effect of AChRe on sperm function. Immunocytochemistry showed that AChRe was expressed in the murine testes and spermatozoa. We found that AChRe was localized only in elongated spermatids from step 12 onwards in the testes. In spermatozoa, AChRe was localized in the head, especially in the anterior region of the acrosome, but only approximately 50% of spermatozoa showed this immunoreactivity. Additionally, we analyzed the effects of ACh and NIC on sperm acrosome reaction (AR) and found that both ACh and NIC suppressed the AR rate, which was restored by an AChRe-specific antagonist. These results suggest that AChRe may be a regulator of mammalian sperm AR.
RESUMO
Sperm migration towards an oocyte in the female reproductive tract is an important step for successful fertilization. Although several sperm-chemotactic factors have been identified in mammals, it is unclear whether these chemoattractants contribute to sperm migration towards an oocyte that is the final destination for sperm. Furthermore, chemoattractants for bovine sperm are still undiscovered even though the follicular fluid attracts sperm in cattle. Here, we demonstrated that a single bovine cumulus-oocyte complex (COC) had the ability to attract sperm, suggesting that the COC secreted sperm chemoattractants. We identified stromal cell-derived factor 1 (SDF1), which was expressed in COCs, and its receptor CXCR4 in sperm, as a candidate. Our results showed that bovine sperm preferentially migrated to the area with a high SDF1 concentration and occasionally showed turn movements by asymmetric flagellar bends during the migration. We also demonstrated that increasing the intracellular Ca2+ concentration via Ca2+ channels was related to SDF1-induced sperm chemotaxis. Finally, a CXCR4 inhibitor significantly suppressed the in vitro bovine sperm migration towards a COC. Taken together, we propose that SDF1 is a chemotactic factor for bovine sperm to regulate their migration towards an oocyte via the CXCR4 receptor.