RESUMO
How does a regime change influence elite mobility? By collecting data on elites after the Meiji Restoration in Japan in 1868, through which Japan transitioned from a feudal regime to a modern regime, we provide new evidence that the impact of the regime change on elite mobility varies across the stages of the regime change. We analyze the impact of the regime change from two aspects: (1) the composition of elites or elite membership and (2) the internal hierarchy within them. The regime change opened an opportunity for commoners to join the elite group. After the Meiji Restoration, the share of elites whose fathers were commoners in the former regime increased, as did the influence of meritocracy on elite ranks. However, once the new regime was established, the elite hierarchy started to reflect the social stratum of the former regime and the influence of meritocracy declined.
Assuntos
Sistemas Políticos , Status Social , Humanos , Japão , Sistemas Políticos/história , História do Século XIXRESUMO
During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.
Assuntos
Comunicação Celular/fisiologia , Células do Cúmulo/metabolismo , Dinoprostona/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/metabolismo , Animais , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Técnicas In Vitro , Modelos Animais , Nitrobenzenos/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores do FSH/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , SuínosRESUMO
BACKGROUND: Bacterial infections of the genital tract are one of the most serious causes of infertility in males. In some human patients with poor semen quality, leukocytospermia has been observed. Because leukocytes express the bacterial-lipopolysaccharide (LPS) responsive Toll-like receptor (TLR) signaling cascade and secrete tumor necrosis factor-α, secreted cytokines comprise one, but probably not the only, class of factors that can impact sperm motility. METHODS AND RESULTS: In this study, we documented that bacterial endotoxins, LPS and peptidoglycan, can be detected in human semen. Furthermore, the addition of endotoxins in the absence of leukocytes directly and significantly reduced the motility and increased the apoptotic rate of both human and mouse sperm and suppressed fertilization by mouse sperm both in vivo and in vitro. The well-known LPS receptor, TLR4, and peptidoglycan receptor, TLR2, were expressed in human and mouse sperm. In Tlr2/4(-/-) double-mutant mice, the negative effects of endotoxins on sperm functions were blocked, suggesting that the bacterial endotoxins mediated activation of TLR-dependent pathways in sperm leading to apoptosis. CONCLUSIONS: Sperm can recognize bacterial endotoxins by TLRs present in their membranes. The activated TLRs reduce sperm motility, induce sperm apoptosis and significantly impair the potential for fertilization.
Assuntos
Apoptose , Endotoxinas/metabolismo , Doenças Urogenitais Masculinas/microbiologia , Espermatozoides/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Animais , Citocinas/biossíntese , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Peptidoglicano/metabolismo , Transdução de Sinais , Espermatozoides/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Sperm motility patterns are continuously changed after ejaculation to fertilization in the female tract. Hyperactivated motility is induced with high glucose medium in vitro or the oviduct fluids in vivo, whereas sperm maintain linear motility in the seminal plasma or the uterine fluids containing low glucose. Therefore, it is estimated that sperm motility patterns are dependent on the energy sources, and the mitochondrial oxidative phosphorylation is activated to produce ATP in low glucose condition. To elucidate these hypotheses, boar sperm was incubated in different energy conditions with the transcription and translation inhibitors in vitro. Sperm motility parameters, mitochondrial activity, ATP level, gene expression and protein synthesis were analyzed. Sperm progressive motility and straight-line velocity were significantly increased with decreasing glucose level in the incubation medium. Moreover, the mitochondrial protein turnover meaning transcription and translation from mitochondrial genome in sperm is activated during incubation. Incubation of sperm with mitochondrial translation inhibitor (D-chloramphenicol) suppressed mitochondrial protein synthesis, mitochondrial activity and ATP level in sperm and consequently reduced the linear motility speed, but not the motility. Thus, it is revealed that the mitochondrial central dogma is active in sperm, and the high-speed linear motility is induced in low glucose condition via activating the mitochondrial activity for ATP generation.
RESUMO
In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.
Assuntos
Células do Cúmulo/metabolismo , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Oogênese/genética , Suínos/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Western Blotting/métodos , Técnicas de Cultura de Células , Gonadotropina Coriônica/farmacologia , Células do Cúmulo/efeitos dos fármacos , Primers do DNA/genética , Relação Dose-Resposta a Droga , Estradiol/análise , Estradiol/farmacologia , Feminino , Líquido Folicular/química , Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/farmacologia , Células da Granulosa/metabolismo , Oogênese/efeitos dos fármacos , Progesterona/análise , Progesterona/farmacologia , RNA Mensageiro/análise , Receptores do FSH/genética , Receptores do LH/genética , Receptores de Progesterona , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superovulação , Testosterona/análiseRESUMO
During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.
Assuntos
Quimiocinas/metabolismo , Exocitose/genética , Regulação da Expressão Gênica , Ovulação/genética , Proteína 25 Associada a Sinaptossoma/genética , Animais , Gonadotropina Coriônica/farmacologia , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Camundongos , Camundongos Mutantes , Ovulação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteína 25 Associada a Sinaptossoma/antagonistas & inibidoresRESUMO
The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.
Assuntos
Meiose/fisiologia , Oócitos/citologia , Isoformas de Proteínas/fisiologia , Receptores de Progesterona/fisiologia , Animais , Sequência de Bases , DNA Complementar , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica , Hormônio Luteinizante/farmacologia , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Isoformas de Proteínas/genética , Receptores de Progesterona/genética , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
It has been reported that LH receptor (LHR) mRNA is not detected in cumulus cells of porcine cumulus-oocyte complexes (COCs) just after collection from small antral follicles. The present study showed that the formation of LHR in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. When the newly synthesized receptors were stimulated by 1.0 microg/ml LH, significantly higher levels of cAMP and progesterone production in cumulus cells were observed as compared with those of COCs cultured with FSH. A loss of proliferative activity of cumulus cells was induced by the additional LH to FSH-containing medium; however, the inhibitory effect was overcome by progesterone receptor antagonist RU486. Furthermore, the addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. These results revealed that during in vitro meiotic maturation of porcine COCs, progesterone secreted by FSH- and LH-stimulated cumulus cells reduced proliferative activity of cumulus cells; the changes of cumulus cells might be involved in inducing meiotic resumption of porcine oocytes.
Assuntos
Hormônio Luteinizante/farmacologia , Oócitos/citologia , Folículo Ovariano/citologia , Animais , Diferenciação Celular , Divisão Celular , AMP Cíclico , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante/farmacologia , Hormônio Luteinizante/genética , Luteolíticos/farmacologia , Meiose , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Progesterona/análise , Receptores do LH/biossíntese , Receptores do LH/genética , Suínos , Fatores de Tempo , Regulação para CimaRESUMO
Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co-injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen-thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen-thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen-thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen-thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.
Assuntos
Fertilização/efeitos dos fármacos , Inseminação Artificial/métodos , Oviductos , Ocitocina/administração & dosagem , Ocitocina/farmacologia , Transporte Espermático/efeitos dos fármacos , Suínos/fisiologia , Animais , Criopreservação , Feminino , Masculino , Preservação do Sêmen , Contagem de Espermatozoides , Estimulação Química , Contração Uterina/efeitos dos fármacos , ÚteroRESUMO
PROBLEM: The aim of this study was to find immune-related genes expressed in cumulus cells of ovulated cumulus oocyte complexes (COCs) and to clear the functional roles during fertilization process. METHOD OF STUDY: Ovulated COCs were collected from oviduct 16 hr after the hCG injections followed by eCG priming. The cumulus cells were used for RT-PCR or western blotting study. COCs were also used for in vitro fertilization study. RESULTS: Cramp, Trf, Lyz2, S100a8, and S100a9 were expressed in cumulus cells during ovulation process. The protein levels of CRAMP or transferrin were detected in ovulated COCs and then secreted into hyaluronan-rich matrix. The high dose of these factors reduced the proliferative activity of E. coli; however, the lower levels of them significantly increased the rate of fertilization in in vitro via the induction of sperm capacitation. CONCLUSION: Cumulus-secreted anti-bacterial factors act on sperm to induce sperm capacitation.
Assuntos
Células do Cúmulo/metabolismo , Fertilização in vitro , Oócitos/metabolismo , Capacitação Espermática/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Catelicidinas/genética , Catelicidinas/metabolismo , Gonadotropina Coriônica/farmacologia , Células do Cúmulo/citologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatozoides/fisiologia , Transferrina/genética , Transferrina/metabolismoRESUMO
Cryopreservation of boar spermatozoa offers an effective means of long-term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70-80%) can be achieved by AI with frozen-thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria-released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll-like receptor-4 (TLR-4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo-capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen-thawed boar sperm.
Assuntos
Criopreservação/veterinária , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Suínos/fisiologia , Animais , Criopreservação/métodos , Congelamento , Inseminação Artificial/veterinária , Masculino , Preservação do Sêmen/métodosRESUMO
Frozen-thawed epididymal spermatozoa have good fertilization capability in vitro; however, their artificial insemination conception rate is less than half of that of frozen-thawed ejaculated spermatozoa. Because the addition of seminal plasma to the thawing solution enhances the in vivo fertilizing ability of frozen-thawed ejaculated spermatozoa, we hypothesized that the reproductive performance of frozen-thawed epididymal spermatozoa could also be improved by the inclusion of seminal plasma. When frozenthawed epididymal spermatozoa were incubated for up to 6 hours, the motility of the sperm significantly decreased in a time-dependent manner. The acrosomal membrane was damaged in the majority of frozen-thawed epididymal spermatozoa. The addition of seminal plasma to the thawing solution significantly decreased the percentage of sperm with abnormal acrosomes and increased their total motility in a dose-dependent manner. Furthermore, the addition of seminal plasma reduced the abundance of a 15-kDa tyrosinephosphorylated protein in frozen-thawed sperm, and the maximum effect was observed at 15% (vol/vol) seminal plasma. When cryopreserved epididymal spermatozoa from 3 different boars were thawed with a 15% (vol/vol) seminal plasma-containing solution, the conception rate and mean litter size obtained by artificial insemination were significantly increased as compared with those in the control without seminal plasma. From these results, we concluded that the addition of seminal plasma to the thawing solution is a key step in obtaining an optimal number of piglets by artificial insemination using frozen-thawed boar epididymal spermatozoa.
Assuntos
Criopreservação/veterinária , Epididimo , Inseminação Artificial/veterinária , Reprodução , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Recuperação Espermática/veterinária , Espermatozoides/fisiologia , Acrossomo/metabolismo , Acrossomo/patologia , Animais , Ejaculação , Feminino , Fertilização , Tamanho da Ninhada de Vivíparos , Masculino , Fosforilação , Gravidez , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Suínos , Fatores de Tempo , TirosinaRESUMO
In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation-like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal-derived materials. In this study, we focused on the increase of intracellular Ca(2+) ([Ca(2+)](i)) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca(2+)](i) indicator, Fluo-3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca(2+) chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post-thawed sperm motility. When the frozen-thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca(2+)](i) and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen-thawed boar sperm.
Assuntos
Bovinos/fisiologia , Quelantes/farmacologia , Criopreservação , Ácido Egtázico/farmacologia , Preservação do Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Cálcio/análise , Ácido Edético/farmacologia , Masculino , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologiaRESUMO
Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 µg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation.
Assuntos
Criopreservação/veterinária , Lipopolissacarídeos/toxicidade , Polimixina B/farmacologia , Substâncias Protetoras/farmacologia , Espermatozoides/microbiologia , Suínos/microbiologia , Animais , Apoptose/efeitos dos fármacos , Feminino , Inseminação Artificial/veterinária , Lipopolissacarídeos/química , Masculino , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.
Assuntos
Crioprotetores , Fertilização , Glicerol , Preservação do Sêmen/métodos , Acrossomo , Animais , Membrana Celular , Criopreservação/métodos , Implantação do Embrião , Feminino , Masculino , Concentração Osmolar , Motilidade dos Espermatozoides , SuínosRESUMO
The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages, and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFkappaB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFkappaB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs) localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.
Assuntos
Células do Cúmulo/efeitos dos fármacos , Citocinas/metabolismo , Ácido Hialurônico/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Células Cultivadas , Quimiocinas/metabolismo , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Fertilização/efeitos dos fármacos , Imunofluorescência , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Espermatozoides/enzimologia , Espermatozoides/metabolismoRESUMO
To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.