RESUMO
Pig pregnancy succeeds thanks to a well-coordinated system ruling both maternal immune activation and embryonic antigen tolerance. In physiological pregnancies, the maternal immune system should tolerate the presence of hemi-allogeneic conceptuses from the pre-implantation phase to term, while maintaining maternal defence against pathogens. Allogeneic pregnancies, as after embryo transfer (ET), depict high embryo mortality during the attachment phase, calling for studies of the dynamic modifications in immune processes occurring at the maternal-foetal interface, for instance, of interferon (IFN)-stimulated genes (ISGs). These ISGs are generally activated by IFN secreted by the conceptus during the process of maternal recognition of pregnancy (MRP) and responsible for recruiting immune cells to the site of embryo attachment, thus facilitating cell-antigen presentation and angiogenesis. We performed RNA-Seq analysis in peri-implantation (days 18 and 24) endometrial samples retrieved from artificially inseminated sows (hemi-allogeneic embryos (HAL) group) or sows subjected to ET (allogeneic embryos (AL) group) to monitor alterations of gene expression that could be jeopardising early pregnancy. Our results showed that endometrial gene expression patterns related to immune responses differed between hemi- or allogeneic embryo presence, with allogeneic embryos apparently inducing conspicuous modifications of immune-related genes and pathways. A decreased expression (Pâ¯<â¯0.05; FCâ¯<â¯-2) of several interferon ISGs, such as CXCL8, CXCL10, IRF1, IRF9, STAT1, and B2M, among others was detected in the endometrium of sows carrying allogeneic embryos on day 24 of pregnancy. This severe downregulation of ISGs in allogeneic pregnancies could represent a failure of ET-embryos to signal IFN to the endometrium to warrant the development of adequate immunotolerance mechanisms to facilitate embryo development, thus contributing to elevated embryo death.
Assuntos
Interferons , Doenças dos Suínos , Animais , Implantação do Embrião , Perda do Embrião/metabolismo , Perda do Embrião/veterinária , Transferência Embrionária/veterinária , Endométrio/metabolismo , Feminino , Gravidez , SuínosRESUMO
This study evaluated the effects of different concentrations (1, 10, 25, 50, and 100 µM) of the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG), during the culture of in vitro-fertilized porcine oocytes. While the highest concentrations of NMPG (50 and 100 µM) were toxic to the developing embryos during the first two days of culture, 25 µM NMPG achieved cleavage rates that were similar to those achieved by the control but did not sustain blastocyst production by Day 7 of culture. Compared to the control culture medium, the culture medium supplemented with 10 µM NMPG increased (P < 0.05) the rates of blastocyst formation, decreased (P < 0.05) the intracellular levels of reactive oxygen substances, and downregulated (P < 0.05) the expression of the oxidative stress related gene GPX1. In conclusion, these results demonstrated that supplementation of porcine embryo culture medium with 10 µM NMPG can attenuate oxidative stress and increase the yield of in vitro production of blastocysts.
Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Suínos/embriologia , Tiopronina/farmacologia , Animais , Meios de Cultura , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
Semen modifies the expression of genes related to immune function along the porcine female internal genital tract. Whether other pathways are induced by the deposition of spermatozoa and/or seminal plasma (SP), is yet undocumented. Here, to determine their relative impact on the uterine and tubal transcriptomes, microarray analyses were performed on the endocervix, endometrium and endosalpinx collected from pre-ovulatory sows 24 h after either mating or artificial insemination (AI) with specific ejaculate fractions containing spermatozoa or sperm-free SP. After enrichment analysis, we found an overrepresentation of genes and pathways associated with sperm transport and binding, oxidative stress and cell-to-cell recognition, such as PI3K-Akt, FoxO signaling, glycosaminoglycan biosynthesis and cAMP-related transcripts, among others. Although semen (either after mating or AI) seemed to have the highest impact along the entire genital tract, our results demonstrate that the SP itself also modifies the transcriptome. The detected modifications of the molecular profiles of the pre/peri-ovulatory endometrium and endosalpinx suggest an interplay for the survival, transport and binding of spermatozoa through, for instance the up-regulation of the Estrogen signaling pathway associated with attachment and release from the oviductal reservoir.
Assuntos
Genitália Feminina/metabolismo , Ovulação/genética , Ovulação/metabolismo , Sêmen/fisiologia , Suínos/genética , Suínos/fisiologia , Animais , Comunicação Celular/genética , Estrogênios/metabolismo , Feminino , Proteína Forkhead Box O1/metabolismo , Inseminação Artificial/veterinária , Masculino , Oviductos/metabolismo , Estresse Oxidativo/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Comportamento Sexual Animal , Transdução de Sinais/genética , Transporte Espermático/genética , TranscriptomaRESUMO
An efficient system to collect large numbers of vital zygotes is a pre-requisite for application of zygote genome-editing technology, including development of efficient models for xenotransplantation using pigs. Owing to the sub-optimal in vitro production of zygotes in pigs, efficient collection of in vivo developed zygotes is required. Timing of ovulation is a key factor to sustain efficiency since the interval between pronuclear formation and the first division is very short in pigs. The weaning-to-estrus interval can, due to its inverse relation with length of estrus and time of ovulation, interfere with ovulation and make it asynchronous, which reduces the probability of obtaining zygotes. This retrospective study compared the effects of three weaning-to-estrus intervals of 3, 4 or 5 days on zygote collection efficiency in a total of 17 trials over a 3-year period including 223 sows. Donor sows in groups of 10-15 animals were super-ovulated with eCG 24â¯h after weaning and those in estrus at 48-72â¯h post-eCG were immediately treated with hCG, followed by insemination 6 and 24â¯h thereafter. Collected structures during laparotomy on Day 2 (Day 0: onset of estrus) were morphologically evaluated and only those with a single cell and two visible polar bodies were considered as zygotes. Zygotes were injected with CRISPR-Cas9 editor mixture and cultured for 6 days to evaluate their developmental ability against non-injected control zygotes. Of all recovered structures (Nâ¯=â¯5,468), 67.4%, 30.8% and 1.8% were zygotes, 2-cell embryos and oocytes-degenerated embryos, respectively. The different weaning-to-estrus intervals did not affect either the percentages of collected zygotes (range: 64.1%-70.0%) or the percentages of sows with zygotes at collection time (range: 69.0%-73.3%). The weaning-to-estrus intervals did not affect the in vitro developmental ability of zygotes. After 24â¯h of culture, 78.1⯱â¯2.0% and 95.1⯱â¯0.6 (Pâ¯<â¯0.05) of injected (Nâ¯=â¯2,345) and non-injected (Nâ¯=â¯335) zygotes, respectively, developed to 2-to-4-cell embryo stage. The total efficiency of the system was 64.1⯱â¯2.2% and 85.8⯱â¯1.5% (Pâ¯<â¯0.05) for injected and non-injected zygotes, respectively. In conclusion, the results indicate that neither the efficiency of collecting in vivo derived porcine zygotes from superovulated sows nor the zygote ability to develop to blastocyst after cytoplasmic genome-editing injection were affected by a weaning-to-estrus interval between 3-to-5 days.
Assuntos
Gonadotropina Coriônica/farmacologia , Oócitos , Suínos/fisiologia , Animais , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Inseminação Artificial/veterinária , Estudos Retrospectivos , Superovulação/efeitos dos fármacos , Suínos/embriologia , Fatores de Tempo , Coleta de Tecidos e ÓrgãosRESUMO
The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000â¯ng/mL, 0â¯=â¯control) to NCSU-23 with 0.4â¯mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100-1000â¯ng/mL, 0â¯=â¯Control-PVA) to a BSA-free medium (NCSU-23 with 0.3â¯mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4â¯mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0⯱â¯10.9 to 70.0⯱â¯5.8%); of day 7-blastocyst rates (range: 46.6⯱â¯10.0 to 56.4⯱â¯8.2%) and of total day 7-blastocyst efficiency (range: 32.3⯱â¯8.3 to 37.2⯱â¯7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1⯱â¯23.2 to 50.5⯱â¯26.4). In Experiment 2, PF4 accelerated embryo development, increasing (Pâ¯<â¯0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500â¯ng/mL (range: 29.9⯱â¯7.8 to 31.8⯱â¯5.5%; Pâ¯<â¯0.05) compared with BSA-control (17.2⯱â¯8.2%) and PF4 1000â¯ng/mL (15.5⯱â¯7.9%); showing similar blastocyst rates (range: 42.0⯱â¯11.5 to 49.3⯱â¯10.0%), total efficiency (28.0⯱â¯8.2 to 32.3⯱â¯7.1%) total cell numbers (range: 42.6⯱â¯19.3 to 45.7⯱â¯23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions.
Assuntos
Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Fator Plaquetário 4/química , Albumina Sérica/química , Suínos/embriologia , Animais , Relação Dose-Resposta a Droga , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator Plaquetário 4/administração & dosagem , Fator Plaquetário 4/farmacologia , Albumina Sérica/administração & dosagem , Albumina Sérica/farmacologiaRESUMO
Recently numerous proteomic approaches have been undertaken to identify sperm and seminal plasma (SP) proteins that can be used as potential biomarkers for sperm function, including fertilization ability. This review aims firstly to briefly introduce the proteomic technologies and workflows that can be successfully applied for sperm and SP proteomic analysis. Secondly, we summarize the current knowledge about boar SP and the sperm proteome, focusing mainly on its relevance to sperm preservation procedures (liquid storage or cryopreservation) and their outcomes in terms of sperm function and fertility.
Assuntos
Proteômica , Preservação do Sêmen/veterinária , Sêmen/química , Espermatozoides/química , Suínos , Animais , MasculinoRESUMO
Short- and medium-term storage of pig embryos has become relevant for commercial application of non-surgical deep uterine embryo transfer (NsDU-ET) in the light of the strict legal and administrative requirements posed by the International Association for Air Transport (IATA) to allow shipment of liquid nitrogen (LN2) containers and the technical drawbacks when using vitrified embryos. Therefore, this study developed an efficient method for the liquid storage of in vivo-derived porcine blastocysts for a moderate duration (48â¯h) without controlled CO2 gassing. We evaluated two storage temperatures (25⯰C and 37⯰C) and three HEPES-supplemented media: the chemically defined media TL-PVA and NCSU-PVA and the semi-defined medium NCSU-BSA. We observed no differences in survival, hatching rate or final developmental stage between the two temperatures, but storage at 25⯰C was more efficient to preserve zona pellucida (ZP) integrity. Blastocysts were successfully stored for 24â¯h in a chemically defined medium. Yet, only 48â¯h storage in NCSU-BSA medium supported blastocyst development. Although all storage conditions resulted in an embryonic developmental delay, blastocysts stored in NCSU-BSA at either tested temperature could hatch and attain the same final developmental stage as control blastocysts when cultured under standard conditions after storage. Moreover, blastocysts stored at 25⯰C for 48â¯h in NCSU-BSA medium could produce pregnancies after surgical transfer. In conclusion, porcine blastocysts maintain their viability and developmental potential after storage in the semi-defined medium NCSU-BSA for at least 48â¯hâ¯at 25⯰C.
Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Suínos/embriologia , Animais , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Gravidez , Fatores de TempoRESUMO
In this study, the effects of addition of the antioxidant ascorbic acid (AsA) were evaluated during porcine in vitro embryo production (IVP) and vitrification. In experiment 1, the effects of AsA supplementation during IVM, IVF and IVC were evaluated, using a total of 2744 oocytes in six replicates. The IVM, IVF and embryo IVC media were supplemented or not (control) with 50 µg/mL AsA in all possible combinations. No significant effects of AsA were detected in any of the maturation, fertilization or embryo development parameters assessed. In experiment 2, we evaluated the effects of adding AsA to vitrification-warming media on the post-warming survival and quality of blastocysts. Day-6 in vitro-produced blastocysts (N = 588) from six replicates were randomly divided in two groups, with vitrification and warming media either supplemented with 50 µg/mL AsA (VW + group) or un-supplemented (VW- control). Addition of AsA increased (P < 0.05) blastocyst survival rate after vitrification compared with that of VW- control embryos. Vitrification and warming increased (P < 0.05) the production of oxygen species (ROS) and reduced (P < 0.05) the glutathione levels in blastocysts. Although VW + blastocysts displayed higher (P < 0.05) ROS levels than those of fresh control blastocysts, the levels were lower (P < 0.05) than those found in VW- control blastocysts. In conclusion, under the experimental conditions, supplementation of IVM/IVF/IVC media with AsA did not improve the embryo production in vitro. By contrast, the addition of AsA to chemically defined vitrification and warming media increased the survival of in vitro-produced porcine blastocysts by decreasing ROS production.
Assuntos
Ácido Ascórbico/farmacologia , Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Suínos/embriologia , Vitrificação/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Criopreservação/veterinária , Técnicas de Cultura Embrionária , FemininoRESUMO
Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8-iso-PGF2α and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.
Assuntos
Cavalos , Radical Hidroxila/farmacologia , Oxirredução , Estresse Oxidativo/fisiologia , Espermatozoides/patologia , Aldeídos/análise , Animais , Apoptose , Caspase 3 , Dinoprosta/análogos & derivados , Dinoprosta/análise , Compostos Ferrosos/farmacologia , Peroxidação de Lipídeos/fisiologia , Masculino , Potencial da Membrana Mitocondrial , Necrose , Motilidade dos Espermatozoides , Espermatozoides/metabolismo , Vitamina K 3/farmacologiaRESUMO
The advancement of porcine embryo transfer (ET) technology is constrained by regulatory hurdles (liquid nitrogen transportation) or, more importantly, the technical obstacles of using vitrified embryos in combination with nonsurgical deep uterine ET technology. Maintaining embryos in culture during transport and prior ET collides with the need of CO2 gassing and the best choice of culture medium. In this work, we describe storage conditions for short-term embryo CO2-free storage that allowed for a majority of in vivo-derived porcine morulae to survive after 3 days of storage in a liquid state, and to develop to the blastocyst stage unhatched, a sanitary prerequisite for ET. The storage conditions included NCSU-23 medium supplemented with bovine serum albumin, where bicarbonate was partially replaced by HEPES to avoid the need for CO2 gassing, and a temperature of 37 °C. These conditions were able to maintain the functionality of the stored embryos (hatching capacity after exposure to conventional culture conditions) and their developmental competence after ET (normal fetuses by day 38 of pregnancy). Use of this strategy of CO2-free storage should allow the shipment of fresh embryos worldwide in the absence of liquid nitrogen.
Assuntos
Transferência Embrionária/veterinária , Mórula/citologia , Suínos/fisiologia , Animais , Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/métodos , Feminino , GravidezRESUMO
In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.
Assuntos
Criopreservação/métodos , Preservação do Sêmen/efeitos adversos , Espermatozoides/patologia , Animais , Membrana Celular/patologia , Congelamento , Cavalos , Masculino , Preservação do Sêmen/métodos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Espermatozoides/metabolismoRESUMO
The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in that oil. These results indicate that a peroxidized MO overlay dramatically decreases embryo production outcomes. This decrease could be associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. It is likely that different oxidant agent(s) and/or other toxic compounds present in the peroxidized MO are responsible for its damaging effects on oocytes and embryos.
Assuntos
Meios de Cultura/química , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Óleo Mineral/química , Oxidantes/farmacologia , Suínos/embriologia , Animais , Células do Cúmulo , Técnicas de Cultura Embrionária , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Óleo Mineral/farmacologia , Oócitos/fisiologia , OxirreduçãoRESUMO
Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.
Assuntos
Citometria de Fluxo/veterinária , Análise do Sêmen/veterinária , Animais , Citometria de Fluxo/métodos , Masculino , Proteômica , Análise do Sêmen/métodos , Espermatozoides/citologiaRESUMO
The improvement in porcine embryo preservation and non-surgical embryo transfer (ET) procedures achieved in recent years represents essential progress for the practical use of ET in the pig industry. This study aimed to evaluate the effects of parity, weaning-to-estrus interval (WEI) and season on reproductive and embryonic parameters at day 6 after insemination of donor sows superovulated after weaning. The selection of donor sows was based on their reproductive history, body condition and parity. The effects of parity at weaning (2 to 3, 4 to 5 or 6 to 7 litters), season (fall, winter and spring), and WEI (estrus within 3 to 4 days), and their interactions on the number of corpus luteum, cysts in sows with cysts, number and quality of viable and transferable embryos, embryo developmental stage and recovery and fertilization rates were evaluated using linear mixed effects models. The analyses showed a lack of significant effects of parity, season, WEI or their interactions on any of the reproductive and embryonic parameters examined. In conclusion, these results demonstrate that fertilization rates and numbers of viable and transferable embryos collected at day 6 of the cycle from superovulated donor sows are not affected by their parity, regardless of the time of the year (from fall to spring) and WEI (3 or 4 days).
Assuntos
Transferência Embrionária/veterinária , Suínos/fisiologia , Animais , Estro , Feminino , Paridade , Gravidez , Fatores de Tempo , DesmameRESUMO
Recent advances in nonsurgical deep uterine (NsDU) embryo transfer (ET) technology allow the noninvasive transfer of porcine embryos into recipients, overcoming the most important impediment for commercial ET in this species. Although many factors in the porcine ET-field have been recently evaluated, many others remain to be explored. We investigated here the future reproductive performance of donors and recipients after artificial insemination subsequent to the default surgical embryo recovery approach and to the NsDU-ET procedure, respectively. Although surgical embryo collection did not influence subsequent farrowing rates (90.5%), litter size decreased severely (8.9 ± 0.8 piglets) compared to presurgery (10.8 ± 0.3 piglets) and control group (10.7 ± 0.3 piglets). In contrast, NsDU-ETs did neither affect fertility nor prolificacy of recipients in the cycle subsequent to ET, regardless of whether they were pregnant after NsDU-ET or not. These results indicate that while the surgical embryo collection procedure compromises the reproductive future of donor sows, the NsDU-ET approach does not affect the reproductive potential of the recipients after reintroduction to the breeding stock of the farm. Further research is thus needed to improve surgical embryo collection.
Assuntos
Transferência Embrionária/veterinária , Coleta de Tecidos e Órgãos/veterinária , Animais , Transferência Embrionária/métodos , Feminino , Tamanho da Ninhada de Vivíparos , Gravidez , Suínos , Coleta de Tecidos e Órgãos/métodosRESUMO
Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.
Assuntos
Aquaporinas/metabolismo , Criopreservação/veterinária , Espermatozoides/fisiologia , Estresse Fisiológico , Animais , Aquaporinas/genética , Regulação da Expressão Gênica , Masculino , Transporte ProteicoRESUMO
Putative changes in E-cadherin and ß-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and ß-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane-bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. ß-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation.
Assuntos
Caderinas/metabolismo , Cães/fisiologia , Endométrio/metabolismo , Ciclo Estral/fisiologia , Prenhez , beta Catenina/metabolismo , Animais , Implantação do Embrião/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica/veterinária , Gravidez , Prenhez/fisiologiaRESUMO
Non-viable sperm ("dead sperm") are present in variable numbers in mammalian ejaculates and their number increase substantially when semen is stored, particularly cryopreserved. This review comparatively highlights, with experimental data in porcine, the role-played by non-viable sperm in the outcome of semen used in assisted reproductive technologies. As well, the review discusses our current understanding of their origin and the pathways involved when their large numbers negative influence the functional lifespan of contemporary viable sperm to eventually cause irreversible dysfunction that reduces their fertility potential and their ability to develop healthy embryos. Finally, it highlights procedures currently available to mitigate these harmful effects.
Assuntos
Sobrevivência Celular , Espermatozoides/fisiologia , Animais , Criopreservação/veterinária , Masculino , Preservação do Sêmen/veterinária , Motilidade dos EspermatozoidesRESUMO
AI is commercially applied worldwide to breed pigs, yielding fertility outcomes similar to those of natural mating. However, it is not fully efficient, as only liquid-stored semen is used, with a single boar inseminating about 2000 sows yearly. The use of liquid semen, moreover, constrains international trade and slows genetic improvement. Research efforts, reviewed hereby, are underway to reverse this inefficient scenario. Special attention is paid to studies intended to decrease the number of sperm used per pregnant sow, facilitating the practical use of sexed frozen-thawed semen in swine commercial insemination programs.
Assuntos
Inseminação Artificial/veterinária , Espermatozoides/fisiologia , Suínos/fisiologia , Animais , Feminino , Inseminação Artificial/métodos , Masculino , Gravidez , Preservação do Sêmen/veterináriaRESUMO
Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleukin-10, pertaining maternal immune tolerance of these foreign cells.