RESUMO
Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mechanisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 × 106 cells/mL) were incubated for 1 h at 38.5 °C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p ˂ 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to influence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to increase SMGT protocols, without altering the sperm viability.
RESUMO
BACKGROUND: Very few studies have evaluated the expression of homeobox A10 (HOXA10) and steroid (estrogen and progesterone) receptors exclusively in deep endometriosis. Conclusions drawn from studies evaluating peritoneal and ovarian endometriosis are usually generalized to explain the pathogenesis of the disease as a whole. We aimed to evaluate the expression of HOXA10, estrogen receptor α (ER-α), progesterone receptor (PR), and PR-B in rectosigmoid endometriosis (RE), a typical model of deep disease. METHODS: We used RE samples from 18 consecutive patients to construct tissue microarray blocks. Nine patients each were operated during the proliferative and secretory phases of the menstrual cycle. We quantified the expressions of proteins by immunohistochemistry using the modified Allred score. RESULT: The HOXA10 was expressed in the stroma of nodules during the secretory phase in 5 of the 18 patients. Expression of ER-α (in 16 of 18 patients), PR (in 17 of 18 patients), and PR-B (17 of 18 patients) was moderate to strong in the glands and stroma of nodules during both phases. Expression of both PR (P = .023) and PR-B (P = .024) was significantly greater during the secretory phase. CONCLUSION: The HOXA10 is expressed in RE, where it likely imparts the de novo identity of endometriotic lesions. The ER-α, PR, and PR-B are strongly expressed in RE, which differs from previous studies investigating peritoneal and ovarian lesions. This suggests different routes of pathogenesis for each of the 3 types of endometriosis.
Assuntos
Endometriose/metabolismo , Endométrio/química , Receptor alfa de Estrogênio/análise , Proteínas de Homeodomínio/análise , Receptores de Progesterona/análise , Doenças Retais/metabolismo , Doenças do Colo Sigmoide/metabolismo , Análise Serial de Tecidos , Adulto , Endometriose/patologia , Endometriose/fisiopatologia , Endométrio/patologia , Endométrio/fisiopatologia , Células Epiteliais/química , Feminino , Proteínas Homeobox A10 , Humanos , Imuno-Histoquímica , Ciclo Menstrual , Doenças Retais/patologia , Doenças Retais/fisiopatologia , Doenças do Colo Sigmoide/patologia , Doenças do Colo Sigmoide/fisiopatologia , Células Estromais/químicaRESUMO
A suggested alternative to improve post-thawed ram semen quality is the addition of seminal plasma (SP). This is thought to be capable of improving sperm resistance to thermal shock, reverting cryocapacitation and helping sperm survival. The aim of this study was to evaluate the effect of frozen-thawed ram semen incubation with SP on mitochondrial activity, acrosomal membrane integrity, necrosis and apoptosis. Frozen/thawed semen was divided into two groups: the SP Group and the control group. After 0, 30 and 60 min, fluorescent probes were added to aliquots from each treatment group and evaluated using flow cytometry. There was no difference between treatment groups in almost all viability parameters evaluated, with exception of the apoptosis, which was found increased in SP group. The increase in incubation period resulted in a decreased percentage of sperm with high mitochondrial membrane potential and acrosomal membrane integrity and an increased percentage of necrotic and apoptotic sperm cells. In conclusion, the present study showed that addition of seminal plasma after thawing cryopreserved ram sperm had no identifiable beneficial effect on sperm quality.
Assuntos
Criopreservação , Sêmen/fisiologia , Espermatozoides/citologia , Espermatozoides/fisiologia , Acrossomo/fisiologia , Animais , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/fisiologia , Modelos Animais , OvinosRESUMO
Immunohistochemistry is a suitable method for the detection of proteins from the Claudin family and several antibodies are commercially available for the detection of Claudin congeners. Immunodetection of Caludin-4 in the paraffin-embedded specimens might be a useful tool for studying the role of these proteins in the cyclic transformation of the endometrium and its role in the endometrial receptivity; furthermore, other components of the junctional zone involved in the transformational process of the endometrium can be detected by means of immunohistochemistry/immunofluorescence with several polyclonal or monoclonal antibodies. The aim of this chapter is to comprehensively overview the materials and methods to perform the endometrial biopsy and to detect Claudin-4 in paraffin-embedded samples of endometrium. Additionally, the interpretation of the results is addressed.
Assuntos
Endométrio/metabolismo , Imuno-Histoquímica/métodos , Proteínas de Membrana/metabolismo , Biotina , Claudina-4 , Amarelo de Eosina-(YS) , Feminino , Imunofluorescência/métodos , Hematoxilina , Humanos , Inclusão em Parafina/métodosRESUMO
Background: Infertility is a disease affecting 10% of the human population in reproductive age and couples experiencing difficulties to achieve pregnancy may benefit from assisted reproduction treatment. According to the Red Latinoamericana de Reproducción Asistida, approximately 34,100 in vitro fertilization (IVF) treatments were performed in Latin America in 2007 and 42.3% (14,428) of these treatments were conducted in Brazil. Standard techniques and treatments for infertility were developed within the last 4 decades and some of them are still under technological improvements or they are frequently considered experimental or "state of art" technology. Usually, these treatments and techniques to overcome infertility were developed by professionals with different backgrounds including physicians, biologists, veterinarians and nurses. We aimed to review the role of veterinarians in translating animal biotechnology to human assisted procreation, developing new assisted reproduction technologies and treatments, and finally acting in clinical embryology laboratories as embryologists or supervisors. Review: Human assisted reproduction landmarks were: (i) the birth of the first in vitro fertilization (IVF) child; (ii) the implementation of intracytoplasmatic sperm injection; and, more recently, (iii) the development of successful protocols of vitrification for cryopreservation of embryos and oocytes. Veterinarians' contribution and participation in the field of human assisted procreation could be distinguished between technological development and/or clinical embryology. Despite of the 3 year delay on the birth of the first in vitro fertilization calf in relation to the birth of the first IVF baby, many steps and protocols of these technological advancements relied on the knowledge acquired from animal models or biotechnologies now used for commercial purposes in animal production and breeding. Furthermore, new embryo culture technologies such as microfluidics environments and controlled atmospheres are initially evaluated in animal models. Another example of the role of veterinarians on the development of human assisted reproduction is the emergence of the widespread concept of ovarian dynamics pattern of follicular waves in monovulatory animals from Theriogenology to Reproductive Medicine in the last decade. The translation of this concept to Reproductive Medicine might allow the development of different approaches of controlled ovarian stimulation. In a human assisted reproduction clinic, the clinical embryology laboratory is responsible for selecting oocytes after transvaginal ovum pick-up, fertilizing these oocytes, cultivating the supposed zygotes until the stage of 8 cell embryos or blastocysts, and embryo preparation for transference to the uterine cavity. Several tasks enrolled in these steps of human IVF are identical to those performed by veterinarians in an animal IVF facility; however, a veterinarian must be familiar to the specific guidelines and needs of a human IVF laboratory. Conclusion: Assisted reproduction treatments are performed by groups of professionals with different academic backgrounds. Veterinarians have been playing an important roles in the development and/or adaptation of several biotechnologies utilized for human procreation; additionally, veterinarians working on animal in vitro fertilization facilities have an adequate formation and skills to act on and/or supervise clinical embryology laboratories after specific education, practice and certification.
Assuntos
Humanos , Animais , Indução da Ovulação/veterinária , Criopreservação/veterinária , Médicos Veterinários/tendências , Injeções de Esperma Intracitoplásmicas/veterinária , Técnicas de Reprodução Assistida/veterinária , Técnicas de Cultura Embrionária/veterinária , Preservação do Sêmen/veterinária , VitrificaçãoRESUMO
To investigate the luteal phase endometrial expression of leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), progesterone receptor (PR), claudin 4 (CLDN4), vascular-endothelial growth factor receptor 3 (VEGFR-3), bone morphogenetic protein 4 (BMP-4) and citokeratin 7 (CK-7), we obtained luteal phase endometrial samples from 52 women. Samples were dated and integrated using a tissue microarray (TMA). Samples were immunostained for LIF, IGF-1, PR, CLDN4, VEGFR-3, BMP-4 and CK-7. Frequencies of positive expressions at the early, mid and late luteal phases were compared by two proportions test. Concomitant expression of these proteins was assessed with Chi-square or Fischer's test. The frequency of LIF was positively correlated to the frequency of IGF-1 (r = 0.99; p < 0.05) and PR (r = 0.99; p < 0.05), and the correlation between IGF-1 and PR tended to be significant (r = 0.98; p < 0.1). The expression of PR was associated with the absence of CLDN4 (p < 0.001). Thus, expression of LIF, IGF-1 and PR are correlated during the luteal phase, and immunohistochemistry for these proteins might be used to assist in the assessment of endometrial maturation. In addition, the expression of CLDN4 and PR was not concomitant, warranting further investigation on the relationship of their endometrial expression.
Assuntos
Endométrio/metabolismo , Fase Luteal/metabolismo , Adulto , Proteína Morfogenética Óssea 4/metabolismo , Distribuição de Qui-Quadrado , Claudina-4 , Feminino , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Queratina-7/metabolismo , Fator Inibidor de Leucemia/metabolismo , Proteínas de Membrana/metabolismo , Seleção de Pacientes , Análise Serial de Proteínas , Receptores de Progesterona/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: To determine whether gonadotropin releasing hormone (GnRH)-agonist or -antagonist induces higher percentages of cumulus cell apoptosis and if the use of either is detrimental to ART outcomes. PATIENTS: Women in a private facility under treatment for IVF had their cumulus cells isolated and analyzed by flow cytometry. Viable, apoptotic, and dead cumulus cell rates related to ovarian stimulation by GnRH-agonist or -antagonist were measured and compared with fertilization and implantation rates. RESULTS: Treatment with GnRH-agonist produced a greater number of follicles than treatment with GnRH-antagonist. No differences in implantation and pregnancy rates were found. While cumulus cell (CC) apoptosis was positively correlated with estradiol on the day of hCG administration, no significant difference in the percentage of apoptotic cells between treatments was detectable. Additionally, implantation rate and the average follicular estradiol production on the day of hCG administration were no different between treatments. CONCLUSIONS: GnRH-agonist or -antagonist treatment protocols induce similar levels of apoptosis in CCs and are not detrimental to ART outcomes.
Assuntos
Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células do Cúmulo/fisiologia , Hormônio Liberador de Gonadotropina/biossíntese , Adulto , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Regulação para Baixo , Implantação do Embrião , Feminino , Fertilização in vitro , Citometria de Fluxo , Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Humanos , Indução da OvulaçãoRESUMO
Incubation time induce damages in sperm cells by necrosis and/or apoptosis. The aim of this study was the evaluation of changes in plasma membrane related to apoptosis and necrosis in bovine sperm cells through 2 hours of incubation. Sperm cells were incubated at 5% (v/v) CO subscribed to 2 in air for 0, 30, 60, 90 and 120 minutes. After each period, sperm cells were incubated with fluorescent probes Yo-pro and propidium iodide (PI) to detect change in plasma membrane related to apoptosis and necrosis respectively. Using Yo-pro/PI assay, three different subpopulations of sperm cells were detected by flow cytometry: a) necrotic sperm cells (PI superscript + and Yo-pro superscript to -/+); b) apoptotic sperm cells (Yo-pro superscript to+ and PI superscript to-) and c) living cells (Yo-pro superscript to- and PI superscript to-). The percentage of live cells (plasma membrane integrity) significantly decreases over 2 hour of incubation, on the other hand, the percentage of necrotic and apoptotic cells increase during incubation. Changes in plasma membrane integrity were correlated to incubation time. While live cells were negatively correlated with the increase of incubation time, necrosis and apoptosis were positively correlated. It was also observed that necrosis was the main damage in sperm cells in all incubation times. In conclusion, incubation time induces changes in plasma membrane integrity related to necrosis and apoptosis, whether necrosis is present in higher quantity in all incubation times.
O tempo de incubação causa danos nas células espermáticas relacionados a necrose e/ou apoptose. O objetivo deste estudo foi avaliar as mudanças na membrana plasmática relacionadas a apoptose e necrose em espermatozóides bovinos durante 2 horas de incubação. Os espermatozóides foram incubados a 5% (v/v) CO subscrito por 2 em ar por 0, 30, 60, 90 e 120 minutes. Depois de cada periodo, as células espermáticas foram incubadas com as sondas fluorescentes Yo-pro e iodito de propideo (PI) para detectar mudanças na membrana plasmática relacionadas a apoptose e a necrose, respectivamente. Usando Yo-pro/PI assay, três subpopulações diferentes de células espermáticas são detectadas pelo citômetro de fluxo: a) células espermáticas em necrose (PI elevado a+ and Yo-pro elevado a-/+); b) células espermáticas em apoptose (Yo-pro elevado a+ and PI elevado a-) e c) células espermáticas vivas (Yo-pro- and PI elevado a-). A porcentagem de células vivas (membrana plasmática integra) significativamente diminui durante 2 horas de incubação, por outro lado, a porcentagem de espermatozóides em necrose e apoptose aumentaram durante a incubação. As mudanças na integridade da membrana plasmática foram correlacionadas com o tempo de incubação. Enquanto as células vivas foram correlacionadas negativamente com o aumento do tempo de incubação, necrose e apoptose foram correlacionadas positivamente. Também foi observado que necrose foi o principal dano causado pelo tempo de incubação nas células espermáticas. Conclui-se que o tempo de incubação causa alteração na integridade da membrana plasmática relacionadas a necrose e apoptose nas células espermáticas, sendo que necrose foi observada em maior quantidade em todos os tempos de incubação.
Assuntos
Animais , Apoptose/fisiologia , Bovinos , Citometria de Fluxo/métodos , Espermatozoides/citologiaRESUMO
Incubation time induce damages in sperm cells by necrosis and/or apoptosis. The aim of this study was the evaluation of changes in plasma membrane related to apoptosis and necrosis in bovine sperm cells through 2 hours of incubation. Sperm cells were incubated at 5% (v/v) CO subscribed to 2 in air for 0, 30, 60, 90 and 120 minutes. After each period, sperm cells were incubated with fluorescent probes Yo-pro and propidium iodide (PI) to detect change in plasma membrane related to apoptosis and necrosis respectively. Using Yo-pro/PI assay, three different subpopulations of sperm cells were detected by flow cytometry: a) necrotic sperm cells (PI superscript + and Yo-pro superscript to -/+); b) apoptotic sperm cells (Yo-pro superscript to+ and PI superscript to-) and c) living cells (Yo-pro superscript to- and PI superscript to-). The percentage of live cells (plasma membrane integrity) significantly decreases over 2 hour of incubation, on the other hand, the percentage of necrotic and apoptotic cells increase during incubation. Changes in plasma membrane integrity were correlated to incubation time. While live cells were negatively correlated with the increase of incubation time, necrosis and apoptosis were positively correlated. It was also observed that necrosis was the main damage in sperm cells in all incubation times. In conclusion, incubation time induces changes in plasma membrane integrity related to necrosis and apoptosis, whether necrosis is present in higher quantity in all incubation times.(AU)
O tempo de incubação causa danos nas células espermáticas relacionados a necrose e/ou apoptose. O objetivo deste estudo foi avaliar as mudanças na membrana plasmática relacionadas a apoptose e necrose em espermatozóides bovinos durante 2 horas de incubação. Os espermatozóides foram incubados a 5% (v/v) CO subscrito por 2 em ar por 0, 30, 60, 90 e 120 minutes. Depois de cada periodo, as células espermáticas foram incubadas com as sondas fluorescentes Yo-pro e iodito de propideo (PI) para detectar mudanças na membrana plasmática relacionadas a apoptose e a necrose, respectivamente. Usando Yo-pro/PI assay, três subpopulações diferentes de células espermáticas são detectadas pelo citômetro de fluxo: a) células espermáticas em necrose (PI elevado a+ and Yo-pro elevado a-/+); b) células espermáticas em apoptose (Yo-pro elevado a+ and PI elevado a-) e c) células espermáticas vivas (Yo-pro- and PI elevado a-). A porcentagem de células vivas (membrana plasmática integra) significativamente diminui durante 2 horas de incubação, por outro lado, a porcentagem de espermatozóides em necrose e apoptose aumentaram durante a incubação. As mudanças na integridade da membrana plasmática foram correlacionadas com o tempo de incubação. Enquanto as células vivas foram correlacionadas negativamente com o aumento do tempo de incubação, necrose e apoptose foram correlacionadas positivamente. Também foi observado que necrose foi o principal dano causado pelo tempo de incubação nas células espermáticas. Conclui-se que o tempo de incubação causa alteração na integridade da membrana plasmática relacionadas a necrose e apoptose nas células espermáticas, sendo que necrose foi observada em maior quantidade em todos os tempos de incubação.(AU)