RESUMEN
Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade the uterus, where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome-editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their roles in trophoblast-guided uterine spiral artery remodeling.
Asunto(s)
Placenta , Placentación , Embarazo , Ratas , Femenino , Animales , Humanos , Placenta/metabolismo , Placentación/genética , Trofoblastos , Útero , Linaje de la Célula/genética , Modelos AnimalesRESUMEN
Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.
Asunto(s)
Placenta , Placentación , Animales , Femenino , Embarazo , Ratas , Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Placenta/metabolismo , Placentación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Trofoblastos , ÚteroRESUMEN
Establishment of the hemochorial uterine-placental interface requires exodus of trophoblast cells from the placenta and their transformative actions on the uterus, which represent processes critical for a successful pregnancy, but are poorly understood. We examined the involvement of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) in rat and human trophoblast cell development. The rat and human exhibit deep hemochorial placentation. CITED2 was distinctively expressed in the junctional zone (JZ) and invasive trophoblast cells of the rat. Homozygous Cited2 gene deletion resulted in placental and fetal growth restriction. Small Cited2 null placentas were characterized by disruptions in the JZ, delays in intrauterine trophoblast cell invasion, and compromised plasticity. In the human placentation site, CITED2 was uniquely expressed in the extravillous trophoblast (EVT) cell column and importantly contributed to the development of the EVT cell lineage. We conclude that CITED2 is a conserved regulator of deep hemochorial placentation.
Asunto(s)
Placenta , Placentación , Proteínas Represoras , Transactivadores , Animales , Femenino , Humanos , Embarazo , Ratas , Placentación/genética , Proteínas Represoras/genética , Transactivadores/genética , Trofoblastos , ÚteroRESUMEN
Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at late zygotene/early pachynema stages, with defects in sex body formation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.
Asunto(s)
Semen , Espermatogénesis , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Animales , Femenino , Masculino , Embarazo , Ratas , Diferenciación Celular , Meiosis , Semen/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Testículo/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismoRESUMEN
Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.
Asunto(s)
Lipoproteínas/metabolismo , Placentación/fisiología , Células Madre/fisiología , Trofoblastos/fisiología , Animales , Sistemas CRISPR-Cas , Células Endoteliales/fisiología , Femenino , Edición Génica , Humanos , Lipoproteínas/genética , Mutación , Placenta/metabolismo , Embarazo , Interferencia de ARN , Ratas , Ratas Sprague-DawleyRESUMEN
Interleukin 33 (IL33) signaling has been implicated in the establishment and maintenance of pregnancy and in pregnancy disorders. The goal of this project was to evaluate the role of IL33 signaling in rat pregnancy. The rat possesses hemochorial placentation with deep intrauterine trophoblast invasion; features also characteristic of human placentation. We generated and characterized a germline mutant rat model for IL33 using CRISPR/Cas9 genome editing. IL33 deficient rats exhibited deficits in lung responses to an inflammatory stimulus (Sephadex G-200) and to estrogen-induced uterine eosinophilia. Female rats deficient in IL33 were fertile and exhibited pregnancy outcomes (gestation length and litter size) similar to wild-type rats. Placental weight was adversely affected by the disruption of IL33 signaling. A difference in pregnancy-dependent adaptations to lipopolysaccharide (LPS) exposure was observed between wild-type and IL33 deficient pregnancies. Pregnancy in wild-type rats treated with LPS did not differ significantly from pregnancy in vehicle-treated wild-type rats. In contrast, LPS treatment decreased fetal survival rate, fetal and placental weights, and increased fetal growth restriction in IL33 deficient rats. In summary, a new rat model for investigating IL33 signaling has been established. IL33 signaling participates in the regulation of placental development and protection against LPS-induced fetal and placental growth restriction.
Asunto(s)
Retardo del Crecimiento Fetal/metabolismo , Interleucina-33/metabolismo , Enfermedades Placentarias/metabolismo , Complicaciones Infecciosas del Embarazo/metabolismo , Transducción de Señal , Animales , Femenino , Retardo del Crecimiento Fetal/etiología , Retardo del Crecimiento Fetal/patología , Interleucina-33/genética , Lipopolisacáridos/toxicidad , Mutación , Enfermedades Placentarias/etiología , Enfermedades Placentarias/patología , Embarazo , Complicaciones Infecciosas del Embarazo/etiología , Complicaciones Infecciosas del Embarazo/patología , Resultado del Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).
Asunto(s)
Quimiotaxis , Caballos/metabolismo , Especies Reactivas de Oxígeno , Capacitación Espermática , Espermatozoides/metabolismo , Animales , MasculinoRESUMEN
STUDY QUESTION: Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? SUMMARY ANSWER: Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. WHAT IS KNOWN ALREADY: Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. STUDY DESIGN SIZE, DURATION: Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. MAIN RESULTS AND THE ROLE OF CHANCE: Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest.
Asunto(s)
Citometría de Flujo/métodos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Tirosina/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Quinasa 2 de Adhesión Focal/metabolismo , Immunoblotting , Masculino , Fosforilación/efectos de los fármacos , Quinolonas/farmacología , Transducción de Señal/efectos de los fármacos , Capacitación Espermática , Motilidad Espermática/efectos de los fármacos , Sulfonas/farmacologíaRESUMEN
Trophoblast stem (TS) cells have the unique capacity to differentiate into specialized cell types, including extravillous trophoblast (EVT) cells. EVT cells invade into and transform the uterus where they act to remodel the vasculature facilitating the redirection of maternal nutrients to the developing fetus. Disruptions in EVT cell development and function are at the core of pregnancy-related disease. WNT-activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human TS cells, activation of canonical WNT signaling is critical for maintenance of the TS cell stem state and its downregulation accompanies EVT cell differentiation. We show that aberrant WNT signaling undermines EVT cell differentiation. Notum, palmitoleoyl-protein carboxylesterase (NOTUM), a negative regulator of canonical WNT signaling, was prominently expressed in first trimester EVT cells developing in situ and upregulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for human TS cell differentiation to EVT cells. Activation of NOTUM in EVT cells is driven, at least in part, by endothelial PAS domain 1 (also called hypoxia-inducible factor 2 alpha). Collectively, our findings indicate that canonical WNT signaling is essential for maintenance of human trophoblast cell stemness and prevention of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for EVT cell differentiation.
RESUMEN
Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TS CV ) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TS CT ) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.
RESUMEN
Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at the zygotene stage, with defects in sex body formation and meiotic sex chromosome inactivation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.
RESUMEN
Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade into the uterus where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 ( Prl7b1 ) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their contributions to trophoblast-guided uterine spiral artery remodeling.
RESUMEN
The extravillous trophoblast cell lineage is a key feature of placentation and successful pregnancy. Knowledge of transcriptional regulation driving extravillous trophoblast cell development is limited. Here, we map the transcriptome and epigenome landscape as well as chromatin interactions of human trophoblast stem cells and their transition into extravillous trophoblast cells. We show that integrating chromatin accessibility, long-range chromatin interactions, transcriptomic, and transcription factor binding motif enrichment enables identification of transcription factors and regulatory mechanisms critical for extravillous trophoblast cell development. We elucidate functional roles for TFAP2C, SNAI1, and EPAS1 in the regulation of extravillous trophoblast cell development. EPAS1 is identified as an upstream regulator of key extravillous trophoblast cell transcription factors, including ASCL2 and SNAI1 and together with its target genes, is linked to pregnancy loss and birth weight. Collectively, we reveal activation of a dynamic regulatory network and provide a framework for understanding extravillous trophoblast cell specification in trophoblast cell lineage development and human placentation.
Asunto(s)
Cromatina , Trofoblastos , Embarazo , Femenino , Humanos , Trofoblastos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Placentación/genética , Diferenciación Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Linaje de la Célula/genética , Placenta/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
Nowadays Molecular Cell Biology (MCB) must be taught as science is practiced. Even though there are several approaches based on scientific practices, a key aspect is to define the purpose of each of these teaching strategies and, most importantly, their implementation. Our goal was to train students to acquire, understand, and communicate new scientific knowledge in the field. The main feature of our new teaching methodology was progressive training in scientific practices associated with a back-and-forward interplay between activities and assessments. The methodology was implemented over 4 years, in students attending the MCB course of the undergraduate degree in Biological Sciences. In the first two modules, the students were prepared to comprehend MCB concepts and techniques and to experience activities based on scientific practices. In the third module, the students analyzed a primary paper in-depth. They were assessed by midterm exams based on a primary paper, written laboratory reports, and the oral presentation of a scientific paper. Our teaching proposal was evaluated through the students' academic performance and by their opinion on the teaching methodology. Most students were satisfied since they improved their acquisition of concepts, their interpretation and integration of scientific knowledge, and developed skills to communicate scientific knowledge in writing and orally. The novelty of transversal interconnections and progressive training in scientific practices provides students with skills in acquiring and understanding new scientific information, even beyond the MCB course.
Asunto(s)
Biología Celular/educación , Evaluación Educacional , Biología Molecular/educación , Estudiantes , HumanosRESUMEN
The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of reactive oxygen species (ROS). In the present study, the aim was to adapt the SSA for frozen-thawed stallion semen samples and evaluate the functional status of those sperm selected using the SSA procedure, and to determine whether this enriched sperm population has a greater capacity to bind to the zona pellucida of cattle oocytes. There were experimental conditions developed to conduct the SSA with stallion sperm. Using these conditions, the indexes of induced acrosome reaction, protein tyrosine phosphorylation, mitochondrial membrane potential, mitochondrial and cytoplasmic reactive oxygen species, and number of sperm bound to the zona pellucida of cattle were greater when the sperm population was selected using the SSA. Consistently, the DNA fragmentation and phospholipase C zeta indexes were less for the selected sperm. In conclusion, stallion sperm selected using chemotaxis utilizing the SSA provides a sperm population of greater quality, which when used may improve the outcomes with use of the ART.
Asunto(s)
Criopreservación/veterinaria , Caballos/fisiología , Preservación de Semen/veterinaria , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiología , Adaptación Fisiológica , Animales , Quimiotaxis , Congelación , Masculino , Reproducibilidad de los ResultadosRESUMEN
The spermatozoon must be physiologically prepared to fertilize the egg, process called capacitation. Human sperm samples are heterogeneous in their ability to capacitate themselves, which leads to variability between samples from the same or different donors, and even along the seasons. Here we studied sperm variation in the capacitation state according to the ability of capacitated spermatozoa to acrosome react upon stimulation (% ARi) and to be recruited by chemotaxis (% Chex). Both indirect indicators of sperm capacitation increased along the incubation time with fluctuations. Those capacitated sperm recruited by chemotaxis showed an ultradian rhythm with a cycle every 2 h, which might be influenced by unknown intrinsic sperm factors. Two infradian rhythms of 12 months for the % ARi and of 6 months for % Chex were observed, which are associated with the joint action of temperature and photoperiod. Thus, to avoid false negative results, human sperm samples are recommended to be incubated for a long period (e.g. 18 h) preferably in spring time. This innovative point of view would lead to better comprehend human reproductive biology and to think experimental designs in the light of sperm cyclicity or to improve sperm aptitude for clinical purposes.
Asunto(s)
Ritmo Infradiano/fisiología , Espermatozoides/fisiología , Ritmo Ultradiano/fisiología , Calcio/metabolismo , Humanos , Espacio Intracelular/metabolismo , Masculino , Potenciales de la Membrana , Espermatozoides/citologíaRESUMEN
Assisted reproductive techniques (ART) have been widely used in farm animals in the last decades. Sexed cryopreserved spermatozoa, ovum pick up, in vitro embryo production and transfer constitute the ART that have revolutionized the dairy industry. However, the efficiency of some of these techniques is still low due in part to sperm quality, which influences fertilization, embryo development and implantation. The Sperm Selection Assay (SSA), based on sperm chemotaxis towards progesterone, provides a sperm subpopulation enriched with spermatozoa that are capacitated, with intact DNA and low level of oxidative stress. Since the SSA selects a sperm subpopulation at optimum physiological state, the application of the SSA may improve the efficiency of the current ART. The aim of this study was to adapt the SSA for unsexed and sexed bovine frozen-thawed semen samples, and then to test whether sperm selection by the SSA improves the cleavage rate of bovine embryos in vitro. The optimal SSA conditions to obtain the higher sperm accumulation percentage given by chemotaxis were the same for both unsexed and sexed semen samples. Thus, sperm accumulation in W2 was significantly higher when: 2 million sperm per mL were placed in W1 (unsexed samples: 12⯱â¯1%, pâ¯=â¯0.002; sexed samples: 14⯱â¯3%, pâ¯=â¯0.02); 1 pM progesterone was placed in W2 (unsexed sample: 9⯱â¯1%, pâ¯=â¯0.009; sexed samples: 11⯱â¯2%, pâ¯=â¯0.02); and to incubate the SSA device for 10â¯min (unsexed samples: 17⯱â¯2%, pâ¯=â¯0.007; sexed samples: 10⯱â¯1%, pâ¯=â¯0.004). We found that the quality of spermatozoa recovered from W2 in unsexed and sexed semen was enhanced. Thus, the capacitation index was significantly increased (unsexed samples: 1.75⯱â¯0.1, pâ¯=â¯0.0001; sexed samples: 1.76⯱â¯0.2, pâ¯=â¯0.004), while DNA fragmentation index was significantly decreased (unsexed samples: 0.33⯱â¯0.07, pâ¯=â¯0.0003; sexed samples: 0.32⯱â¯0.04, pâ¯=â¯0.002). Moreover, the cleavage index of oocytes fertilized with either unsexed or sexed SSA-selected sperm was significantly improved (unsexed samples: 3.2⯱â¯0.4, pâ¯=â¯0.0001; sexed samples: 2.3⯱â¯0.33, pâ¯=â¯0.03). Thus, we show that the SSA can be used to recruit a bovine sperm subpopulation at optimal functional state regardless of whether the sample is previously sexed, and that this optimal state improves bovine embryo cleavage rate.