Specific lectin binding sites during in vitro capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx and plasma membrane undergo outstanding modifications during fertilization. However, it is unclear how in vitro capacitation time and acrosome reaction affect the specific location of boar sperm glycoconjugates. This study aimed to identify lectin binding patterns and to describe the sequential changes during different in vitro capacitation times (1 and 4 h) and acrosome reaction in boar spermatozoa. With Aleuria aurantia agglutinin (AAA), most uncapacitated cells were labelled in the postacrosomal region. Nevertheless, after 1 h of in vitro capacitation and the acrosome reaction, most AAA binding sites were in the acrosomal region. With Concanavalin A (ConA), most sperm were labeled in the postacrosomal region before and after capacitation. After the acrosome reaction induction, this pattern changed to a highly stained acrosomal and postacrosomal regions. Peanut agglutinin (PNA) binding sites were in the acrosomal region in uncapacitated and capacitated sperm. In acrosome reacted sperm after 4h capacitation, the most frequent pattern showed remaining positive labeling in the central area of the head. With Pisum sativum agglutinin (PSA), most uncapacitated cells showed a postacrosomal region staining. Nevertheless, faint stained all over the head and highly acrosomal region labelling was observed in the major part of capacitated and acrosome reacted sperm respectively. With Wheat germ agglutinin (WGA), the most representative pattern in uncapacitated, capacitated and acrosome reacted sperm was labelled in the acrosomal region. Regarding capacitation time, the most significant changes in the most representative pattern were observed in acrosome reacted spermatozoa after 4 h of in vitro capacitation.This research was supported by AGL2015-70159-P, PGC2018-094781-B-100 and PEJ2018-002736-P (MCINN/AEI/FEDER,UE)

Estudio de la expresión génica y polimorfismos relacionados con la espermatogénesis y la infertilidad

Según la Organización Mundial de la Salud (OMS), la infertilidad es un problema de salud global que afecta a millones de personas en todo el mundo. La infertilidad se define como la incapacidad de concebir tras 12 meses de relaciones regulares sin protección. La prevalencia puede alcanzar el 10,5% de la población española, afectando a unas 900.000 parejas en dicho país. Objetivos Esta tesis se divide en dos diferenciados capítulos, que tienen como objetivo común aumentar el conocimiento de los mecanismos moleculares implicados en la reproducción y, por tanto, mejorar el diagnóstico de la infertilidad y las tasas de reproducción asistida. En el primer capítulo se analiza la expresión génica de testículo para comprender los procesos moleculares implicados en el desarrollo del espermatozoide. Para ello, utilizamos un modelo de primate no humano, el babuino (Papio anubis). El segundo capítulo pretende mejorar los métodos de selección embrionaria, basándose en los polimorfismos presentes en genes potencialmente implicados en la implantación y desarrollo embrionario temprano. De esta forma se pretende aumentar las posibilidades de éxito en las transferencias de embriones. Metodología Para abordar el primer capítulo, se utilizaron 8 muestras testiculares, 4 de babuinos inmaduros y 4 de maduros. El estado de madurez se corroboró mediante características anatómicas e histológicas. El ARN de cada muestra se analizó mediante la tecnología RNA-seq. Los resultados obtenidos se procesaron mediante un análisis bioinformático, donde las secuencias se pseudoalinearon con los transcriptomas de referencia (Panubis1.0 y hg38) y se compararon entre los grupos (maduros e inmaduros). Los genes expresados diferencialmente (logFC≥|2| y FDR<0,01) se utilizaron para evaluar su posible papel en la espermatogénesis. En el segundo capítulo, se utilizaron muestras de ADN de 131 embriones, que habían sido sometidos a una biopsia. Por un lado, se diseñó un panel Ampliseq, eligiendo 33 genes relacionados con la implantación y desarrollo gestacional temprano. Con este panel se analizaron todos los embriones y se evaluaron los polimorfismos detectados en función del tipo de mutación, genotipo, localización en el gen, posible fenotipo patogénico y su prevalencia en la población. Por otro lado, se utilizó la tecnología KASP para evaluar los polimorfismos c.677C>T y c.1298A>C del gen MTHFR de todos los embriones transferidos. Los resultados de estos análisis genéticos se compararon con los resultados clínicos tras la transferencia embrionaria. Resultados y conclusiones El análisis transcriptómico RNA-seq de los testículos de babuino anotó un total de 15.297 genes distintos del genoma Papio anubis. El análisis de expresión diferencial entre testículos maduros e inmaduros detectó 2029 genes sobreexpresados y 555 subexpresados en los testículos maduros. Algunos de los nuevos genes que aparecieron en este estudio son SPATA18, SPATC1, TMEM262, ARRDC5, SPATA31D1, TMEM239, LEMD1, FAM187B, C3orf22, C11orf42, TMEM31, TSPAN16, CT83, C7orf61 y SLC9C2. Se detectaron un total de 1897 genes no codificantes, donde destacan SPACA6, LINC00303 y LINC01121. Tras la evaluación de los polimorfismos genéticos de los embriones humanos (panel Ampliseq), se destacaron ARHGAP21, ENG, STAT3, PMM2 y GATA4 como posibles genes implicados en la implantación y desarrollo embrionario temprano, pudiendo ser utilizados como marcadores de selección embrionaria. Se ha detectado un posible efecto deletéreo de las variantes polimórficas homocigóticas chr17:40481601 C>T (STAT3) y chr16:8904956 G>A (PMM2). Además, se ha detectado un posible efecto deletéreo en otras mutaciones no detectadas hasta ahora en la población, incluso en heterocigosis, como chr10:24909241 C>T, chr10:24909164 G>A y chr10:249090 TC>T (ARHGAP21), o chr9:130578295 CAG>C (ENG). No se pudo confirmar la relación entre los polimorfismos del gen MTHFR y el resultado clínico tras la transferencia embrionaria. Sin embargo, se ha descrito por primera vez un embrión portador de la combinación polimórfica c.677TT y c.1298AC del gen MTHFR, que no implantó.According to the World Health Organisation (WHO), infertility is a global health issue affecting millions worldwide. Infertility is defined as the inability to conceive after 12 months of regular unprotected intercourse. The prevalence of infertility may be as high as 10.5% of the Spanish population, affecting approximately 900,000 couples in this country. Objectives The present thesis is divided into two different chapters, both of which have, as a common aim, the increase of knowledge about the molecular mechanisms involved in reproduction and hence, the improvement of infertility diagnosis and the assisted reproduction rates. The first chapter analyses the gene expression of the testis to understand the molecular processes involved in the development of a cell as specialised as the spermatozoon. For this purpose, we used a non-human primate model, the baboon (Papio anubis). The second chapter intends to improve the methods of selection of the best embryo to transfer, based on the polymorphisms present in genes potentially involved in early human embryonic development. This way we try to increase the chances of success after embryo transfers. Methodology To tackle the first chapter, 8 testicular samples, 4 from immature baboons and 4 from mature baboons, were used. The maturity state of each sample was checked using anatomical and histological characteristics. The RNA of each sample was analysed using RNA-seq technology. The results obtained were then analysed by bioinformatic procedures, where the sequences were pseudoaligned with the reference transcriptomes (Panubis1.0 and hg38) and compared between groups (mature and immature). The differentially expressed genes (logFC≥|2| and FDR<0.01) were used to evaluate their possible role in spermatogenesis. In the second chapter, DNA samples from 131 embryos, that had undergone biopsy, were used. On one hand, we design an Ampliseq panel, choosing 33 genes related to implantation and early gestational development. All the embryos were analysed using this panel, and the polymorphisms detected were evaluated in function of the mutation type, genotype, as well as the location in the gene, the possible pathogenic phenotype or its presence in the population. On the other hand, the KASP technology was used to evaluate the polymorphisms c.677C>T and c.1298A>C of the MTHFR gene in all the embryos transferred. The results from all these genetic analyses were compared with the clinical results after the embryo transfer. Results and conclusions The RNA-seq transcriptomic analysis of baboon testes has annotated a total of 15,297 distinct genes in the Papio anubis genome. The differential expression analysis between mature and immature testis detected 2029 genes overexpressed and 555 underexpressed in mature testis. Some of the novel genes that appeared in this study were SPATA18, SPATC1, TMEM262, ARRDC5, SPATA31D1, TMEM239, LEMD1, FAM187B, C3orf22, C11orf42, TMEM31, TSPAN16, CT83, C7orf61 and SLC9C2. A total of 1897 non-coding genes were detected, where SPACA6, LINC00303 and LINC01121 stand out. After the evaluation of genetic polymorphisms of human embryos (Ampliseq panel), ARHGAP21, ENG, STAT3, PMM2 and GATA4 were highlight as possible genes involved in implantation and early embryonic development that can be used as human embryo selection markers. It has been detected a possible deleterious effect of homozygotic polymorphic variants chr17:40481601 C>T (STAT3) and chr16:8904956 G>A (PMM2). Moreover, a possible deleterious effect in implantation has been detected in other mutations not detected so far in the population even in heterozygosis. They are chr10:24909241 C>T, chr10:24909164 G>A and chr10:24909090 TC>T of ARHGAP21 gene, or chr9:130578295 CAG>C of ENG gene. The relationship between MTHFR gene polymorphisms and the clinical outcome after embryo transfer could not be confirmed. However, an embryo carrying the polymorphic combination c.677TT and c.1298AC of MTHFR gene, which did not implant, has been described for the first time

New Insights into the Mammalian Egg Zona Pellucida

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