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)

In vitro culture of ovine embryos up to early gastrulating stages

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview

Analysis of Minor Proteins Present in Breast Milk by Using WGA Lectin

Breast milk is a complex and dynamic biological fluid and considered an essential source of nutrition in early life. In its composition, the proteins have a relevant biological activity and are related to the multiple benefits demonstrated when compared with artificial milks derived from cow’s milk. Understanding human milk composition provides an important tool for health care providers toward the management of infant feeding and the establishment of breastfeeding. In this work, a new technique was developed to increase the knowledge of human milk, because many of the components remain unknown. To isolate minor proteins present in breast milk by using WGA lectin, breast milk was centrifuged to remove cells and separate the fat phase from the serum phase. The serum obtained was separated into two groups: control (n = 3; whole serum sample from mature milk) and WGA lectin (n = 3; sample processed with WGA lectin to isolate glycosylated proteins). The samples were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). A total of 84 different proteins were identified from all of the samples. In the WGA lectin group, 55 different proteins were isolated, 77% of which had biological functions related to the immune response. Of these proteins, there were eight WGA lectin group exclusives, and two had not previously been described in breast milk (polyubiquitin-B and POTE ankyrin domain family member F). Isolation by WGA lectin is a useful technique to detect minor proteins in breast milk and to identify proteins that could not be observed in whole serum

Metabolites involved in cellular communication among human cumulus-oocyte-complex and sperm during in vitro fertilization

Background: Fertilization is a key physiological process for the preservation of the species. Consequently, different mechanisms affecting the sperm and the oocyte have been developed to ensure a successful fertilization. Thus, sperm acrosome reaction is necessary for the egg coat penetration and sperm-oolema fusion. Several molecules are able to induce the sperm acrosome reaction; however, this process should be produced coordinately in time and in the space to allow the success of fertilization between gametes. The goal of this study was to analyze the metabolites secreted by cumulus-oocyte-complex (COC) to find out new components that could contribute to the induction of the human sperm acrosome reaction and other physiological processes at the time of gamete interaction and fertilization. Methods: For the metabolomic analysis, eighteen aliquots of medium were used in each group, containing: a) only COC before insemination and after 3 h of incubation; b) COC and capacitated spermatozoa after insemination and incubated for 16–20 hours; c) only capacitated sperm after 16–20 h in culture and d) only fertilization medium as control. Six patients undergoing assisted reproduction whose male partners provided normozoospermic samples were included in the study. Seventy-two COC were inseminated. Results: The metabolites identified were monoacylglycerol (MAG), lysophosphatidylcholine (LPC) and phytosphingosine (PHS). Analysis by PCR and in silico of the gene expression strongly suggests that the cumulus cells contribute to the formation of the PHS and LPC. Conclusions: LPC and PHS are secreted by cumulus cells during in vitro fertilization and they could be involved in the induction of human acrosome reaction (AR). The identification of new molecules with a paracrine effect on oocytes, cumulus cells and spermatozoa will provide a better understanding of gamete interaction.This study was supported by grant GV/2009/097 from Department of Education, Generalitat Valenciana, Vicerrectorado de Investigación, University of Alicante, Alicante, Spain (Vigrob-137), the Spanish Ministerio de Economía y Competitividad AGL2012-40180-C03-02 and Fundación Seneca (04542/GERM/06)

Protein Identification of Spermatozoa and Seminal Plasma in Bottlenose Dolphin (Tursiops truncatus)

17 Pág. Departamento de Reproducción animalProteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.This research was supported by the Spanish Ministry of Science and Innovation (PID2019-106380RB-I00/AEI/10.1303/501100011033) and PGC2018-094781-B-I00 (MCINN/AEI/FEDER, UE), and Oceanográfica Foundation.Peer reviewe

El oviducto : análisis transcriptómico, identificación de proteínas secretadas y aplicaciones en reproducción artificial

El oviducto es la región anatómica del tracto genital femenino que conecta el útero y los ovarios. Este órgano juega un papel importante en el transporte de gametos y su almacenamiento, incluyendo el reservorio espermático. Además, en él tienen lugar la fecundación y las primeras fases del desarrollo embrionario. En la primera parte de este trabajo se planteó una técnica que permite la separación de subpoblaciones de espermatozoides porcinos en función de su afinidad por azúcares relacionados con el reservorio espermático y se evaluó la capacidad de unión de estos espermatozoides por la zona pelúcida (ZP), para evaluar el potencial de esta selección en técnicas de reproducción asistida. En la segunda parte de la tesis, se llevó a cabo un estudio de transcriptómica del oviducto de la coneja antes y después de la ovulación y durante el desarrollo embrionario preimplantacional para identificar factores clave en cada una de estas etapas. Se empleó la separación celular mediante citometría de flujo (sorting) para seleccionar las subpoblaciones espermáticas porcinas pre-incubadas en medio no capacitante con ovalbúmina y el trisacárido Lewisa fluorescentes. La afinidad por la ZP se evaluó mediante incubación en medio capacitante de cada subpoblación espermática y su correspondiente control con ZPs aisladas a partir de ovocitos aspirados de ovarios obtenidos en matadero. La cantidad de espermatozoides unidos a la ZP se evaluaron con ayuda de Hoescht 33342 mediante microscopía de fluorescencia. Las diferencias temporales en el transcriptoma del oviducto se evaluaron mediante análisis de micromatrices y RT-qPCR, haciéndose las correspondientes preparaciones inmunohistoquímicas y análisis de proteínas secretadas al fluido oviductal (FO) mediante western blot. Los resultados de la primera parte muestran que se puede separar subpoblaciones espermáticas en función de su afinidad por sondas glucídicas, y que estas poblaciones tienen diferente afinidad por la ZP, lo que les confiere diferente capacidad para fecundar un ovocito. Los resultados del análisis mediante micromatrices muestran un total de 602 transcritos diferencialmente expresados entre las etapas analizadas (pre-ovulatoria, post-ovulatoria, oviducto con embriones de ocho células y oviducto con mórulas). Además, se identificaron tres proteínas del (FO) con una expresión diferente durante las etapas estudiadas: la osteopontina (SPP1), cuya expresión es mayor en la etapa post-ovulatoria (en presencia de zigotos); el neuropéptido Y (NPY), secretado antes de la ovulación; la metalopeptidasa 7 (MMP7), que deja de expresarse en las últimas fases del desarrollo pre-implantacional. Como conclusiones de la primera parte de la tesis: 1) existen varias subpoblaciones espermáticas con distintas afinidades por azúcares que poseen diferente afinidad por la ZP, 2) es posible separar las subpoblaciones espermáticas en base a su afinidad por glicoconjugados mediante sorting, pero su afinidad por la zona pelúcida se compromete, 3) es necesario implementar un método de separación diferente al de sorting que no disminuya la afinidad de los espermatozoides por la zona pelúcida. En cuanto a la segunda parte de la tesis: 1) el transcriptoma del oviducto de la coneja presenta diferencias espaciales y temporales durante la ovulación y el desarrollo embrionario pre-implantacional; 2) Varias proteínas del FO varían entre la cópula y el final del desarrollo embrionario pre-implantacional, incluyendo SPP1, NPY y MMP7; 3) La respuesta a la ovulación y la cópula presenta ciertos mecanismos moleculares conservados entre diferentes especies de mamíferos, favoreciendo la expresión de SERPINE1, SPP1 y PTGS2; 4) La respuesta del oviducto de los mamíferos a la presencia de embriones involucra una modulación local del sistema inmune. Aún así, no se ha esclarecido un mecanismo de respuesta universal debido a la discrepancia de resultados entre especies.The oviduct is the anatomical region of the female genital tract which connects the utherus with the ovaries. This organ plays a relevant role in gamete transport and storage, including the sperm reservoir. Moreover, fecundation and the first stages of embryo development take place in the oviduct. In the first part of this thesis, a technique on boar spermatozoa separation was developed on the bases of affinity for sugars involved in the sperm reservoir. A study on the ability of these spermatozoa to bind the zona pellucida (ZP) was done to evaluate a possible use on assisted reproduction techniques. In the second part of the thesis, a transcriptomics study of the oviduct of the rabbit doe was performed before and after ovulation, and during the preimplantational embryo development to identify key factors of each stage. Among the experimental techniques used, there was flow cytometry cell sorting to select the sperm subpopulations pre-incubated with fluorescent ovalbumin and the sugar Lewisa. Affinity for ZP was evaluated by incubating in capacitating medium each sperm subpopulation and its correspondent control with isolated ZPs from oocytes obtained from ovaries of a local abatoir. The quantity of sperm bound to the ZP was evaluated with Hoescht 33342 by fluorescence microscopy. The temporal differences in the transcriptome of the oviduct were evaluated by microarray analysis and RT-qPCR, doing the correspondent immunohistochemistry techniques. The analysis of secreted proteins to the oviductal fluid (OF) by western blot. Results show that it is possible to separate sperm subpopulations based on their affinity for sugar probes, and these subpopulations have a different affinity for the ZP, so they have a distinct fertilization potential. Microarray analysis show 602 transcripts differentially expressed between the analysed stages (pre-ovulatory, post-ovulatory, 8-cell containing oviduct and morulae containing oviduct). Furthermore, three oviductal proteins with a differential expression during the analysed stages were identified: osteoponting (SPP1), whose expression is highest in the post-ovulatory stage; neuropeptide Y (NPY), secreted before ovulation; metallopeptidase 7 (MMP7), whose expression is repressed during the last stages of pre-implantational development. To conclude with the first part of the thesis: 1) there are several sperm subpopulations with different affinities for sugars which have different affinities for the ZP, 2) it is feasible to separate these sperm subpopulations by flow cytometry cell sorting, but its affinity for the ZP is compromised, 3) it is mandatory to implement a method of sperm selection not involving sorting in order to avoid disminishing the affinity for the ZP. About the second part of the thesis: 1) rabbit doe oviductal transcriptome shows spatial and temporal differences through ovulation and pre-implantational development; 2) Several OF protein concentrations fluctuate along copulation and the end of pre-implantational development, among those: SPP1, NPY and MMP7; 3) Response to ovulation and copulation involves certain molecular mechanisms conserved across different mammalian species, favouring the expression of SERPINE1, SPP1 and PTGS2; 4) Response to embryo presence within the oviduct involves a local regulation of the immune system. However, it has not been discovered a clear universal mechanism of response due to discrepancy of results among species

Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates

Departamento de Reproducción Animal​ (INIA)Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated in vitro, hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching in vitro culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to in vitro produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching in vitro culture system able to directly determine developmental potential circumventing the need of experimental animals.Research on conceptus elongation carried out by the authors and the publication costs of this review were funded by the projects StG-757886-ELONGAN from the European Research Council and AGL2017-58739-R from the Spanish Ministry of Economy and Competitiveness (MINECO). Images shown on Figure 2 were taken on an equipment funded by the project ECQ2018-005184-P from MINECO. PR-I is funded by a Ramón y Cajal Contract from MINECO (RYC2018-025666-I).The authors thank the slaughterhouses Transformación Ganadera de Leganés SA, Matadero Madrid-Norte SA, and Matadero Mondejano SL (especially to Reyes Prieto Cabañas) for gently providing the ovaries required for research on ungulate lineage differentiation markers.Peer reviewed13 Pág

The human cumulus cell transcriptome provides poor predictive value for embryo transfer outcome

9 Pág.Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence?This work was funded by the projects IND2017/BIO-7748 from the Madrid Region Government, and AGL2017-84908-R and PID2020-117501RB-I00 from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). A.M.-M. was funded by project IND2017/BIO-7748, and I.L.-T. by an FPI fellowship by MINECO.Peer reviewe

The Sperm Olfactory Receptor OLFR601 is Dispensable for Mouse Fertilization

11 Pág. Departamento de Reproducción Animal​ (INIA)Fertilization involves the fusion of two gametes by means of yet unknown membrane binding and fusion events. Over the last years, many sperm proteins have been uncovered to play essential roles in sperm-egg fusion in mammals, but their precise role in fertilization remains unknown, being unclear how these proteins interact with each other or with other yet unknown sperm proteins. The aim of this study has been to identify possible sperm proteins interacting with TMEM95, a protein essential for fertilization located in the sperm membrane. A list of 41 sperm proteins that were pulled down with TMEM95 and identified by mass spectrometry did not include other sperm proteins known to play a role in fertilization, suggesting an independent role of TMEM95 in fertilization. Between these lists, OLFR601 is allocated to the acrosomal region and may mediate affinity for an odorant involved in fertilization. However, Olfr601 disruption did not impair the sperm fertilization ability, suggesting that its function may be redundant with that of other sperm proteins.This work was supported by the projects AGL 2017-84908-R and PID 2020-117501RB-I00 to B-AP and PID 2020-114109GB-I00 to J-MM, funded by the Spanish Ministry of Science and Innovation and FEDER funds from the European Union.Peer reviewe