Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Hamze, Julieta Gabriela; Jiménez-Movilla, María; Romar, Raquel

doi:10.1530/rep-20-0123

Cited by 3 publications

(8 citation statements)

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“…The choice of employing a model featuring (Alternate Protocol 1) or lacking cumulus cells will depend on the availability of material and the final objective of the study. Our results show that the sperm-binding pattern is similar using both versions of the model (Hamze et al, 2019(Hamze et al, , 2020b. The advantage of using the most complete version of the model (i.e., beads coated with cumulus cell over the protein layer) is that it is a more physiological approach and might be a better option to reveal subtle features of sperm in certain studies, or from certain individuals (animals or humans).…”

Section: Introductionmentioning

confidence: 69%

“…Moreover, a higher number of sperm per bead is expected when comparing the experimental group (protein-coated beads) with the control group (beads conjugated with non-transfected CHO cell medium). This indicates that sperm can specifically bind to the studied recombinant proteins (porcine ZP2 and IZUMO1 receptor, JUNO; 'figure 1D' in Hamze et al, 2020b;'figure 2B' in Hamze et al, 2020a). Therefore, toxicological studies could be carried out by adding toxic compounds to the incubation medium and monitoring for potential changes in sperm binding.…”

Section: Of 24mentioning

confidence: 87%

“…The experimental groups would be the protein-coated beads and control beads from Basic Protocol 1. For example, in Hamze, Jiménez-Movilla, & Romar (2020b) there were four experimental groups: (i) ZP2-coated beads, (ii) ZP3-coated beads, (iii) ZP4-coated beads, and (iv) control beads (where beads were not conjugated to any recombinant protein).…”

Section: Of 24mentioning

confidence: 99%

“…The choice of protein expression system (Strategic Planning) is essential to ensure the functionality of the designed recombinant protein. If the protein is a mammalian glycoprotein, it is advisable to use a mammalian cell line such as CHO to ensure proper glycosylation, as described in this work and in our previous studies with porcine ZP recombinant proteins (Hamze et al, 2019(Hamze et al, , 2020b and bovine IZUMO1 receptor (JUNO protein; Hamze et al, 2020a). Moreover, when designing the protein construct, it is recommended to add the histidine tail (6×His) at the N-or C-terminus, to try to avoid altering protein domains.…”

Section: Critical Parametersmentioning

confidence: 99%

“…Thanks to the ability of this model to decoy sperm, once the beads are coated with proteins (and cumulus cells, if desired), they can be subjected to sperm-binding assays, with the objective of gaining knowledge of the molecular basis of gamete interaction, selecting sperm with the highest fertilizing ability, or even predicting sperm fertility in the future (Hamze et al, 2019(Hamze et al, , 2020a(Hamze et al, , 2020b.…”

Section: Introductionmentioning

confidence: 99%

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Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

2020

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We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus‐oocyte complex. Once generated, this 3D model can be used in a sperm‐binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm‐binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS‐PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm‐binding assay Alternate Protocol 2: Sperm preparation by the swim‐up method

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Section: Introductionmentioning

confidence: 69%

Section: Of 24mentioning

confidence: 87%

Section: Of 24mentioning

confidence: 99%

Section: Critical Parametersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

2020

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Oolemma Receptors in Mammalian Molecular Fertilization: Function and New Methods of Study

Jiménez-Movilla

Hamze

Romar

2021

Front. Cell Dev. Biol.

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Fertilization is a key process in biology to the extent that a new individual will be born from the fusion of two cells, one of which leaves the organism in which it was produced to exert its function within a different organism. The structure and function of gametes, and main aspects of fertilization are well known. However, we have limited knowledge about the specific molecules participating in each of the steps of the fertilization process due to the transient nature of gamete interaction. Moreover, if we specifically focus in the fusion of both gametes’ membrane, we might say our molecular knowledge is practically null, despite that molecular mechanisms of cell-to-cell adhesion are well studied in somatic cells. Moreover, between both gametes, the molecular knowledge in the egg is even scarcer than in the spermatozoon for different reasons addressed in this review. Sperm-specific protein IZUMO1 and its oocyte partner, JUNO, are the first cell surface receptor pair essential for sperm–egg plasma membrane binding. Recently, thanks to gene editing tools and the development and validation of in vitro models, new oocyte molecules are being suggested in gamete fusion such as phosphatidylserine recognition receptors. Undoubtedly, we are in a new era for widening our comprehension on molecular fertilization. In this work, we comprehensively address the proposed molecules involved in gamete binding and fusion, from the oocyte perspective, and the new methods that are providing a better understanding of these crucial molecules.

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Genome-Wide Association Study Reveals Novel Candidate Genes Influencing Semen Traits in Landrace Pigs

Zhuang,

Li,

Yang

et al. 2024

Animals

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Artificial insemination plays a crucial role in pig production, particularly in enhancing the genetic potential of elite boars. To accelerate genetic progress for semen traits in pigs, it is vital to understand and identify the underlying genetic markers associated with desirable traits. Herein, we genotyped 1238 Landrace boars with GeneSeek Porcine SNP50 K Bead chip and conducted genome-wide association studies to identify genetic regions and candidate genes associated with 12 semen traits. Our study identified 38 SNPs associated with the analyzed 12 semen traits. Furthermore, we identified several promising candidate genes, including HIBADH, DLG1, MED1, APAF1, MGST3, MTG2, and ZP4. These candidate genes have the potential function to facilitate the breeding of boars with improved semen traits. By further investigating and understanding the roles of these genes, we can develop more effective breeding strategies that contribute to the overall enhancement of pig production. The results of our study provide valuable insights for the pig-breeding industry and support ongoing research efforts to optimize genetic selection for superior semen traits.

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Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Cited by 3 publications

References 34 publications

Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

Oolemma Receptors in Mammalian Molecular Fertilization: Function and New Methods of Study

Genome-Wide Association Study Reveals Novel Candidate Genes Influencing Semen Traits in Landrace Pigs

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