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Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection

Identifikátory výsledku

  • Kód výsledku v IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F21%3A00550572" target="_blank" >RIV/86652036:_____/21:00550572 - isvavai.cz</a>

  • Výsledek na webu

    <a href="https://bio-protocol.org/e4193" target="_blank" >https://bio-protocol.org/e4193</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.21769/BioProtoc.4193" target="_blank" >10.21769/BioProtoc.4193</a>

Alternativní jazyky

  • Jazyk výsledku

    angličtina

  • Název v původním jazyce

    Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection

  • Popis výsledku v původním jazyce

    Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation, instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the uterotubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37 degrees C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time-and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating (Ded et al., 2020). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm.

  • Název v anglickém jazyce

    Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection

  • Popis výsledku anglicky

    Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation, instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the uterotubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37 degrees C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time-and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating (Ded et al., 2020). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm.

Klasifikace

  • Druh

    J<sub>imp</sub> - Článek v periodiku v databázi Web of Science

  • CEP obor

  • OECD FORD obor

    10608 - Biochemistry and molecular biology

Návaznosti výsledku

  • Projekt

    Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

  • Návaznosti

    P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Ostatní

  • Rok uplatnění

    2021

  • Kód důvěrnosti údajů

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Údaje specifické pro druh výsledku

  • Název periodika

    Bio-protocol

  • ISSN

    2331-8325

  • e-ISSN

  • Svazek periodika

    11

  • Číslo periodika v rámci svazku

    20

  • Stát vydavatele periodika

    US - Spojené státy americké

  • Počet stran výsledku

    3

  • Strana od-do

    e4193

  • Kód UT WoS článku

    000713687100010

  • EID výsledku v databázi Scopus

    2-s2.0-85119158509