Počet záznamů: 1
Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection
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SYSNO ASEP 0550572 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Collection of in vivo Capacitated Sperm from Different Locations Along the Reproductive Tract of Time-Mated Female Mice by Microdissection Tvůrce(i) Děd, Lukáš (BTO-N) RID
Chung, J.-J. (US)Celkový počet autorů 2 Číslo článku e4193 Zdroj.dok. Bio-protocol. - : Bio-protocol - ISSN 2331-8325
Roč. 11, č. 20 (2021)Poč.str. 3 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova Mice ; Oviduct ; Sperm ; Microdissection ; Timed mating ; Microscopy Vědní obor RIV EB - Genetika a molekulární biologie Obor OECD Biochemistry and molecular biology CEP GJ20-17403Y GA ČR - Grantová agentura ČR ED1.1.00/02.0109 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy Způsob publikování Open access Institucionální podpora BTO-N - RVO:86652036 UT WOS 000713687100010 EID SCOPUS 85119158509 DOI 10.21769/BioProtoc.4193 Anotace 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. Pracoviště Biotechnologický ústav Kontakt Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Rok sběru 2022 Elektronická adresa https://bio-protocol.org/e4193
Počet záznamů: 1