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

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 utero-tubal 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°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.

*For correspondence: Lukas.Ded@ibt.cas.cz; jean-ju.chung@yale.edu [Abstract] 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°C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca 2+ . 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.
[Background] The discovery of sperm capacitation occurred in the 1950s when researchers failed to fertilize eggs in vitro by directly adding sperm to the oocytes without any prior incubation in media of similar composition close to the oviductal fluid (Austin, 1951;Chang, 1951). Since then, a vast majority of the existing information about the physiological and molecular processes surrounding capacitation was obtained using in vitro systems, which were first used in the 1970s for the conception of the first child through in vitro fertilization (IVF) (Steptoe and Edwards, 1976). Nowadays, up to 10% of children vivo capacitation, sacrifice the female mice with the confirmed vaginal plugs either by CO2 asphyxiation or cervical dislocation.
2. Under the well-lit stereo microscope, grip the fat pad around the ovary and stretch gently to clearly identify the membranous boundary between the ovary and the oviduct (line 1) and the oviduct and the uterus (line 2) (Figure 1). 3. Carefully cut through the membrane around the proximal oviduct to slightly de-coil the oviduct first (line 2), followed by the membrane around the bursa and just outside infundibulum (line 1), and lastly, cut through the uterus just outside UTJ (line 3) (Figure 1).  and pick individual sperm with an aspirator connected to a filter in the middle and 50-ul glass microcapillary by mouth pipetting (Figure 3C and 3D). 9. Depending on the speed of dissection and handling of the tissue, there will be other cell types, such as discharged epithelial cells and red blood cells (RBC), and debris together with sperm cells ( Figure 3E). As sperm cells are smaller than these somatic cells (except fine debris), transferring the liquid to the 2 nd droplet by avoiding these contaminants can help to obtain a preparation more enriched with sperm cells. and apply 50 µl per well (3-well slide) or 10 µl per well (8-well slide). Incubate the slide/coverslip in a humid chamber for ~1 h. Rinse the coating solution with water and keep the slides in the chamber prior to sperm deposition. The time between the coating and sperm deposition should be as short as possible. Ideally, the whole procedure should be tested to optimize the cell attachment and reduce the background for the subsequent immunofluorescent microscopy due to the lot-to-lot variation of fibronectin.
2. Place the picked-up sperm to 10 µl PBS droplet on the fibronectin-coated 8-well Teflon slides ( Figure 4A: At a minimum, an average of 10 sperm per sample should be obtained). If too much tissue material is transferred by micropipette, a PBS washing step can be added (Figure 3E).

Data analysis
Obtained microscopic slides can be subjected to classical procedures for immunofluorescent staining, followed by confocal or super-resolution imaging. Shown here is an example of raw data