Yu Matsumoto; Ban Sato; Masafumi Inui; Manato Sunamoto; Natsuko Kawano; Kenji Miyado

doi:10.21769/BioProtoc.5544

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In Press文章, 已正式接受发表，还没有分配卷号和期号。

Peer-reviewed

Methods for Collecting and Analyzing Post-Ejaculatory Uterine Fluid and the Uterus in Mice

YM Yu Matsumoto email

In Press, 发布时间: 2025年11月24日 DOI: 10.21769/BioProtoc.5544 浏览次数: 59

评审: Anonymous reviewer(s)

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Cover of microPublication Biology, featuring study using the protocol.

Nov 2025

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Abstract

In mammals, the semen is ejaculated into the female reproductive tract, and the sperm travel to the oviduct to fertilize the egg. A comprehensive understanding of the pre- and post-ejaculatory intrauterine environment is one of the key points for overcoming infertility; however, the dynamics of the intrauterine environment and its physiological role in the uterus, namely in the internal fertilization process, remain unclear. Conventional methods for collecting uterine fluids from the uterus post-ejaculation of mice show challenges regarding the ambiguous ejaculation timing. Here, we established a method for a mating environment with exact ejaculation timing. We also created a simple method for collecting pre- and post-ejaculatory uterine fluid without using forceps. Our methods achieved time-dependent biochemical and histological analyses of uterine fluids to provide fundamental information regarding protein composition and uterine structure changes during pre- and post-ejaculation. This protocol is suitable for analyzing temporal changes in reproductive phenomena, thereby contributing to elucidating the physiological role of the uterus in the process of intrauterine fertilization.

Key features

• This protocol is used for the simple collection of pre- and post-ejaculatory uterine fluid.

• Changes in the pre- and post-ejaculatory intrauterine environment can be examined by controlling the dissection time of females after ejaculation.

• An estrous female can be determined without a vaginal smear test in this protocol.

• This protocol can be used to analyze the protein composition of post-ejaculatory uterine fluid and is applicable to analyze sperm within the uterus post-ejaculation.

Keywords: Mating

Internal fertilization

Intrauterine environment

Biochemical analysis

Histological analysis

Reproductive biology

Graphical overview

Methods for collecting and analyzing post-ejaculatory uterine fluid and the uterus

Background

The female reproductive tract comprises the vagina, uterus, oviducts, and ovaries. Mammals reproduce by internal fertilization, whereby the male ejaculates semen into the uterus or vagina. Ejaculated sperm fertilize eggs through a complex process in the female reproductive tract [1]. Mice, which share mechanistic features with humans in reproductive phenomena such as internal fertilization and implantation, are extremely useful as model animals. Previous studies in mice have reported that regulation of sperm migration in the uterotubal junction (UTJ) and sperm storage and capacitation in the oviduct are essential subprocesses of fertilization [2–4]. Although a recently published article suggested that an abnormal fluid environment within the post-ejaculatory uterus contributes to infertility [5], the role of the post-ejaculatory uterus in internal fertilization remains unclear compared to the UTJ and oviduct. To address this issue, it is necessary to establish protocols that allow evaluation of pre-ejaculatory, post-ejaculatory (immediately post-ejaculation or pre-fertilization), and post-ejaculatory (post-fertilization) intrauterine environment. The conventional protocol for collecting post-ejaculatory uterine fluid (eUF) from the uterus post-ejaculation is limited in that it cannot collect eUF immediately after post-ejaculation. Therefore, we propose a simple method for collecting pre-and post-ejaculatory (immediately post-ejaculation) uterine fluid and uterus, followed by biochemical and histological analyses. This protocol provides a novel approach to study dynamics of the uterus, sperm (motility, capacitation, and survival), and fertilization in internal fertilization, which is expected to contribute to elucidating the physiological role of the uterus.

Materials and reagents

Biological materials

1. ICR male and female mice (Japan SLC, Inc.)

Reagents

1. Ethanol 70% (Yoshida Pharmaceutical Company, Ecosyoueta Disinfectant Solution, catalog number: 14987288980046)

2. Pierce^TM bovine serum albumin standard ampules, 2 mg/mL (Thermo Fisher Scientific, catalog number: 23209)

3. Pierce^TM BCA Protein Assay kit (FUJIFILM Wako Pure Chemical Corp., catalog number: 297-73101)

4. Nunc^TM MicroWell^TM 96-well, Nunclon Delta-treated, flat-bottom microplate (Thermo Fisher Scientific, catalog number: 167008)

5. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M314835406-91)

6. Sodium dodecyl sulfate (SDS) (FUJIFILM Wako Pure Chemical, catalog number: 191-07145)

7. Glycerol (FUJIFILM Wako Pure Chemical, catalog number: 075-00616)

8. Bromophenol blue (FUJIFILM Wako Pure Chemical, catalog number: 029-02912)

9. Tris(hydroxymethyl)aminomethane (Tris) (NACALAI TESQUE, Inc., catalog number: 35406-91)

10. Hydrochloric acid (HCl) (FUJIFILM Wako Pure Chemical, catalog number: 080-01066)

11. Acrylamide/bis solution 37.5:1 at 30% (Bio-Rad Laboratories, Inc., catalog number: 1610158)

12. N,N,N',N'-Tetramethylethylenediamine (TEMED) (FUJIFILM Wako Pure Chemical, catalog number: 205-06313)

13. Ammonium persulfate (APS) (FUJIFILM Wako Pure Chemical, catalog number: 802811)

14. Urea (FUJIFILM Wako Pure Chemical, catalog number: 219-00175)

15. Glycine (FUJIFILM Wako Pure Chemical, catalog number: 077-00735)

16. Protein molecular weight marker (FUJIFILM Wako Pure Chemical, catalog number: 234-02464)

17. Coomassie brilliant blue R-250 (CBB-R250) (Thermo Fisher Scientific, catalog number: 20278)

18. Acetic acid (FUJIFILM Wako Pure Chemical, catalog number: 017-00251)

19. Methanol (FUJIFILM Wako Pure Chemical, catalog number: 131-01826)

20. 10× Phosphate-buffered saline (PBS) (TOHO Co., Ltd., catalog number: 12-9423-5)

21. Bouin's solution (FUJIFILM Wako Pure Chemical, catalog number: 023-17361)

22. Sucrose (FUJIFILM Wako Pure Chemical, catalog number: 196-00015)

23. Tissue-Tek^® Cryomold^® (Sakura Finetek Japan Co., Ltd., catalog number: 4557)

24. Optimal cutting temperature (OCT) compound (Sakura Finetek Japan, catalog number: 45833)

25. Liquid nitrogen

26. New hematoxylin solution type M (Muto Pure Chemicals, catalog number: 30142)

27. Eosin Y solution at 1% (Muto Pure Chemicals, catalog number: 32002)

28. Ethanol 100% (Muto Pure Chemicals, catalog number: 43105)

29. Xylene (Muto Pure Chemicals., catalog number: 43122)

30. Mounting medium (New M·X) (Matsunami Glass Industry Co., Ltd., catalog number: FX00500)

Solutions

1. Tris/HCl (pH 6.8, pH 8.8), 1 M (see Recipes)

2. 6× Sample buffer (see Recipes)

3. SDS 10% (see Recipes)

4. APS 25% (see Recipes)

5. Stacking gel solution (see Recipes)

6. Separation gel solution (see Recipes)

7. Running buffer (see Recipes)

8. CBB staining solution (see Recipes)

9. CBB decolorizing solution (see Recipes)

10. Eosin solution (see Recipes)

Recipes

1. Tris/HCl (pH 6.8, pH 8.8), 1 M

Reagent	Final concentration	Quantity or volume
Tris	1 M	60.57 g
Adjust to pH 6.8 and pH 8.8 with HCl
Distilled water	n/a	<500 mL
Total	n/a	500 mL

Note: pH 6.8 and pH 8.8 Tris/HCl solutions are prepared separately.

2. 6× sample buffer

Reagent	Final concentration	Quantity or volume
Tris/HCl (pH 6.8), 1 M	350 mM	3.5 mL
2-Mercaptoethanol	30% (v/v)	3 mL
SDS	10% (w/v)	1 g
Glycerol	30% (v/v)	3 mL
Bromophenol blue	0.06% (w/v)	6 mg
Distilled water	n/a	0.5 mL
Total	n/a	10 mL

3. SDS 10% (w/v)

Reagent	Final concentration	Quantity or volume
SDS	10% (w/v)	20 g
Distilled water	n/a	<200 mL
Total	n/a	200 mL

4. APS 25% (w/v)

Reagent	Final concentration	Quantity or volume
APS	25% (w/v)	2.5 g
Distilled water	n/a	<10 mL
Total	n/a	10 mL

5. Stacking gel solution

Reagent	Quantity or volume
30% Acrylamide/bis solution 37.5:1	375 μL
Tris/HCl (pH 6.8), 1 M	468.75 μL
SDS, 10% (w/v)	37.5 μL
TEMED	3 μL
Distilled water	2856.25 μL
Total	3740.6 μL

6. Separation gel solution

Reagent	Quantity or volume
30% Acrylamide/bis solution 37.5:1	2000 μL
Tris/HCl (pH 8.8), 1 M	2812.5 μL
SDS, 10% (w/v)	75 μL
TEMED	6 μL
Distilled water	2587.5 μL
Total	7481 μL

7. Running buffer

Reagent	Final concentration	Quantity or volume
Tris	25 mM	3.03 g
SDS	0.1% (w/v)	1 g
Glycine	192 mM	14.4 g
Distilled water	n/a	<1,000 mL
Total	n/a	1,000 mL

8. CBB staining solution

Reagent	Final concentration	Quantity or volume
CBB R-250	0.25% (w/v)	0.5 g
Acetic acid	10% (v/v)	20 mL
Methanol	50% (v/v)	100 mL
Distilled water	n/a	80 mL
Total	n/a	200 mL

9. CBB decolorizing solution

Reagent	Final concentration	Quantity or volume
Acetic acid	7.5% (v/v)	15 mL
Methanol	25% (v/v)	50 mL
Distilled water	n/a	135 mL
Total	n/a	200 mL

10. Eosin solution

Reagent	Quantity or Volume
1% Eosin Y solution	100 mL
60% ethanol	500 mL
Acetic acid	0.6 mL
Total	600.6 mL

Laboratory supplies

1. Disposable latex gloves (ASKUL, catalog number: WE59721)

2. Microcentrifuge tubes 1.5 mL (Greiner Bio-One Co., Ltd., catalog number: 616201)

3. Centrifuge tubes 15 mL (AS ONE, catalog number: 4-3632-01)

4. Pipette tips 10 μL (WATSON, catalog number: 110-207C)

5. Pipette tips 200 μL (WATSON, catalog number: 110-705C)

6. Pipette tips 1,000 μL (WATSON, catalog number: 110-7-6C)

7. Gel loading tips 200 μL (BM Equipment Co., Ltd., catalog number: 010-Q)

8. Kimwipes (NIPPON PEPAR CRECIA Co., Ltd., catalog number: 62020)

9. Paper towels (ASKUL, catalog number: 1944368)

10. Dish 60 mm in size (AGC TECHNO GLASS Co., Ltd., catalog number: 1010-060)

11. Absorbent paper (ATTO Corp., catalog number: CB-06A)

12. Glass slide (Muto Pure Chemicals Co., Ltd., catalog number: 513617)

13. 24 mm × 50 mm cover glass (thickness: 0.13–0.17 mm) (Matsunami Glass Industry, catalog number: C024501)

Equipment

1. Personal protective equipment (e.g., mask, goggles, and lab coats)

2. Precision balance (Mettler Toledo, model: PB602-S)

3. P-20 pipette (Gilson, model: F123600)

4. P-200 pipette (Gilson, model: F123601)

5. P-1000 pipette (Gilson, model: F120602)

6. Small straight scissors (Natsume Seisakusho, model: B-12)

7. Large straight scissors (Natsume Seisakusho, model: B-3)

8. Tweezers (AS ONE, model: 2-529-12)

9. Plastic cages (Clea Japan, Inc., model: CL-0103-2 Mouse TPX)

10. Water bottles, rubber stoppers (Clea Japan, Inc., model CL-0904)

11. SpectraMax (Molecular Devices, model: SpectraMax^® iD5e)

12. 1 mm dual mini gel cast (glass plates, seal gasket, comb, and clips) (ATTO, model: AE-6401)

13. pH meter (HIRIBA, model: F-72)

14. Beaker glass, 500 mL (AS ONE, model: 2-5091-06)

15. Magnetic stirrer (Thermo Fisher Scientific, model: Magnetic stirrer RT Basic-12)

16. Stirrer (AS ONE, model: 3-6657-02)

17. Electrophoresis system (ATTO, model: WSE-1100)

18. Rocking mixer (AS ONE, model: 1-5829-22)

19. Cryostat (Thermo Fisher Scientific, model: CryoStar NX50)

20. Microscope (Keyence Corp., model: BZ-X700)

Software and datasets

1. Microsoft Excel version 16.102.1 (Microsoft Corporation)

2. BioRender (https://www.biorender.com/)

Procedure

文章信息

稿件历史记录

提交日期: Sep 27, 2025

接收日期: Nov 16, 2025

在线发布日期: Nov 24, 2025

版权信息

如何引用

Matsumoto, Y., Sato, B., Inui, M., Sunamoto, M., Kawano, N. and Miyado, K. (2025). Methods for Collecting and Analyzing Post-Ejaculatory Uterine Fluid and the Uterus in Mice. Bio-protocol 15(24): e5544. DOI: 10.21769/BioProtoc.5544.