Embryomax human tubal fluid

Manufactured by Merck Group

Sourced in United States

EmbryoMax Human Tubal Fluid is a laboratory product designed to mimic the natural environment of the human fallopian tube. It is formulated to support the growth and development of human embryos in an in vitro setting.

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Human Tubal Fluid EmbryoMax

8 protocols using embryomax human tubal fluid

In Vitro Fertilization Procedure with Mouse Sperm

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Twelve-week-old CD1 male mice (

lowercase italic n equals 2

) were euthanized, and the epididymis and vas deferens were harvested via abdominal dissection and placed in preequilibrated EmbryoMax human tubal fluid (HTF) medium (Cat. no. MR-070-D; Sigma). Dissection needles were used to retrieve sperm. Following a 10-min incubation, sperm was counted using a Makler chamber. From each male donor, 1 million sperm were added to a preequilibrated in vitro fertilization dish containing human tubal fluid (HTF) medium. Dishes were incubated for 1.5 h before the addition of COCs. Sperm and oocytes were then incubated together for 4 h. Following insemination, oocytes were washed through four droplets of preequilibrated potassium simplex optimized medium (KSOM; Cat. no. MR-121-D; Sigma) under Ovoil (Cat. no. 10029; Vitrolife).

Pascuali N., Pu Y., Waye A.A., Pearl S., Martin D., Sutton A., Shikanov A, & Veiga-Lopez A. (2024). Evaluation of Lipids and Lipid-Related Transcripts in Human and Ovine Theca Cells and an in Vitro Mouse Model Exposed to the Obesogen Chemical Tributyltin. Environmental Health Perspectives, 132(4), 047009.

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In Vitro Fertilization of Mouse Oocytes

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Superovulation was performed using 5-week-old C57BL/6J donors and 4-week-old B6D2F1 donors. They were injected with 5 IU eCG (n = 10 for each strain) or 100 μL IAS (n = 5 for each strain), followed by 5 IU hCG after 47 h. Oocytes were then collected for IVF on the second day of hCG injections. Two male mice (12 weeks old) from each strain were euthanised by cervical dislocation. The cauda epi didymides were collected and pre-incubated in a modified Krebs-Ringer Bicarbonate solution (Cat# 4002, Sigma-Aldrich) medium with 1.0 mg/mL polyvinyl alcohol (Cat# P8136, Sigma-Aldrich) and 0.75 mM methyl-β-cyclodextrin (Cat# C4555, Sigma-Aldrich) covered with light mineral oil (Cat# M8410, Sigma-Aldrich) at 37°C for 1 h to induce capacitation. Fertilisation was performed by adding 400-800 sperm/μL into EmbryoMax human tubal fluid (Cat# MR-070, Sigma-Aldrich) containing cumulus-oocyte complexes and cultured at 37°C, 5% CO 2 for 3 h. The above experiment was performed at the same time. Fertilisation rate was calculated at 24 h after insemination using the following formula:

Zhao X., Huang J.X., Zhang H., Gong X., Dong J., Ren H.L, & Liu Z. (2021). A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing. Reproduction, fertility, and development, 33(14).

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Murine ICSI: Oocyte Collection and Embryo Transfer

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Metaphase II‐arrested oocytes were collected from superovulated BDF1 females (3–4 weeks) or C57BL/6J females (3–4 weeks). Cumulus cells were removed using hyaluronidase (Sigma, #H3884) at 37 °C for 5 min. The cauda epididymis was washed twice with Dulbecco's phosphate buffered saline (DPBS, Gibco, #14190094), then directly put in 1 mL of EmbryoMax Human Tubal Fluid (Merck Millipore, #MR‐070‐D). Cell suspensions were exposed to ultrasound for 1–2 min. Spermatozoa without tail were picked up into a blunt piezo‐driven pipette with a tip of 10–15 μm diameter. A single sperm head was injected into a single oocyte in a droplet of M2 medium (Sigma, #M7167) containing 5 μg/mL cytochalasin B (Sigma, #C6762) using a pipette with a tip of 10–15 μm diameter, and a piezo micromanipulator controller (Japan Prime Tech, #PMAS‐CT150). Injected oocytes were maintained in KSOM medium (Merck Millipore, #MR‐106‐D) at 37 °C with 5% CO₂ in air. Two‐cell embryos were transferred into the oviducts of pseudopregnant ICR females. Offspring were born on day 19.5 of gestation.

Zhou H., Zeng Z., Koentgen F., Khan M, & Mombaerts P. (2019). The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation. Genesis (New York, N.y. : 2000), 57(6), e23295.

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Mouse Oocyte Isolation and IVF Protocol

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Superovulation was induced in 4-week-old C57BL/6 NCrl females by injection of 5-IU pregnant mare serum gonadotropin and 5-IU human chorionic gonadotropin 46 or 24 hours before oocyte isolation, respectively. Oocytes were isolated from oviducts, and cumulus cells were removed with by treatment with hyaluronidase (0.5 mg/ml; Sigma-Aldrich). When IVF experiments were performed in the absence of the ZP, cumulus-free oocytes were additionally treated for 20 s with acidic Tyrode’s solution (Sigma-Aldrich). For isolation of sperm, both epididymides isolated from a single male mouse were mechanically disrupted in cryoprotective agent (CPA) buffer (18% raffinose and 3% milk powder in sterile water), and sperms were allowed to swim out for 15 min. After counting, 5 × 10⁵ sperm cells were added to the oocytes in EmbryoMax Human Tubal Fluid (Merck Chemicals GmbH). After 5 hours, oocytes were transferred to M16 medium (Sigma-Aldrich). Binding of sperm cells to the oocytes was monitored 1 hour after insemination. Embryo development was assessed microscopically 24 and 48 hours after fertilization.

Contreras W., Wiesehöfer C., Schreier D., Leinung N., Peche P., Wennemuth G., Gentzel M., Schröder B, & Mentrup T. (2022). C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly. Science Advances, 8(31), eabo6049.

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In Vitro Fertilization in Mice

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Superovulation in females was induced as described above. HTF medium (EmbryoMax Human Tubal Fluid; Merck Millipore) was mixed 1:1 with mineral oil (Sigma-Aldrich) and equilibrated overnight at 37°C. Sperm were capacitated for 90 min in TYH medium supplemented as indicated above. On the day of preparation, 100 μl drops of HTF were covered with the medium/oil mixture and 10⁵ sperm were added to each drop. Cumulus-enclosed oocytes were prepared from the oviducts of superovulated females and added to the drops. After 4 h at 37°C and 5% CO₂, oocytes were transferred to fresh HTF. The number of 2-cell stages was evaluated after 24 h.

Balbach M., Hamzeh H., Jikeli J.F., Brenker C., Schiffer C., Hansen J.N., Neugebauer P., Trötschel C., Jovine L., Han L., Florman H.M., Kaupp U.B., Strünker T, & Wachten D. (2020). Molecular Mechanism Underlying the Action of Zona-pellucida Glycoproteins on Mouse Sperm. Frontiers in Cell and Developmental Biology, 8, 572735.

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Investigating Mouse Embryo Development

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Sperm were collected from the epididymis of 8- to 9-month-old male ICR mice and capacitated in EmbryoMax Human Tubal Fluid (HTF, Merck Millipore) for 40 minutes at 37°C and 5% CO₂. The oocyte-cumulus complexes were collected from superovulated 5- to 6-week-old or 7- to 8-week-old female mice and placed in HTF at 37°C in an atmosphere of 5% CO₂. An appropriate amount of capacitated sperm was added into the HTF liquid drops, which contained oocyte-cumulus complexes. After 6 hours, mouse zygotes were microinjected with siRNA or cRNA. siRNAs and cRNAs were as follows: Rsl1d1 siRNA (siRsl1d1, 5′–CCUCAGAUGUAUGUCUCUUTT–3′, 40 μM); synonymous mutated Rsl1d1 cRNA (1,000 ng/μL); cRNA of mClover3-RSL1D1 (1,000 ng/μL) and H₂B-mcherry (1,000 ng/μL); human KPNA7 cRNA (3,000 ng/μL); mouse Kpna7 cRNA (3,000 ng/μL); human KPNA2 cRNA (1,000 ng/μL); and mouse Kpna2 cRNA (1,000 ng/μL). After injection, zygotes were transferred to KSOM medium (Nanjing Aibei Biotechnology) for culture. Embryos were assessed at 24 hours, 48 hours, 60 hours, 72 hours, and 108 hours after fertilization. Fluorescence was observed 3 hours after injection for the RSL1D1 nuclear transport assays.

Wang W., Miyamoto Y., Chen B., Shi J., Diao F., Zheng W., Li Q., Yu L., Li L., Xu Y., Wu L., Mao X., Fu J., Li B., Yan Z., Shi R., Xue X., Mu J., Zhang Z., Wu T., Zhao L., Wang W., Zhou Z., Dong J., Li Q., Jin L., He L., Sun X., Lin G., Kuang Y., Wang L, & Sang Q. (2023). Karyopherin α deficiency contributes to human preimplantation embryo arrest. The Journal of Clinical Investigation, 133(2), e159951.

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Modulating Embryo Development via miR-451

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A total of 4–10 pl of the miR-451 inhibitor (50 μmol·l^− 1) was injected into the cytoplasm of mouse MII oocytes. An equal volume of NC inhibitor was injected into the control oocytes. Approximately 60 oocytes were injected each time, and each injection experiment was repeated at least thrice. After injection, the oocytes were introduced into M2 medium for 8 h and then used for IVF. The oocytes injected with miR-451 inhibitor or NC inhibitor were placed in 500 μl EmbryoMax Human Tubal Fluid (Millipore, Billerica, MA, USA) medium under mineral oil. After preincubation of fresh sperm, 100 μl of the sperm suspension (final concentration: 10,000–20,000 spermatozoa·ml^− 1) was added to the drop containing oocytes. The fertilization dishes were incubated at 37 °C in 5% CO₂ and 95% humidified air for at least 5 h. The inseminated oocytes were then cultured in EmbryoMaxKSOM (Millipore) medium. The 2-cell formation rate and blastocyst rate were recorded at days 2 and 4 post-fertilization.

Li X., Zhang W., Fu J., Xu Y., Gu R., Qu R., Li L., Sun Y, & Sun X. (2019). MicroRNA-451 is downregulated in the follicular fluid of women with endometriosis and influences mouse and human embryonic potential. Reproductive Biology and Endocrinology : RB&E, 17, 96.

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Super Ovulation and In Vitro Fertilization

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For super ovulation, 2–3 months old wild-type females were primed intraperitoneally (IP) with 5 IU of equine chorionic gonadotropin (eCG) hormone. Exactly 52 hours later, the primed females were injected IP with 5 IU of human chronic gonadotropin (hCG) hormone. Fourteen hours after the hCG injection, the females were sacrificed to obtain ovulated eggs. Ovaries with attached oviducts with swollen ampullae were immersed in mineral oil in a dish that also contained drops of Human Tubal Fluid (HTF) media (Millipore: EmbryoMax^® Human Tubal Fluid). The ampullae were disrupted and the cumulous oocytes complexes (COCs) were dragged from the mineral oil into a 285 μL drop of HTF media. Prior to this egg collection, spermatozoa from each cauda epididymis of 14-3-3ε conditional knockout and litter mate WT mice were extracted in 1 mL HTF media as described earlier. Following capacitation at 37°C for 1 hour in a 5% CO₂ incubator, 15 μL of the sperm suspension was added to the COCs and incubated for 4 hours at 37°C under 5% CO₂. Eggs were then washed 2 times in fresh drops of 200 μL of HTF and incubated overnight at 37°C and 5% CO₂. The following day, 2-cell stage embryos were then scored and counted.

Eisa A., Dey S., Ignatious A., Nofal W., Hess R.A., Kurokawa M., Kline D, & Vijayaraghavan S. (2020). The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility. Andrology, 9(1), 312-328.

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