Penicillamine

After 20–24 h of IVM, COCs were washed twice in TL medium before being transferred in groups of 5–48 μl droplets under mineral oil. The IVF droplets consisted of modified TL medium supplemented with fatty-acid-free BSA (0.6% w/v), pyruvic acid (0.2 mM), heparin (2 μg/mL) and gentamycin (50 mg/mL). COCs were transferred to IVF droplets 15 min prior to adding the spermatozoa. To stimulate sperm motility, penicillamine (2 mM; Sigma-Aldrich), hypotaurine (1 mM; Sigma-Aldrich) and epinephrine (250 mM; Sigma-Aldrich) were added to each droplet. The selected spermatozoa were counted using a hemocytometer and diluted with IVF medium to obtain a final concentration of 1 × 10⁶ sperm/mL. Finally, 2 μL of the sperm suspension was added to the droplets containing the matured COCs. The fertilization medium was incubated at 38.5°C for 18 h in a humidified atmosphere of 95% air and 5% CO₂. Presumptive zygotes were denuded by treatment with 0.1% bovine testicular hyaluronidase.

In vitro-matured COCs were fertilized with frozen-thawed bovine sperm from a batch that had previously been used in our laboratory. COCs (10/drop) were transferred to 50 μL FERT-TALP medium (IVL02, Caisson Laboratories, 836 South 100 East Smithfield, UT 84335) supplemented with 0.1 mg/mL Heparin (Sigma H0519), 1 mM Hypotaurine (Sigma H1384), 250 mM Epinephrine (Sigma E4642), 2 mM Penicillamine (Sigma P4875), and 1x antibiotic solution (ICN 1670049 MP Biomedicals, Fisher Scientific Company, Ottawa, ON, USA). Semen was thawed at 36 °C for 1 min, and motile spermatozoa were separated using percoll gradients (Allgrad^® 90% and 45% LifeGlobal group, 393 Soundview Rd, Guilford, CT 06437) by centrifugation at 780× g for 5 min at room temperature. The pellet was diluted with 300 μL of FERT-TL media, and the final concentration was adjusted at 2 × 10⁶ sperm/mL, and added in each drop containing COCs [11 (link)]. COCs and semen were incubated for 18 to 20 h at 38.5 °C in a humidified atmosphere of 5% CO₂ in air [23 (link),41 (link)].

Bucillamine was purchased from FUJIFILM Wako Chemicals USA Corporation (Richmond, VA, USA) or Santa Cruz Biotechnology (Dallas, TX, USA); penicillamine, MESNA, and minocycline from Sigma; afatinib and afatinib dimaleate from Selleck (Houston, TX, USA. Antibodies against EGFR and pEGFR (Y1068) were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG antibodies were purchased from GE Healthcare (Chicago, IL, USA). The A431 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37 °C.

To investigate whether sperm entry point
(SEP) is random or preferential in bovine,
oocytes (n=311) were submitted to in vitrofertilization (IVF) as described previously (10 (link)).
In brief, frozen-thawed and washed sperm from
a single Holstein sire of proven in vitro fertility
were used for fertilization after capacitation by
the swim-up procedure. Spermatozoa (1×10⁶/
ml sperm) and matured COCs (40-45 COCs/200
μl) were co-incubated in modified fertilization-
Tyrode’s albumin lactate pyruvate (TALP,
handmade) medium containing 0.01 mM
heparin (Sigma, USA), 0.2 mM penicillamine
(Sigma, USA) and 0.1 mM hypotaurine
(Sigma, USA) for 18 hours at 39.5˚C, 6%
CO₂ in humidified air. Fertilized oocytes at
4-5 hours post fertilization (hpf) were fixed,
stained with Hoechst-33342 and visualized
with a fluorescence-assisted micromanipulator
microscope (Olympus, Japan). Schurmann et al.
(14 (link)) demonstrated 4-6 hpf as the optimum timepoint
of maximum early fertilization. To map
the SEP, eggs were rotated under constant UVlight
until MII-spindle (the approved reference
point of SEP) was positioned to 3 O’clock.
Then, the spatial relationship between SEP and MII-spindle was measured as described
by Motosugi et al. (Fig .2) (15 (link)). In this scheme,
zones I and IV are the closest and the furthest
from the MII-spindle, respectively.

The following reagents were used in the study: Hank’s medium (SIGMA, USA), gentamicin (SIGMA, USA), penicillin (SIGMA, USA), streptomycin (SIGMA, USA), PBS (SIGMA, USA), TCM-199 HEPES (SIGMA, USA), foetal bovine serum (FBS) (GIBCO, Scotland), mineral oil (SIGMA, USA), FSH (SIGMA, USA), β-oestradiol (SIGMA, USA), sodium pyruvate (MERCK, France), BSA fraction V (SIGMA, USA), BSA fraction FAF (SIGMA, USA), penicillamine (SIGMA, USA), hypotaurine (SIGMA, USA), epinephrine (SIGMA, USA), High Pure RNA Isolation Kit (Roche Diagnostics), SYBR Green (Roche Applied Science), Hybond-ECL nitrocellulose membranes (Amersham Biosciences), Trypan blue (SIGMA) antibody against HSP70 (Abcam, ab6535), antibody against OVGP1 (Abcam, ab74544), rabbit anti-mouse secondary antibodies (Santa Cruz Biotechnology, sc-2005), goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, sc-2004), ECL Plus (Amersham Life Sciences), Fluoromount (Sigma USA).

Cell fractionations were performed with Cell Fractionation Kit (CST9038). Kinase inhibitors Wortmannin (Selleck S2758), and LY294002 (Selleck S1105) were used at the indicated doses. Etoposide (Sigma, E1383) and doxorubicin (Selleck, S1208), insulin (Invitrogen 41400‐045), EGF (Sigma E9644), PDGF (Sigma SRP3123) and IGF (Sigma SRP3069) were used at the indicated doses. MG132 (Enzo Life Science, BML‐PI102) was used at the indicated doses. TTM and penicillamine were purchased from Sigma. Metals including CuSO₄, ZnCl₂, Fe(NO₃)₃, Ni(NO₃)₂, AgNO₃, LiCl, MnCl₂, CaCl₂, and MgCl₂ were purchased from Fisher Scientific. Copper‐PDC beads were purchased from Affiland. Metal resin was generated with the metal ions with Glutathione‐Sepharose slurry (Pierce) or NTA Agarose (Qiagen 30310) for 1 h in room temperature and washed twice with phosphate buffered saline (PBS) buffer.

Cystine, sodium chloride, sodium cyanide, sodium nitroprusside, tiopronin, N-acetylcysteine, and penicillamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical/reagent grade and dissolved in ultra-pure deionized water from a Milli-Q system. A Cystine stock solution (700 g/L, 2.9 mM) was prepared daily by dissolving 0.35 g of Cystine and 8.33 g of NaCl in 0.5 L of ultra-pure deionized water, followed by the addition of 3 M NaOH to achieve a final pH of 8.0.