Mr 051 f

Recombinant GDF9 protein (50 μg/mL, R&D, 739-G9-010/CF) or BSA (50 μg/mL, Sigma-Aldrich, V900933) as a control were labeled with rhodamine using the Pierce NHS-rhodamine antibody labeling kit (53031, ThermoFisher) according to the manufacturer’s protocol^{60 (link)}. To collect the oocytes, the COCs were isolated by puncturing the antral follicles from C57BL/6 mice at PD23 after 46 h of PMSG treatment (5 IU, i.p. injection). After treated with 0.3% hyaluronidase (Merck, MR-051-F) in medium, denuded oocytes were performed microinjection, and approximately 10 pL of labeled protein or rhodamine solution were injected into one oocyte with a FemtoJet 4× electric microinjector (Eppendorf). After 15 or 30 min of injections, the oocyte was imaged under an Andor Dragonfly spinning-disc confocal microscope with the previously described indexes.

To collect empty ZP from oocytes, C57BL/6 mice at PD23 with 5 IU of pregnant mare serum gonadotropin (PMSG; Sansheng Biological Technology) for 46 h and the ovaries were punctured to obtain cumulus-oocyte complexes. The cumulus cells were digested with 0.3% hyaluronidase medium (Merck, MR-051-F) to obtain denuded oocytes, which were then incubated with 10% high osmotic glucose solution to separate the ZP and the oocyte. Under the microinjection system, the ZP was torn to release the oocyte, and then the empty ZP was collected. The protein (~5 μg) from ~3000 pieces of ZP was extracted by RIPA extraction buffer for a single protein MS assay, which was repeated three times.
Protein digestion was performed using the filter-aided sample preparation (FASP) method with modifications as previously described^{53 (link),54} . Nanospray electrospray (ESI)-MS was performed on a Thermo Q-Exactive high-resolution mass spectrometer (Thermo Scientific) with a 70,000 MS scan resolution, 17,500 MS/MS scan resolution, and top-10 MS/MS selection. Raw data from the mass spectrometer were preprocessed with Mascot Distiller 2.4 for peak picking. The resulting peak lists were searched against the Uniport Mouse database using the Mascot 2.5 search engine. Scaffold PTM was used to evaluate phosphorylation sites of the Mascot search results using the Ascore algorithm.

To detect mouse fertility, 5-week-old female Oo-Rdx^−/− mice and control mice were continuously mated with 8-week-old C57BL/6 fertile males for 40 weeks. The numbers of pups and litters were recorded, from which fertility rates were determined.
For superovulation, Oo-Rdx^−/− and control mice (PD23) were intraperitoneally injected with 5 IU of PMSG, (Sansheng Biological Technology, Ningbo, China) followed by 5 IU human chronic gonadotropin (hCG, Sansheng Biological Technology, Ningbo, China) 46 h later. After an additional 16 h, oocytes were collected from oviducts, and the numbers of oocytes for each animal were counted after digestion with 0.3% hyaluronidase (Merck, MR-051-F).