Culture plate inserts

To measure axonal outgrowth during development, flies were reared at 25°C and were dissected in fresh PBS. A minimum of 5 fly brains (10 sLNv projections) per genotype were fixed in 4% formaldehyde and stained with an anti-GFP antibody (Molecular Probes; 1:500), according to standard methods (Ayaz et al., 2008 (link)). For the axonal regrowth analysis after injury, flies were reared at 18°C (to minimize developmental effects), and shifted to 25°C 1 day prior to injury. Whole brain explants on culture plate inserts were prepared as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)). In brief, culture plate inserts (Milipore) were coated with laminin and polylysine (BD Biosciences). Fly brains were carefully dissected out in a sterile Petri dish containing ice cold Schneider's Drosophila Medium (GIBCO), and transferred to one culture plate insert containing culture medium (10 000 U/ml penicillin, 10 mg/ml streptomycin, 10% Fetal Bovine Serum, and 10 μg/ml insulin in Schneider's Drosophila Medium (GIBCO). sLNv axonal injury was performed using an ultrasonic microchisel controlled by a powered device (Eppendorf) as described (Ayaz et al., 2008 (link); Koch and Hassan, 2012 (link)) and dishes were kept in a humidified incubator at 25°C. Four days later, cultured brains were fixed and immunohistochemical staining was performed as for freshly dissected samples.

Ovaries from day10 mice were placed on culture plate inserts (Millipore) and cultured in 400 μl of DMEM/F12 containing 0.1% BSA, 0.1% Albumax II, insulin-transferrin-selenium, 0.05mg/ml L-ascorbic acid and penicillin-streptomycin under a membrane insert to cover ovaries with a thin layer of medium. Ovaries were treated with 1–10 μM of MHY1485 and cultured for 4 days with medium change after 2 days of culture. Control ovaries were treated with solvent only. At the end of culture, ovaries were fixed with formalin before weighing. Some ovaries were paraffin-embedded and cut into continuous sections at 8 μm thickness. Sections were stained with hematoxylin and eosin for follicle counting, and in each section, only follicles with clearly stained oocyte nucleus were counted to avoid recounting of the same follicle.

The ovary tissue of patients (n = 11) of non-ovarian factors containing primordial follicles were collected using excision surgery at the Department of Obstetrics and Gynecology in Shanghai Changzheng Hospital. All the participants are transsexual patients that had normal ovarian function and were not under hormonal treatment before the surgery. This study is approved by the Institutional Medical and Ethical Review Committee of Second Military Medical University. Clinical characteristics of cases were shown in Table 1. After being cut into 1 mm³ cubes, cortical tissues were placed on culture plate inserts (Millipore) and cultured in 400 μl of DMEM/F12 (Gibco) containing 10% human serum albumin (CSL Behring), 1% penicillin-streptomycin solution (HyClone), 0.3 IU/ml FSH, and 0.05 mg/ml L-ascorbic acid under a membrane insert to cover ovaries with a thin layer of medium. Ovaries were treated with 1–20 μM MHY1485 (10 mM in 1 ml DMSO, APExBIO, United States) and cultured for 3 h before immunoblotting analysis. Control ovaries were treated with solvent only. Other pairs of ovaries were cultured for 2 days with a medium change after 24 h of culture. Ovaries were fixed with formalin for histological analysis or prepared for transplantation before weighing.

For LPS treatment in vitro, ovaries from 10-day-old mice were placed on culture plate inserts (Millipore Corporation) and cultured in 400 µl of DMEM/F12 culture medium (Solarbio) supplemented with 10% FBS (Biological Industries) and 1% penicillin–streptomycin 100× solution (HyClone) in an incubator at 37 °C with 5% CO₂ as previously reported^{69 (link)}. A thin layer of medium covered the ovaries on the inserts. Ovaries were treated with 10 µg ml⁻¹ LPS with media changes every 2 d for 4 d. At the end of the culture, ovaries were fixed in Bouin’s solution, paraffin-embedded and cut into continuous sections before staining with H&E. The growing and primordial follicle percentages were analyzed in these sections as previously reported^{70 (link)}.