Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Significance This study improves our understanding of the role of Müllerian inhibiting substance (MIS/AMH) in antagonizing primordial follicle activation. By applying scRNA-seq methods to neonatal mouse ovaries, we revealed transcriptional signatures associated with MIS treatment in ovarian cell types present during the first wave of folliculogenesis and follicle development. We showed that MIS inhibits pregranulosa cell differentiation and the proliferation of ovarian surface epithelium and stromal cells. MIS also uncoupled granulosa and germ cell maturation, leading to abnormal development of activated follicles. These findings identify markers and pathways related to primordial follicle quiescence that could be targeted in contraception, preservation of ovarian reserve during aging or chemotherapy, or synchronization of preantral follicle growth for IVF.

Total RNA was isolated from the cultured ovaries and various gene levels were analyzed by qPCRs. Primer sequences are presented in Tables S3 and S4.

ELISA
ELISA was used to determine AAV9-MIS (human) concentration in mouse and rat serum. A 96well high protein-binding capacity plate (Nunc MaxiSorp) was coated with 5µg/ml of in house produced mouse anti-MIS antibody (6E11) overnight at 4°C. Then, the plate was washed 3 times with PBS/0.01% Tween-20 and saturated with a PBS/2% BSA solution for 2hr at room temperature.
The LR-MIS standard was added at 35ng/ml and 8 serial 1:2 dilutions were made before adding serum samples previously diluted at 1:100 for 2h. Rabbit polyclonal anti-MIS antibody (MGH6), was added at 1:4 000 and incubated for 2hr. Then a secondary HRP labeled antibody (Donkey antirabbit IgG conjugated HRP; Jackson ImmunoResearch Laboratories) was diluted at 1:70 000 and added during 1hr. Lastly, plate was washed five times and revealed by enzyme substrate (TMB).
Absorbance was read at 450 nm after stopping the enzymatic reaction by the addition of sulfuric acid. To validate our ELISA, MIS ELISA kit from Beckman and coulter (AMH Gen ELISA kit; ref : A73818) test was realized in parallel according the manufacturer protocol.

Immunofluorescence (IF) and immunohistochemistry (IHC)
Dissected ovaries or embryos were fixed in 4% (wt/vol) paraformaldehyde at 4 °C (for embryos histology) or in 10% neutral buffered formalin at room temperature overnight (for ovarian histology, RNAish and immunofluorescence). Tissues were embedded in paraffin blocks in an automated tissue processor (Leica #TP1020). 5μm ovarian sections were used for hematoxylin and eosin (H&E) staining, immunofluorescence (IF), and RNAish using the RNA scope™ (ACD Bio) system.
Human ovarian blocks were procured by the Massachusetts General Hospital, Gynecological Pathology Department through an IRB approved protocol (IRB 2007P001918) (6).