Fig 2: Danazol promoted the maturation of follicles and thickening of the uterine wall in neonatal female rats. (A) Serum levels of FSH, LH, E2, and leptin in the two rat groups were detected by ELISA (n = 6). (B) HE staining of the uterus (×50, scale bar = 200 mm; ×200, scale bar = 100 mm) and ovary of two rat groups (×50, scale bar = 500 mm; ×200, scale bar = 100 mm) (n = 6). CL, corpus luteum; MF, mature follicle. (C) The thickening of the uterine wall and the number of corpora lutea were calculated. (D) The mRNA expression of FSHR and LHR in uterine tissues were detected by qRT-PCR (n = 3). (E) The expression of GnRH in ovarian tissues was detected by immunohistochemical staining (n = 6). (F) The mRNA levels of GnRH, GnRHR, netrin-1, and KISS1 in hypothalamus tissues were detected by qRT-PCR (n = 6).

Fig 3: Comparison of GnRH (gonadotropin releasing hormone) and Npffr1 expression levels between MKRN3‐modified rabbits and wild‐type (WT) rabbits. (A) Hypothalamic IF (immunofluorescence) staining results. DAPI (2‐(4‐Amidinophenyl)‐6‐indolecarbamidine dihydrochloride): Nuclear staining. Scale bar = 50 μm. (B) Pituitary IF staining results. DAPI: Nuclear staining. Scale bar = 100 μm. (C) The IF result statistics of GnIH (gonadotropin inhibiting hormone) (n = 6) (corresponding to Figure 4A). ns: p> 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (D) The IF results of Npffr1 were statistically analyzed (n = 6) (corresponding to Figure 4B). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (E) qPCR results: Detection of mRNA levels of NPFFR1 in the hypothalamus of MKRN3‐modified rabbits and WT rabbits (n = 6). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (F) Hypothalamic IHC (immunohistochemistry) staining results. Scale bar = 200 μm. (G) Pituitary IHC staining results. Scale bar = 200 μm. MKRN3 m+/p–: 3‐month‐old paternal mutant MKRN3‐modified rabbits; WT, 3‐month‐old WT rabbits.

Fig 4: CPP (central precocious puberty) pathological examination of paternal MKRN3‐modified rabbits. (A) H&E (hematoxylin and eosin) staining results: It can be seen that the uterine wall thickness of 3‐month‐old MKRN3‐modified rabbits is about twice that of WT rabbits of the same age; the number of developing follicles (blue arrow) in the ovaries of 3‐month‐old MKRN3‐modified rabbits is significantly higher than that of WT rabbits of the same age. Scale bar = 500 μm. (B) Statistical analysis of uterine body section diameter (corresponding to Figure 3A) (n = 6). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (C) Statistics of the number of follicles in ovarian development (corresponding to Figure 3A) (n = 6). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (D) H&E staining results: Compared with the WT of the same age, it can be seen that the testes of 3‐month‐old MKRN3 gene–modified rabbits have more mature sperm development. Scale bar = 50 μm. (E) The qPCR results of the hypothalamic GnRH (gonadotropin releasing hormone) mRNA levels between 3‐month‐old MKRN3 gene–modified rabbits and WT rabbits of the same age (n = 6). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (F) IHC (immunohistochemistry) staining results of GnRH in the hypothalamus. Scale bar = 50 μm. (G) Statistics of GnRH‐positive cells in IHC staining results (corresponding to Figure 3F) (n = 6). ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. MKRN3 m+/p–, paternal mutant MKRN3‐modified rabbits; WT, wild‐type rabbits.