Mitofusin 1 is required for female fertility and to maintain ovarian follicular reserve

a Fertility of Mfn1？/？ and WT female mice (8-week-old) was assessed by mating with WT males of proven fertility (male/female; 1:2) for 12 weeks. The average number of delivered pups per litter per mouse was recorded. Data presented as mean？±？SEM. N？=？7 for each genotype; ***p？<？0.001 vs. WT using t-test. b Mfn1？/？ and WT female mice (8-week-old) were superovulated by PMSG and hCG, and the number of mature (MII) oocytes collected from oviducts was counted. Data represent mean？±？SEM. N？=？6 for each genotype; ***p？<？0.001 vs. WT using t-test. c and d Ovaries from Mfn1？/？ and WT mice (8-week-old) primed with PMSG (d) or unprimed (c) were fixed, embedded, sectioned, and stained with hematoxylin and eosin. Follicle counts using 3 mice ovaries for each genotype were performed. No antral follicles were found in Mfn1？/？ ovaries, with or without PMSG stimulation. e, f, g and h Representative micrographs ovarian sections from Mfn1？/？ and WT mice (8-week-old) with (f and h) or without (e and g) PMSG stimulation. i The number of secondary follicle subtypes per ovary was quantified as described in Materials and Methods section (n？=？4 mice per genotype). j The diameters of secondary follicle subtypes per ovary were measured (n？=？4 mice per genotype). k Secondary follicle-enclosed oocytes and granulosa cells were collected from Mfn1？/？ and WT ovaries. qRT-PCR was performed to determine Gdf9 and Bmp15 mRNA expression in Mfn1？/？ and WT oocytes. l qRT-PCR analysis of ActivinβB, Follistatin, and kit-ligand in Mfn1？/？ and WT granulosa cells was performed. m, n PCNA immunofluorescence assays and the quantification of PCNA-positive granulosa cells in Mfn1？/？ and WT ovaries are shown. o Serum FSH levels in sera of Mfn1？/？ and WT mice (n？=？7–11 per genotype). Data presented as mean？±？SEM. *p？<？0.05, **p？<？0.01 vs. WT using t-test. T4: follicles with two layers of cuboidal granulosa cells, T5a: follicles with three layers of granulosa cells, T5b: follicles with many layers of granulosa cells but no antrum. Data represent mean？±？SEM. *p？<？0.05 vs. WT using t-tes