Extended Data Fig. 6: In vitro fertilization and cohousing suggest germline transmission directly contributes to F1 phenotypes.

(a) Schematic of the cohousing strategy to empirically determine whether prior maternal exposure to an nABX male or his environment influences offspring phenotype independently of germ cells via, for example, coprophagy or microbiome transfer. Females were maintained within a control or nABX male cage for 4 days, which recapitulates the maximum time period used for natural matings throughout the study (mating within 1–4 days). Mating was prevented by removing males during the evening whilst leaving the females within the male environment, including exposure to faeces, microbes and chemical cues. After 4 days, independent control males were used for mating to examine the functional effect of prior cohousing with an nABX males. (b) Survival of F1 offspring from mothers mated with control males but pre-exposed to control or nABX males/environments. Significance by Mantel–Cox test (log-rank). (c) Neonatal birth weight, p-value by nested two-tailed t-test and bar indicate mean with 95% C.I. (d) Growth curves of F1 offspring from mothers mated with control males but pre-exposed to control or nABX male environments. ns = non-significant by nested two-tailed t-test analysis. (e) Schematic of experimental strategy for in vitro fertilization using control or dysbiotic (nABX) sperm donors. Oocytes from C57BL/6J females were pooled and split evenly to be fertilized by freshly harvested sperm from CON or nABX-treated (6wk) C57BL/6J males. IVF from CON and nABX males was performed in parallel. Fertilized embryos were transferred to recipient foster dams and subsequently analysed for F1 effects postnatally. (f) Growth curve of F1 offspring from IVF transferred to CD1 surrogate dams shows mean birth- and postnatal- weight is reduced when fertilized by nABX donor sperm. The prevalence of the severe growth restriction phenotype, characterized by extremely low body weight (Z-score < −3) by P15, can be observed in nABX outliers. Shown right is all data points from IVF embryos at P15. p-value by nested two-tailed t-test. (g) Growth curves of independent IVF offspring transferred to C57BL/6J surrogate dams. p-value by nested two-tailed t-test. Note CD1 mothers foster larger pups than C57BL/6J mothers, despite offspring being genetically-identical pure C57BL/6J (compare scale in Fig B & C), owing to higher quality in utero/maternal care from CD1. Nevertheless, we still observed a recapitulation of the F1 body weight phenotypes as observed in natural C57BL/6J matings in both in utero backgrounds.

Source Data