Extended Data Fig. 10: Paternal leptin deficiency induces testicular phenotypes and major intergenerational expression changes.
From: Paternal microbiome perturbations impact offspring fitness
(a) Bar chart showing sperm count in young (6wk old) ob/ob males that lack leptin signalling for an equivalent time period as 6wk-treated nABX males, where leptin is also impaired (WT = 3; ob/ob = 3, sperm samples). Bars show mean with S.D and significance by two-tailed t-test. (b-c) Bar chart showing (b) body weight and (c) testis/body weight ratio of ob/ob males at 6-week-old. Note that despite increased body weight linked with elevated satiety, testes weight is still reduced in absolute terms in ob/ob males (see Fig. 3i) and as a ratio to body weight, indicating a reproductive response. (d) Percentage natural matings that led to a successful pregnancy from control or ob/ob males. The lack of ob/ob pregnancies could indicate infertility due to direct sperm defects, or to indirect effects, such as impaired behaviour or physical capacity. These can be distinguished by IVF (see panel E-F), which indicates failure of ob/ob natural mating at this age is due to indirect effects, as IVF from ob/ob sperm was equally efficient as from WT. (e) Schematic showing the experimental design to test potential F1 impacts of dysregulated paternal leptin signalling. wt/ob mice were intercrossed to generate ob/ob and wt/wt males and sperm from such littermates was then isolated and used to fertilse WT oocytes, with no difference observed in the fertilization rate or development to blastocyst, indicating viable sperm from ob/ob and wt/wt males. (f) Principal component analysis of single-embryo transcripomes from E4.5 (left) and E5.5 (right) blastocysts. Each embryo was generated by parallel fertilizations using control or leptin-deficient sperm. Triplicate independent males were used per condition, across duplicate independent IVF experiments. A reproducible shift in gene expression patterns and thus phenotype, is observed depending on the father’s leptin levels. (g) Expression of Leptin in early embryos is undetectable. This argues against a potential haplo-insufficient effect on gene expression in wt/ob embryos derived from leptin-deficient (ob/ob) sires. Instead it points to a paternally inherited defect in sperm. (h) Gene ontology pathways of differentially expressed genes in embryos from leptin-deficient fathers. The test statistic reported as p-value. (i) Representative differentially expressed genes in blastocysts, depending on the paternal leptin status (WT = 9; ob/ob = 10, blastocysts). Each data point indicates log expression in an individual embryo at E4.5.
