Sex-dependent regulation of vertebrate somatic growth and aging by germ cells

Abstract

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Fig. 1.. Germ cell removal modulates life span and body size in N. furzeri.

(A) Germ cell–removed N. furzeri embryo by injecting morpholino antisense oligo (MO) against dnd at 12 days postfertilization. Germ cells were visualized by EGFP expression in Tg (vasa:EGFP) transgenic strain. (B) Adult control (Cont) and germ cell–removed N. furzeri (GR). (C) Gonads of control (Cont) and germ cell–removed N. furzeri (GR). (D) Life span of control and germ cell–removed N. furzeri. Left: Survival curves of control (Cont, black) versus germ cell–removed female (GR, magenta). Right: Survival curves of control (Cont, black) versus germ cell–removed male (GR, green). (E) Quantifications of body length and body weight of control (Cont) and germ cell–removed (GR) N. furzeri. Animals measured at each time point are different individuals. P values are from Welch’s t test (*P < 0.05; **P < 0.01; ***P < 0.001). Bars and error bars represent mean ± SD.

Fig. 2.. Germ cell removal does not cause sex reversal but affects steroidogenesis in N. furzeri.

(A) Heteroduplex mobility assay for genotyping of gdf6, the sex-determining gene of N. furzeri. In genotypic female, a single 134-bp PCR product from X chromosome is detected. In genotypic male, 125-bp PCR product from Y chromosome is mixed, and thus four bands including two kinds of hetero dimers are detected. (B) RT-PCR analysis of male markers, dmrt1 and cyp11c, and female markers, foxl2 and cyp19a1a in adult gonads at young age, actb is used as internal control. (C and D) Quantification of (C) estradiol and (D) 11-ketotestosterone levels extracted from the whole body of N. furzeri at young and old age. Cont, control. GR, germ cell removed. P values are from Welch’s t test (*P < 0.05; **P < 0.01; ***P < 0.001). Bars and error bars represent mean ± SD.

Fig. 3.. Low estrogen and increased IGF-1 signaling is associated with accelerated aging in germ cell–removed females.

(A) GO term analysis of down-regulated genes in the female livers. Red box indicates “cellular response to estrogen stimulus” and “response to estrogen.” N. of genes, number of genes. BP, biological process. (B) Expression levels of fibrinogens; fga, fgb, and fgg in the female livers by RNA-seq. (C) Expression levels of key genes of fatty acid biosynthesis (acaca) and triglyceride biosynthesis (scd and dgat2) in the female liver by RNA-seq. (D) Liver sections of old females stained with Oil Red O and counterstained with hematoxylin. Right: Quantification of lipid accumulation in the liver. (E) Sum of blood LDL and VLDL levels in females. (F) Expression levels of igf1 in the liver by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). (G) Schematic illustration of IGF-1 signaling and its downstream genes in the skeletal muscle. (H) Expression levels of IGF-1 signaling target genes (fbxo32 and trim63) in the skeletal muscle by RNA-seq. (I) Liver sections of females stained with senescence-associated β-galactosidase (SA-βgal) and counterstained with nuclear fast red. Right: Quantification of SA-βgal–stained area in the liver. (J) Expression level of cellular senescence marker cdkn2ab in the female livers. Cont, control. GR, germ cell removed. P values are from Welch’s t test (*P < 0.05; **P < 0.01; ***P < 0.001) in (D), (E), (F), (I), and (J). FDRs are from quasi-likelihood methods in edgeR (*FDR < 0.05; **FDR < 0.01; ***FDR < 0.001) in (B), (C), and (H). Bars and error bars represent mean ± SD. FDR, false discovery rate. CPM, count per million.

Fig. 4.. Increased vitamin D signaling contributes to somatic growth and aging prevention in germ cell–removed males.

(A) Muscle sections of males immuno-stained with the Pax3/7 antibody (magenta), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and autofluorescence (green). Yellow arrowheads indicate Pax3/7⁺ cells. Right: Quantification of Pax3/7⁺ cell number. (B) Expressions of satellite cell marker genes (pax7 and myod) in the male skeletal muscle by RT-qPCR. (C) Skin sections of males stained with the Masson’s trichrome stain. Right: Quantification of the skin collagen fiber thickness. (D) Expression level of col1a1a in the male skin by RT-qPCR. (E) Micro-CT images of bone vertebrae of old males. Right: Quantification of the bone volume and bone mineral density. (F) Expression level of vitamin D activating enzyme cyp2r1 in the male liver by RNA-seq. CPM, count per million. FDRs are from quasi-likelihood methods in edgeR (**FDR < 0.01). (G and H) Expression levels of vitamin D signaling target gene vdrb (skeletal muscle) (G) and cyp24a1 (skin) (H) in males by RT-qPCR. (I) Schematic illustration of alfacalcidol (alfa) treatment. (J) Alfa-treated males at 11 weeks after treatment initiation. Right: Quantification of the body length of males at 0 and 11 weeks after treatment initiation. wpt, weeks post-treatment initiation. (K) Skin sections of alfa-treated males at 4 months old stained with Masson’s trichrome stain. Right: Quantification of the thickness of skin collagen fiber. (L) Expression of col1a1a in the alfa-treated male skin at a young age by RT-qPCR analysis. P value is determined from Dunnett’s multiple comparisons test (*P < 0.05). (M and N) Life span of control and alfa-treated males (M) and females (N). Survival curves of control (black) versus alfa-treated animals (orange). P values are from Welch’s t test (*P < 0.05; **P < 0.01) in (A), (B), (C), (D), (E), (G), (H), (J), and (K). Bars and error bars represent mean ± SD.