Developmental basis of sexually dimorphic digit ratios

Abstract

Males and females generally have different finger proportions. In males, digit 2 is shorter than digit 4, but in females digit 2 is the same length or longer than digit 4. The second- to fourth-digit (2D:4D) ratio correlates with numerous sexually dimorphic behavioral and physiological conditions. Although correlational studies suggest that digit ratios reflect prenatal exposure to androgen, the developmental mechanism underlying sexually dimorphic digit development remains unknown. Here we report that the 2D:4D ratio in mice is controlled by the balance of androgen to estrogen signaling during a narrow window of digit development. Androgen receptor (AR) and estrogen receptor α (ER-α) activity is higher in digit 4 than in digit 2. Inactivation of AR decreases growth of digit 4, which causes a higher 2D:4D ratio, whereas inactivation of ER-α increases growth of digit 4, which leads to a lower 2D:4D ratio. We also show that addition of androgen has the same effect as inactivation of ER and that addition of estrogen mimics the reduction of AR. Androgen and estrogen differentially regulate the network of genes that controls chondrocyte proliferation, leading to differential growth of digit 4 in males and females. These studies identify previously undescribed molecular dimorphisms between male and female limb buds and provide experimental evidence that the digit ratio is a lifelong signature of prenatal hormonal exposure. Our results also suggest that the 2D:4D ratio can serve as an indicator of disrupted endocrine signaling during early development, which may aid in the identification of fetal origins of adult diseases.

Fig. 1.

Ontogeny of the 2D:4D ratio. (A) Male (Left) and female (Right) adult human hands showing dimorphic 2D:4D ratios. Horizontal line marks the distal tip of the second digit (D2). Note that the fourth digit (D4) is longer than 2D in the male but shorter than 2D in the female. (B) Mouse foot skeleton at postnatal day (P) 21. Digits are numbered, and vertical lines show axes of measurement for proximal (p) and middle (m) phalanges of 2D and 4D. (C) The mean 2D:4D ratio at P21 is smaller in males (n = 30) than in females (n = 28). (D) Whole-mount in situ hybridization showing Sox9 mRNA (purple stain), which marks the precartilagenous condensations, in right footplate at embryonic day (E) 12.5. Yellow lines show axes measured. (E) The mean 2D:4D ratio is the same for males (n = 21) and females (n = 19) at E12.5. (F) E17 mouse right foot stained with alcian blue to show cartilage. (G) The mean 2D:4D ratio in E17 males (n = 18) is significantly smaller than females (n = 22) in right hind paws. Error bars show ± SEM. *P < 0.05.

Fig. 2.

AR and ER-α distribution in developing digits. (A–C, E–G, I–K, and M–O) Immunolocalization of AR (red, A–C and E–G) and ER-α (green, I–K and M–O) in mouse digits at E12.5 and 14.5. Blue signal is DAPI. Stage is indicated at left, sex is indicated at top, and 2D and 4D are numbered. White boxes in A, E, J, and N are shown at high magnification in C, G, K, and O, respectively. (D, H, L, and P) Quantitative analysis of AR-positive (D and H) and ER-α–positive (L and P) cells with nuclear (Nu) and cytoplasmic (Cy) staining. Note that 4D has the highest levels of activated (nuclear) AR in males and females at E12.5 (C and D) and E14.5 (G and H), although levels drop in the female between these stages. ER-α staining is also higher in 4D at both stages (L and P). ER-α is mostly cytoplasmic at E12.5 (K and L), but by E14.5, most of the ER-α has gone to the nucleus (O and P). Error bars show ± SEM. *P < 0.05. (Scale bars: 100 μm, A, B, I, and J; 30 μm, E, F, M, and N; 10 μm, C, G, K, and O.)

Fig. 3.

Experimental manipulation of prenatal androgen and estrogen signaling alters 2D:4D ratios via digit- and phalanx-specific growth. (A) 2D:4D ratios in right hindlimbs of AR (n = 6) and ER-α (n = 7) mutant mice at P0. Control males (n = 12) were derived from the same litters as mutants. (B) 2D:4D ratios in mice treated with flutamide (anti-AR), fulvestrant (anti-ER), DHT, or estradiol. Digits were measured at P21. All prenatal treatments were done from E12.5 to 15.5, except for fulvestrant, which was administered at E12.5 and 14.5 to avoid labor induction. Postnatal treatment was done from P0 to 3. (C) 2D:4D ratios of E17 mouse embryos treated prenatally from E12.5 to 15.5. (D) Digit length index (digit length/tibia length) of E17 mice shows that flutamide or estradiol treatment shortens the male 4D, and fulvestrant or DHT treatment lengthens the female 4D. The 2D index shows no significant response. (E and F) Phalangeal length index (phalanx length/tibia length) for 4D proximal (E) and middle (F) phalanges. (E) The male proximal phalanx index of 4D is shortened by flutamide or estradiol treatment. The female proximal phalanx index is not significantly altered by fulvestrant or DHT treatment. (F) The male middle phalanx index of 4D is shortened by flutamide treatment, and the female middle phalanx index is lengthened by fulvestrant or DHT treatment. Error bars show ± SEM. *P < 0.05.

Fig. 4.

AR and ER have digit-specific effects on cell proliferation. (A–D and F–I) BrdU immunolocalization (red) and DAPI staining (blue) of longitudinal sections through proximal and middle phalanges of 2D and 4D of right hindlimbs at E16. Phalanges are oriented with proximal to the left. (Scale bars: 50 μm.) (E and J) Mitotic indices calculated from sections as represented in A–D and F–I (n = 4 embryos per group) show that antiandrogen treatment (flutamide) decreases cell proliferation in 4D of males (E), whereas antiestrogen treatment (fulvestrant) increases cell proliferation in 4D of females (J). Error bars show ± SEM. *P < 0.05.

Fig. 5.

AR and ER have digit-specific effects on expression of skeletogenic genes. Results of quantitative RT-PCR expression analysis of 90 skeletogenic genes in 2D and 4D are shown. Graphs show relative transcript levels in primordia of 4D compared with 2D, which was assigned a value of 0. The 19 genes showing statistically significant differences (P < 0.05) of >40% are shown. See Table S1 for results of all 90 genes. Error bars show ± SEM, and asterisks denote significant differences.

Fig. 6.

Developmental basis for the sexually dimorphic 2D:4D ratio. Models for the development of sexually dimorphic digit proportions are shown. AR (blue circles) and ER (pink circles) are present in the digit condensations of male and female embryos, with higher levels found in 4D. (A) In males, digits are exposed to high levels of circulating androgen and low levels of circulating estrogen, which results in preferential binding and activation of AR (AR^A represents the androgen bound to the AR). High AR activity and low ER activity (ΔAR^A/Δer) in males leads to differential gene expression profiles in 4D relative to 2D (green indicates genes higher in 4D, and red indicates genes higher in 2D). In turn, chondrocyte proliferation is increased in the proximal phalanx of 4D, which results in elongation of 4D relative to 2D, leading to a lower 2D:4D ratio. (B) In females, digits are exposed to high levels of estrogen and low levels of androgen, leading to preferential binding and activation of ER (ER^E). Low AR activity and high ER activity (Δar/ΔER^E) induces an opposite shift in the skeletogenic gene expression profile of 4D relative to 2D (indicated by gene names in green and red, as above). Higher levels of activated ER cause decreased chondrocyte proliferation in the middle phalanx of 4D, which reduces its growth relative to 2D and results in a higher 2D:4D ratio.