High mobility group A2 (HMGA2) deficiency in pigs leads to dwarfism, abnormal fetal resource allocation, and cryptorchidism

Abstract

Expression of HMGA2 is strongly associated with body size and growth in mice and humans. In mice, inactivation of one or both alleles of Hmga2 results in body-size reductions of 20% and 60%, respectively. In humans, microdeletions involving the HMGA2 locus result in short stature, suggesting the function of the HMGA2 protein is conserved among mammals. To test this hypothesis, we generated HMGA2-deficient pigs via gene editing and somatic cell nuclear transfer (SCNT). Examination of growth parameters revealed that HMGA2^-/+ male and female pigs were on average 20% lighter and smaller than HMGA2^+/+ matched controls (P < 0.05). HMGA2^-/- boars showed significant size reduction ranging from 35 to 85% of controls depending on age (P < 0.05), and organ weights were also affected (P < 0.05). HMGA2^-/+ gilts and boars exhibited normal reproductive development and fertility, while HMGA2^-/- boars were sterile due to undescended testes (cryptorchidism). Crossbreeding HMGA2^-/+ boars and gilts produced litters lacking the HMGA2^-/- genotype. However, analysis of day (D) D40 and D78 pregnancies indicated that HMGA2^-/- fetuses were present at the expected Mendelian ratio, but placental abnormalities were seen in the D78 HMGA2^-/- concepti. Additionally, HMGA2^-/- embryos generated by gene editing and SCNT produced multiple pregnancies and viable offspring, indicating that lack of HMGA2 is not lethal per se. Overall, our results show that the effect of HMGA2 with respect to growth regulation is highly conserved among mammals and opens up the possibility of regulating body and organ size in a variety of mammalian species including food and companion animals.

Keywords: HMGA2; dwarfism; gene editing; organ size; swine.

Fig. 1.

HMGA2 protein in gene-edited fetal fibroblasts. HMGA2 protein levels for HMGA2^+/+, HMGA2^−/+, HMGA2^−/−, and HMGA2^−/−Stra8 fetal fibroblasts were detected by Western blot. Proteins were extracted from primary D40 fetal fibroblasts of all three HMGA2 genotypes generated from the breeding of HMGA2^−/+ founders. HMGA2^−/−Stra8 samples were isolated from primary D43 fetal fibroblasts generated by SCNT. HMGA2^+/+ had twice as much protein as HMGA2^−/+ samples, suggesting there was no compensation from the remaining active allele (SI Appendix, Fig. S2). There was no protein detected from HMGA2^−/− or HMGA2^−/−Stra8 fibroblasts. β-Actin was used as internal control. HEK293 lysate was used as positive control.

Fig. 2.

Fetal weights (D40 and D78) of HMGA2 gene-edited pigs. Pregnancies were generated by crossbreeding HMGA2^−/+ gilts and boars. (A) Fetuses with all expected genotypes were detected at D40 at the expected Mendelian frequency. Weights of HMGA2^−/+ fetuses were comparable to those of HMGA2^+/+ fetuses, but the HMGA2^−/− fetuses were significantly smaller (30% reduced) compared with the other two genotypes. Two females and one male HMGA2^−/− fetuses were collected, all size-restricted. (B) At D78, HMGA2^−/+ fetuses were 24% smaller and HMGA2^−/− were 72% smaller than HMGA2^+/+ fetuses. Both HMGA2^−/+ and HMGA2^−/− male and female fetuses were affected (nine females:three males for HMGA2^−/+ and one female:three males for HMGA2^−/−).

Fig. 3.

Placental morphology in HMGA2 concepti at D78 of gestation. (A) HMGA2^−/+ placentas were healthy and well-vascularized, while HMGA2^−/− placentas showed lack of absorptive structures (areolae; arrowheads compared with arrows) and vascularization. (B) Histological analysis of placenta and endometrium from HMGA2^+/+, HMGA2^−/+, and HMGA2^−/− fetuses. Villi were severely degenerated in HMGA2^−/− placenta compared with other genotypes. Furthermore, poor contact of villi between uterine endometrium (Endo) and chorionic membrane (Chorio) was detected for HMGA2^−/− (arrow), suggesting the connection between uterus and placenta was deficient. (Scale bars: 100 μm.)

Fig. 4.

Growth comparison of HMGA2^−/− SCNT, HMGA2^−/+ SCNT, HMGA2^+/+ SCNT, and HMGA2^+/+ naturally bred (NB) boars. Measurements for weight (A), chest circumference (B), shoulder height (C), and crown–rump length (D) were taken every 2 wk over a 26-wk period. HMGA2^+/+ NB, n = 5; HMGA2^+/+ SCNT, n = 6; HMGA2^−/+; n = 6; HMGA2^−/−, n = 3 (one HMGA2^−/− died at 22 wk so last 4 wk n = 2). Error bars indicate SEM.

Fig. 5.

Visual comparison of representative HMGA2^+/+, HMGA2^−/+, and HMGA2^−/− boars. At 26 wk of age boars were photographed in front of the same measuring board for to facilitate size comparisons. Board numbering is in centimeters. (A) HMGA2^+/+ NB, (B) HMGA2^+/+ SCNT, (C) HMGA2^−/+ SCNT, (D) HMGA2^−/− SCNT. Note drastic reduction in size and stocky appearance of the HMGA2^−/− boar. NB, naturally bred.

Fig. 6.

Testis histology and HMGA2 expression in control and age-matched HMGA2^−/−Stra8 testis. (A) Testis from control and HMGA2^−/−Stra8 pigs at postnatal D4 and 4 mo of age. At D4, cell structure and arrangement of seminiferous tubules was the same as in age-matched HMGA2^+/+ testis. However, at 4 mo, none of seminiferous tubules in the cryptorchid HMGA2^−/−Stra8 testis showed germ-cell differentiation, while a few tubules in HMGA2^+/+ testis showed evidence of spermatogenesis. (Scale bars: 100 μm.) (B) Testes from an HMGA2^−/+ boar at 9 mo of age compared with the testis of an age-matched HMGA2^+/+ boar, both showing evidence of spermatogenesis. (C) RT-PCR analysis of 4-mo HMGA2^−/−Stra8 and age-matched HMGA2^+/+ testis. Lanes: 1, GAPDH; 2, transgenic β-geo; 3, endogenous HMGA2 partial CDS (detects only endogenous HMGA2); 4, pig STRA8 CDS; 5, transgenic HMGA2 (detects only mStra8-driven HMGA2); and 6, HMGA2 full-length CDS (detects both endogenous and transgenic HMGA2). Results support inactivation of the endogenous HMGA2 and expression of the transgenic HMGA2 driven by the mStra8 promoter in the HMGA2^−/−Stra8 testis.