Slc2a8 deficiency in mice results in reproductive and growth impairments

Abstract

SLC2A8, also known as GLUT8, is a facilitative glucose transporter expressed in the testis, brain, liver, heart, uterus, ovary, and fat. In this study we examined the effect of Slc2a8 deficiency on mouse gamete, preimplantation embryo, and implantation phenotype, as well as postnatal growth and physiology. For this model, the transcriptional start site and exons 1-4 were targeted and a lack of protein expression was confirmed by Western immunoblot. Oocytes obtained from Slc2a8(-/-) mice demonstrated abnormal metabolism and ATP production. In addition, deletion of Slc2a8 resulted in impaired decidualization, a critical step in the differentiation of endometrial stromal cells (ESCs), necessary for implantation. This indicates a role for SLC2A8 in decidualization, which is supported by Slc2a8 mRNA expression in both mouse and human ESCs, which increases dramatically in response to hormonal changes occurring during the process of implantation. Ovarian transplantation studies confirm that lack of SLC2A8 affects both the embryo and the implantation processes. This phenotype leads to decreased litter size, and smaller pups at weaning that continue to display an abnormally small growth phenotype into adulthood. The Slc2a8 null mice display decreased body fat by magnetic resonance imaging, and, interestingly, they are resistant to a diet high in fat and carbohydrates.

FIG. 1.

Targeted disruption of Slc2a8. A) Schematic representation of the wild-type Slc2a8 allele, the targeting vector used, the Slc2a8 targeting construct, and the targeted Slc2a8 gene are illustrated. Note the neomycin resistance gene replaces exons 1−4 of the Slc2a8 gene. Additionally, the location of the external 5′ probe used to in Southern blot analysis of resistant ES clones and Slc2a8-deficient mice is indicated. BI, BglI; H, HindIII; BHI, BamHI; P, PvuI. B) Southern blot analysis of EcoRV-digested genomic tail DNA from the progeny of Slc2a8^+/− mice. C) RT-PCR. Lane 1, positive control; lane 2, marker; lanes 3–5, Slc2a8^+/+ testes, heart, liver; lanes 6–8, Slc2a8^−/− testes, heart, liver.

FIG. 2.

Impaired protein expression in Slc2a8^−/− mice. A) Western blot of SLC2A8 expression in testes protein. SLC2A8 is absent in testes from Slc2a8^−/− mice. B) Immunofluorescence staining for SLC2A8 in paraffin-sectioned testes. SLC2A8 protein is not present in the Slc2a8^−/− mice. Original magnification ×10.

FIG. 3.

Male and female gonadal phenotype. A) No changes in overall testes morphology observed in Slc2a8^−/− mice. B) Slc2a8^−/− mice exhibit a trend toward reduced sperm motility (n.s.). C) No changes in overall ovary morphology observed in Slc2a8^−/− mice. D) ATP is significantly decreased in Slc2a8^−/− oocytes by ∼25%, n = 30. E) Hadh2 is significantly decreased in Slc2a8^−/− oocytes by ∼23%, n = 20. Original magnification ×4 (A and C). Error bars are ± SEM.

FIG. 4.

Aberrant uterine decidualization in Slc2a8^−/− mice. H&E staining of Day 6.5 implantation sites in wild-type (A) and Slc2a8^−/− mice (B). Immunofluorescence staining of Cox2 in Day 6.5 implantation sites; the site of the implanted embryo is marked as EM. Negative control (C), wild-type (D), Slc2a8^−/− (E). Quantitative real-time of wild-type and Slc2a8^−/− Day 6.5 implantation sites for Cox2 (F) and Prp (G). Interimplantation areas were excised and used for negative control. *P < 1.0 × 10⁻⁶; **P < 0.001. Error bars are ± SEM.

FIG. 5.

In vivo artificial decidualization is incomplete in Slc2a8^−/− mice. A) Representative image of control horn (left) and deciduoma (right) in the Slc2a8^+/+ (left) and Slc2a8^−/− (right) uterus. B) The Slc2a8^−/− decidualized horn weighed significantly less than the Slc2a^+/+ decidualized horn compared to control horns. Error bars are ± SEM.

FIG. 6.

Ovarian transplants result in reduced litter sizes. A) Cartoon depicting experimental overview. B) Compared to wild-type × wild-type matings, both groups of mice who underwent the ovarian transplant show decreased number of pups per litter similar to Slc2a8^−/− × Slc2a8^−/− matings. Error bars are ± SEM.

FIG. 7.

Slc2a8^−/− mice exhibit aberrant growth. A) Slc2a8^−/− mice are smaller at 3 wk of age and into adulthood. Male mice are shown but females exhibit the same pattern. B) Weights of pups at Postnatal Day 1 are similar to those of Slc2a8^+/+ mice. C) Slc2a8^−/− mice consume the same amount of food as Slc2a8^+/+, indicating this is not related to a decrease in calorie consumption. Error bars are ± SEM.

FIG. 8.

Overall glucose homeostasis is not affected under normal conditions and maintained during high-fat feedings. A) Slc2a8^−/− mice have less body fat by magnetic resonance imaging than Slc2a8^+/+ mice (n.s.). B and C) GTTs and ITTs are similar to those of Slc2a8^+/+ mice. D) Slc2a8^−/− mice do not gain significant body fat on a high-fat diet compared to Slc2a8^+/+ mice. E and F) Slc2a8^−/− mice maintain a normal GTT and ITT on a high-fat diet. Error bars are ± SEM.