Subfertility in mice harboring a mutation in betaB2-crystallin

Abstract

Purpose: betaB2-crystallin is one of the most abundant proteins of the adult ocular lens of mammals although it is expressed at lower levels in several extralenticular locations. While mutations in betaB2-crystallin are known to result in lens opacities, alterations in tissues besides the lens have not been previously investigated in these mutants. Since we found mice harboring the Crybb2Phil mutation bred poorly, here we assess the contribution of betaB2-crystallin to mouse fertility and determine the expression pattern of betaB2-crystallin in the testis.

Methods: The expression pattern of betaB2-crystallin in the testis was analyzed by rt-PCR, western blotting, and immunohistochemistry. The fecundity of wildtype and Crybb2Phil mice was analyzed by quantitative fertility testing. The morphology of testes and ovaries was assessed by hematoxylin and eosin staining.

Results: In the mouse testis, betaB2-crystallin mRNA is found at low levels at birth, but its expression upregulates in this tissue as the testis is primed to initiate spermatogenesis. Western blotting detected betaB2-crystallin protein in sperm obtained from mice, cattle, and humans while immunolocalization detected this protein in developing sperm from the spermatocyte stage onward. Male and female mice homozygous for a 12 nucleotide inframe deletion mutation in betaB2-crystallin are subfertile when analyzed on a Swiss Webster derived background due to defects in egg and sperm production. However, mice harboring the same mutation on the C57Bl/6 genetic background did not exhibit any defects in reproductive function.

Conclusions: betaB2-crystallin is expressed in developing and mature sperm and mice of both sexes harboring the Philly mutation in the betaB2-crystallin gene are subfertile when analyzed on a Swiss Webster genetic background. While these data are suggestive of a role for betaB2-crystallin in fertility, definitive determination of this will await the creation of a betaB2-crystallin null mouse.

Figure 1

Defects in homozygous STOCK Crybb2^Phil testis. A: Gross appearance of a Swiss-Webster mouse testis. B: Gross appearance of a STOCK Crybb2^Phil testis. C: Hematoxylin and eosin stained paraffin section of a Swiss-Webster mouse testis. D: Hematoxylin and eosin stained paraffin section of a homozygous STOCK Crybb2^Phil testis.

Figure 2

βB2-crystallin protein is expressed in the testis and mature sperm. A, B: βB2-crystallin localization in the 4 week old mouse testis. A: βB2-crystallin (brown stain) is found at the basal surface of some but not all seminiferous tubules in cells having the round nuclei and scant cytoplasm diagnostic of the leptotene and zygotene spermatocytes. B: βB2-crystallin is only weakly detected in basal cells with ovoid nuclei characteristic of spermatogonia, but is easily observed in rounded acrosomal vesicles in contact with the nuclei of spermatids (arrowheads). C: Semi-quantitative rt-PCR analysis of βB2-crystallin mRNA expression in the testes during postnatal development. L-lens, 1,3,8,12,15,17, age of testis in days post natal. D: Western blot analysis of βB2-crystallin protein expression in sperm. WL-wildtype lens, PL- homozygous Crybb2^Phil lens, WS- wildtype mouse sperm, PS- homozygous Crybb2^Phil mouse sperm, BS- bovine sperm, HS- human sperm. A, bar=200 μm; B, bar=70 μm.

Figure 3

Defects in homozygous STOCK Crybb2^Phil ovary. A: Gross appearance of a Swiss-Webster mouse ovary. B: Gross appearance of a STOCK Crybb2^Phil ovary. C: Hematoxylin and eosin stained paraffin section of a Swiss-Webster mouse ovary. D: Hematoxylin and eosin stained paraffin section of a homozygous STOCK Crybb2^Phil ovary.