Inactivation of a sperm motility gene by insertion of an epidermal growth factor receptor transgene whose product is overexpressed and compartmentalized during spermatogenesis.

Abstract

Transgenic mice were generated with a human epidermal growth factor (EGF) receptor cDNA driven by the chicken 13-actin gene promoter. One line (AE24) that exhibited a unique expression pattern in which dramatically elevated levels of EGF receptor RNA were found only in the testis was established, suggesting that the 13-actin promoter was being influenced by an adjacent testis-specific enhancer. EGF receptor RNA was detected in primary spermatocytes, whereas the synthesis of receptor protein was restricted to elongate spermatids, indicating that transgene expression was under translational control. At spermiation, the EGF receptor was sequestered in residual bodies and excluded from mature sperm by a compartmentalization mechanism. About half of AE24 homozygous males were sterile because of sperm paralysis, whereas heterozygous males and females of either genotype were completely fertile. Electron microscopic analysis of sperm flagella from sterile AE24 homozygotes revealed an aberrant axonemal structure in which outer doublet microtubules were missing from the middle piece, resembling changes observed in the sperm of some infertile humans. Flagellar axonemal disassembly was observed in the vas deferens and epididymis but not in the testis, suggesting that outer doublets were assembled in a grossly normal manner but possessed a latent instability. These results demonstrate that in the AE24 mouse line the EGF receptor transgene was integrated into and inactivated an endogenous autosomal gene, causing sperm flagellar axonemal disruption and male sterility.

Immotile Sperm and Infertility in Mice Lacking Mitochondrial Voltage-dependent Anion Channel Type 3

“…Two of the outer dense fibers that are associated with microtubules 3 and 8 terminate within the principal piece and form the longitudinal columns of the fibrous sheath that partition the axoneme into two unequal compartments. It has been proposed that loss of doublets 4 -7 represent sliding of microtubules during attempted motility, with extrusion of half of the axoneme (35). Although, based on the results presented here, expression of VDAC3 protein appears ubiquitous, VDAC1 and VDAC2 reportedly are expressed in a more limited fashion, with VDAC1 restricted to Sertoli cells and VDAC2 found in secondary spermatocytes and in round and elongated spermatids (36).…”

Identification and Characterization of SNX15, a Novel Sorting Nexin Involved in Protein Trafficking

“…The sequences of primers used to construct SNX15, SNX15A, and the SNX15 mutant cDNAs are available upon request. pCIS2 expression vector encoding full-length human receptor cDNAs for insulin (12), EGF (13), and PDGF (14) have been previously described (12,15). Mutant human PDGF ␤-receptors were kindly provided by Dr. A. Kazlauskas.…”

Identification of a Family of Sorting Nexin Molecules and Characterization of Their Association with Receptors

“…The epitope-tagged molecules were then cloned into either pCDNA(3.1Ϯ) (Invitrogen Corp., Carlsbad, Calif.) or pCMV5myc-1 expression vectors (2) by standard techniques. Full-length human insulin receptor (33), PDGF receptor (40), and EGF receptor (28) were cloned into pCDNA (3.1Ϯ) (Invitrogen Corp.) or pCIS vectors as previously described (33,34). Full-length human leptin receptor short and long isoforms (11,22,37) were cloned into pCIneo vector (Promega Corp., Madison, Wis.) by standard techniques.…”