Deletion of the Parkin coregulated gene causes male sterility in the quaking(viable) mouse mutant

Abstract

Quaking(viable) (qk(v)) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the qk(v) mutant is a deletion of approximately 1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the qk(v) mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the qk(v) allele. This observation suggested that the sperm differentiation defect in qk(v) is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the qk(v) mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in qk(v) males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.

Fig. 1.

Pacrg transcript is highly expressed in testis in both mouse and humans. (A) Northern blot on multiple mouse tissues shows that Pacrg mRNA is detected only wild-type testis (testis lane wt). The Pacrg mRNA is completely absent in testes from qk^v mutant animals (testis, qk^v/qk^v lane). (Lower) Hybridization with an actin probe as loading control. (B) Northern blot on human tissues shows that PACRG mRNA is most abundant in testis. Lower levels of expression are also detected in brain, liver, small intestine, pancreas, prostate, and ovaries. (Lower) Hybridization with an actin probe is shown as a loading control.

Fig. 2.

Pacrg developmental and cellular expression during mouse spermatogenesis. (A) Northern blot analysis of Pacrg in neonatal testes shows the onset of expression of Pacrg at 14 dpp. Higher levels of Pacrg transcript are detected from 20 dpp onward, when sperm differentiation initiates. (Lower) Hybridization with an actin probe as loading control. (B) In situ hybridization analysis by using a Pacrg RNA probe on adult mouse testis shows expression in the seminiferous epithelium. Roman numerals indicate the stage(s) assigned to each seminiferous tubule (×200 magnification). (C) Cellular localization of Pacrg mRNA by in situ hybridization on testis seminiferous tubules at different stages (indicated by Roman numerals in the bottom right corner, ×400 magnification). The dotted curves delineate the approximate boundary between the spermatocyte (toward the basal membrane) and the spermatid cellular layers (toward the lumen) in each of the stages showed.

Fig. 3.

Transgenic expression of Pacrg restores spermiogenesis in qk^v males. (A) Northern blot analysis on testis total RNA from qk^v/qk^v Tg(Pacrg) transgenic (Tg), qk^v/qk^v mutant (qk^v), and unaffected control mice (c). (Lower) Hybridization with an actin cDNA probe as a loading control. (B) Periodic acid Schiff staining of testes sections from qk^v/qk^v mutant and from a qk^v/qk^v Tg(Pacrg) transgenic littermate. The arrow points to sperm tails in the lumen of the seminiferous tubules in the qk^v/qk^v Tg(Pacrg) testes.

Fig. 4.

The Pacrg protein is present in mature spermatozoa. (A) Western blot analysis on protein extracts from testis (Left) and isolated sperm (Right) from mutant (qk^v) or control wild-type (wt) animals. The arrowheads identify the 28-kDa band corresponding to Pacrg in both testes and sperm; the asterisk indicates the cross-reacting band present in the testis extract. (B) Immunofluorescence on methanol-fixed epididymal spermatozoa with anti-Pacrg antiserum (Left, ×200 magnification; Inset, ×400 magnification). White arrowheads and yellow arrows identify the Pacrg staining (green fluorescence) in the postacrosomal region and in the midpiece of the sperm flagellum, respectively. No specific fluorescence is detected in sperm incubated with anti-Pacrg sera preabsorbed with 5 μg/ml of the antigen peptides (Right). Sperm were counterstained for nucleic acids with 4′,6-diamidino-2-phenylindole (blue fluorescence).

Fig. 5.

Pacrg is evolutionarily conserved in protozoan species. Protein sequence alignment of mouse Pacrg protein with gene products from protozoa. Residues conserved in the majority of sequences are highlighted in red, conservative amino acid substitutions are highlighted in yellow, and residues identical in all sequences are highlighted in green. The consensus sequence is shown in the lower line of the alignment. The arrowhead indicates the partially conserved cysteine residue within the putative prenylation motif. The protein sequences were obtained by conceptual translation of DNA GenBank and dbEST database entries from each species, corresponding to the following accession nos.: Mouse, AK005771; Chlamydomonas, BG856901; Trichomonas, CD664326; Trypanosoma, AC091330; Giardia, BAB56145; Leishmania, AAF69760.1. Alignment was generated by using clustalw version 1.8 (see Materials and Methods).