Genomic Analysis of the Hook1 Gene in the azh/azh Mutant

On the basis of the deletion in the Hook1 cDNA detected by RTPCR, a genomic fragment spanning exons 9-12 was amplified with wild type, heterozygote (+/azh) and homozygote azh/azh DNA. In this case, a deletion in the azh/azh mutant was observed when the PCR fragments were compared to those of wild type mouse. The heterozygote DNA presented both bands.

Several research groups have worked with the azh/azh mutant because of its unique feature of displaying an abnormal phenotype that affects exclusively the spermatozoa head shape, quality that makes this mutant mouse the subject in reproduction investigations concerning the study of head shape alterations. The general method used until now to identify the homozygous azh/azh from the heterozygous or wild type litter mates is through testicular biopsy, but it is clear that such procedure is not totally effective. In fact, for this research, an acquired heterozygous +/azh mouse resulted to be a wild type mouse, confirmed by the genomic PCR together with the analysis of the generated offspring. Now, we propose that a deletion of the Hook1 gene can be responsible for the mutant phenotype azh/azh. Therefore, by simple PCR, it might be possible to genotype the mice coming from a litter with the azh allele background effectively and rapidly. Until now, 59 mice coming from five breeding crosses have been amplified with a relation of genotype-phenotype of 100%, using only a small piece of tail. By this way, it was clearly demonstrated that both heterozygous and homozygous mice presented the deletion, and that the deletion segregated with the phenotype.

Interestingly, Meistrich et al. (1994) speculated that the azh mutation should arise from a deletion at a regulatory locus. Besides, they believe it is possible that the more cylindrical, less flattened spermatozoa heads, the most of which lack the pointed hook, can be some causes of the reduced fertility, since they would be unable to concentrate penetrating forces in as small an area as wild type sperm do. This sperm form defect together with the possibility that altered enzyme levels in the acrosome of sperm suggest that in the low fecundity observed in the azh/azh mouse can be due to inability to fertilize ova. These hypotheses could be right, but certainly the disassembly easiness displayed by the sperm head and tail as well as the reduced acrosome reaction seen by club-shaped sperm in azh/azh, can probably also play a role in the reduced fertility observed in the azh mutation. Concerning this matter, some unexplained human infertility cases have been reported to be related to the fragility in the connection between head and tail and with abnormal spermatozoa head shape (megalohead and pinhead) (Kahraman et al., 1999; Kamal et al., 1999). It would be interesting to analyse DNA of these patients to look for possible mutations in the human Hook1 gene.

By analysing the nature of the defect present in the azh/azh mouse, it has been observed that in longitudinal sections of developing spermatids tails of azh/azh, the midpiece revealed a structurally intact axoneme surrounded by dislodged mitochondria at a focal point where the abnormal accumulation of dense material was observed (Mochida et al., 1999). This accumulation is not present in wild type spermatids. This finding strongly suggest that there could be a blockage in the transport of some material in the mitochondrial sheath during the spermatid stage related to the azh/azh mutation, since this accumulation is not present in wild type and heterozygous mice. What is more, some dense material (named linkers) have been reported to be between the nuclear envelope and the manchette, attaching individual microtubules of the manchette to one another as well as microtubules of the nuclear envelope. These rod-like linkers were not seen in spermatids after elongation occurred and the manchette has been displaced caudally.

They may have the task of maintaining parallel the manchette with respect to the nuclear envelope and helping the manchette to “zipper” down the nuclear envelope, thereby changing its shape. These linkers may be microtubule-associated proteins (MAPs) or microtubule motor proteins (cytoplasmic dyneins or kinesin) that bind microtubules and stabilize and link them to each other. Nothing is cleared about the linkers in the azh/azh mutant (Russell et al., 1991). According to Walenta et al. (2001), a function of Hook proteins as microtubule linker proteins is also consistent with the analysis of Drosophila hook1 loss-of-function mutations in endocytic trafficking. Drosophila hook1 appears to be responsible for the assembly or stability of mature MVBs (Walenta et al., 2001) and probably be microtubule linkers necessary for the anchoring of organelles in their characteristic positions (Nielsen, et al., 2000). Concerning this, it is noticeable to keep in mind that the manchette serves as a path for movement of organelles during the spermiation.

Mochida et al. (1999) speculated that the sperm tail abnormalities such as bending and looping of the midpiece of the tail of azh spermatozoa could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation (1999). These probable deficiencies in assembly of peri-axonemal proteins could be related to the dysfunction of the Hook1 protein, which is truncated in the azh/azh mouse due to a deletion of the exons 10 and 11.

On the other hand, Hook1 could also be a microtubule-dependent motor protein that provides a solid framework for transport of organelles along microtubules. Concerning this, accumulation of dense material was observed in the flagella of Chlamydomonas fla14 mutant homolog to the 8 kDa dynein light chain (Pazour et al., 1998) similarly as dense material was seen to accumulate in the principal piece of the azh mutant spermatozoa. In fact, it has been speculated that vesicular transport between compartments is organized along microtubules: trafficking from early to late endosomes depends on microtubules, as does retrograde and anterograde trafficking between the Golgi complex and the ER (Gruenberg and Howell, 1989, Lippincott-Schwartz, 1998).

Concerning this, the actin cytoskeleton plays an active role in the engulfment of phagocytosed material (Allen and Aderem, 1995). An intriguing connection between the function of Drosophila hook1 and actin cytoskeleton is provided by the visible bristle phenotype of hook1 mutations. Mutations in the forked, chickadee and singed genes cause malformations of bristles similar to hook mutations. The chickadee and singed genes encode Drosophila homologues of the actin-binding proteins profilin and fascin.

Profilin is a family of actin-binding proteins. It is involved in diverse functions such as maintaining cell structure integrity, cell motility, tumor cell metastasis, as well as growth factor signal transduction. Some members of this family are ubiquitous, but there is one form specific for kidney and testis. Fascin is expressed in several tissues including eye, brain, uterus, and in Drosophila the female mutants are sterile. These proteins are required for the formation of actin bundles in the early stages of extension of the bristle shaft, but the function of these actin-binding proteins is not restricted to the formation of bristles (Cant et al., 1994). To date the role of these proteins in phagocytosis or endocytosis of transmembrane ligands has not been tested.

In this work, the mouse Hook1 gene was characterized and it was demonstrated the importance of this gene in spermatogenesis and that loss of Hook1 function results in an azh/azh mutant phenotype. Also, from the findings found in this work, it can be speculated that mutations of the human Hook1 gene can be responsible for a genetically cause of male infertility in humans.