Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 1. Evidence for a structural deficit in the axoneme.

Male mice had previously been generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was inactivated by the substitution of four exons encoding the ATP-binding site (P1-loop) with the neomycin resistance gene, giving a putative non-functional gene product. We have used additional techniques of electron microscopy to determine what effect the truncated, non-functional heavy chain has on the assembly of the inner dynein arm complex. From a comparison of MDHC7-/- with the wild-type morphology, we have found that the expected loss of a C-terminal (globular) domain is associated with inner dynein arm 3, a change from two visible "heads" to one. This deficit was seen in replicas of rapidly-frozen, deeply-etched spermatozoa, and was confirmed in filtered images of 20-nm-thin sections, cut in longitudinal planes. Assembly of the other IDAs appeared unaffected. This study is the first to reveal the location of a specific dynein heavy chain within the 96-nm repeat pattern of the inner dynein arms of the mammalian axoneme.

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 2. A developmental explanation for the asthenozoospermia.

Male mice had been previously generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was disrupted. MDHC7-/- animals show asthenozoospermia and are sterile. Very few of their spermatozoa can achieve forward progression, but for those that can, we add here the information (1) that the three-dimensional aspects of their movement are normal; (2) that their maximum velocity is less than that of wild-type controls; and (3) that they are entirely unable to penetrate media of raised viscosity (25-4,000 cP). However, the large majority of the spermatozoa can achieve only a low amplitude vibration. In these sperm we find, using electron microscopy, that the outer dense fibres retain attachments to the inner surface of the mitochondria. Such attachments are present in normal epididymal mouse spermatozoa but are broken down as soon as the sperm become motile on release from the epididymis. The attachments are presumed to be essential during midpiece development and, afterwards, to require a threshold level of force to loosen them and so permit the sliding displacements necessary for normal bending. We presume that the disruption of the inner dynein arm heavy chain gene, MDHC7, means that there is insufficient force to overcome the attachments, for all but a few spermatozoa.

Basal sliding and the mechanics of oscillation in a mammalian sperm flagellum.

The mechanism of oscillation in cilia and flagella has been a long-standing mystery. This article raises the possibility of a mechanical explanation based on new findings relating to where in the flagellum microtubule sliding can occur--and where it cannot occur. All theoretical analyses of flagellar bending have until now made the assumption that sliding displacements at the base of the flagellum cannot occur. One consequence of this has been the need to accept that sliding must be transmitted through propagating bends, an idea that has been tolerated even though it becomes paradoxical if bends are the result of resistance to sliding. Our observations, of spermatozoa from the chinchilla, have led us to a contradictory view. We have shown directly, by light microscopy and by two methods of electron microscopy, that basal sliding does occur. Also, evidence from video microscopy indicates that a propagating bend cannot transmit sliding through it. We have analyzed a movement pattern in which the beat frequency increases fourfold in a phasic manner. Our analysis of this suggests that new bends terminate when no further sliding is possible. At this point the bend direction immediately reverses. That is, the flagellar beat frequency increases when there is a limitation to sliding. One can see directly the alternation in basal sliding direction under these circumstances. This suggests a mechanism for the initiation of a new bend in the opposite direction to the bend just completed: we propose that the initiating trigger is the reversal of elastic deformations at the base, which reverses the direction of interdoublet sliding.

Microtubule displacements at the tips of living flagella.

We have observed that the flagellar axoneme of the Chinese hamster spermatozoon undergoes periodic changes in length at the same frequency as the flagellar beat. The amplitude of the length oscillation recorded at the tip is maximally about 0.5 microm or 0.2% of the total length. In some favourable cells, it was possible to see the opposing "halves" of the axoneme moving at the tip in a reciprocating manner and 180 degrees out-of-phase. This behaviour, when analysed quantitatively, is broadly consistent with predictions made from the sliding-doublet theory of ciliary and flagellar motility and thus it constitutes an additional verification of the theory, for the first time in a living cell. However, on close examination, there is a partial mismatch between the timing of the length oscillation and the phase of the beat cycle. We deduce from this that there is some sliding at the base of the flagellum, sliding that is accommodated by elastic compression of the connecting piece. Micrographic evidence for such compression is presented.