A comparative light and electron microscopic study was done on cauda epididymal spermatozoa from + /tx, T/ +, T/tx, C5TBL/6J, BALB/c and randomly breeding Swiss Albino mice. The results show that all of the males contain abnormal spermatozoa and that all contain the same types of defective gametes. No unique defect was found which can be correlated with the increased transmission frequency of the tx -bearing allele.

In normal matings male mice heterozygous for specific tx alleles ( + /tx or T/P) transmit the tx -bearing spermatozoa in a frequency greater than 50 %. Yanagisawa (1965) suggested that this non-Mendelian transmission ratio would occur if all of the tx -bearing spermatozoa from heterozygous males were ultra-structurally normal while some, or most, of the + -or T-bearing spermatozoa were ultrastructurally defective. This hypothesis was based on data obtained from an ultrastructural study of vasa deferentia spermatozoa from T/t1, T/ + and + / + males. In this study, Yanagisawa found that the gametes had only tail aberrations (missing, excessive or disorganized doublets and/or dense fibers); and that these defective gametes were limited, with the exception of one abnormal spermatozoon in the T/+ males, to the mice heterozygous for the t1 allele.

On the basis of these results, Yanagisawa proposed that the presence of a tx allele caused spermatozoan tail abnormalities; and since the tx spermatozoa bear a selective advantage, he further suggested that the abnormal spermatozoa were the T-bearing gametes. The tail defects of the T-bearing spermatozoa would render them less motile than tx -bearing gametes and thus less likely to reach the site of fertilization. As a consequence, the morphologically normal tx -bearing spermatozoa would be at a selective advantage in fertilizing ova, resulting in the increased transmission frequencies. Olds (1971, 1973) examined epididymal spermatozoa from fertile heterozygous (T/tw32, T/tw18), as well as from sterile tw32/tw18, males. She also found that these males contained spermatozoa with tail defects. In this latter study, however, spermatozoa from + / + and T/ + males were not examined.

The evidence in support of Yanagisawa’s hypothesis is, therefore, based upon a limited number of observations and is still incomplete. The present light and electron microscopic studies were undertaken to determine if a consistent and specific abnormality could be found in spermatozoa obtained from heterozygous (T/tx; + /tx) males and if this abnormality was limited to only those spermatozoa obtained from males heterozygous for the tx allele. We reasoned that either a positive or negative correlation between the presence of a tx allele and a specific spermatozoan defect would serve to focus the search for the primary cause of the transmission ratio distortion.

Cauda epididymal spermatozoa were obtained from six-month-old T/t12, T/tw32, T/t6 + /t12, + /tw32, +/t6, T12/ +, T32/ +, T6/ +, BALB/c, C57BL/6J and randomly breeding Swiss Albino mice. These same animals were also used for spermiogenesis studies reported elsewhere (Hillman & Nadijcka, 1978). All of the males were tested for fertility and found to be normal fertile (Dunn & Bennett, 1969). Each of the heterozygous (T/tx, +/tx) males exhibited high transmission frequencies of the tx -bearing gametes. The transmission frequency of t12 and tw32 averaged 0 · 75 and that of t6 averaged 0 · 78.

Three males of each genotype and strain were sacrificed by cervical dislocation, and their cauda epididymides were extirpated and placed separately into sperm Ringer’s solution (pH 7 · 4). The epididymides were teased apart to free the spermatozoa. For observations at the light-microscope level, an aliquot of each of these epididymal samples was used to score both the specific types of gross abnormalities and the percentage of spermatozoa which showed each specific type of abnormality. For these determinations, filtered 1 % aqueous Eosin Y in sperm Ringer’s was added in a 1:10 ratio to the aliquot containing the spermatozoa (Wyrobek, Heddle & Bruce, 1975). After a 30 min staining period, spermatozoa were placed on slides, air dried and the slides were mounted. One thousand spermatozoa were scored from each male. Comparisons between the frequency of abnormal spermatozoa in random-bred and inbred strains and in random-bred and mutant genotypes were made using a contingency χ 2 test. Because of the technical limitations imposed by electron microscopy, it was necessary to utilize these light microscopic observations in order to quantitate the frequencies of abnormal spermatozoa.

The remainder of each spermatozoan sample was placed into a centrifuge tube and the spermatozoa were pelleted (800 rev./min for 5 mins). The supernatant fraction was decanted. The spermatozoa were resuspended in 3 % glutaraldehyde in 0 · 1 M-PO4 buffer (pH 7 · 4, for 1 h), repelleted by centrifugation, and resuspended in 0 · 1 M-PO4 buffer. After 1 h the spermatozoa were recentrifuged. The pellet was treated, in turn, with 1 % OsO4 in Millonig’s buffer (pH 7 · 3, 1 h) and Millonig’s buffer (pH 7 · 3, 1 h). The spermatozoa were then dehydrated through alcohols and embedded in epon.

The cauda epididymides from the remaining three mice of each genotype and strain were extirpated, fixed in 3 % glutaraldehyde and cut into 1 mm segments. Each segment was processed for electron microscopy. The spermatozoa within these segments served as a control for the centrifuged spermatozoa to eliminate the possibility that any of the observed ultrastructural abnormalities occurred as a result of the preparative procedures. Ultrathin sections of the epididymides and of the centrifuged spermatozoa were stained with uranyl acetate (Watson, 1958) and lead citrate (Venable & Coggeshall, 1965) and examined with a Philips 300 electron microscope.

The excellent ultrastructural studies of mammalian spermatozoa by Fawcett & Ito (1965), Fawcett & Phillips (1969), Stefanini, Oura & Zamboni (1969), Zamboni & Stefanini (1971) and Fawcett (1975) have been utilized both for distinguishing abnormal spermatozoa and for the morphological terminology used in the present report. The numbering system of the flagellar doublets and their associated outer dense fibers follows the numbering pattern proposed by Afzelius (1959).

THE LIGHT MICROSCOPIC STUDIES

Although the majority of the spermatozoa from each male were normal, all of the males contained abnormal epididymal gametes (Table 1). The data show that C57BL/6J and BALB/c males contained higher frequencies of abnormal gametes than did T/tx, T/ +, and + /tx males. These observations confirm the subjective ranking of these same males based on the ease of finding abnormal spermatids in randomly selected thin sections of their seminiferous tubules (Hillman & Nadijcka, 1978). The data also show that males which are heterozygous for the t alleles, each of which is transmitted in an increased frequency, have either the same frequency of abnormal spermatozoa, (T/t6, T/tw32, + /tw32, + /t12) or a decrease in the frequency of abnormal spermatozoa (+/t3, T/t12) when compared with the frequency of abnormal spermatozoa in random-bred males. Moreover, among the T/+ males where there is no distorted transmission frequency we have found one group (T12/ + ) in which the frequency of abnormal spermatozoa is the same as in random-bred males, one group (T6/ + ) in which the frequency is significantly lower, and one group (Tw32/ + ) in which the frequency is significantly higher. Taken together, these data clearly indicate that the cause of increased transmission frequencies of the tx -bearing gametes cannot be attributed to an increased frequency of spermatozoan abnormalities in tx -heterozygous males.

Table 1.

The frequency of abnormal spermatozoa in different genotypes and strains of mice

The frequency of abnormal spermatozoa in different genotypes and strains of mice
The frequency of abnormal spermatozoa in different genotypes and strains of mice

It can be noted from Table 2 that the higher frequency of abnormal spermatozoa in the inbred strains, C57BL/6J and BALB/c, results primarily from the large numbers of spermatozoa with abnormal heads. In earlier studies, Krzanowska (1972) reported that inbred strains of mice ‘differed significantly in the incidence of spermatozoa with morphologically abnormal heads, with the highest percentage found in C57BL/6J’. Although Krzanowska did not examine spermatozoa from BALB/c males, our data lend support to her observation that strains and genotypes differ in the incidence of abnormal spermatozoa and that C57BL/6J contain high numbers of aberrant gametes.

Table 2.

Distribution of abnormal spermatozoa in different strains and genotypes of mice

Distribution of abnormal spermatozoa in different strains and genotypes of mice
Distribution of abnormal spermatozoa in different strains and genotypes of mice

Table 2 also shows that all classes of aberrant spermatozoa were present in males from each of the strains and genotypes represented. No specific defects were found in males which were heterozygous for the tx alleles. These observations support the results of the light microscopic studies by Bryson (1944), Rajasekarasetty (1954), and by Braden & Gluecksohn-Waelsch (1958). These investigators found no unique gross defect(s) of the spermatozoa obtained from fertile T/tx and + /tx males which could account for the non-Mendelian transmission ratio of the tx -bearing gametes.

General observations

No differences were observed between the types of abnormalities found in the spermatozoa subjected to centrifugation and in those fixed within the epididymides. The ease of finding aberrant spermatozoa in each male was directly correlated with the incidence of abnormal spermatozoa found in the light-microscopic studies. All of the males contained abnormal spermatozoa and all groups of males contained spermatozoa with the same types of ultrastructural defects.

Head abnormalities

The head defects can be grouped into four categories, the same as those which can be distinguished at the light microscope level. These are (1) misshaped heads, (2) microheads, (3) bifurcated and bifid heads, and (4) heads contained in cytoplasmic droplets.

Bryson (1944) and Rajasekarasetty (1954) described, and presented camera lucida drawings of, the types of irregularly shaped (misshaped) heads of spermatozoa obtained from + / + and various tx -bearing mice. The types of abnormally shaped heads found in the present light and electron microscopic study agree with those described in both of the former studies and will, therefore, be discussed only briefly in this report. Spermatozoa with misshaped heads are present in a higher frequency, in all males, than are spermatozoa with any other head defect (Table 2). The aberrant head shapes which we observed are myriad. However, in most of the misshapen heads the distance from the implantation fossa to the most anterior tip of the head is greatly reduced (Fig. 1) when compared with this distance in a normal spermatozoon (Fig. 2). This reduction in length causes the head to appear wedge-or club-shaped.

Fig. 1.

A longitudinal section through the misshaped head of a spermatozoon. The distance from the implantation fossa to the most anterior part of the head is reduced compared with the normal (compare with Fig. 2). × 11000.

Fig. 1.

A longitudinal section through the misshaped head of a spermatozoon. The distance from the implantation fossa to the most anterior part of the head is reduced compared with the normal (compare with Fig. 2). × 11000.

Fig. 2.

A longitudinal section through the head and neck of a normal spermatozoon, × 11000.

Fig. 2.

A longitudinal section through the head and neck of a normal spermatozoon, × 11000.

Spermatozoa which are classified as ‘microhead abnormals’ have heads which, in addition to being smaller than normal, are usually misshapen (Fig. 3). Bryson (1944) and Rajasekarasetty (1954) also found microheaded spermatozoa in both control and tx -bearing males. Although the cause of this defect is not known, it is possible that the microheaded spermatozoa develop from abnormal spermatids containing nuclei which are smaller than normal (see fig. 14, Hillman & Nadijcka, 1978).

Fig. 3.

A microheaded spermatozoon with an abnormal acrosome. The neck and midpiece appear normal, × 11000.

Fig. 3.

A microheaded spermatozoon with an abnormal acrosome. The neck and midpiece appear normal, × 11000.

Spermatozoa with bifurcated and bifid heads, the third head abnormality, were found in all males. Such spermatozoa typically have a head with two apices, and these heads often have lateral nuclear extensions (Fig. 4). These apices and extensions can be covered by a single continuous acrosome; or each apex can be covered by a separate acrosome. Those spermatozoa which contain multiple acrosomes may develop from a uninucleated spermatid in which two or more proacrosomal vesicles and granules become associated with the single nucleus. Conversely, the bifurcated spermatozoa which contain a single acrosome may develop from binucleated spermatids. Both of these types of defective spermatids; cells with duplicated proacrosomal vesicles and cells which contain multiple nuclei which share a single acrosome; have been observed in all of the males used for this study (Hillman & Nadijcka, 1978).

Fig. 4.

A longitudinal section through a bifurcated spermatozoon. Note that each apex is covered by a separate acrosome. × 14000.

Fig. 4.

A longitudinal section through a bifurcated spermatozoon. Note that each apex is covered by a separate acrosome. × 14000.

The fourth class of spermatozoan head abnormalities is composed of spermatozoa whose heads are contained in cytoplasmic droplets. Usually the heads are bizarre. There are similarities between these aberrant heads and the aberrant spermatid heads which develop from multinucleated cells in which the nuclei share a common acrosome (Bryan & Wolosewick, 1973; Hillman & Nadijcka, 1978). We presume, therefore, that these defective spermatozoa originate from conjoined multinucleated spermatids. The multinucleated heads are contained within cytoplasmic droplets; and frequently, thin sections of these droplets contain multiple transverse or longitudinal sections of the tail (Fig. 5). The head and multiple sections of the tail(s) are not surrounded by a plasma membrane and are not contained in vacuoles. We propose that the head and tail(s) are contained within the residual spermatid cytoplasm. This is based on the observation that the cytoplasm contains, in addition to the spermatozoan structures, no organelles except smooth-surfaced tubular and vesicular elements. These structures are characteristic of those found in the cytoplasmic droplet of mammalian epididymal spermatozoa (Bloom & Nicander, 1961 ; Fawcett & Ito, 1965).

Fig. 5.

A section through a cytoplasmic droplet which contains a bizarre head and six repeats of the midpiece of the tail. In one section of the midpiece (→) the doublets and outer dense fibers are abnormally arranged and in another section (----→) doublets 4 – 7 and their associated outer dense fibers are missing. Note the presence of ectopic outer dense fibers and doublets in the cytoplasm of the droplet, × 20000.

Fig. 5.

A section through a cytoplasmic droplet which contains a bizarre head and six repeats of the midpiece of the tail. In one section of the midpiece (→) the doublets and outer dense fibers are abnormally arranged and in another section (----→) doublets 4 – 7 and their associated outer dense fibers are missing. Note the presence of ectopic outer dense fibers and doublets in the cytoplasm of the droplet, × 20000.

The cytoplasmic droplets may also contain the head of a spermatozoon which developed from a uninucleated spermatid (Fig. 6). Such heads may be normally or abnormally shaped, and the droplet may also contain sections of a tail, or tails. Because of our observation that the presence of numerous cross sections of tails within a common cytoplasm is usually a result of either the coiling or the folding of a single tail rather than to the presence of multiple tails, we assume that the multiple cross-sections of tails found in sections of these aberrant spermatozoa are also from a single tail which is coiled or folded (see below).

Fig. 6.

A longitudinal section through a spermatozoon head which is contained within a cytoplasmic droplet. The acrosome is not contiguous with the nucleus, × 18000.

Fig. 6.

A longitudinal section through a spermatozoon head which is contained within a cytoplasmic droplet. The acrosome is not contiguous with the nucleus, × 18000.

Acrosomal defects

All of the spermatozoa which have aberrantly shaped heads also have abnormal acrosomes (Figs. 1, 3 – 5). It was suggested by Hollander, Bryan & Gowen (1960) that the cause of the abnormally shaped heads of spermatozoa from ps/ps males can be traced to faulty acrosome development in the spermatid. Later, Hunt & Johnson (1971) found that the formation of an abnormal acrosome is a specific phenotypic defect of spermiogenesis in p6H/p6H and p25H/p25H sterile mice. In these sterile genotypes the spermatozoan heads were misshapen, and Hunt & Johnson concluded that the irregularities of the head shape were caused by the aberrantly formed acrosomes. It is probable therefore that the aberrantly shaped spermatozoan heads, common to all of the mice examined in the present study, are also related to faulty acrosomal development during spermiogenesis.

Multiple defects

Although the neck and tail are normal in most of the spermatozoa with abnormally shaped heads, we have found spermatozoa having both head and tail defects in all males examined (see Table 2: multiply defective sperm). While certain tail defects (coiled, folded or double tails) can be readily distinguished at the light-microscope level, other tail defects can be resolved only at the ultrastructural level. For example, spermatozoa with abnormal heads often have disorganized or missing flagellar components. Therefore, because of the technical limitations imposed by both light and electron microscopy, the exact incidence of multiply defective spermatozoa cannot be accurately determined.

Neck defects

Spermatozoa having visible defects in the neck region were never observed. The only abnormality common to all of the males and attributable to defects in the neck region was the presence of tails without heads and the reverse. The separation in all of these spermatozoa occurred between the implantation fossa and the capitulum of the connecting piece (Fig. 7). This abnormality was not included as a category of spermatozoan abnormalities in the light-microscopic study since it was infrequently observed and since the possibility existed that the separation of the head from the neck occurred as a result of processing. The observation, however, is similar to the ‘decapitated sperm defect’ which has been described in Guernsey cattle, which is genetic in origin and which results in sterility. In these cattle, the separation is associated with the migration of the cytoplasmic droplet along the midpiece (Hancock, 1955; Blom & Birch-Andersen, 1970).

Fig. 7.

A longitudinal view of a spermatozoon showing the separation of the head from the flagellum. The separation always occurs between the implantation fossa and the capitulum of the neck, × 10000.

Fig. 7.

A longitudinal view of a spermatozoon showing the separation of the head from the flagellum. The separation always occurs between the implantation fossa and the capitulum of the neck, × 10000.

Tail defects

Spermatozoa with double tails are one of the least frequent types of abnormalities which we observed but are found in all of the groups of males (Table 2). There are difficulties, however, in establishing the true double nature of sperm tails at the ultrastructural level. In order to establish the reality of the duplication which we apparently observe in longitudinal tail sections (Fig. 8) it is necessary to trace the tail(s) through serial sections to the implantation fossa or fossae. This is true even when the duplicated elements are contained within a common plasma membrane. In a number, but not all, of the observed putative cases of double tails, we have established the duplication through serial sections which show that there are two flagella and that the capitulum of each neck is connected to a single nucleus at a separate implantation fossa (Fig. 9). Double tails are more easily categorized as such in transverse sections. According to Gibbons (1963), the two diverging arms of subunit A normally extend in a clockwise direction. Thus, in a transverse section which contains portions of two axonemal complexes, if the arrangements of the substructures of the axonemes are isomorphic, the probability is high that the section is from a double-tailed spermatozoon.

Fig. 8.

A longitudinal view through two midpieces which are surrounded by a single plasma membrane. In this longitudinal view it is not possible to determine if the section is from a spermatozoon with a double or folded tail, × 13000.

Fig. 8.

A longitudinal view through two midpieces which are surrounded by a single plasma membrane. In this longitudinal view it is not possible to determine if the section is from a spermatozoon with a double or folded tail, × 13000.

Fig. 9.

A longitudinal section of a double-tailed spermatozoon. Note the presence of the cytoplasmic droplet between the two tails, × 11000.

Fig. 9.

A longitudinal section of a double-tailed spermatozoon. Note the presence of the cytoplasmic droplet between the two tails, × 11000.

The necessity for following serial sections in order to establish true tail duplication arises from our observations that apparently duplicated sperm tails found in ultrathin sections are actually portions of a single tail which is folded or coiled within a common cytoplasm. For example, Fig. 10 shows a spermatozoon which has a single tail with a folded midpiece. Note that the outer surfaces of the folded tail are limited by the plasma membrane, while the inner surfaces are devoid of a cell membrane. A less propitious longitudinal section of this particular spermatozoon would contain only the two closely apposed parts of the folded single midpiece (cf. Figs. 8 and 10) and would be scored erroneously as a double-tailed spermatozoon. We have observed folding at all levels of the spermatozoon tail, with the extent of this folding being variable. Transverse sections through a tail, folded in the region of the principal piece, would appear as in Fig. 11. Such folding is readily established if the two axonemal complexes are enantiomorphic.

Fig. 10.

A longitudinal section through a spermatozoon with a folded tail, × 6000.

Fig. 10.

A longitudinal section through a spermatozoon with a folded tail, × 6000.

Fig. 11.

A transverse section through a tail folded in the area of the principal piece. Note the enantiomorphism of the two axonemal complexes, × 40000.

Fig. 11.

A transverse section through a tail folded in the area of the principal piece. Note the enantiomorphism of the two axonemal complexes, × 40000.

Folding does not always result in the affected sections of the tail being closely apposed to each other (Figs. 12, 13). In some spermatozoa the tail is curved rather than folded and the space between the curved tail segments contains cytoplasm in which the remnants of the organelles of the cytoplasmic droplet are present. In some cases this cytoplasm is vacuolated (Fig. 13). It is possible that this vacuolization is a normal method for disrupting the droplet and that it continues until the residual cytoplasm is totally removed. Under these conditions the tail would straighten, and the spermatozoon would become a morphologically normal gamete.

Fig. 12.

A longitudinal section through a curved spermatozoon tail. Unlike folded tails, in curved tails no segments of the tail are closely apposed to each other. CD, Cytoplasmic droplet, × 36000.

Fig. 12.

A longitudinal section through a curved spermatozoon tail. Unlike folded tails, in curved tails no segments of the tail are closely apposed to each other. CD, Cytoplasmic droplet, × 36000.

Fig. 13.

A longitudinal section through a spermatozoon with a curved tail. Note the vacuoles in the cytoplasmic droplet, × 7500.

Fig. 13.

A longitudinal section through a spermatozoon with a curved tail. Note the vacuoles in the cytoplasmic droplet, × 7500.

When the tail is folded, a transverse section contains only an apparent duplication of the tail. When the tail is coiled, a transverse section contains multiple repeats of the same tail. The coiling, like folding, can involve either a specific section (Fig. 14) or different sections of the flagellum. As a consequence, the component tail parts found in a transverse section of a coiled tail will vary (Fig. 15).

Fig. 14.

A longitudinal section of a principal piece, a portion of which is coiled within the cytoplasmic droplet, × 13000.

Fig. 14.

A longitudinal section of a principal piece, a portion of which is coiled within the cytoplasmic droplet, × 13000.

Fig. 15.

This micrograph contains one transverse view of a curved tail (arrow) and one transverse view of a coiled tail. The latter contains sections of both the midpiece and the principal piece, × 7500.

Fig. 15.

This micrograph contains one transverse view of a curved tail (arrow) and one transverse view of a coiled tail. The latter contains sections of both the midpiece and the principal piece, × 7500.

The cause of the coiling or folding of spermatozoan tails within a common cytoplasm is unknown. The phenomenon has, however, been described in other mammalian species (Blom & Birch-Andersen, 1966, Blom, 1966, 1968 -Jersey cattle; Aughey & Renton, 1968 -Ayrshire bull; Ross, Christie & Kerr, 1971 — human; Kojima, 1975-boar). It has been suggested that adverse conditions (e.g. hypotonicity -Drevius, 1963, Drevius & Eriksson, 1966; lowered temperatures -Pedersen & Lebech, 1971) cause tails to coil within a common cytoplasm. Although it cannot be ruled out that some of the coiling and folding found in our light-microscopic studies may have resulted from the preparative techniques, adverse conditions were avoided in the processing of the spermatozoa for ultrastructural studies. Because spermatozoa with coiled and folded tails have been found in all of the epididymal spermatozoan samples examined at the ultrastructural level but have not been found in ultrastructural studies of the testes (Hillman & Nadijcka, 1978), we suggest that the coiling or folding of spermatozoan tails within a common cytoplasm is a ubiquitous defect of mouse epididymal spermatozoa. Hancock (1972) suggested that these defects ‘may occur as a result of the fusion of different areas of the plasma membrane where these become apposed to one another as a result of coiling and bending of the sperm tail’. Causal evidence, however, is not available; and the phenomenon remains unexplained.

Axonemal and outer dense fiber defects

Identical types of axonemal defects have been observed in normal, folded and coiled tails in spermatozoa of all males. Transverse sections of the midpiece of an unaffected tail show a 9 + 2 arrangement of flagellar doublets (Fig. 16). In extreme cases the midpiece will lack either all of the doublets or all of the doublets except for the central pair (Fig. 17). Transverse sections of the principal piece which lack all doublets or which contain only the central doublet have also been found. In less extreme cases, only certain doublets are missing. These are usually doublets 4 and 7 (Fig. 18) or 4 – 7 (cf. Figs. 19 and 20). Olds (1971, 1973) found that doublets 4-7 were frequently missing in epididymal spermatozoa from T/tw18, T/tw32 and tw18/ tw32 males. This defect is also found in the epididymal spermatozoa from pink-eyed sterile mice (Hunt & Johnson, 1971) as well as in the epididymal spermatozoa of other mammalian species (e.g. Jersey cattle; Blom & Birch-Andersen, 1966).

Fig. 16.

A transverse view through the midpiece showing the normal 9 + 2 arrangement of doublets, × 27000.

Fig. 16.

A transverse view through the midpiece showing the normal 9 + 2 arrangement of doublets, × 27000.

Fig. 17.

A transverse view through an abnormal midpiece. All of the doublets except the middle pair are missing, × 27 000.

Fig. 17.

A transverse view through an abnormal midpiece. All of the doublets except the middle pair are missing, × 27 000.

Fig. 18.

A section through a spermatozoon with a coiled tail. The section through the midpiece is normal. The three sections through the principal piece are abnormal. In one section (1), all the doublets are missing; in the second (2), doublets 4 and 7 are missing and doublet 5 is misplaced; in the third (3), doublets 4 and 7 are missing but the remaining doublets are normally arranged. This micrograph shows that tail defects may be spatially limited and may vary at different levels of the same tail. × 33500.

Fig. 18.

A section through a spermatozoon with a coiled tail. The section through the midpiece is normal. The three sections through the principal piece are abnormal. In one section (1), all the doublets are missing; in the second (2), doublets 4 and 7 are missing and doublet 5 is misplaced; in the third (3), doublets 4 and 7 are missing but the remaining doublets are normally arranged. This micrograph shows that tail defects may be spatially limited and may vary at different levels of the same tail. × 33500.

Fig. 19.

A transverse view through the principal piece of a spermatozoon tail. This section shows the normal arrangement of the axonemal complex and the associated outer dense fibers at this level, × 41000.

Fig. 19.

A transverse view through the principal piece of a spermatozoon tail. This section shows the normal arrangement of the axonemal complex and the associated outer dense fibers at this level, × 41000.

Fig. 20.

A transverse view through a defective principal piece. Doublets 4 – 7 are missing, × 41000.

Fig. 20.

A transverse view through a defective principal piece. Doublets 4 – 7 are missing, × 41000.

Because of the technical limitations inherent in electron microscopy, it is impossible to trace the axial filament through the entire length of an uncoiled or unfolded tail; and it is therefore impossible to determine if a defect found in one area of the tail extends for the entire length of the structure. In cross-sections of coiled or folded tails, however, the axonemes at one level may contain the normal 9 + 2 doublet arrangement while doublets are missing or misplaced at a second level of the same axoneme. For example, the midpiece of the coiled tail in Fig. 18 is normal. The axonemal components of the principal piece, however, show different defects at different levels. This observation suggests that axonemal defects may be spatially limited and need not extend throughout the entire length of uncoiled or unfolded tails.

Fig. 21 shows additional ubiquitous flagellar abnormalities -excessive doublets, misplaced doublets, and misplaced outer dense fibers -which are found in both normal and abnormal (folded or coiled) tails. This transverse section through a coiled tail contains a portion of the midpiece, three sections of the principal piece and one section of the end piece. One section of the principal piece and the section of the end piece contain excessive numbers of doublets which are irregularly arranged. The remaining two sections of the principal piece lack doublets. In the midpiece, there is only one recognizable doublet. The outer dense fibers are irregularly arranged in both the midpiece and the principal piece. In this same figure, as well as in Fig. 18, ectopic doublets can be found in the cytoplasm. Frequently ectopic outer dense fibers are also found in the cytoplasm. This ectopic arrangement of outer dense fibers can be seen in both transverse (Fig. 5) and longitudinal sections (Fig. 22).

Fig. 21.

A transverse view of a coiled tail. The cytoplasmic droplet contains one section of the middle piece, three sections (numbered) of the principal piece and one section of the end piece. Only one doublet is present in the midpiece and the outer dense fibers are abnormally arranged. In principal piece 1, there are excessive doublets and abnormally arranged outer dense fibers; in principal piece 2, four doublets are missing and the dense fibers are abnormally arranged; and in principal piece 3, doublets are missing. In the last, the doublet subunits are separated from each other. In the section of the end piece the doublets are abnormally arranged and there is an extra doublet. Note the ectopic doublets in the cytoplasm, × 40000.

Fig. 21.

A transverse view of a coiled tail. The cytoplasmic droplet contains one section of the middle piece, three sections (numbered) of the principal piece and one section of the end piece. Only one doublet is present in the midpiece and the outer dense fibers are abnormally arranged. In principal piece 1, there are excessive doublets and abnormally arranged outer dense fibers; in principal piece 2, four doublets are missing and the dense fibers are abnormally arranged; and in principal piece 3, doublets are missing. In the last, the doublet subunits are separated from each other. In the section of the end piece the doublets are abnormally arranged and there is an extra doublet. Note the ectopic doublets in the cytoplasm, × 40000.

Fig. 22.

A longitudinal section through either a double or folded tail. Ectopic outer dense fibers are present (arrow), × 13000.

Fig. 22.

A longitudinal section through either a double or folded tail. Ectopic outer dense fibers are present (arrow), × 13000.

Abnormal mitochondrial sheath configuration

All of the animals contained spermatozoa in which mitochondria did not form the normal helical arrangement around the midpiece axoneme and its associated outer dense fibers. Fig. 23 shows typically disorganized mitochondria as they appear in cross sections through the midpiece, and Fig. 24 shows an aberrant arrangement of mitochondria in a longitudinally sectioned spermatozoon. The normal mitochondrial configuration can be seen in Fig. 16 (cross section) and in Fig. 12 (longitudinal section). Abnormal mitochondrial configurations similar to those observed here have been reported as a characteristic of the ‘decapitated sperm defect’ in Guernsey bulls (Blom & Birch-Andersen, 1970).

Fig. 23.

A transverse section through a midpiece which contains abnormally arranged mitochondria, × 17500.

Fig. 23.

A transverse section through a midpiece which contains abnormally arranged mitochondria, × 17500.

Fig. 24.

A longitudinal section through a midpiece in which the mitochondria are abnormally arranged, × 17500.

Fig. 24.

A longitudinal section through a midpiece in which the mitochondria are abnormally arranged, × 17500.

  1. All mice from both wild-type and mutant strains contain abnormal epididymal spermatozoa and all contain the same types of defective gametes.

  2. The total incidence of abnormal spermatozoa as well as the incidence of specific spermatozoan aberrations differs among the various strains and genotypes.

  3. The present findings do not support the hypothesis of Yanagisawa (1965) that the increased transmission frequency of tx -bearing spermatozoa from heterozygous (T/tx, + /tx) males is related to the abnormal axoneme structure of the T-and +-bearing gametes. Missing or excessive axonemal structures and other tail defects are ubiquitous to all of the males examined and, therefore, cannot be considered either to be caused by the presence of the t allele or to be related to the increased transmission frequency of the tx -bearing gametes.

This research was supported by United States Public Health Service Grants Nos. HD 00827 and HD 09753. The authors would like to thank Dr Ralph Hillman for his help in the preparation of this manuscript and Marie Morris and Geraldine Wileman for their technical assistance.

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