/ . Embryol. exp. Morph. Vol. 44, pp. 263-280, 1978
Printed in Great Britain © Company of Biologists Limited 1978
263
A study of spermatozoan defects in wild-type
and T:t-bearing mice
By NINA H I L L M A ^ A N D MARY NADIJCKA1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
A comparative light and electron microscopic study was done on cauda epididymal
spermatozoa from + [tx, Tj +, T/tx, C57BL/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.
INTRODUCTION
In normal matings male mice heterozygous for specific tx alleles ( + /tx or
T/tx) transmit the ^-bearing spermatozoa in a frequency greater than 50 %.
Yanagisawa (1965) suggested that this non-Mendelian transmission ratio would
occur if all of the ^-bearing spermatozoa from heterozygous males were ultrastructurally 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, Tj + 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 ^-bearing gametes and thus less likely to reach the
site of fertilization. As a consequence, the morphologically normal F-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 (TjtwS2, Tjtxm), as well as from sterile
tir32ltlcl8, males. She also found that these males contained spermatozoa with
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pennsylvania
19122, U.S.A.
264
N. HILLMAN AND M. NADIJCKA
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.
MATERIALS AND METHODS
Cauda epididymal spermatozoa were obtained from six-month-old T/t12,
T/F 32 , T\t\ + /t12, +/*"»*, +/t\ T12I + , T32/ + , T«l + , 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 ?x-bearing gametes. The transmission frequency
of tx% and twZ% 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 x2 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-PO 4 buffer (pH 7-4, for 1 h), repelleted by centrifugation, and resuspended in 0-1 M-PO 4 buffer. After 1 h the spermatozoa were
Spermatozoon defects
265
Table 1. The frequency of abnormal spermatozoa in different
genotypes and strains of mice
Total counted for each item listed: 3000.
Normal
Random bred
BALB/c
C57BL/6J
r//66
+//
T6/ +
Tit " 3 2
+/r 32
JV32/ +
r//1212
+ //
r i 2 /+
Abnormal
No.
Freq.
No.
Freq.
2244
.1800
1746
74-8
600
58-2
731
77-3
791
730
73-8
70-7
770
75-5
72-9
756
1200
1254
807
681
627
810
786
879
690
735
813
25-2
400**
41-8**
26-9
22-7*
20-9**
270
26-2
29-3**
23-0*
24-5
27-1
2193
2319
2373
2190
2214
2121
2310
2265
2187
* Significantly different from random-bred males at the 005 level.
** Significantly different from random-bred males at the 0001 level.
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).
266
N. HILLMAN AND M. NADIJCKA
Table 2. Distribution of abnormal spermatozoa in different strains
and genotypes of mice
Abnormal heads
Abnormal tails
Total
1 UlctI
A
Coiled
Heads
no.
in
or
MisMultiple with
shaped Micro Bifid Droplets Total folded ]Double Total defects* defects
Random
bred
BALB/c
C57BL/6J
Tit"
+ //6
TB
Tit!t'32
+ //w32
Tw32/ +
TJt12
+ //12
r i2 /+
128
22
9
15
174
534
9
543
39
756
681
75
15
3
42
45
801
687
342
420
6
3
348
423
51
144
1200
1254
27
507
444
408
6
6
3
513
450
411
87
18
21
807
681
627
12
19
9
328
393
399
138
60
156
810
786
879
17
15
6
330
375
456
54
48
81
690
735
813
624
150
183
9
3
9
3
148
18
11
18
207
213
195
273
17
12
42
344
316
279
252
18
12
15
12
21
48
333
324
374
390
237
255
198
9
9
15
9
12
18
51
36
45
306
312
276
313
360
450
21
18
* The category ' multiple defects' includes those spermatozoa which had both head and
tail defects.
RESULTS AND DISCUSSION
T H E L I G H T M I C R O S C O P I C 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/tG, Tjtu'32, + /tu'32, + It12)
or a decrease in the frequency of abnormal spermatozoa (+/te, 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 (r 12 / + ) in which the frequency of abnormal
spermatozoa is the same as in random-bred males, one group (T"6/ +) in which
the frequency is significantly lower, and one group (!T"'32/ + ) in which the
frequency is significantly higher. Taken together, these data clearly indicate that
the cause of increased transmission frequencies of the ?x-bearing gametes cannot
be attributed to an increased frequency of spermatozoan abnormalities in
/^-heterozygous males.
Spermatozoon defects
267
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 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 + jtx males which could account for the non-Mendelian transmission ratio of the ^-bearing gametes.
T H E U L T R A S T R U C T U R A L STUDIES
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 lightmicroscopic 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 ?x-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
268
N. HILLMAN AND M. N A D I J C K A
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). x 11000.
Fig. 2. A longitudinal section through the head and neck of a normal spermatozoon,
x 11000.
Fig. 3. A microheaded spermatozoon with an abnormal acrosome. The neck and
midpiece appear normal, x 11000.
Fig. 4. A longitudinal section through a bifurcated spermatozoon. Note that each
apex is covered by a separate acrosome. x 14000.
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.
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 P-bearing males. Although the cause of this defect is not
Spermatozoan defects
269
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,
x 20000.
Fig. 6. A longitudinal section through a spermatozoon head which is contained
within a cytoplasmic droplet. The acrosome is not contiguous with the nucleus,
x 18000.
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, x 10000.
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).
Spermatozoa with bifurcated and bifid heads, the third head abnormality,
were found in all males. Such spermatozoa typically have a head with two
18
EMB 44
270
N. HILLMAN AND M. NADIJCKA
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).
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).
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).
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 &
Go wen (1960) that the cause of the abnormally shaped heads of spermatozoa
frompsjps males can be traced to faulty acrosome development in the spermatid.
Later, Hunt & Johnson (1971) found that the formation of an abnormal
and
acrosome is a specific phenotypic defect of spermiogenesis in pm/pm
Spermatozoan defects
111
p25Hjp25ii 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 & BirchAndersen, 1970).
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
18-2
272
N. HILLMAN AND M. N A D I J C K A
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, x 13000.
Fig. 9. A longitudinal section of a double-tailed spermatozoon. Note the presence
of the cytoplasmic droplet between the two tails, x 11000.
Fig. 10. A longitudinal section through a spermatozoon with a folded tail, x 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, x 40000.
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
Spermatozoan defects
273
are isomorphic, the probability is high that the section is from a double-tailed
spermatozoon.
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.
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.
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).
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
274
N. HILLMAN AND M. N A D I J C K A
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, x 36000.
Fig. 13. A longitudinal section through a spermatozoon with a curved tail. Note the
vacuoles in the cytoplasmic droplet, x 7500.
Fig. 14. A longitudinal section of a principal piece, a portion of which is coiled
within the cytoplasmic droplet, x 13000.
Spermatozoan defects
275
\
15
18
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, x 7500.
Fig. 16. A transverse view through the midpiece showing the normal 9 + 2 arrangement of doublets, x 27000.
Fig. 17. A transverse view through an abnormal midpiece. All of the doublets
except the middle pair are missing, x 27000.
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,
x 33500.
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
276
N. HILLMAN AND M. N A D I J C K A
19
21
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, x 41000.
Fig. 20. A transverse view through a defective principal piece. Doublets 4-7 are
missing, x 41000.
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 fn principal
Spermatozoan defects
277
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/twls, T/r 3 2 and twl8ltwZ2 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).
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
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, x 40000.
Fig. 22. A longitudinal section through either a double or folded tail. Ectopic outer
dense fibers are present (arrow), x 13000.
Fig. 23. A transverse section through a midpiece which contains abnormally
arranged mitochondria, x 17500.
Fig. 24. A longitudinal section through a midpiece in which the mitochondria
are abnormally arranged, x 17500.
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N. HILLMAN AND M. NADIJCKA
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).
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).
CONCLUSIONS
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 ^-bearing spermatozoa from
heterozygous (T/P, + jtx) 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 F-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.
Spermatozoon defects
279
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{Received 9 September 1977)