Human Reproduction, Vol.24, No.4 pp. 775–781, 2009
Advanced Access publication on December 20, 2008 doi:10.1093/humrep/den456
ORIGINAL ARTICLE Andrology
Age- and substrain-dependent sperm
abnormalities in BALB/c mice and
functional assessment of abnormal
sperm by ICSI
Hiroshi Ohta 1, Yuko Sakaide, and Teruhiko Wakayama
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku,
Kobe 650-0047, Japan
1
Correspondence address. Tel: þ81-78-306-3049; Fax: þ81-78-306-3095; E-mail: [email protected]
background: Male BALB/c mice produce a high proportion of morphologically abnormal sperm. Although the BALB/c male may be a
useful model of human male infertility, it remains unclear whether the sperm abnormality rate (SAR) is affected by age or the BALB/c substrain.
methods: SARs (head shape) were assessed in three BALB/c substrains (A, AnN, ByJ) at 7 and 9 weeks and 6–10 months of age (c.100
sperm/male). The functional ability of abnormal sperm produced from 7-week-old and 6–10-month-old males was determined in BALB/c
AnN mice by ICSI.
results: The SAR (quasi-normal plus abnormal sperm) was lower in BALB/c A than in the other two strains (P , 0.05). Further, the
SARs of BALB/c AnN and ByJ strains at 7 weeks old were high and decreased rapidly by 9 weeks, suggesting that early spermatogenesis (i.e.
the first wave of spermatogenesis) produced low-quality sperm. ICSI experiments indicated that 2-cell stage embryos which developed from
morphologically abnormal sperm from both the first wave of spermatogenesis and the older mice (6 –10 months) were similar to those from
morphologically normal sperm in terms of producing progeny, although the number of 2-cell embryos was slightly lower (P , 0.05, x2 test)
than with normal sperm.
conclusions: Although the SARs of BALB/c mice are affected by both age and substrain, the embryos that developed from morphologically abnormal sperm have normal genetic potential in terms of production of progeny.
Key words: BALB/c / ICSI / morphology / sperm / tetrazoospermia
Introduction
Spermatogenesis is a highly regulated process that takes place in the
seminiferous tubules, where morphological alterations lead to the
formation of differentiated sperm (Russell et al., 1990a). Spermatogenesis can be subdivided into three main phases: spermatogonial
proliferation, meiosis of spermatocytes and spermiogenesis of
haploid spermatids (Russell et al., 1990b). During spermiogenesis,
the round haploid spermatids undergo an elongation phase and
transformation of the germ cell in which the majority of the
somatic histones are replaced, first by transition proteins and then
protamines, packing the DNA into the sperm cell nucleus (SassoneCorsi, 2002).
A proportion of the spermatozoa produced by males of many
mammalian species are morphologically abnormal. A high incidence
of abnormal sperm may cause lower fertility or abnormal embryos,
since a high incidence of abnormal karyotypes was found in
embryos that had been inseminated with morphologically abnormal
sperm (Kishikawa et al., 1999). Therefore, it was suggested that morphologically abnormal sperm have some abnormalities in terms of producing progeny. In the mouse, some lines produce a high proportion
of abnormal sperm, although they are fertile, e.g. C57BL/Kw, PL/
J2-azh/azh, KE and BALB/c inbred strains (Krzanowska, 1981; Kot
and Handel, 1987; Krzanowska, 1988; Pogany and Balhorn, 1992).
These mouse lines would be useful models for analyzing the correlation between sperm morphology and fertilization or development.
In the mouse, spermatogenesis begins after birth; approximately 35
days are required for the development of mature sperm. This initial
spermatogenesis after birth is referred to as the first wave of spermatogenesis. Krzanowska (1981) assessed the sperm abnormality rate
& The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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776
(SAR) during development in the C57BL/Kw and KE mouse lines and
found that the SAR was higher in young males (6 or 7 weeks old) and
decreased rapidly by 8–10 weeks of age. This suggests that the first
wave of spermatogenesis produced morphologically low-quality
sperm, which may harbor abnormalities such as genetic or epigenetic
defects. However, it was unclear whether the morphologically abnormal spermatozoa produced from the first wave of spermatogenesis
could produce progeny because it was difficult to assess the function
of the abnormal sperm by mating or IVF experiments because of the
co-occurrence of normal sperm.
Male haploid germ cells can be injected directly into oocytes using
the microinsemination technique ICSI (for review see Ogura et al.,
2005; Yanagimachi, 2005). To date, this technique has been used in
fields such as basic research, animal production and human infertility
treatment. Since the ICSI technique allows the selection of specific
sperm for fertilization, it would also be useful in examining the functional integrity of morphologically abnormal sperm. Although a previous study using ICSI indicated that at least some abnormal sperm
have the ability to produce progeny (Burruel et al., 1996), it remains
unclear whether this ability is similar to that of morphologically
normal sperm.
In this study, we examined the SAR of the BALB/c mouse line,
which is commonly used in research and produces a high proportion
of abnormal sperm. We assessed the SARs of three BALB/c substrains, i.e. A, AnN and ByJ, to determine whether there is a difference
in the SARs among the substrains. The age dependence of the SAR
was also assessed. We also examined the development of oocytes
that received via ICSI abnormal sperm from both the first wave of
spermatogenesis and from older males.
Materials and Methods
Mice
Male BALB/c ByJ and BALB/c A mice were purchased from CLEA
(Tokyo, Japan). Male BALB/c AnN mice were purchased from Charles
River Japan. Female BDF1 (6– 8 weeks old) and ICR mice were purchased
from SLC (Hamamatsu, Japan).
All animal experiments were conducted in accordance with the Guide
for the Care and Use of Laboratory Animals. All experiments were
approved by the Institutional Committee on Laboratory Animal Experimentation of the RIKEN Kobe Institute.
Determination of the SAR
To assess the SAR, sperm samples were prepared from the three BALB/c
mouse strains. Briefly, mice aged 7 and 9 weeks and 6 – 10 months were
sacrificed by cervical dislocation, and sperm was retrieved from the cauda
epididymis. The sperm was fixed with 4% paraformaldehyde (PFA) for
30 min, resuspended in 50% glycerol/phosphate-buffered saline (PBS)
and mounted on glass slides for microscopic observation. The sperm
was categorized into three types by head shape according to the study
of Burruel et al. (1996): normal (Fig. 1A), quasi-normal (Fig. 1B and C)
and abnormal (Fig. 1D – F). The SAR includes quasi-normal and abnormal
sperm. Three mice were analyzed in each substrain and age. Approximately 100 spermatozoa were analyzed and classified for each male.
Ohta et al.
Figure 1 Classification of abnormal sperm from BALB/c mice, as in
Burruel et al. (1996).
(A) Morphologically normal sperm. (B and C) Quasi-normal sperm. (D –F)
Morphologically abnormal sperm.
ICSI and embryo transfer
ICSI was performed using BALB/c AnN males aged 7 and 9 weeks and
6 – 10 months. BDF1 females were induced to superovulate by consecutive
injections of equineCG (5 IU) and hCG (5 IU) 48 h apart. At 14 h
post-hCG injection, cumulus– oocyte complexes were collected from
the oviducts. Oocytes were freed from the cumulus cells by adding
0.1% bovine testicular hyaluronidase (ICN Biochemicals, Costa Mesa,
CA, USA). After the cumulus cells had dissociated from the oocytes,
the oocytes were rinsed twice with CZB medium (Chatot et al., 1990).
Approximately 2 ml of sperm suspension were mixed with a drop of
HEPES-Human Tubal Fluid medium containing 10% (w/v) polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA, USA). The sperm head was separated from the tail by applying a few piezo-pulses to the sperm neck
region and then injected into the oocyte as described by Kimura and Yanagimachi (1995). Sperm with abnormal (Fig. 1D – F) or normal (Fig. 1A)
head morphology was identified under 400 magnification and selectively
injected into oocytes following the methods of a previous report (Burruel
et al., 1996). Oocytes that were successfully injected with sperm were
incubated in CZB medium at 378C under 5% CO2 in air. When
embryos reached the 2-cell stage, they were transferred to oviducts of
0.5-day post-coitum (dpc) pseudo-pregnant ICR females. Briefly, females
were anesthetized by Avertin injection (640 mg/kg), and 10 – 15
embryos were transferred into a pseudo-pregnant female via both oviducts (i.e. 5 – 8 embryos/oviduct). After the mice recovered from the
operation, they were housed until 18.5 dpc.
Assessment of blastocysts
We examined expression of markers for trophectoderm (TE; Cdx2) and
inner cell mass (ICM; Oct 3/4), and the number of cells in the blastocysts.
The number of cells in blastocysts was estimated by counting the total
number of nuclei and the number of TE nuclei using propidium iodide
(PI) staining and immunostaining for Cdx2, respectively. The number of
cells in the ICM was estimated roughly as the total number of nuclei
minus the number of TE nuclei. Briefly, blastocysts cultured for 96 h
777
Sperm abnormalities in BALB/c mice
Table I SARs of three BALB/c substrains during mouse development
BALB/c substrain
Age
Type of sperm (%)*
..................................................................................
Normal
Quasi-normal
SAR (%)#
Abnormal
.............................................................................................................................................................................................
A
7 weeks
9 weeks
6 –10 months
72.5 + 2.4
81.8 + 2.9
66.5 + 4.6
22.4 + 0.2
16.3 + 3.0
31.3 + 3.5
5.1 + 1.9
1.9 + 0.7
2.3 + 0.4
27.4 + 2.0a,d’
18.2 + 2.4a’,e’
33.5 + 3.8a,f’
AnN
7 weeks
9 weeks
6 –10 months
33.5 + 11.5
71.9 + 3.9
51.5 + 5.0
37.2 + 6.0
24.1 + 4.2
38.5 + 4.5
29.2 + 3.7
4.0 + 1.6
10.0 + 0.8
66.5 + 9.5b,d
28.1 + 3.9b’,e
48.6 + 5.1b,f
ByJ
7 weeks
9 weeks
6 –10 months
43.7 + 0.6
71.5 + 0.6
49.9 + 2.1
40.6 + 1.0
19.9 + 0.5
38.4 + 4.9
15.6 + 0.9
8.6 + 0.3
11.7 + 3.2
56.3 + 0.5c,d
28.5 + 0.4c’,e
50.0 + 1.7c,f
*Three males were used for each age and substrain; the mean percentage + SD is shown. #The SAR includes quasi-normal and abnormal sperm, as defined in Burruel et al. (1996); the
mean percentage + SD is shown. a,a’;b,b’;c,c’;d,d’;e,e’;f,f’P , 0.05 (Tukey’s honest significant difference test).
after insemination were fixed with 4% PFA, washed with PBS containing
1% bovine serum albumin and incubated with an anti-Cdx2 monoclonal
antibody (1:200; BioGenex, San Ramon, CA, USA). Primary antibody
binding was visualized using goat anti-mouse immunoglobulin G conjugated
with Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA). After
immunostaining for Cdx2, blastocysts were incubated with PBS containing
1 mg/ml PI. Serial confocal images were taken using a fluorescence confocal microscope system (Yamagata et al., 2005), and three-dimensional
images of blastocysts were reconstructed using MetaMorph software
(Universal Imaging, Downingtown, PA, USA). Ten blastocysts from each
of three males (i.e. 30 blastocysts for one group) were stained, and the
number of cells was counted to determine the total number of cells
(PI-positive) and number of TE cells (Cdx2-positive). In some experiments, immunostaining for Oct3/4 was also performed using the same
procedure as for Cdx2 staining, using anti-Oct3/4 monoclonal antibody
(1:200; Santa Cruz Biotechnology, Inc., CA, USA).
Statistical analysis
Tukey’s honest significant difference test (Tables I and II) and Student’s
t-test (Table III) were performed. The embryo development efficiencies
in Tables IV and V were analyzed using an arcsine transformation followed
by one-way analysis of variance (ANOVA). A post hoc procedure using
Tukey’s honest significant difference test was used for multiple comparisons. The embryo development efficiencies given in Tables IV and V and
the Supplementary Table were also analyzed using the x2 test. P , 0.05
was considered statistically significant.
Results
SARs in BALB/c mouse substrains
To examine whether the SAR in BALB/c mice was affected by the
substrain or age, sperm from the BALB/c substrains A, AnN and
ByJ was assessed in 7- and 9-week-old and 6–10-month-old males
by microscopic observation. At 7 weeks of age, the SARs of BALB/
c substrains A, AnN and ByJ were (mean + SD) 27.4 + 2.0, 66.5 +
9.5 and 56.3 + 0.5%, respectively (Table I). The SARs were similar
at 7 weeks and 6–10 months of age (33.5 + 3.8% for A, 48.6 +
5.1% for AnN and 50.0 + 1.7% for ByJ; Table I). At 9 weeks of age,
the SARs of the three BALB/c substrains were markedly decreased:
18.2 + 2.4% in A, 28.1 + 3.9% in AnN and 28.5 + 0.4% in ByJ.
Table II Assessment of blastocysts that developed
from oocytes injected by ICSI with morphologically
abnormal sperm from 7-week-old and 6 –10-month-old
males
Type of
sperm
injected,
age of
mouse (n)
Total number
of cells (PI) (n)
Number of TE
cells (Cdx2)
(n)
Number of
ICM cells
(PI-Cdx2)
(n)
........................................................................................
Abnormal, 7
weeks (3)
46.4 + 10.9 (30)
38.4 + 11.8 (30)
7.9 + 5.3 (30)
Abnormal,
6– 10
months (3)
41.8 + 8.7 (30)
34.1 + 8.8 (30)
7.7 + 3.8 (30)
Normal, 9
weeks (3)
43.3 + 6.5 (30)
34.0 + 7.3 (30)
9.3 + 4.5 (30)
There were no significant differences among sperm types and age for each variable
measured (Tukey’s honest significant difference test). Ten blastocysts were examined
for each male, and three males were used in each group (i.e. 30 blastocysts/group). The
mean + SD for 30 blastocysts is shown. Cdx2 is a marker for trophectoderm (TE). PI,
propidium iodide; ICM, inner cell mass.
Thus, the SARs in BALB/c males were high in young males (7 weeks
old), decreased rapidly by 9 weeks of age and increased again at 6–10
months of age. This suggests that the first wave of spermatogenesis
produced morphologically low-quality sperm at similar frequencies
as observed in aged males. In addition, the SAR was lower in
BALB/c substrain A than in the other two substrains (P , 0.05,
Table I). Thus, the SARs of BALB/c mice are affected by both age
and genetic background.
Assessment of preimplantation embryos
which developed from oocytes inseminated
with morphologically abnormal sperm
We examined the functional ability of the morphologically abnormal
sperm produced both from the first wave of spermatogenesis (7
weeks old) and older (6 –10 months old) males by ICSI, and compared
that with morphologically normal sperm produced by 9-week-old
778
Ohta et al.
animals. Since the SARs of BALB/c substrains AnN and ByJ were
higher than that of BALB/c substrain A (Table I), we used the
BALB/c AnN substrain for the ICSI experiment. More than 85% of
the oocytes that received abnormal sperm from both young and
older males formed pronuclei with a frequency similar to that of
normal sperm from 9-week-old animals (Tables IV and V, one-way
Table III Characterization of progeny delivered after
injecting the oocytes with abnormal sperm from young
(7 weeks) and old males
Type of
sperm
injected, age
of mouse (n)
Body weight
(n) (g)
Placenta
weight (n) (g)
Gender
ratio
(M:F)
........................................................................................
Abnormal, 7
weeks (4)
1.46 + 0.10 (56)
0.15 + 0.02 (56)
25:31
Abnormal, 6 –
10 months (5)
1.46 + 0.15 (52)
0.15 + 0.02 (52)
25:27
Normal, 9
weeks (3)
1.52 + 0.13 (56)
0.17 + 0.02 (56)
26:30
There were no significant differences between sperm type and age for each variable
measured (Student’s t-test). Data are mean + SD.
ANOVA), indicating that the oocyte activating factors are retained in
these morphologically abnormal sperm. Approximately 70 –81% of
zygotes developed to the blastocyst stage, regardless of the type of
sperm used for insemination (Table IV, one-way ANOVA and the
x2 test). In Table V, however, the x2 test detected a significant difference between morphologically abnormal sperm and normal sperm,
suggesting that the in vitro development of the ICSI embryo may be
affected by sperm morphology.
We assessed blastocyst-stage embryos to determine whether
abnormal preimplantation development occurs in blastocysts that
develop from morphologically abnormal sperm. All of the blastocysts
were positive for Cdx2 (Fig. 2); therefore, we counted the number of
Cdx2-positive cells as the number of TE cells. The total number of
cells, the number of TE cells and the estimated number of ICM cells
in the blastocysts were similar among all blastocysts examined
(Table II). In addition, most of the blastocysts had Oct3/4-positive
cells in the ICM, regardless of the type of sperm used: 42/44
(Fig. 2D –D00 ), 30/31 (figure not shown) and 22/23 (figure not
shown) blastocysts which developed from 7-week-old and 6–
10-month-old abnormal sperm, and 9 week-old normal sperm,
respectively, were positive for Oct3/4. Therefore, there was no
obvious abnormality in zygotes that developed from morphologically
abnormal sperm.
Table IV In vitro development of mouse oocytes injected by ICSI with morphologically abnormal sperm from 7-week-old
and 6–10-month-old males
Type of
sperm
injected, age
of mouse (n)
No. of
embryos
injected
No. of embryos with
PN (%) (mean + SD)
No. of 2-cell stage
embryos (%)
(mean + SD)
No. of 4- to 8-cell stage
embryos (%)
(mean + SD)
No. of M/B stage
embryos (%)
(mean + SD)
.............................................................................................................................................................................................
Abnormal, 7
weeks (3)
127
117 (92.1) (92.8 + 2.5)
116 (91.3) (92.1 + 2.4)
106 (83.5) (85.5 + 9.9)
97 (76.4) (76.3 + 17.7)
Abnormal, 6 –
10 months (3)
113
108 (95.6) (95.4 + 0.6)
107 (94.7) (94.6 + 1.3)
96 (85.0) (86.7 + 9.9)
80 (70.8) (75.3 + 16.1)
Normal, 9
weeks (3)
107
100 (93.5) (97.1 + 3.9)
98 (91.6) (91.0 + 4.5)
96 (89.7) (80.2 + 11.6)
87 (81.3) (73.9 + 6.5)
There were no significant differences among sperm type and age for each variable measured [using one-way analysis of variance (ANOVA) or x2 test]. PN, pronuclei; M/B, morula/
blastocyst.
Table V Full-term development of progeny following embryo transfer
Type of sperm
injected, age of
mouse (n)
No. of oocytes
that survived
after injection
No. of embryos that
formed PN (%)
(mean + SD)
No. of 2-cell embryos (%)
(mean + SD)
No. of
embryos
transferred
No. of progeny (%)#
(mean + SD)
.............................................................................................................................................................................................
Abnormal, 7
weeks (4)
162
139 (85.8)a (84.7 + 6.3)
116 (71.6)b (68.4 + 18.4)
116
56 (48.3) (40.0 + 24.4)
Abnormal, 6 –10
months (5)
141
131 (92.9) (93.2 + 2.3)
117 (83.0)b’,c (80.6 + 16.2)
117
52 (44.4) (37.5 + 23.2)
Normal, 9 weeks
(3)
119
117 (98.3)a’ (98.1 + 1.7)
111 (93.2)b’,c’ (92.3 + 7.0)
111
56 (50.5) (47.5 + 17.8)
Oocytes were injected by ICSI with morphologically abnormal sperm from 7-week-old and 6–10-month-old males. There were no significant differences among sperm types and age for
each variable measured using one-way ANOVA. a,a’;b,b’;c,c’P , 0.05 (x2 test). #Per cent of number of transferred embryos shown in parenthesis.
779
Sperm abnormalities in BALB/c mice
Figure 2 Characterization of blastocysts that developed from oocytes that received abnormal sperm from young (7 weeks old) and older (6– 10
months old) male mice.
Light micrographs of blastocysts 96 h after insemination with morphologically abnormal sperm prepared from 7-week-old (A) and 6– 10-month-old (B) males, and with
normal sperm prepared from 9-week-old males (C). Each blastocyst was stained with propidium iodide (PI, A0 –C0 ) and Cdx2, marker for trophectoderm (A00 – C00 ). Each
of the merged photographs is shown in A000 –C000 . In (D – D00 ), blastocysts at 120 h after insemination with morphologically abnormal sperm from 7-week-old mice were
stained with Oct3/4 antibody, marker for inner cell mass (D0 ). A light micrograph (D) and a merged micrograph (D00 ) are also shown. Scale bars: 100 mm.
Functional assessment of abnormal sperm
produced from 7-week-old and 6 – 10-monthold males using ICSI and embryo transfer
To determine the functional ability of morphologically abnormal
sperm, we carried out embryo transfer experiments. The 2-cell
stage embryos (Fig. 3A) derived from oocytes injected with morphologically abnormal sperm prepared from 7-week-old and 6–
10-month-old males were transferred into pseudo-pregnant females.
We compared the birth rates with those obtained using normal
sperm prepared from 9-week-old males. Approximately 50% of the
embryos developed to full term in the case of morphologically
normal sperm (Table V). The oocytes that received abnormal sperm
from 7-week-old and 6 –10-month-old males had similar birth rates
(Table V). No major abnormality, including body weight, placenta
weight or sex ratio, occurred in the progeny, even when they developed from morphologically abnormal sperm (Fig. 3B, Table III).
Therefore, the abnormal sperm from young and older males had
the same ability to produce progeny after microinsemination as
normal sperm.
Discussion
We examined the SAR in the BALB/c mouse substrains A, AnN
and ByJ, and found that the SARs were affected by both substrain
and age. The SAR was higher in the AnN and ByJ than the A substrain
for the ages examined. In addition, the SAR of 7-week-old mice was
higher than that of 9-week-old mice, suggesting that low-quality
sperm was produced in the first wave of spermatogenesis after
birth. Further, the ICSI technique allowed us to determine that
morphologically abnormal sperm prepared from both 7-week-old
and 6–10-month-old males had genetic potentials similar to
morphologically normal sperm (9 weeks of age) in terms of producing
progeny, although it is possible that the in vitro development of
780
Figure 3 Production of progeny from oocytes that were injected
with abnormal sperm from 7-week-old mice.
(A) 2-cell stage embryos 24 h after the injection of oocytes with abnormal
sperm prepared from 7-week-old males. (B) Newborn mice delivered from
the zygotes of (A) after transfer to pseudo-pregnant females.
ICSI embryos was slightly affected by sperm morphology (Table V). To
our knowledge, this is the first comparison of the function of
morphologically abnormal sperm with normal sperm during
development.
We showed that the 2-cell stage embryos that developed from
morphologically abnormal sperm had a similar developmental ability
to that of embryos that developed from morphologically normal
sperm. However, some defects may be present during the in vitro
development of oocytes injected with morphologically abnormal
sperm produced from 7-week-old mice: although we could not find
a significant difference in the one-way ANOVA analysis in Table V,
the x2 test detected a significant difference at the P , 0.05 level. A
similar tendency was also found when the data for the 2-cell stage
of development in Tables IV and V were combined (Supplementary
Table). This discrepancy may arise from the difference in the sample
numbers used in the statistical analysis. In the ANOVA analysis, the
range of sample numbers in the Supplementary Table was 6 –8 and
its average power was 0.05, suggesting that type II error may occur
in this analysis (if we attempt to obtain data with sufficient power,
30– 50 males are required for each group). In contrast, the sample
number is sufficient for comparisons between the number of
embryos using the x2 test (more than 200 for each group). Therefore,
some abnormality may arise through the use of morphologically
abnormal sperm. Importantly, more than 70% of the oocytes injected
with morphologically abnormal sperm developed to the 2-cell stage,
which then developed to term at a frequency similar to that of
embryos derived from normal sperm (c.44–48%, and 50%, respectively), suggesting that the resulting embryos were functionally similar
in terms of producing progeny.
A previous study suggested that the SAR is higher in young males
(6 or 7 weeks old) and decreases rapidly by 8–10 weeks of age
(Krzanowska, 1981). This observation strongly suggests that the first
wave of spermatogenesis produces morphologically low-quality
sperm. Consistent with this, Yoshida et al. (2006) showed that the
first wave of spermatogenesis differed from the second wave. Specifically, the first wave of spermatogenesis occurred directly from gonocytes (precursors of spermatogonia), without the self-renewal of
spermatogonia (Yoshida et al., 2006). Therefore, we hypothesized
that morphologically abnormal sperm produced from the first wave
of spermatogenesis in this study would also have abnormalities, such
Ohta et al.
as chromosomal or epigenetic defects, that affect the normal development of progeny. However, only a minor change was found in the in
vitro development of ICSI embryos when we used morphologically
abnormal sperm produced from the first wave of spermatogenesis.
This suggested that normal parental chromosomes, at least in terms
of producing progeny, were formed from a large proportion of the
morphologically abnormal sperm that was produced during the first
wave of spermatogenesis.
Here, we demonstrated that the resulting 2-cell stage embryos
produced using morphologically abnormal sperm had similar
genetic potential to those derived from morphologically normal
sperm in terms of producing progeny. This finding is perhaps counterintuitive. It highlights that the prediction of sperm function may be
difficult or impossible based on morphology alone. Our results indicate that sperm morphology alone cannot be used as an indicator
for the development of embryos after insemination. That is, it is
possible that morphologically normal sperm may have abnormal
chromosomes and vice versa. Long-term studies of the progeny
resulting from insemination with morphologically abnormal sperm
are necessary to confirm this. Whether these results are relevant
to the use of morphologically abnormal or otherwise dysfunctional
sperm in human infertility treatment is also worthy of detailed
evaluation.
The SAR of BALB/c mice differed by substrain. The BALB/c line
was originally established as the Bagg albino by Bagg in 1913 and
was then provided to Snell in 1932, who named the line BALB/c
(Potter, 1985; Supplementary Figure). Friis and Andervont independently bred the line and established BALB/c A and BALB/c An,
respectively (Potter, 1985; CLEA). BALB/c AnN and BALB/c ByJ
were developed from the BALB/c An line by the National Institutes
of Health and The Jackson Laboratory, respectively (Potter, 1985).
Thus, it appears that BALB/c A was the earliest substrain separated
from the other two lines (Potter, 1985; CLEA; Charles River Japan;
Supplementary Figure). Consistent with this history, the SAR of
BALB/c A mice was lower than that of BALB/c AnN and BALB/c
ByJ mice (Table I), suggesting that these lines of substrains are phenotypically and genetically different. Genetic mutations that affect sperm
morphology may have accumulated in the BALB/c AnN and BALB/c
ByJ substrains. To understand human male infertility in terms of abnormal sperm morphology, it may be helpful to examine the genetic
mutations that occurred in the BALB/c AnN and BALB/c ByJ
substrains.
Acknowledgements
We thank the Laboratory for Animal Resources and Genetic Engineering for housing the mice.
Funding
This work was supported by grants for Scientific Research in Priority
Areas (15080221) and the Project for the Realization of Regenerative
Medicine (research field: technical development of stem cell manipulation) to T.W. by the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
781
Sperm abnormalities in BALB/c mice
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Submitted on June 3, 2008; resubmitted on October 15, 2008; accepted on
November 19, 2008