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360
P. ESPONDA
THE JOURNAL
AND M.OF
MORENO
EXPERIMENTAL ZOOLOGY 282:360–366 (1998)
Acrosomal Status of Mouse Spermatozoa in the
Oviductal Isthmus
PEDRO ESPONDA* AND MARTA MORENO
Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain
ABSTRACT
The principal purpose of this study was to establish the acrosomal status of mouse
spermatozoa stored in the isthmus of the oviduct after natural mating. Scanning electron microscopy of oviducts fixed about 6 hr before the estimated ovulation time showed numerous
acrosome-intact spermatozoa attached to the mucosal surface of the oviduct, or trapped in
the oviductal crypts. Nevertheless, an unambiguous assessment of the state of the acrosome
requires transmission electron microscopy. Using this method, it was observed that the
acrosome was intact in the 81% of spermatozoa attached to the mucosal surface but in only
31% of spermatozoa that were free in the lumen. Most of the free spermatozoa showed swelling or disruption of the acrosome. This result might indicate that the in vivo spermatozoonoviductal mucosa interaction maintains the acrosome intact. Alternatively, it could mean that
only ejaculated spermatozoa with a normal acrosome can establish such a mucosal relationship. J. Exp. Zool. 282:360–366, 1998. © 1998 Wiley-Liss, Inc.
In most vertebrates with internal fertilization,
sperm storage occurs in the female genital tract
to a variable degree. In some reptiles, fertile spermatozoa are stored there for years (Howarth, ’74;
Gist and Jones, ’87). In birds, according to species, this may occur for days or weeks in the
uterovaginal glands of the female tract (Howarth,
’74; Baskt, ’87). Among mammals, the female tract
of some marsupials can store spermatozoa for periods of several days (Rodger and Bedford, ’82;
Taggart and Temple-Smith, ’91), and in some bats
this can extend for months (Racey, ’79). In shrews,
spermatozoa are sequestered for a short period
by either isthmic crypts or the “bubble-like” ciliated crypts in the oviduct ampulla without attachment to the epithelium (Bedford et al., ’97a,c).
However, for many mammals, a short interval of
sperm storage in the isthmus region of the oviduct seems to be the rule (Harper, ’94), a phenomenon that has been preferentially examined
in the hamster (Smith et al., ’87; Smith and
Yanagimachi, ’90, ’91), the rat (Shalgi and Kraicer,
’78), sheep (Hunter et al., ’82; Hunter and Nichol,
’83), rabbit (Overstreet and Cooper, ’79), cow
(Hunter and Wilmut, ’84), sow (Fléchon and
Hunter, ’81), mare (Parker et al., ’75; Bader, ’82),
human (Croxatto et al., ’75), and mouse (Suarez,
’87). In this group, in general, soon after mating
a few thousand spermatozoa pass through the
uterotubal junction and remain confined to the
isthmus of the oviduct. At around the time of ovulation, a very small proportion of these spermato© 1998 WILEY-LISS, INC.
zoa ascend to the site of fertilization in the ampulla (Harper, ’94). After their arrival in the isthmus, some spermatozoa lie free in the lumen, but
others attach to the epithelium of the isthmus
crypts. In the hamster, the percentage of living
spermatozoa appears to be higher in the fraction
attached to the isthmic epithelium than in that
free in the lumen (Smith and Yanagimachi, ’90).
Furthermore, some data suggest that sperm capacitation may be completed during storage in the
isthmus (Smith and Yanagimachi, ’91). On the
other hand, numerous in vitro experiments, using monolayers of oviduct epithelial cells, have
shown that the attachment to the oviductal epithelium promotes the hyperactivated motility and
the fertilizing ability of spermatozoa (Pollard et
al., ’91; Anderson and Killian, ’94; Chian and
Sirard, ’95; Dobrinski et al., ’97; Smith and
Nothnick, ’97). In vitro experiments have also established that the spermatozoon-epithelium association is related to, or would induce, sperm
capacitation (Anderson and Killian, ’94; Chian and
Sirard, ’95; Lefebvre and Suarez, ’96). These results suggest that the phenomenon of sperm storage in the mammalian oviduct represents an
Grant sponsor: DGICYT (Spain); Grant number: PB93-0117.
*Correspondence to: Dr. Pedro Esponda, C. Investigaciones
Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. E-mail:
[email protected]
Received 23 September 1997; Accepted 16 March 1998
ACROSOME IN OVIDUCTAL SPERM
important step for the achievement of mammalian fertilization.
The acrosomal status is a significant factor
when considering the fertilizing capacity of mammalian spermatozoa. In the mouse, only acrosomeintact spermatozoa are able to fertilize the egg
(Wassarman, ’87; Yanagimachi, ’94), and several
reports have pointed out that the acrosomal reaction phenomenon occurs only after the spermatozoa contact the zona pellucida, which contains
factors to initiate the reaction (Bleil and Wassarman, ’83; Yanagimachi, ’94). Moreover, in numerous mammals it has been shown that the
acrosomal reaction seems to be a prerequisite for
the spermatozoa to penetrate the zona (Yanagimachi, ’94). Thus, the acrosomal status would appear to be an important correlate of fertilizing
capacity.
In an attempt to clarify the functional status of
the two sperm populations in the oviduct isthmus,
we have analyzed the acrosomal status of those
spermatozoa free in the lumen and those attached
to the epithelium of the isthmus at various times
after natural mating.
MATERIAL AND METHODS
Swiss albino mice housed in the animal facilities of C. Investigaciones Biológicas (Madrid,
Spain) were fed with food and water ad libitum
and were maintained at a constant temperature
and with a light-dark cycle of 12 hr–12 hr. Males
of proved fertility and virgin females were used.
Females were injected with 4 IU of PMSG and 44
hours later with 5 IU of human chorionic gonadotropin (HCG). After HCG injection, each female
was placed with a male, and the presence of a
vaginal plug was checked. Mated females were
killed by ether at different times after HCG injection, and the uterus and oviduct were promptly
fixed for light or electron microscopy. Fixation was
conducted at 37°C in order to avoid muscle contractions of the female tract, which can displace
spermatozoa. Females were anesthetized, and before they died, the genital tract was gently exposed. The fixing solution was spread on the
abdominal cavity, and then part of the uterus and
the whole oviduct were sectioned and immersed
in the fixative. After 30 min, the uterine end and
the complete oviduct were isolated and fixed during other 30 min.
For light microscopy, samples were fixed in 2%
p-formaldehyde in phosphate buffer (pH 7.3), dehydrated, and embedded in paraffin. Sections
stained with hematoxylin-eosin were studied us-
361
ing bright- or dark-field microscopy. For electron
microscopy, tissues were fixed in a solution of 2%
glutaraldehyde, 1% p-formaldehyde, and 0.1 M
sucrose in 0.5 M cacodylate buffer (pH 7.3).
Samples were thoroughly washed with the buffer
and post-fixed in 2% osmium tetroxide in cacodylate buffer. Then they were treated in two ways.
First, for scanning electron microscopy, pieces of
oviducts were dehydrated in graded series of
alcohols, dried with CO2 in a critical-point drying
apparatus and coated with gold. For transmission
electron microscopy, samples were dehydrated in
alcohol and embedded in an epoxy resin. Semithin
sections were stained with 0.2% toluidine blue and
observed under a light microscope in order to locate spermatozoa in the oviductal isthmus. Ultrathin sections cut in an LKB ultratome, and
stained with uranyl acetate and lead citrate, were
studied in either a Hitachi Scanning S-2100 or a
Philips 400 electron microscope. The acrosomal
characteristics (acrosome intact or modified) of
spermatozoa were checked and recorded under
transmission electron microscopy, and the twotailed Fisher’s exact probability test applied to
these data.
RESULTS
Hematoxylin-eosin-stained sections showed that
spermatozoa invade the lower oviduct region soon
after coitus. Under bright-field microscopy, the recognition of spermatozoa was difficult (Fig. 1), but
in dark field, the sperm nuclei appeared strongly
brilliant and were easily identifiable (Fig. 2). This
clearly demonstrated the presence of sparse spermatozoa inside the oviductal isthmus from 6 hr
before the estimated time of ovulation (Fig. 2),
when many spermatozoa were still present in the
uterus. The scarcity and location of spermatozoa
observed in these sections coincide with the results of previous articles on spermatozoon transport in the mouse (Austin, ’60; Harper, ’94).
The use of scanning electron microscopy clearly
demonstrated the characteristics of the oviductal
spermatozoa attached to the mucosal surface. In
these preparations, those with an intact acrosome
were clearly distinguishable from spermatozoa
with modified acrosomes (Fig. 3). The intact state
of the acrosome was defined by the presence of the
acrosomal ridge and the arched profile of the apical
region of the spermatozoon head (Figs. 3–6). By contrast, modified acrosomes show an irregular surface (Fig. 3). Observations of oviducts at different
time periods before ovulation showed that numerous spermatozoa attached to the mucosal surface
362
P. ESPONDA AND M. MORENO
Fig. 1. Section of the caudal region of an oviduct fixed 2
hr before ovulation. Two crypts are indicated (C) in which
sperm heads are not clearly distinguished. Hematoxylin-eosin
staining. ×425.
Fig. 2. Same as Fig. 1 but observed under dark-field microscopy. Sperm heads can be seen clearly because their nuclei are strongly refringent.
Fig. 3. Scanning electron microscopy of an oviduct fixed 4
hr before ovulation. A spermatozoon shows an intact acrosome
(arrowhead); the other two exhibit modified acrosomes. ×4,350.
Figs. 4 and 5. Oviducts fixed 4 hr before ovulation.
Acrosome-intact spermatozoa are in close relation with the
numerous microvilli (mv) present in the crypts. 4: ×5,200; 5:
×4,900.
Fig. 6. Scanning electron microscopy of an oviduct fixed
1 hr before ovulation. Several spermatozoa lying on the oviductal surface can be seen. All show intact acrosomes (arrowheads). ×6,900.
ACROSOME IN OVIDUCTAL SPERM
displayed intact acrosomes (Figs. 4–6). The attachment of the spermatozoon head to the microvilli
of the oviductal crypts was sometimes particularly
noticeable, and in these cases spermatozoa also
showed intact acrosomes (Figs. 4 and 5).
Because scanning electron microscopy did not
permit an accurate analysis of the free spermatozoa or the beginning of acrosome modifications,
transmission electron microscopy was used to analyze the morphology of isthmic spermatozoa. This
revealed that the 81.08% of spermatozoa associated with the oviductal epithelial surface had an
intact acrosome (Table 1) (Figs. 7 and 8), whereas
only 31.25% of luminal spermatozoa had intact
acrosomes (Table 1). This differential was seen at
all times before ovulation. However, spermatozoa
attached to the mucosa showed a slight increase
of modified acrosomes when they were close to
the time of ovulation (Table 1). Attached spermatozoa appeared to be embraced by the abundant
microvilli and in contact with numerous lucid
vacuolar structures in the crypt lumen (Fig. 8).
These vacuoles undoubtedly represent the serous
material secreted by isthmus oviductal cells described in other mammals (Harper, ’94). Modification of acrosome structure was usually first
reflected in a swallowing (Fig. 11), with some disruptions of the plasma and external acrosomal
membranes (Figs. 9 and 10), and with a nonhomogeneous acrosomal content (compare Figs. 10 and
11 with Figs. 7 and 8).
DISCUSSION
The acrosomal characteristics of spermatozoa
stored in the lower oviduct have been analyzed in
very few mammals. In the Dasyurid marsupial
Sminthopsis, transmission electron microscopy
techniques have demonstrated that spermatozoa
stored in the crypts of the oviductal isthmus had
intact acrosomes at ovulation time (Breed et al.,
’89), a fact that also was evident in some Insecti-
363
vora spermatozoa stored in the oviduct ampullar
crypts (Bedford et al., ’97a,c). Light microscopy
studies in the rabbit have shown that 36% and
66% of oviductal spermatozoa had modified acrosomes when they were collected 1.5 and 12 hr
postcoitus, respectively (Overstreet and Cooper,
’75). In contrast, spermatozoa collected from the
uterus at the same time periods showed that only
7.8 to 13.9% of them exhibited modified or reacted
acrosomes (Overstreet and Cooper, ’75). An accurate analysis of rabbit spermatozoa collected solely
from the oviductal ampulla at fertilization time
demonstrated that 98% possessed intact acrosomes (Suarez et al., ’83). Scanning electron microscopy of bovine and pig spermatozoa in the
lower oviduct demonstrated that the majority were
acrosome intact when observed before ovulation,
but many observed shortly before or after ovulation displayed vesiculation of the acrosomal surface (Hunter et al., ’87, ’91). Mouse spermatozoa
from the entire oviduct analyzed by light microscopy showed that 43% of them presented modified acrosomes (Klemm and Engel, ’91). These
results suggest that the lower oviduct produces a
detrimental effect on the acrosome stability of
some spermatozoa (Overstreet and Cooper, ’75).
This study indicates that the spermatozoon-epithelium relationship protects the acrosome in the
mouse. The 81% of spermatozoa attached to the
oviductal epithelium exhibited intact acrosomes,
whereas almost 70% lying free in the isthmus lumen showed modified acrosomes. How the epithelial relationship supports acrosomal integrity is
not known, though Hunter (’94) has commented
that some oviductal factors, for example, glycoproteins, could be acting in this way.
The in vivo site of the acrosomal reaction is
still a matter of controversy (Wassarman, ’87).
This fact is probably due to the rare in vivo ultrastructural analyses assayed. In this sense,
transmission electron microscopy is probably
TABLE 1. Transmission electron microscopical observations of the acrosomal status of spermatozoa stored
in the oviductal isthmus at different times before ovulation*
Hours before
ovulation
0–2
2–3
3–6
%
N
5†
6
5
Sperm attached
to the epithelium
IA
MA
11
10
9
81.08
4
2
1
18.92
*N = number of females analyzed; IA = intact acrosomes; MA = modified acrosomes.
†
Two females were used at ovulation time.
1
P = Two tailed Fisher’s exact probability test.
Sperm free
in the lumen
IA
MA
P1
2
2
4
31.25
9
7
8
68.75
0.0154
0.0092
0.0150
3 × 10–6
364
P. ESPONDA AND M. MORENO
Fig. 7. Transmission electron microscopy of an oviductal
crypt fixed 3 hr before ovulation. A spermatozoon head with
an intact acrosome (a) is closely connected with the numerous microvilli of the crypt. (n: sperm nucleus). ×22,500.
Fig. 8. A similar image to that shown in Fig. 7, but from
an oviduct fixed at ovulation time. The intact acrosome (arrowhead) is clearly seen. Note the numerous microvilli and
clear vacuoles, which engulfed the spermatozoon. n, sperm
nucleus. ×15,200.
Figs. 9–11. Figs. 9 and 11 show two spermatozoa free in
the oviductal lumen displaying modified acrosomes (arrow
head in 9 and a in 11). Fig. 10 shows a higher magnification
of the acrosome of Fig. 9. In both cases, acrosomes appeared
swollen, with some membrane disruptions (arrows in Fig. 10)
and containing a nonhomogeneous material. Fixation 2 hr
before ovulation. Fig. 9, ×11,000; Fig. 10, ×34,000; Fig. 11,
×25,200.
ACROSOME IN OVIDUCTAL SPERM
the only method that permits discriminating
plasma and acrosomal membrane characteristics. In the shrew Suncus murinus, an in vivo
analysis has shown that all the spermatozoa in
the cumulus oophorus had reacted, suggesting
that the cumulus would stimulate the reaction
(Bedford et al., ’97b). In the hamster, in vivo
and in vitro experiments have shown that in
some spermatozoa among the cumulus cells, the
acrosomes were swollen, wrinkled, or totally
absent (Yanagimachi and Noda, ’70). Also in the
rabbit, electron microscopical analysis of oocytes
fertilized in vivo indicated that many spermatozoa lying between the granulosa cells already
displayed initial stages of the acrosomal reaction (Bedford, ’68). In the cow, an electron microscopical study showed that the acrosomal
reaction probably occurs when spermatozoa contact the zona pellucida (Crozet, ’84). In the
mouse, transmission electron microscopy of in
vivo fertilized oocytes leads to the assumption
that the acrosomal reaction occurs outside the
zona pellucida (Stefanini et al., ’69; Zamboni,
’72), and similar descriptions were made using
oocytes fertilized in vitro (Saling et al., ’79;
Wassarman, ’87). In general, in the mouse it is
accepted that only acrosome-intact spermatozoa
can bind to the zona, and that is the zona that
stimulates the acrosomal reaction (Wassarman,
’87). In mammals the site of acrosomal reaction
could vary to some extent among species. Nevertheless, the general picture is that spermatozoa
must face egg vestments with an intact acrosome
and must complete the acrosomal reaction before
entering the zona (Yanagimachi, ’88, ’94). In this
regard, our results show that oviductal storage
maintains the acrosome intact, contributes to the
maintenance of fertilization ability, and operates
as a sort of selective mechanism for the gametes
attached to the isthmus epithelium. On the other
hand, our observations reinforce the belief that
the spermatozoon-epithelium relationship is a
phenomenon related to sperm-fertilizing ability
(Smith and Yanagimachi, ’90, ’91).
Spermatozoon storage in the oviduct represents
another important phase in the events occurring
to the spermatozoon before fertilization. Initially,
the epididymis induces spermatozoon maturation.
During ejaculation spermatozoa interact with
macromolecules secreted by the male accessory
glands and must ascend through the uterus. In
general, a few spermatozoa are able to reach the
oviduct, and some of them are engulfed by the
crypts where sperm capacitation probably occurs.
Soon after ovulation, some of these spermatozoa
365
that have preserved their acrosomes intact ascend
to the ampulla and fertilize the oocyte.
ACKNOWLEDGMENTS
We thank J. M. Bedford (Cornell University
Medical College, New York) for valuable suggestions and comments on the manuscript. This work
was partially supported by grant PB93-0117 from
DGICYT (Spain).
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