4. Sperm reservoirs
A functional interaction between spermatozoa and the female tract is also crucial to the establishment and maintenance of sperm reservoirs, which are needed when mating
or insemination precedes ovulation by many hours or days. Within the female genital tract, regions that accumulate and retain a population of viable spermatozoa for an
extended period of time are considered to function as sperm reservoirs. In cattle, sheep and goats, the cervix with its cervical mucus maintains a population of viable spermato-
zoa that is thought to provide a continued source of spermatozoa for the upper tract Ž
. Hawk, 1983 . A cervical population adequate to achieve 100 fertilization is estab-
Ž .
lished within 30–60 min of mating in ewes Hunter and Nichol, 1993 , and motile Ž
spermatozoa can be found in cervical mucus as long as 3.5 days after mating Hunter .
and Nichol, 1983 . In rabbits, the vagina, cervix and uterus each maintain a population Ž
. of progressively motile spermatozoa for at least 16 h after mating Cooper et al., 1979 ,
Ž by which time ovulation has occurred and 98 of eggs are fertilized Overstreet and
. Cooper, 1978b . Motile spermatozoa may persist in the lumen of the dog uterus for as
Ž .
long as 11 days Doak et al., 1967 . In addition, spermatozoa accumulate in the uterine glands of the bitch within 15 min after mating and are found there consistently for 6
days and with variable frequency for up to 9 days, when mating occurs on the first day Ž
. of estrus Doak et al., 1967 . The disappearance of spermatozoa from the uterine glands
prior to the loss of motile luminal spermatozoa, suggests that the glands supplement the Ž
. luminal population Doak et al., 1967 . In the hare, spermatozoa have been found deep
in the uterine glands and at the UTJ at 17 days of pregnancy, and this conservation of spermatozoa is thought to account for the superfetation that occurs in this species
Ž .
Martinet and Raynaud, 1975 . In horses, pregnancy can result from a single mating or insemination up to 6 days
Ž .
prior to ovulation Day, 1942; Burkhardt, 1949 . Although this indicates the presence of Ž .
a sperm reservoir in the mare, its anatomical location s has not been confirmed. However, within 4 h after preovulatory insemination, equine spermatozoa accumulate at
Ž .
the UTJ Scott et al., 2000 , and spermatozoa can be found there 18 hours after Ž
. insemination Scott, unpublished observations . The appearance of the spermatozoa and
their intimate contact and orientation with the epithelium of the UTJ are suggestive of Ž
. sequestration there Fig. 2 .
In the pig, the UTJ and lower oviductal isthmus are considered to be functional sperm reservoirs, because spermatozoa may survive in these regions for as long as 72 h
Ž .
Hunter, 1988 . Stable numbers of spermatozoa persist at the UTJ for 24 h following Ž
. artificial insemination Rigby, 1966 , and are found within the terminal folds of this
Ž .
region for at least 24 h following coitus Flechon and Hunter, 1981; Mburu et al., 1997 . Based on staining patterns with fluorescent probes, more than 40 of the spermatozoa
Ž recovered by flushing the UTJ after ovulation have intact plasma membranes Mburu et
. al., 1996 and are presumably viable.
During the sustained phase of sperm transport, competent spermatozoa that cross the UTJ prior to ovulation will accumulate in the lower isthmus of the oviduct and remain
Ž there until ovulation is imminent. In sheep Hunter et al., 1982; Hunter and Nichol,
. Ž
. Ž
. 1983 , pigs Hunter, 1984 , and cattle Hunter and Wilmut, 1984 , the technique of
Fig. 2. Scanning electron micrograph of equine spermatozoa fixed in situ at the UTJ of a mare, 18 h after insemination. These spermatozoa are located deep in an epithelial fold of the uterine papilla and show a
characteristic side by side arrangement and orientation that is suggestive of a specific interaction with the Ž
. epithelium of this region bar s 2 mm .
surgical transection of the isthmus at various intervals between mating and ovulation was used to demonstrate that fertilization competent spermatozoa do not progress
beyond the first 1.5–2 cm of the lower isthmus until the periovulatory period. In the rabbit, which does not ovulate spontaneously, the isthmic reservoir will accumulate
normal numbers of spermatozoa following artificial insemination, but spermatozoa are
Ž not found in the upper oviduct unless ovulation is induced Overstreet and Cooper,
. 1979 . Thus, the lower isthmus not only functions as a sperm reservoir, but also appears
to regulate sperm ascent to the ampulla. 4.1. Mechanisms of sperm storage
A close relationship between spermatozoa and the luminal epithelium of the female tract appears to be important for the survival of spermatozoa in reservoirs. This
relationship is epitomized in the female bat, which may store viable spermatozoa in the uterus, UTJ, andror oviducts for many weeks to months, depending on the species
Ž .
Racey, 1979 . Regardless of species, or location or duration of storage, during the period of storage, spermatozoa are found displaying a typical linear, side by side
Ž arrangement, with their heads closely associated with the luminal epithelium Racey,
. 1979; Krutzsch et al., 1982; Racey et al., 1987; Krishna, 1997 . These spermatozoa are
structurally intact. This relationship appears to be established soon after insemination Ž
. and very early in the period of storage Racey et al., 1987 , and it is not found in
Ž .
females inseminated prior to the defined breeding season Krishna, 1997 . The universal nature of these observations in female bats suggests that the observation of similar
morphological associations in other mammalian species may also depict sperm storage Ž
. in a reservoir e.g., Fig. 2 .
In domestic animal species, observations of spermatozoa in situ following insemina- tion have demonstrated similar sperm–epithelium interactions in regions of sperm
accumulation. Using scanning electron microscopy, intimate sperm–epithelium contact Ž
. has been visualized in the UTJ and lower isthmus of the cow Hunter et al., 1991 , and
Ž .
Ž pig Flechon and Hunter, 1981; Mburu et al., 1997 , and at the UTJ of the horse Scott
. et al., 2000 . Spermatozoa have been found to line up in groups within epithelial crypts
Ž .
of the lower isthmus of the hamster Smith et al., 1987; Smith and Yanagimachi, 1990 , where they appear to ‘‘dig in’’ to the mucosa with their hooked heads, indicating a
Ž .
physical attachment Smith and Yanagimachi, 1990 . This close contact appears to maintain sperm viability, since spermatozoa with close epithelial contact maintain
Ž flagellar activity and survive longer than luminal spermatozoa Smith and Yanagimachi,
. Ž
. 1990 . Suarez 1987 observed similar interactions between living mouse spermatozoa
and the oviductal epithelium in excised oviducts and proposed two possible mechanisms of sperm retention in the isthmus: adherence to the epithelium or immobilization. In the
rabbit, motility is suppressed in sperm recovered from the lower isthmus in native fluid,
Ž .
and this is a proposed mechanism for their retention there Overstreet et al., 1980 . In cattle, a combination of a narrow lumen and the presence of mucus may contribute to
Ž .
sperm retention in the lower isthmus Suarez et al., 1997 . During the period of sperm storage in female bats, epithelia intimately contacted by
spermatozoa are ultrastructurally and histochemically different from epithelia without Ž
. sperm contact
Krutzsch et al., 1982; Krishna, 1997 . Differences have also been observed for epithelia of storage sites between the period of storage and the time of
Ž .
ovulation or arousal from hibernation Krutzsch et al., 1982; Krishna, 1997 . These observations suggest a functional change in the epithelia of storage sites that may
regulate sperm attachment and release. Support for a regulation of sperm physiology or attachment by epithelia comes from
in vitro studies of spermatozoa co-cultured with monolayers of oviductal epithelial cells Ž
. OEC . For example, when equine spermatozoa are incubated with OEC, a lower
intracellular calcium concentration is found in attached spermatozoa compared with spermatozoa in media, suggesting that attachment is associated with a modulation of
Ž .
calcium Dobrinski et al., 1996 . The authors proposed that reduced intracellular calcium levels might prevent premature capacitation and acrosome reaction in spermatozoa
Ž .
during storage Dobrinski et al., 1996 . 4.2. Ascent of competent spermatozoa to the site of fertilization
Fertilization competence in spermatozoa is gained after a period of residence within the female tract. Functional changes in sperm cell physiology, collectively called
capacitation, are induced or facilitated by sperm interaction with the luminal fluids and epithelial surfaces during transit, and enable spermatozoa to acrosome react, penetrate
Ž .
the zona pellucida and fuse with the oolemma see Yanagimachi, 1994 . Associated with capacitation are changes in sperm motility, termed hyperactivation, that are character-
Ž .
ized by a highly vigorous asymmetrical flagellar motion see Yanagimachi, 1994 . Hyperactivated motility generates thrusting forces considered great enough to enable a
Ž sperm to gain release from storage and to penetrate the vestments of the oocyte see
. Katz et al., 1989 . In ruminants and primates, capacitation is initiated in the cervix with
the removal of sperm surface proteins as spermatozoa swim through the microstructure Ž
. of the cervical mucus see Drobnis and Overstreet, 1992 . The final stages of capacita-
Ž .
tion appear to be completed in the lower isthmus Smith and Yanagimachi, 1990 .
There is a temporal relationship between the resumption of sperm progress to the oviductal ampulla and the occurrence of ovulation. This coordination may be regulated
Ž .
by the changing hormonal profile that occurs at ovulation see Hunter, 1988 . Release of spermatozoa from the isthmic reservoir is associated with capacitation and the develop-
Ž .
ment of activated motility Overstreet and Cooper, 1979 . Forward progress within the confined spaces of the oviduct is enhanced in spermatozoa that display hyperactivated
Ž .
motility Katz and Yanagimachi, 1980 , which suggests it plays a key role in sperm migration to the ampulla, but also, oviductal contractility is likely to assist sperm transit
Ž .
see Katz and Yanagimachi, 1980 . Spermatozoa ascend from the lower isthmus sooner Ž
. if mating occurs closer to ovulation Smith et al., 1987 , and this corresponds to a
Ž .
shortened length of time required for capacitation Smith and Yanagimachi, 1989 .
5. The elimination of excess spermatozoa
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