Cwnp. Biarhm Phnzai. Vol 7OA. pp. 387 to 395. 1981 030%96?9,81 011387-OW2.

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Printed in Great Brick. All rights rewed Copyright 0 1981 Pergamon Prrrc Lid

STUDIES ON PROLONGED SPERMATOZOA SURVIVAL IN

CHIROPTERA-I. THE ROLE OF UTERINE FREE
FRUCTOSE IN THE SPERMATOZOA STORAGE
PHENOMENON

ELIZABETHG. CRICHTON,PHILIP H. KRUTZSCI~and

WILLIAM A. WIMSATT’
Department of Anatomy, College of Medicine. University of Arizona, Tucson, AZ 85724 and ‘Section of
Genetics and Development. Bradfield Hall, Cornell University. lthaca, NY 14853. U.S.A.

(Receired 30 Murc,h 1981)

Abstract-i. The fructose content of the reproductive tracts of M&s /ff~~~~~.~and M. relifer was
recorded at intervals throughout the annual reproductive cycle.
2. The data indicate that seminal Ruid ~parti~ulariy the ampullary glands) contributes to the uterine
fructose cuntent and that the uterus is also fructo~eni~.
3. Preliminary experiments tested the role of steroids and of stress-related hormones on uterine
fructose.
4. Fructose was not present in the epididymides.
5. While fructose of seminal and uterine origin probably plays a role in prolonged sperm survival, the
possibility that other metabolizable substrates are present and of use for spermatozoa retention in the
female reproductive tract should be investigated.

Since spermatogenesis, copulation and fertilization Racey & Potts (1970) did not reveal any unique cyto-
are closely synchronized events in most mammals, logical features of intrauterine sperm as compared
spermatozoa are not usually called upon to maintain with epididymal sperm (Fawcett & Ito, 1965) that
their viability for long periods of time. However, might be responsible for their prolonged survival in
many species of vespertilionid and rhinolophid bats the female tract.
deviate from this theme, interposing a long period of The alternative, that bat spermatozoa or the repro-
sperm storage between copulation in autumn and fer- ductive tract separately or in consort, may possess
tilization in spring (Hartman, 1933). One of the most special physiological adaptations that allow for their
intriguing reproductive adaptations of these bats is active participation in the phenomenon, finds support
the remarkable ability of the spermatozoa to survive from observations that spermatozoa are orientated
for long periods, up to several months in torpid bats. towards and assume a close intimacy with the endo-
without loss of viability or fertilizing capacity metrial and oviductal epithelium (Courrier, 1921:
(Matthews, 1937; Folk, 1940; Wimsatt, 1942, 1944; Nakano, 1928; Rendez, 1929; Hiraiwa & Uchida,
Kleiman & Racey, 1969; Racey, 1972. 1973, 1975. 1955; Racey et ul., 1975; Krishna & Dominic. 1978).
1979). Prolonged sperm survival, although not unique In some instances, spermatozoa have been seen
to bats, reaches its greatest development in this order indenting or penetrating the epithehum. bringing
of mammals where both the male (epididymides) and them close to cellular organelles (see Plate 8. Fig. 17,
female (uterine corpus, tubo-uterine junction and Wimsatt ef ill., 1966) and glycogen granules (Napoli-
vagina) reproductive tracts participate in the phenom- tano et ul., 1963). This relationship has also been con-
enon. firmed in several additional studies (Racey & Potts,
Early suggestions that prolonged viability may be a 1970; Racey et ul., 1975; Mori & Uchida, 1980;
direct (and passive) response to hypothermia have Richardson CI al., 1980). Sperm orientation towards
largely been refuted by the realization that many spe- the adjacent epithelium has also been recorded in
cies may be more active during hibernation than pre- other sperm-storing vertebrates (e.g. guppy: Jalabert
viously supposed (Strelkov, 1962; Ransome, 1971: & Billard, 1969; domestic hen: Van Krey ef ul., 1967;
Racey, 1973) and by the recent discovery of sperm garter snakes: Fox. 1956; Hoffman & Wimsatt, 1972).
storage in several species of tropical and neotropical Although there is no proof that the survival of these
nonhibernating (but not necessarily nonheterother- spermatozoa is in any way favored by this relation-
mic) VerspertiIionids (~opalakr~shna & Madhavan, ship, it appears that their structural integrity is main-
1971, i978; Medway, 1972; Racey PI ul., 1915: Myers, tained during storage.
1977; Krishna & Dominic, 1978). Crowding of stored Racey (1975) has cautioned against ascribing
spermatozoa, producing local high pC02 levels that special significance to the spermatozo~~n-uterus orien-
may favor reduced motility and metabolism (Rendez, tation after having simulated a similar tropism by
1929; Schwab, 19.52) is unlikely to be the principal confining semen in drops of paraffin oil. Nevertheless,
mechanism favoring prolonged longevity. Electron considerable evidence has accumulated which lends
microscopic studies by Wimsatt et ~1. (1966) and support to the existence of a functional interrelation-

387
388 ELIZABETH G. CRK-HT~N et cd.

ship between the female reproductive tract epithelial became visible for removal (April to June). Anlpuilary
lining and the orientation of stored spermatozoa (see glands, seminal vesicles. prostate glands and caudae epidi-
review by Racey, 1979). While a function in capacita- dymides were analyzed separately, either individually or
pooled into groups of up to five depending on organ
tion has been attributed to the close sperm-uterine
weight. Samples were homogenized in cold IO”.,, trichloro-
relatioIlship in some species (Austin, 1969), obser- acetic acid (TCA) and filtered. The filtrate was then ana-
vations suggest that a nutritional interrelationship lyzed for fructose according to the resorcinol calorimetric
may more likely underlie intimate sperm-uterine as- procedure of Roe (1934). as modified by Lindner & Mann
sociations. Evidence giving credence to this specu- (1960). No allovvance was made to correct for the small
lation relates to the presence of glycogen in the uter- amount of phosphofructose probably lost due to its cxtrac-
ine (and epididymal) epithelium (Nakano, 1928; Wim- tion by TCA (Mann. 1964).
satt, 1949: Racey & Potts, 1970; Racey, 1975) while
certain enzymes of glycolysis and the pentose and sor-
To determine the effect of arousal on the uterine fructose
bitol pathways have been located in the spermatozoa
levels. bats were removed from hibernation and kept active
as well as the epithelia from both the epididymides
at room temperature (72’ F) for 24 hr prior to sacrifice.
and uterus (Pipi.~tre~lus p~F~.str~l~~s, Racey, 1975;
Myofis /ucifugu.s, Wimsatt, 1969). These pathways to
sperm nutrition are well defined in some vertebrate Sex steroid hormones were dissolved in a 0.2 ml aliquot
(Hers, 1957: Lorenz, 1958; Lake et cd., 1962; Mann, of absolute alcohot and brought to final dilution with corn
1964) and invertebrate systems (Anderson & Per- oil. Injections (O.l~ml) were given subcutaneously in the
sonne, 1969). It is possible that the intimate sperm.. interscapular region. Animals received a total dose of
uterine contact may represent a means whereby either 30 I_cgestrogen (5 pg/day per 6 days); 750 gg pro-
nutrients from the endometrium are accessible to gesterone (250 ng/every second dayithree times); 20 fig
estrogen (5 pg/day per 4 days) plus 500 btcg progesterone
stored spematozoa.
(250 &day per 2 days) or 1500 pg testosterone (250 &day
It is well established that spermatozoa utilize ex- per 6 days). Control animals were given oil alone in similar
ogenous fructose for their nutritional needs under schedules. Bats were kept active and fed for the duration of
conditions where oxygen supplies are too low to these experiments, except in one instance when the experi-
maintain the aerobic utilization of certain endogenous mental protocol required the animals to be returned to
substrates (i.e. intracellular lipids). Since fructose is a hibernation.
principat constituent of the male accessory reproduc-
tive organs (Mann, 1946, 1949, 1964) and as such,
Bats were ovariectomized or sham-operated under ether
becomes available to sperm upon ejaculation, it is
anesthesia. Oviducts were ligated with silk thread at the
Logical to implicate this nutrient as a possible sup-
utero-tubal junction and the ovaries removed. Animals
porting substance for uterine (ejaculated) spermato- were stabilized for several days prior to further experimen-
zoa. Bats are no exception to many other mammals in tal procedures.
containing fructose as a component of the seminal
plasma (Rajalakshmi & Prasad, 1970; Racey. 1974; Adrend trnd pifuifrtry hormone supplemrniufion
Krutzsch et al., 1976; Mokkapati & Dominic, 1976). In an experiment performed in November, IOOpg epi-
Wimsatt (1969) and Racey (1975) suggest that the neahrine or 0.10 iu ACTH (Armour) were iniected (0.10 ml)
uterus of hibernating bats may also contain fructose. in~ra~rit~neaily into active (n = 17) or hibernating
The present study therefore was undertaken in order (n = 20) bats which were sacrificed 4 hr later.
to confirm the presence of fructose in the female
reproductive tract and to determine if this fructose is
To exclude the possibility that the fructose being
uterine-generated (as opposed to male-contributed via measured in the uteri during the sperm storage period
semen) and if it exhibits an annual cycle which is was that deposited in seminal plasma as a result of autum-
influenced by the steroid milieu. nal and later copulations, a population of presumed nonin-
seminated M. ~~~~~~~~~us was collected on August 19. These
aMATERIALS AND METHODS bats (n = 10) were mduced to hibernate in the laboratory
and later (October 2) were sacrificed and their uteri ana-
lyzed for fret fructose alongside a uterine sample obtained
Four hundred adult A4rofi.s /t~c~~igus (New York State) at that time from a naturally hibernating population
and 250 adult M. t&fir (Arizona) were used in this study. (n = 5, presumed inseminated). In another experiment,
Each month, excepting May and June, bats were removed M. relifrr collected on August 23 (n = 5) were hibernated
from nature into the laboratory, where they were main- in captivity until sacrificed and analyzed alongside a
tained in an environment chamber simulating the con- sample collected fresh on November 29 (n = 5, presumed
ditions from which they had been collected. Water was inseminated).
provided U& fihirtrtn, and the bats were usually sacrificed
within a day or two of their collection. Those intended for of tisswfiw
P r~~~~~~ffi~J~1 light micwscopy cmd fiisfclchetnistr?
retention in captivity in an active state for a longer period Fixation was by immersion in lo”,, buffered formalin.
were provided meal worms (larval Tcnehrio) and water url cold Rossman’s fluid or aqueous Bouin’s fluid prior to de-
lihirmr. hydration, clearing in benzene and paraffin embedding.
Serial sections were cut at 6b(rn and stained with Harris’
haematoxylin and eosin or by the Ma&anus PAS tech-
Bats were removed from the environment chamber to nique for polysaccharides (Pearse, 1968). Diastase drgestinn
room temperature about an hour prior to sacrifice. Repro- was carried out on alternate slides to establish that PAS
ductive organs were removed from sacrificed animals, freed reactivity was due to the presence of glycogen.
of extraneous tissue and weighed on a torsion balance to Tissues for enzyme analysis were removed from the ani-
the nearest tenth of a milligram. Female tracts were ana- mal, placed immediately on a microtome chuck. sur-
lyzed minus the ovaries and embryonic mass when this rounded by OCT compound (Tissue TEK) and frozen.
 Fructose in sperm storage
Sections were cut from these preparations at iOpm m a
cryostat (- 20°C) and mounted on glass slides. For the
analysis of acid and alkaline phosphatase activities, the
azo-dye coupling techniques of Pearse (1968), were used;
the enzymes were localized by Fast Garnet GEK and Fast
Red TR salts, respectively.
The Student Newman-Keuls test was applied to deter-
mine whether differences between the data from the
various sampling times were significant. Differences were
considered significant at the P < 0.05 level.
RESULTS
Attnuul fiuctogeniccycle
Mules. Male M. lucijiigus exhibited a fructogenic
cycle in the accessory sex glands (ampullary, prostate
and seminal vesicles) that varied seasonally (Fig. 1).
The ampullary gland was the major fructose-produc-
ing accessory sex organ. In this gland, fructose
reached its maximum levef (September/October) at
the onset of hibernation (almost 5.0mg fructose/g
tissue). Fructose remained significantly elevated until
male bats emerged from hibernation (April). Follow-
ing the return of bats to an active metabolic state,
ampullary gland fructose content decreased markedly
(I .4 mg fructose/g tissue during May and June) in
keeping with the involution of the entire gland. The
prostate gland also showed an annual fructose cycle-
not as dramatic, however, as the ampullary gland, and
furthermore, the organ produced less sugar. Fructose
content of the prostate rose at the beginning of hiber-
nation (2.2mg fructose/g tissue in September) and,
with some fluctuations remained higher throughout
the sperm storage period than at other times in the
year. The seminal vesicles also elaborated fructose,
but at even lower levels (0.5-1.5 mg fructose/g tissue)
and with less seasonal fluctuation than the ampullary
and prostate glands. When .the differences between
seasonal levels of fructose in either the prostate or
seminal vesicles, were analyzed, no significance was
recorded (P < 0.05).
Although our annual data from M. velifer were not
complete (we lacked late winter and early spring
,',,'s'o'N'D'J' F’,.,‘A’M’J
Months
Fig. I. Annual fructose cycle in the accessory organs of
male M. ~f~~~~~~.~. O-----Cl Ampullary gland; m----II
Prostate gland; 0.1 .O Seminal vesicles. Rec. = recru-
descence; Cop. = copulation; Invol. = involution. Vertical
bars represent the standard error of the mean. Numbers at
each point represent the number of samples (sample = l-5
bats pooled).
389
E”
J ASONDJF
Months
Fig. 2. Annual fructose cycle in the accessory organs of
male M. uelifir. n-----O Ampullary gland; B---N Pros-
tate gland; #+...+ 0 Seminal vesicles. Rec. = recrudes-
cence; Cop. = copulation; Invol. = involution. Vertical
bars represent the standard error of the mean. Numbers at
each point represent the number of samples (sample = l-5
bats pooled).
values), a small elevation in fructose was recorded in
the prostate and ampullary glands, from trace levels
during July and August to 2.0-3.0 mg fructose/g tissue
at the onset of hibernation in October (Fig. 2). Levels
were reduced in May (1.S mg fructose/g tissue).
Seminal vesicle fructose values, though much lower,
demonstrated a similar though less well marked trend
in annual cyclicity. An analysis of the differences
between monthly means revealed no significance
(P < 0.05).
Fructose was not identified in the caudae epididy-
mides in either species.
Fe&es. An analysis of the female reproductive
tracts of both species demonstrated fructose in the
uterus, although at much lower levels than recorded
from the accessory sex glands in males. Fructose
underwent cyclic changes seasonally (Figs 3 and 4). In
M. velifrr, uterine fructose levels rose from baseline
values in June (0.03 mg fructose/g uterus). July
(0.06 mg fructose/g uterus) and early August to a
maximum level in late August (1.26 mg fructose/g
1 9
Months
Fig. 3. Annual uterine fructose cycle in female M. ~~~~~g~,~.
O-----O Monthly fructose levels. An. = anestrus;
Cop. = copulation; Preg. = pregnancy; Lact. = lactation.
Vertical bars represent the standard error of the mean.
Numbers at each point represent the number of samples
(sample = l-5 bats pooled).
390 ELIZABETH G. CRICHTON et ui.

analyzed I hr after arousal. When these experiments

were performed near the end of natural hibernation
(April), bats aroused from hibernation 24 hr prior to
analysis and bats that remained hibernating until the
time of sacrifice in each case contained significantly
less uterine fructose than bats brought to room tem-
perature about an hour prior to analysis (Table 1).
b,I9

Results of experiments to determine the role of the

ovary in the fructogenic cycle are given in Table 2 (M.
~~~~~~gus)and Table 3 (M. celifh). Ovariectomy at the
end of the sperm storage period, without subsequent
steroid therapy, reduced uterine fructose levels below
Months
those to be expected at this time of the year, whereas
Fig. 4. Annual uterine fructose cycle in female M. wlfcr. uterine fructose was raised after supplementation by
Cl-----U Monthly fructose levels. An. = anestrus: testosterone and by progesterone alone or in combi-
Cop. = copulation; Preg. = pregnancy; Lact. = lactation. nation with estrogen. The fructose values obtained
Vertical bars represent the standard error of the mean. were not significantly different (P < 0.05). except in
Numbers at each point represent samples (sample = f--5
the one experiment in which testosterone was admin-
bats pooled).
istered to ovariectomized bats.
uterus). Eighty percent of specimens of M. luc$uguyus In February (during the sperm storage period), only
sampled in July were without uterine fructose. The estrogen in combination with progesterone effected a
remaining two lactating samples (n = 6) from July marked rise in uterine fructose levels of active intact
and four nonlactating samples (n = 20) from early bats. Progesterone alone reduced fructose below con-
August contained more than 1.2 mg fructoseig uterus, trol levels, while estrogen alone raised fructose
while the other 11 samples assayed during August slightly. A comparison between fructose values
contained smaller amounts of fructose. Uterine fruc- obtained after hormone treatment and normal values
tose decreased in September in both species, but once showed that none of the differences was significant.
again rose in November, attaining a maximum value Towards the end of the sperm storage period (April).
of 1.36 mg fructose/g uterus in December in M. luci- all steroid hormone treatments lowered uterine fruc-
fiqus. Fructose values dropped sharply in January in tose below the control value in intact. aroused or hi-
M. lt~~~~giis, while 804, of ,M. wlifer sampled at this bernating bats, testosterone producing the least
time contained no uterine fructose. Levels rose again reduction.
during February in M. wlifer, but remained low in M. A sample of eight M. rle[fer obtained in August and
lucifugus until April, the time of arousal and concep- used as oil-injected controls for a steroid supplemen-
tion. A comparison of the averages of monthly uterine tation experiment contained no uterine fructose.
fructose levels showed that, although differences Ovariectomy at this time did not alter these values,
existed, they were not significant. but hormone supplementation to intact bats pro-
Specimens of M. vei$'er assayed in May included duced small increases in uterine fructose, with pro-
two nonpregnant animals with greatly elevated fruc- gesterone effecting the most pronounced elevation
tose levels (4.5 and 1.4 mg fructoseig uterus). If these (Table 3). A comparison between means showed that
two bats are treated as a separate sample. their uter- none of the differences was significant.
ine fructose content was significantly elevated above
other bats (all pregnant), obtained at the same time
(X = 0.54 mg fructose/g uterus). ACTH and epinephrine were ~ldministcred to active
and ~libernatin~ female ;M. ~I~~~~~~~~~s.These hormones
significantly depressed uterine fructose levels in active
During the early sperm storage period (November- bats; in hibernating animals. ACTH depressed uterine
December), M. luc@gus brought from the hibernating fructose, but epinephrine significantly raised it
state about an hour before analysis had significantly (Table 4).
more uterine fructose (x = 1.063 mg fructose&
uterus) than bats sacrificed immediately from hiber-
nation (x = 0.323 mg fructoseig uterus). Bats ana- In an assay performed on October 2. presumed
lyzed after 24 hr of arousal had slightly less uterine noninseminated M. !~ccifu~rr.s held captive since
fructose (x = 0.986mg fructose/‘g uterus) than those August 19 (at which time a sample contained 0.46 mg
fructose/g uterus) contained 0.71 mg fructose/g uterus
Table 1. Effect of hibernation and arousal on fructose while presumed inseminated bats collected on
levels (mgig uterus) in the bat uterus. Each value rep- October 2 had 0.81 mg fructoseig uterus. These values
resents the mean of 3-7 samples (sample = 5 bats pooled) did not differ signi~cantly.
In another assay performed on M. u~i+r, the
Time Aroused Normal Hibernating
August-collected, presumed noninseminated bats still
April 0.055 0.710 0.183 contained no uterine fructose, whereas the November-
November 0.986 1.063 0.323 collected, presumed inseminated bats contained
0.66 mg fruct0se.g uterus.
 Fructose in sperm storage 391

Table 2. Uterine fructose levels (mg/g tissue) in response to ovariectomy and to steroid
administration to intact and ovariectomized bats (Myotis lucifugus) during the mid-sperm
storage period (February) and towards its termination (April). n = number of samples
(sample = 3-5 bats pooled)

Intact
Aroused Intact Intact Ovariectomized
_(Feb) Aroused Hib. Aroused
Hormone n X range n (Apr) n (Apr) n (Apr)

Estrogen 5 0.32 2 0.56 3 0.18 1 0.16

(GO.70) ((M.36)

Progesterone 3 0.04 3 0.19 2 0.34 1 0.72

(0.17) (O-0.33)

Estrogen & 5 0.76 4 0.39 3 0.17 1 0.72

progesterone (fS2.72) (0.21LO.56) ((rO.34)

Testosterone 3 0.63 3 0.55 1 2.86

(0.51LO.76) (0.31LO.71)

Control 4 0.25 1 0.73 2 0.62 1 0.14

(crO.55)
Normal, 7 0.32 22 0.78
seasonal
value

Table 3. Uterine fructose levels (mg/g tissue) in response to ovariectomy and to

steroid administration to intact bats (Mq’otis uelifer) just prior to the sperm storage
period (August). (Each value represents the average of 3-5 analyses)

Estrogen &
Ovariectomy Estrogen Progesterone progesterone Testosterone Control

0 0.28 0.79 0.13 0.22 0

(SO.43) (Ck1.69) (crO.50) (W.49)

effect of steroids on ocarian histology Histochemical localization of enzymes

Ovaries from active M. lucfugus that served as con- Histochemical analysis of the female tract during
trols in steroid supplementation experiments con- the sperm storage season demonstrated the presence
ducted in February contained many multilaminar fol- of acid and alkaline phosphatases in the epithelia of
licles, some of which were antral. In contrast, there the utero-tubal junction and uterine corpus. Glyco-
were no antral follicles in the ovaries of bats sacrificed gen, however, was only present in the epithelial cells
following steroid therapy. Estrogen administration in the utero-tubal junction and caudae epididymides.
alone or in combination with progesterone strongly The caudae epididymides reacted strongly for alkaline
stimulated the growth of interstitial cell tissue. There phosphatase when spermatozoa were present, but
were few follicles present in the ovaries of estrogen- were negative for this enzyme when spermatozoa were
supplemented bats. Exogenous estrogen and pro- absent. Conversely, acid phosphatase was absent
gesterone together and progesterone alone resulted in when spermatozoa were present, but the epididymal
the formation of many large bilaminar and multi- epithelium was positive for acid phosphatase once the
laminar follicles. sperm storage season had ended and the caudae epi-
didymal lumina were empty of spermatozoa.

DISCUSSION

Table 4. Effect of ACTH and epinephrine on fructose We have demonstrated fructose in the reproductive
levels (mg/g uterus) in the bat uterus during the sperm tracts of both male (epididymides excepted) and
storage period. (Each value represents the mean of 3 female Myotis in amounts that vary seasonally. In the
analyses). male, we find that the annual fructogenic cycle corre-
lates positively with accessory gland hypertrophy.
Metabolic state ACTH Control Epinephrine Fructose rises near the end of the spermatogenic
phase and maintains maximal levels throughout the
Active 0.15 1.04 0.06
Hibernating 0.11 0.32 1.13
hibernation (sperm storage) phase of the male repro-
ductive cycle, a time when accessory glands are hyper-
392 ELIZABETH G.
trophied. Fructose levels are lowest in June and July,
a time when males are undergoing their annual sper-
matogenic cycle but are yet without epididymal sperm
and enlargement of the involuted accessory sex glands
has not begun. Contrary to many other mammalian
species and the tropical bat Cynopterus sphinx (Mok-
kapati & Dominic, 1976), the seminal vesicles of
Myoti,s contribute the least amount of fructose to the
seminal fluid. In M_rotis, as in Scotophilus heuthi,
Tuphozous 1ongimunu.s and Pteropus gigunteus (Raja-
lakshmi & Prasad. 1970; Mokkapati & Dominic,
1976), the ampullary glands are the most active male
reproductive organs in fructose production. Fructose
is not available to epididymal sperm. as evidenced by
the negligible quantity of this sugar in this organ.
In the female, it is likely that part or all of the rising
uterine fructose values during the early sperm storage
period result from periodic infusions of seminal
fructose. Although insemination reaches its climax in
SeptemberrOctober (prior to hibernation), it prob-
ably occurs sporadically throughout hibernation, due
to the continued proestrous state and resultant recep-
tivity of the female.
In August in M. lucifugus. uterine fructose probably
reflects early copulatory activity, since it is then that
plasma testosterone levels reach a peak and the ac-
cessory gland epithehum attains maximum height
(Gustafson, 1977). These indicators, as well as direct
measurement of seminal fructose, suggest that the
fructose measured is of seminal fluid origin.
Further evidence that some of the uterine fructose
is of male origin is as follows. In M. lucr~ugus, a deep
hibernator, uterine fructose declines steeply to prehi-
bernation levels during January and remains at this
level throughout the rest of the sperm storage period.
Copulatory activity during this time is presumably
minimal in M. lucifugus as compared with M. odifrr,
whose intermittent hibernating behavior permits
copulation. That such may be the case is reflected in
the presence of fructose in January and February.
Since the accessory glands of Myoris remain secretory
and hypertrophied throughout the sperm storage
period, seminal fructose is likely supplied continu-
ously to those females receiving periodic insemina-
tions during hibernation. Nevertheless, successful fer-
tilization is known to operate without such additional
infusions (Matthews, 1937: Folk, 1940; Wimsatt,
1942. 1944; Kleiman & Racey. 1969; Racey. 1972,
1973). Further copulation may also occur at arousal
and briefly thereafter, as suggested by the highly vari-
able data obtained from female M. lucifigus at this
time (April).
The data obtained from samples of M. cd@ in
May similarly suggest a predominately seminal con-
tribution to the pool of uterine fructose. Two non-
pregnant bats with significantly higher uterine fruc-
tose values could represent such recently inseminated
individuals.
Female bats isolated from males prior to the mat-
ing season also yielded data supporting our convic-
tion that some female reproductive tract fructose is
male-contributed. For example, M. /ucifugus was iso-
lated in mid-August and shown in October to have
uterine fructose values virtually identical to those in
uteri of bats brought freshly from the field; such cor-
respondence suggests that the summer isolated
CRICHTON ef 01.
females had already been inseminated at the time of
capture, M. cd@, however, is unlikely to mate as
early as August; thus bats collected at this time and
shown lacking in uterine fructose in November prob-
ably had not received a seminal contribution.
Nevertheless, we believe that some of the fructose
recorded is of female reproductive tract origin. This
belief arises from experiments in which bats were
allowed to remain hibernating until the time of sacri-
fice. These experiments (November and April) showed
that bats activated about an hour prior to sacrifice
(normal method) generated significant amounts of
uterine fructose as compared with the small amount
present in the hibernating state. (Low uterine fructose
levels during hibernation are logical concomitants of
reduced needs of any spermatozoa that may be
present). A prolonged period of arousal (24 hr) was
followed by a reduction in uterine fructose in experi-
ments performed in April. Prolonged arousal might
lead to a demand by stored spermatozoa for increased
uterine fructose; such need could only be met by ac-
celerated fructogenesis. In experiments carried out in
November, fructose levels did not decline significantly
following 24 hr of arousal. Thus, the capacity to toler-
ate such metabolic demand was greater at the begin-
ning of sperm storage (November). At that time, per-
iodic arousals normally would occur in nature, and a
built-in mechanism to accommodate to these meta-
bolic fluctuations would be important to sperm sur-
vival.
Thus, uterine fructose noted during the sperm stor-
age season could be self-generated, male-contributed,
or both. The steady accumulation of uterine fructose
demonstrated in M. luc$ugu,s from September to
December may reflect the synthesis of uterine fructose
in readiness for spermatozoan maintenance, the ac-
cumulation of aliquots of seminal plasma fructose, or
both. Conceivably also. the steep decline in uterine
fructose from December to January in M. Iucjfugus,
followed by its retention at pre-hibernation levels
throughout the remainder of the sperm storage
period, reflects gradual dilution and dumping of
uterine or seminal fructose. Or the drop could be
explained by utilization by stored spermatozoa of
fructose at a rate that exceeded supply or synthesis.
Hibernation in M. cc/@ in Arizona would likely be
interrupted by periods of arousal. depending on local
conditions. Uterine fructose would be expected to
show a less well marked correlation with sperm stor-
age. Thus, the highly variable results obtained during
January and February could reflect either periodic
arousals permitting copulatory activity or fluctuations
in the metabolic demands made on uterine fructo-
genesis by alternately active and torpid intrauterine
spermatozoa.
Preliminary experiments were designed to see if the
ovary and ovarian steroids play a role in regulating
uterine fructose. The results were inconclusive. Never-
theless, elevated uterine fructose in M. lucifugus after
administration of exogenous testosterone leads us to
consider a possible role for steroids on uterine fructo-
genesis. Androgens are known to be required for fruc-
tose production in the male (Mann, 1964). Indications
are that in female rats, male accessory organ trans-
plants can be induced to produce fructose following
either direct administration of exogenous testosterone
 Fructose in sperm storage 393

or indirect stimulation of ovarian androgenesis by where they commonly take on a close association
steroids (Price et ul., 1949, 1955). Synthetic progester- with epithelial cells. It is not known whether the
one is held to have androgenic activity when given in luminal environment of the female reproductive tract
large doses. In some species, androgens are the princi- of the bat is aerobic (as it is for other mammalian
pal steroids secreted by the ovarian stroma during the species: Bishop, 1956; Nevo, 1965) or anaerobic dur-
follicular phase (Rice & Savard, 1966; Hilliard et nl., ing sperm storage (hibernation). Thus, the relative im-
1974) and it is through their aromatization that estro- portance of glycolytic vs oxidative metabolism has
gen is produced in the granulosa. not been determined for bat intrauterine spermatozoa
Although the testes of hibernating M. luc~figus are and the preference for fructose over other oxidizable
not spermatogenic during the winter, testosterone is substances (e.g. pyruvate, alcohols, organic acids) or
present in the peripheral plasma (Gustafson & She- glycolysable substrates (glucose, mannose) that may
mesh, 1976). The origin of this plasma testosterone is be present, is unknown. It is tempting to think that
unknow~l, since ultrastructural studies of the Leydig luminal conditions are anaerobic, since during hiber-
cells suggest that they are nonsecretory once the sper- nation the bat has very low respiratory and pulse
matogenic and mating seasons have passed (Gustaf- rates, depressed body temperature and dramatically
son, 1979). An extragonadal site of androgen synthesis slowed metabolism. These factors suggest that oxygen
during testicular involution has been proposed in is in limited supply and that intrauterine spermatozoa
studies on interscapular brown adipose tissue continue to draw upon anaerobic mechanisms for
(Krutzsch & Wells. 1960). Support has come from metabolism during their prolonged storage. It is
histochemical demonstrations of 17~-hydroxysteroid interesting to note that Bishop (1971) speculated that
dehydrogenase activity within the multilocular fat capacitation in spermatozoa corresponds to a shift in
cells during the sperm storage season, and the epidi- metabolism from post-insemination glycolysis to oxi-
dymis. an established site of androgen synthesis in the dative phosphorylation, a process that probably could
rat (Hamilton, 1971), has also been implicated in the not occur in the bat until its emergence from hiber-
bat, by A5-3 fi-hydroxysteroid and 17p-hydroxy- nation and return to a constant, active metabolic
steroid dehydrogenase activity (Crichton & Krutzsch, state, (the time of ovulation). Of further interest is the
unpublished observations). Although the origin of finding of Rogers & Yanagimachi (1975) that the
androgen in the male hibernating bat remains acrosome reaction (and therefore capacitation) is
unclear, perhaps the low levels of steroid synthesis retarded in the spermatozoa of the guinea pig by high
signify the physiologic inhibition of prolonged torpor concentrations of fructose (and glucose). These obser-
rather than the absence of appropriate synthetic path- vations suggest the possible existence of an environ-
ways. ment-controlled mechanism in the bat that would
Aside from problems associated with obtaining delay capacitation. Such delay would in turn conserve
physiologically homogeneous samples of bats, vari- energy in stored spermatozoa until they are required
ations in uterine fructose levels may also reflect the for fertilization.
effects of capture and handling stress. In order to The female bat is in persistent estrus throughout
evaluate this possibility, bats were stimulated directly sperm storage: the reproductive tract may, therefore,
by the injection of epinephrine or indirectly by the contain elevated or reduced levels of other substances
activation of the adrenals with exogenous ACTH. The that, in some mammalian species at least, are relevant
speculation that stress, through elevation of adrenal to sperm survival, e.g. hypotaurine (Van der Hors: &
and pituitary hormones, might promote carbohydrate Brand, 1969; Jones, 1978) and ~lycerylphosphoryicho-
synthesis was substantiated, but only in epinephrine- line diesterase (White & Wallace, 1961; Wallace &
stressed hibernating bats. Both epinephrine and White, 1965; Wallace et a[., 1964, 1965).
ACTH reduced uterine fructose significantly in active Racey (1973) suggests that the burden of sperm
bats suggesting that the fructose values obtained in storage in bats falls more heavily on one sex or the
this study may have been lowered as the result of our other, depending on the species. Our studies suggest
handling of animals, but at least such handling was that factors operating in the male to promote sperm
performed in a consistent manner. survival may differ from those prevailing in the female;
Small quantities of fructose, or some fructose furthermore, different mechanisms may operate at dif-
precursor, may therefore be elaborated by the ferent points of the sperm storage period. Fructose,
female reproductive tract in preparation for or in while showing an annual cyclicity in the uterus, does
response to the presence of spermatozoa, and sperm not bear a strong relationship with the sperm storage
may derive some of the energy needed for their pro- period and it may only play a role in sperm survival
longed intrauterine stay from the metabolism of this during the initial storage phase. Fructose is not
sugar. However, it would appear that most of the present in the epididymis, and thus cannot account
fructose present in the uterus during the period when for prolonged sperm survival in the male.
spermatozoa are stored comes from seminal fluid. A recent review by Harrison (1977) points out that
smce uterine fructose levels decline rapidly in M. luci- in certain species, seminal plasma (containing fruc-
firgus once copulation has ended and torpor is under- tose) is unable to penetrate the cervix and “. . there-
way. In the anaerobic environment created by fore it cannot act as a long-term direct source of
n~lmerous recently instilled spermatozoa, such fruc- energy for the cells (spermatozoa). ” Fructose may,
tose would likely serve as the principal metabolic sub- however, contribute significantly to the acetyl stores,
strate. As hibernation continues, spermatozoa dis- which become available for normal oxidation if carni-
perse. Large numbers of them are probably voided, tine is present. For some species of bats, it is certain
while the remainder are seen in the lumen of the that spermatozoa are delivered and stored in the
uterus and its glands and in the utero-tubal junction, vagina. In Myotis. however. sperm are rarely found
394 ELIZABETH G. CRICHT~Ner (11.
there in large numbers. It may be that, in this species,
spermatozoa are delivered directly into the body of
the uterus, thereby maintaining contact with the
seminal fluid and its utilizable substrates.
These observations suggest that further research
might be productively directed at defining other
metabolizable substrates present and utilizable for
spermatozoan survival in the bat uterus. In particular,
glucose should be investigated as a possible substrate
for the metabolism of stored spermatozoa. It is the
main carbohydrate present in the female reproductive
tract (Pedron et ul., 1975) and it has bekn implicated
as an energy source for mouse spermatozoa during
capacitation (Hoppe, 1976). It seems equally relevant
here to postulate mechanisms that could prolong
sperm retention and survival by suppressing the meta-
bolic rate of gametes, (e.g. metallic zinc; Crichton,
Krutzsch & Chvapil, in press) or inhibiting uterine
motility and the activity of the phagocytic system
(Hunter et al., 1971a, 1971b). Such mechanisms, acting
separately or in unison, could conceivably extend the
life span of spermatozoa within the female reproduc-
tive tract by providing their nutrition and suppressing
their metabolic demands.
Acknnwledgfmenrs-This study was supported by NSF
Grant PCM 77-Q4020 to P. H. Krutzsch. The technical
assistance of Dr M. Coetzee is gratefully acknowledged.
We wish also to thank Dr Jay B. Angevine, Jr for his
editorial assistance with the manuscript and Mr G. Kwie-
cinski for his assistance in the acquisition of specimens of
M. lu~$ugus.
REFERENCES
ANDERSON W. A. & PERSONNEP. (1969) The cytochemical
localization of sorbitol dehydrogenase activity in sper-
matozoa of He/ix aspersa. .I. Microsc. 8, 97-102.
AUSTINC. R. (1969) Sperm capacitation-biological signifi-
cance in various species. S&ring Symposium on Mechan-
isms Involved in Concepprion. (Edited by RASPEG.). Perga-
mon-Vieweg, Braunschweig.
BISHOP D. W. (1956) Oxygen concentrations in the rabbit
genital tract. Proc. Third Int. Congr. Anim. Reprod. pp.
53-55.
BISHOPD. W. (1971) Sperm transport in the fallopian tube.
In Pathways to Conception (Edited by SHERMANA, I.),
pp. 99-106. Thomas, Springfield.
COURRIER R. (1921) Sur la rble physioio~que des
s&c&ions utCrine et tubaire chez la chauve-souris hiber-
name. C.r. SPanc. Sot. Bioi. 84, 572-574.
CRICHTON E. G., KRUTZSCHP. H. & CHVAPILM. Studies
on prolonged spermatozoa survival in Chiroptera-II.
The rote of zinc in the spermatozoa storage phenom-
enon. Camp. Biochem. Pkvsiol. In press.
FAwcEn D. W. & fT0 S. (1965) The fine structure of bat
spermatozoa. Am. J. Anar. 116, 567-610.
FOLKG. E. (1940) The longevity of sperm in the female bat.
Anut. Rec. 76, 103-109.
Fox W. (1956) Seminal receptacles of snakes. Anat. Rec.
124, 5 19-540.
GOPALAKRISHNA A. & MADHAVANA. (1971) Survival of
spermatozoa in the female genital tract of the Indian
vespertilionid bat, Pipisfr~~~~s ~~~~o~~i~u~ ~hrysof~7r~,~
(Wroughton). Proc. Indian Aeud. SC;. B ‘73, 43-49.
GOPALAKR~SHNA A. & MADHAVANA. (1978) Viability of
inseminated spermatozoa in the Indian vespertilionid
bat Scotophilus henthi (Horsefield), Indian J. exp. Biol.
16, 852-854.
GUSTAFSON A. W. (1977) Observations on the male access-
ory apparatus in the hibernating vespertilionid bat,
Myotis lucifugus lucifugus during the annual reproduc-
tive cycle. Anat. Rec. 187, 595.
GUSTAFSON A. W. (1979) Male reproductive patterns in hi-
bernating bats. J. Reprad. Ferr. 56, 317--331.
GUSTAF~~NA. W. & SHEMESH M. (1976) Changes in plasma
testosterone levels during the annual reproductive cycle
of the hibernating bat, Myotis fucifugus iucifigus with a
survey of plasma testosterone levels in adult male verte-
brates. Biol. Reprod. 15, 9--24.
HAMILTON D. W. (1971) Steroid function in the mammalian
epididymis. J. Reprod. Fert. Suppl. 13, 89-97.
HARRISONR. A. P. (1977) The metabolism of mammalian
spermatozoa. In Fronriers in Reprodu~tiun und Ferfj~~t~
Conrrof II. (Edited by GREEPR. 0. & KOBLINSKY M. A.),
pp. 379401. MIT Press, Cambridge, Massachusetts.
HARTMANC. G. (1933) On the survival of spermatozoa in
the female genital tract of the bat. Q. Rec. Biol. 8,
185-193.
HERS H. G. (1957) Le Metaholisme du Frucfose. Edition
Arscia, Bruxeltes.
HILLIARDJ., SCARAMUZZIR. J., PANG C., PEWARDIR. &
SAWYERC. H. (1974) Testosterone secretion by rabbit
ovary in vice. Endocrinolog) 94, 267-271.
HIRAIWAY. K. & UCHIDAT. (1955) Fertilization in the bat
Pipisrrellus abramus ubramus (Temminck) II. On the
properties of semen stored in the uterus. Sci. Bull. Fuc.
Agric. Kpushu Univ. 30, 255-266.
HOFFMANL. H. & WIMSATTW. A. (1972) Histochemical
and electron microscopic observations on the sperm
receptacles in the garter snake oviduct. Am. J. Anclt. 134,
71-96.
HOPPEP. C. (1976) Glucose requirement for mouse sperm
capacitation in vitro. Biot. Reprod. 15, 39-45.
HUNTERA. G.. BARKERL. D. S., JOHNSONW. L., FAHNINC~
M. L. & SCHULTZR. H. (1971a) Antigenicity, toxicity
and cross reactions of male bat (My&is ~c~~i~ff~~.~) repro-
ductive organs. .I. Reprod. Ferf. 24, 171-177.
HUNTERA. G., JOHNSONW. L.. BARKERL. D. S., FAHNIN~;
M. L. & SCHULTZR. H. (1971b) Bat seminal vesicle pro-
tein: its characterization and physiological properties. J.
Reprod. Fert. 24, 179-186.
JALABERT B. & BILLARDR. (1969) Etude ultrastructurale du
site de conservation des spermatozoides dans I’ovaire de
Poccifia ~eri~~~ufa (Poisson TClkostCen). Ann. Bioi. anim.
Biochim. Biophys. 9, 273-280.
JONESR. (1978) Comparative biochemistry of mammalian
epididymal plasma. Comp. Biochem. Physiol. 61B,
365-370.
KIXIMAN D. G. & RA~EY P. A. (1969) Observations on
noctule bats breeding in captivity. Lrt~x IO, 65-77.
KRISHNAA. & DOMINICC. J. (1978) Storage of spermato-
zoa in the female genital tract of the vespertilionid bat,
Scotophilus heurhi. J. Reprod. Ferr. 54, 319-321.
KRUTZSCHP. H. & WELLSW. W. (1960) Androgenic ac-
tivity in the interscapular brown adipose tissue of the
male hibernating bat (Myo:oris lucijlgu.s). Proc. Sot. exp.
Biol. Med. 105, 578-581.
KRL~TZSCH P. H., WATSONR. H. & Lox C. D. (1976)
Reproductive biology of the male leaf-nosed bat, Mac-
rof~s watrrhousii in southwestern United States. Anat.
Rec. 184. 61 l-636.
LAKEP. E., LORENZF. W. & REIMANW. D. (1962) Further
investigations of the carbohydrate metabolism of cock
spermatozoa. Narrvc: Lond. 194, 545-547.
LINDNERH. R. & MANN T. (1960) Relationship between
the content of androgenic steroids in the testes and the
secretory activity of the seminal vesicles in the bull, J.
Endocr. 21, 341-.360.
LORENZF. W. (1958) Carbohydrate metabolism of cock
spermatozoa. Nnrure, Lond. 182, 397-398.
MANN T. (1946) The origin and function of seminal fruc-
tose. Biochem. J. 40,481490.
 Fructose in sperm storage 395

MANN T. (1949) Metabolism of semen. Adr. Enzym. 9, RANSOMER. (1971) The effect of ambient temperature on
329-390. the arousal frequency of the hibernating greater horse-
MANN T. (1964) The Biochemistry oj’Semen und of rhe Mull shoe bat, Rhinolophus ferrum-equinum, in relation to site
Reproductice Tracr. Methuen, London. selection and the hibernation site. J. Zool., Lond. 164,
MATTHEWS L. H. (1937) The female sexual cycle in the 353-371.
British horse-shoe bats, Rbjno~op~~s ,~err~m-e~uinum RENDEZ E. (1929) Das Verhalten der Saugetierspermato-
jns~~~~~ri.~~rrett-Hamilton and R. ~~jpp~).~~der~~s rninuft~s zoen zwischen Begattung und Befruchtung. Z. Zellforsch.
Montagu. Truns. wol. Sot. Land. 23, 224-266. mikrosk. Anar. 9, 734749.
hfEDWAY Lord (1972) Reproductive cycles of the Aat- RICE B. F. & SAVARD K. (1966) Steroid hormone formation
headed bats T,v/onyc~teris path \‘pus and T. robu.stula in the human ovary: IV. Ovarian stromal compartment:
(Chiroptera: Vespertilioninae) in a humid equatorial en- Formation of radioactive steroids from acetate-l-14C
vironment. Zoctl. I. Linn. Sot. 51, 33-61. and action of gonadotropins. J. C/in. Endocr. Metab. 26,
MOIC~~APATIS. & DO%UNITC. J. (1976) Sites of production 593-609.
of fructose and citric acid in the accessory reproductive R~~HXRDSOI\T E. G., KRI~TZSCH P. H. & NAGLE R. B. (1980)
glands of three species of male chiropterans. Biol. TEM and SEM observations on the prolonged sperm-
Reprod. 14, 621-629. uterine relationship in hibernating bats. Anat. Rec. I%,
h40~1 T. & LJcrrm~ T. A. (1980) Sperm storage in the 156p157A.
reproductive tract of the female Japanese long-fingered ROE J. H. (1934) A calorimetric method for the determi-
bat, Minioprerus .schreihersii firliyirzo.str.s. J. Reprod. Fert. nation of fructose in blood and urine. J. biol. Chem. 107,
S&429-433. 15.-22.
hJYERS P. (1977) Patterns of reproduction of four species of ROGERS B. J. & YANAGIMACH~ R. (1975) Retardation of
vespertilionid bats in Paraguay. L’nir. Ctrlif: P&i. Zoo/. guinea pig sperm acrosome reaction by glucose: the
107, t-41. possible importance of pyruvate and lactate metabolism
NAKANV 0. (1928) ijber die Verteilung des Glykogens bei in capacitation and the acrosome reaction. Biol. Reprod.
den zyklischen Veranderungen in den Geschlechts-orga- 13, 568-575.
nen der Fledermaus. Und iiber die Nahrungsaufnahme SCHWAB H. (1952) Beobachtungen iiber die Begattung und
der Spermien in dem weiblichen Geschlechtswege. Folio. die Spermakonservierung in den Geschlechtsorganen bei
Anal. /up. 6, 717-828. weiblichen Flederm%usen. Z. mikrosk.-anat. Forsch. 58,
NAPOLITA~[) L. M.. KRUTZSCH P. H. & WIMSATT W. A. 326-357.
(1963) Electron microscopy of the sperm-uterine re- STRELKOV P. P. (1962) The peculiarities of reproduction in
lationship in a hibernating bat. Anui. Rec. 145, 365. bats (Vespertilionidae) near the northern border of their
NEV~ A. C. (1965) Dependence of sperm motility and res- distribution. Proc. Int. Symp. Merh. Mamma/. Inoesr.
piration on oxygen concentration. J. Reprod. Ferr. 9, Brno. 1960 (Edited by KRATOCHVIL J. & PELIKAN J.).
103m 107. pp. 306311. Praha, Czechoslovakia.
PEAKSE A. G. E. (1968) Histor,hemistry Theoreticul and VAN DER HORST C. J. & BRAND A. (1969) Occurrence of
Applied, Vol. I. Williams & Wilkins, Baltimore. hypotaurine and inositol in the repioductive tract of the
PEDR~N N., GINER J.. HICKS J. J. & ROSADO A. (1975) ewe and its regufation by pregnenolone and progester-
Comparative glycolytic metabolism of sperm from nor- one. Nature, Lond. 223, 67-78.
mal, asthenospermic and oligospermic men. Fert. & VAX KREY H. P., OCASAWARA F. X. & PANGBORN J.
Steril. 26, 309~-3 13. (1967) Light and electron microscopic studies of possible
PRICE D., MANN T. & LUTWAK-MANN C. (1949) Some sperm gland emptying mechanisms. PO&. Sci. 46,
metabolic effects of androgenic substances. Nuture. Land. 69-78.
164, 95@-95 I, WALLACE J. C. & WHITE I. G. (1965) Glycerylphosphoryl-
PRICE D.. MAKN T. & LUTWAK-MANN C. (1955) The choline diesterase in female reproductive tract. J.
stimulating effect of female hormones on the metabolic Rrprod. Fert. 9, 163-176.
activity and histological structure of male rat accessory WALLACE J. C., STONE G. M. & WHITE I. G. (1964)
reproductive glands. Anur. Rec. 122, 363-375. Influence of some estrogens and progestogens on the
RACEY P. A. (1972) Viability of bat spermatozoa after pro- activity of glycerylphosphorylcholine diesterase in rins-
longed storage in the epididymis. J. Reprod. Ferr. 28, ings of the rat uterus. J. Endocr. 29, 175-184.
309 -311. WALLACE J. C., STONEG. M. & WHITE I. G. (1965) Effect of
RACEY P. A. (1973) The viability of spermatozoa after pro- the oestrous cycle and of oestradiol on the breakdown of
longed storage by male and female European bats. seminal glycerylphosphorylcholine by secretions of the
Period. Biol. 75, 201&205. rat uterus. Ausr. J. biu/. Sri. IS, 88.-96.
RACEY P. A. (1974) The reproductive cycle in male noctule WHITE I. G. & WALLACE J. C. (1961) Breakdown of
bats N~crulus noctula. J. Reprod. Fert. 41, 169-182. seminal glycerylphosphorylcholine by secretions of
RACEY P. A. (1975) The prolonged survival of spermatozoa the female reproductive tract. Narure, Lond. 189, 843
in bats. In Tke Biolog? qf rhe Male Gamete. (Edited by -844.
DUCKETT J. G. & RACEY P. A.), pp. 385416. Academic WIMSATT W. A. (1942) Survival of spermatozoa in the
Press. London. female reproductive tract of the bat. Anat. Rec. 83,
RACEY P. A. (1979) The prolonged storage and survival of 299m 307.
spermatozoa in Chlroptera. .I. Reprod. Ferr. 56, 391-402. WIMSA~ W. A. (1944) Further studies on the survival of
RACEY P. A. & POTTS D. M. (1970) Relationship between spermatozoa in the female reproductive tract of the bat.
stored spermatozoa and the uterine epithelium in the Anut. Rec. 88, 193-204.
pipistrelle bat (Pipistrellu.~ pipi.stre//us). J. Reprod. Frrt. WIMSATT W. A. (1949) Cytochemical observations on the
22, 57-63. fetal membranes and placenta of the bat, Mpofis luci-
RAC.EY P. A., Suzuai F. & MEDWAY Lord (1975) The re- fuytrs iucifufuyus. Am. J. Anar. 84, 63--142.
lationship between stored spermatozoa and the oviduc- WIMSATT W. A. (1969) Some interrelations of reproduction
tal epithelium in bats of the genus T+trcreris. In The and hibernation in mammals. Symp. SW. exp. Biof. 23,
Bio&~ c$ Spermatozou. (Edifed by &A&Z E. S. E. & 51 l-549.
THIBAULT C. G.), pp. 123-133. Karger, Basel. WIMSATT W. A., KRUTZSCH P. H. & NAPOLITANO L. (1966)
RAJALAKSHMIM. & PRASAD M. R. N. (1970) Sites of forma- Studies on sperm survival mechanisms in the female
tion of fructose, citric acid and sialic acid in the access- reproductive tract of hibernating bats. 1. Cytology and
ory glands of the giant fruit bat, Preropus giganteus ultra-structure of intra-uterine spermatozoa in Myotis
giganreus (3riinnich). J. Endocr. 46, 413416. luc@gus. Am. J. An&. 119, 25-60.