Human Reproduction vol 11 no 7 pp 1451-1456, 1996
Evaluation of motility, fertilizing ability and embryonic
development of murine epididymal sperm after
coculture with epididymal epithelium
A.Bongso1 and A.Trounson
Human and Animal Reproductive Biology Laboratories, Institute of
Reproduction and Development, Monash University, Clayton,
Victoria, Australia and Department of Obstetrics and Gynaecology,
National University of Singapore, Lower Kent Ridge Road,
Singapore 0511
'To whom correspondence should be addressed at: Department of
Obstetrics and Gynaecology, National University Hospital, Lower
Kent Ridge Road, Singapore 0511
Murine sperm from the caput, corpus and cauda epididymis
were cocultured with epididymal epithelial cells of their
own region or more distal regions, in the presence and
absence of androgens (testosterone and dihydrotestosterone). Epithelial cell cultures were used 3 or 10 days after
preparation in a complex tissue culture medium (Chang's)
as plated tubules. The coculture studies involving spermatozoa and oocytes with epithelial cells were carried out in T6
medium. Motility of caput spermatozoa was maintained
for 24 h in the presence of day 3 corpus and cauda epithelial
cells and hormones but not under other conditions. Likewise, the motility of corpus spermatozoa was maintained
for 24 h in the presence of day 3 cauda epithelial cells and
hormones but not other conditions. Fertilization of zonaintact oocytes by epididymal spermatozoa was not affected
by their coculture for 24 h with epithelial cells but fertilization rates for zona-free oocytes were increased for caput
spermatozoa cocultured with more distal epithelial cells.
Fertilization rates for both zona-intact and zona-free
oocytes were increased for corpus spermatozoa cocultured
with more distal cauda epithelial cells. The developmental
capacity of embryos derived from caput spermatozoa was
not significantly increased by coculture with epithelial cells
but those derived from corpus spermatozoa cocultured
with cauda epithelial cells were significantly increased. We
conclude that the presence of more distal epithelial cells of
the mouse epididymis maintains motility in culture,
increases the ability of caput and corpus spermatozoa to
fertilize zona-free oocytes and increases the developmental
capacity of embryos formed from corpus spermatozoa.
These observations demonstrate the function of epididymal
regions in the maturation of murine spermatozoa for
fertilization and embryo development
Key words: embryonic development/epididymal epithelium
coculture/fertilizing ability/motility/murine spermatozoa
Introduction
Many classical texts in reproductive biology have suggested
that mammalian spermatozoa must traverse the entire length
© European Society for Human Reproduction and Embryology
of the epididymis to mature, gain motility and acquire fertilizing
capacity (Blandau and Rumery, 1964; Gaddum, 1968; Bedford,
1975; Cooper, 1986; Amann, 1987; Blaquier et al., 1988). It
has been shown in a variety of domestic and laboratory animal
species that under androgen control, the epithelium of the
epididymis plays an important role in encouraging spermatozoa
to develop their full progressive motility, ability to bind to the
zona pellucida and to undergo fusion with the oolemma
(Cooper, 1986; Moore, 1990). In most animals that have been
studied thus far, spermatozoa from the caput epididymis exhibit
only weak, side to side vibrating movements without linear
progression and are incapable of fertilizing oocytes. However,
recently it has been suggested that the journey through the
epididymis may not be an absolute requirement for sperm
fertilizing capacity (Silber, 1995). Pregnancies in the human
have been recently reported where a single testicular spermatozoa after microinjection directly into the ooplasm (intracytoplasmic sperm injection, ICSI) resulted in fertilization, cleavage
and implantation. Interestingly however, 57% of these
pregnancies resulted in abortion (Toumaye et al, 1994), which
has raised doubts about die developmental competence of
such embryos In the ICSI technique the natural barriers for
fertilization are bypassed (zona pellucida and oolemma) as
motility is not required. However, for in-vivo or in-vitro
fertilization, motility is still one of the most important parameters for fertilization and it is evident that this is acquired
during maturation of spermatozoa in the tubules of the
epididymis.
Some components of maturation of spermatozoa in the
hamster and human can be mimicked in vitro by coculturing
immature spermatozoa with plated epididymal tubules and
protein extracts of epididymal epithelium (Orgebin-Crist and
Jahad, 1979; Gonzales-Echeverria et al, 1984; Moore and
Hartman, 1986; Moore et al, 1992). It was shown in the
hamster that sperm maturation was accompanied by the transfer
of specific epitopes from the epididymal epithelial apical
surface to the sperm surface (Moore et al, 1992).
In addition to undergoing maturational changes in morphology, epididymal spermatozoa were shown to develop progressive motility in vivo in the mouse as they progress from the
caput to the cauda epididymis (Lacham-Kaplan and Trounson,
1991). Vibrating motility in the proximal and distal caput
gradually changed to circular non-progressive motility in the
corpus followed by forward progressive motility in the cauda
epididymis. Lacham-Kaplan and Trounson (1994) showed that
insemination of munne oocyte-cumulus complexes with caput
spermatozoa yielded very poor fertilization rates (<10%)
when compared to corpus spermatozoa (<30%) and cauda
spermatozoa (>90%). Additionally, blastulation rates in diese
1451
A.Bongso and A.Troanson
three groups were also proportionately different, suggesting
developmental incompetence of caput and corpus inseminated
murine oocytes. Interestingly, the same workers (LachamKaplan, 1995) demonstrated that if immature murine spermatozoa were injected directly into the ooplasm (ICSI), the same
developmental incompetence was observed with both caput
and corpus but not cauda spermatozoa. These studies suggest
that, in the mouse, traversing of caput spermatozoa through
the corpus and cauda is essential for maturation not only in
terms of motility but also for sperm fertilizing capacity.
The maturation of epididymal spermatozoa in vitro after
coculture with the epithelium of the proximal cauda epididymis
has been investigated in the hamster (Moore and Hartman,
1986; Moore et aL, 1986). However, similar investigations
have not been extensively earned out in humans. A detailed
study was undertaken to evaluate murine sperm maturation in
a coculture system involving cells and tubular fragments from
different parts of the murine epididymis. It was hoped that the
results of this investigation will shed some light on human
epididymal sperm maturation, since such direct investigations
on the human will be difficult due to a paucity of research
material.
Materials and methods
Culture of epididymal tubules
Adult male mice (CBAXC57) were killed by cervical dislocation and
their entire intact epididymides and testes were collected into M2
medium (Sigma, St Louis, MO, USA). The tesus was then separated
from the epididymis, all fatty and connective tissue dissected away,
and the membrane surrounding the entire epididymis cut open to
expose all the parts of the epididymis. The distal caput, corpus and
cauda regions as illustrated in the diagrams of Pavlok (1974) and
Soler et al. (1994) were carefully separated and washed well in M2
medium. The spermatozoa within the tubules of each part of the
epididymis were gently teased out using fine watch-maker forceps
and the empty tubules then transferred to small pern dishes containing
10 mg collagenase (type 1A, Sigma) per ml of M2 medium and
incubated at 37°C in 5% CO 2 in air for 2 h. Following collagenase
treatment it was easy to separate individual tubules mechanically
with watch-maker forceps or by swirling the petri dishes. The tubular
fragments in collagenase were then centrifuged at 300 g for 10 min,
the supernatant decanted and tubules washed once more with M2
medium to remove all traces of collagenase. The clean tubules were
then transferred to Chang's medium (Gibco, Grand Island, NY,
USA) supplemented with 200 mM glutamine, penicillin (100 IU/ml),
streptomycin (100 (ig/ml), testosterone (200 nM), dihydrotestosterone
(1 nM), transferrin (5 ug/ml) and retinol (1 |!g/ml) (Sigma). Chang's
medium is a complex medium enriched with 8% newborn calf serum,
sodium pyruvate, ribo- and deoxyribonucleosides, vitamins, amino
acids and polypeptides Tubules approximately 7 mm long were slit
open under the stereomicroscope with 26 gauge needles fitted to
tuberculin syringes so as to expose the inner epithelium. Thirty such
tubular fragments were then seeded in 0.5 ml of the hormone
supplemented Chang's medium in each well of four-well plastic
dishes (Nunclon, Copenhagen, Denmark) and the dishes incubated at
37°C in 5% CO2 in air. The fragments were evenly spread in each
well. Similar to the observations made for the hamster by Moore
et aL (1986), the murine tubules everted to form circles of tissue
with epithelium on the outside within 2 h of incubation. The dishes
1452
were not disturbed for 24 h so as to allow attachment of the tubular
fragments to the plastic. Chang's medium supplemented with the
male hormones was replaced every 48 h.
Recovery and preparation of spermatozoa
Twelve- to 14-week-old F[ hybrid males were used for recovery of
spermatozoa. Their epididymides as according to the diagrams of
Pavlok (1974) and Soler et aL (1994) were separated after death and
the distal caput, corpus and cauda carefully dissected away as
according to the diagrams of Pavlok (1974) and Soler et al. (1994),
and transferred mto separate dishes of T6 medium (Whittingham,
1971), ensuring that spermatozoa from the different regions did not
mix during the dissection procedure. With the help of watch-maker
forceps the tubules from each epididymal region were gently squeezed
to express spermatozoa into the medium and the residual tubules
discarded. The spermatozoa were incubated in the T6 medium
supplemented with 200 nM testosterone, 1 uM dihydrotestosterone,
5 ug transfernn/ml and 1 ug reUnol/ml for 2 h at 37°C in 5% CO2
m air.
Coculture of epididymal spermatozoa with epithelial culture
Three- and 10-day plated cultures were used in this study. Just prior
to the coculture with spermatozoa, all the Chang's medium was
removed and the plated tubules washed carefully with T6 medium
supplemented with 200 nM testosterone, 1 uM dihydrotestosterone,
5 ug transferrin/ml and 1 jig retinol/ml and then covered with fresh
0.3 ml of T6 medium supplemented with the same male hormones.
To maintain osmolality and prevent evaporation the medium was
overlayered with endotoxin-tested mineral oil (Sigma, USA). The
culture volume was reduced to 0.3 ml to concentrate any possible
factors released by the tubular epithelial cells and Chang's medium
was replaced with T6 medium because preliminary studies had shown
that motihty and fertilization rates with ejaculated munne spermatozoa
were reduced in Chang's medium. Moreover, T6 medium has been
specifically designed for the culture of mouse ova (Whittingham,
1971). Sperm suspension (50 ul) was seeded into each well and
cocultured with the epithelial tubules up to 72 h. Control wells
without tubules and containing 0.3 ml of T6 medium with hormones
were also seeded with the same concentration of spermatozoa. Each
well had sperm concentrations ranging from 2 to 4X10 6 spermatozoa/
ml. Besides controls, distal caput spermatozoa were separately cocultured with distal caput epithelial cells, corpus epithelial cells and
cauda epithelial cells; corpus spermatozoa cocultured with corpus
and cauda epithelial cells and cauda spermatozoa were cocultured
with cauda epithelial cells.
Measurement of sperm maturation
Sperm motility
The percentage sperm motihty (number of motile spermatozoa/total
motile and non-motile spermatozoa X100) was determined at 0, 6,
12, 24, 48 and 72 h for five groups per replicate. Motility for caput,
corpus and cauda spermatozoa was taken to mean any spermatozoa
that showed flagellation or movement of the tail however vigorous
with or without forward progressive motility. The 5 groups for each
spermatozoa-epidielial coculture combination were (1) a 3-day tubular
culture + hormones, (2) a 3-day tubular culture - hormones, (3) a
10-day tubular culture + hormones, (4) spermatozoa alone from the
region under test in medium + hormones but without tubular cells
(control) and (5), cauda spermatozoa alone in medium + hormones
but without tubular cells (control). Four replicates were studied for
each sperm-cell combination with 200 spermatozoa scored per sample
for motihty.
Sperm evaluation and epldidymal epithelium coculture
In-vitro fertilization and cleavage
Female F\ hybrid mice (CBAXC57) were superovulated firstly with
an intrapentoneal administration of 5 IU pregnant mare's serum
gonadotrophin (PMSG) (Folligon: Intervet Ltd, St Leonards, NSW,
Australia) followed 48 h later with 5 IU human chorioruc gonadotrophin (HCG) (Chorulon: Intervet Ltd). Oocyte-cumulus complexes
were recovered 14 h later and placed in 100 uJ droplets of T6 medium
under oil at 37°C in 5% CO 2 in air To these drops, 100 |il spermatozoa
incubated for 24 h in epididymal cell co-cultures or 100 uJ of
spermatozoa incubated for 24 h in medium alone (controls) were
added The spermatozoa concentration per drop was 4x lf^/ml and
10 oocyte-cumulus complexes were cultured per drop. After 5 h of
spermatozoa-oocyte interacuon, the oocytes were washed in several
drops of T6 medium and examined for fertihzauon (presence of two
pronuclei, 2PN) using Nomarski's inverted optics. Thereafter the
percentage of embryos reaching two-cell, four-cell, compacted morula
and blastocyst stages were determined From the same mice an
approximately equal number of oocytes were also denuded of their
surrounding cumulus and zonae with 0 1% hyaluronidase (Sigma)
and 0.5% Pronase (Sigma) respectively. Zona-free oocytes were
exposed to 24 h-incubated spermatozoa from the cocultures and
controls in the same manner as that described previously for oocytecumulus complexes. However, to avoid high rates of polyspermy in
this group, sperm concentrations were reduced to 1X lO^ml for every
10 zona-free oocytes per drop. Feruhzauon rates after 5 h exposure to
spermatozoa and the percentages of two-cell, four-cell and compacted
morula stages reached were calculated Fertilization rates and
embryonic behaviour for both zona-intact and zona-free oocytes
were studied for three replicates of each sperm-epididymal cell
combinauon.
Statistical analysis
X2 analyses were used to compare differences in significance between
experiments and controls for sperm mouhty, fertilization rates and
embryonic development Transformed data were also analysed using
Student's /-test
Results
Morphology of epididymal cultures
Within 2 h in culture the tubular fragments from distal caput,
corpus and cauda regions formed circular rings of tissue with
epithelium everted to the outside. After 1-2 days of culture,
microvilli were observed on the apical surfaces of the columnar
epithelium These remained for as long as 10 days in culture.
Outgrowths of epithelioid-like cells started to plate out from
the epithelium of each fragment and after joining those
outgrowths of neighbouring balls of tissue, an almost confluent
epithelioid monolayer was produced in 7-10 days of culture.
Three-day cultures had numerous epithelioid outgrowths but
confluency was not observed. When androgens were not added
to the medium, epithelial outgrowths continued to form from
balls of tissue but there were fewer microvilh-like structures
and a considerable number of cells were observed floating in
the medium.
Sperm motility
Only 40-45% of the spermatozoa recovered from the distal
caput epididynus at 0 h were motile (Table I), and the motility
was more of a vibratory or twitching motion with complete
Table I Motility of distal caput epididymal sperm in distal caput, corpus
and cauda epididymal epithelial coculture*
Moule: spermatozoa (%)
Groups
3 day D caput cells + hormone
3 day D caput cells - hormone
10 day D caput cells + hormone
Distal caput sperm alone
Cauda sperm alone
3 day corpus cells + hormone
3 day corpus cells - hormone
10 day corpus cells + hormone
Distal caput sperm alone
Cauda sperm alone
3 day cauda cells + hormone
3 day cauda cells - hormone
10 day cauda cells + hormone
Distal caput sperm alone
Cauda sperm alone
Oh
6h
12 h
24 h
48 h
45
40
44
44
92
42
38
41
40
89
44
41
46
48
92
43
40
36
42
85
58
40
38
40
80
55
40
40
40
84
40
30
21
38
67
60
36
20
35
65
58
35
24
30
70
20
18
0
21
31
63 b
28
0
0
0
0
0
12
0
0
0
0
10
0
0
0
0
15
22c
35
60"
30
0
20*
41
"Mean of four replicates
b
*P < 0.001
^P < 0 001.
D = distal
Table II Motility of corpus epididymal sperm in corpus and cauda
epididymal epithelial coculture1
Groups
3 day corpus cells + hormone
3 day corpus cells - hormone
10 day corpus cells + hormone
Corpus sperm alone
Cauda sperm alone
3 day cauda cells + hormone
3 day cauda cells - hormone
10 day cauda cells + hormone
Corpus sperm alone
Cauda sperm alone
Motile sperm (%)
Oh
6h
12 h
24 h
48h
72 h
51
54
56
55
90
60
58
55
52
85
50
50
48
50
87
65
53
45
48
82
40
36
31
42
80
66
50
38
45
78
30
28
0
31
45
68 b
40
0
30c
43
10
6
0
8
18
12
0
0
0
0
10
0
0
0
0
8
2
0
1
20
"Means of four replicates
b >
*/ < 0 01.
absence of forward progression When distal caput spermatozoa
were cocultured with distal caput cells (3 day as well as
10-day culture, with and without hormones) no significant
improvement of sperm motility was observed compared to
controls (caput spermatozoa alone) over a 24 h period (Table I).
The percentage motility of distal caput spermatozoa in controls
and 3-day coculture dropped to approximately half at 24 h
(18-21%) (Table I). By 48 h, caput spermatozoa in all groups
were immotile, unlike the cauda spermatozoa, 12% of which
survived this length of time.
When distal caput spermatozoa were cocultured with 3 day
old corpus epithelial cells supplemented with hormones, sperm
motility increased from 42% at 0 h to 63% at 24 h and then
became totally immotile at 48 h (Table I). However, distal
caput sperm motility in 3-day cocultures - hormones, 10 day
cocultures + hormones and controls decreased gradually from
0 h through 24 h Distal caput spermatozoa in the 10-day
coculture + hormones suffered drastic drops in motility to
almost half their values at 12 h and to total immotility at 24 h,
1453
A.Bongso and A.Trounson
Table V. Fertilization and embryonic behaviour of oocytes inseminated with
corpus epididymal sperm cocultured with corpus and cauda epididymal
epithelial cells for 24 h
Table ED Motility of cauda epididymal spenn in cauda epididymal
epitbelial coculture*
Groups
Motile spenn (%)
3 day cauda cells + hormone
3 day cauda cells - hormone
10 day cauda cells + hormone
Cauda sperm alone
Distal caput sperm alone
Oh
6h
12 h
24 h
48b
72 h
91
90
85
94
40
89
88
80
86
38
85
80
76
78
30
55
50
2
46
18
30
20
0
26
0
15
10
0
8
—
'Mean of four replicates
Table IV Fertilization and embryonic behaviour of oocytes inseminated
with distal caput epididymal spenn cocultured with distal caput, corpus and
cauda epididymal epithelial cells for 24 h
Groups
Oocytes1
2PN
2-cell
4-cell
Morulae
Groups
Zona-intact
Corpus cells*
Control
Zona-free
Corpus cells'"
Control
Zona-intact
Cauda cells'1
Control
Zona-free
Cauda cells'"
Control
Oocytes* 2PN
2-cell
4-cell
Morulae Blastocyst
100
102
30(30)
32 (31)
93(28)
90 (28)
79(22)
71 (20)
51(11)
48 (10)
35(4)
28 (3)
105
105
28 (27)
24 (23)
94 (25)
91 (21)
81 (20)
75 (16)
55 (11)
50 (8)
-
105
110
50 (53)c 94 (50)e 80 (40)c 60 (24)' 40 (10/
30 (33)d 88 (29/ 76 (22)f 55 (12)f 32 ( 4 /
100
100
45 (45)c 93 (42)' 84 (35)' 64 (22/
24(24) d 88(21) f 80(17/ 54(9/
-
•Mean of three replicates.
*3 day cocultures + hormone
P < 0 01
c4
Zona intact
D caput cellsb
Control
Zona-free
D caput cellsb
Control
Zona-intact
Corpus cellsb
Control
Zona-free
Corpus cellsb
Control
Zona-intact
Cauda cells'"
Control
Zona-free
Cauda cells'"
Control
tfp < 0 05
100
96
0
0
98
100
8(8)
6(6)
90
82
110
103
100
100
100
100
0
0
22(24)°
6(6) d
0
0
20 (20)c
10 (10)d
0
0
100 (8)
100(6)
0
0
100 (24)
100 (6)
0
0
95 (19)
100 (10)
0
0
38(3)
33(2)
0
0
67 (16)
60(3)
0
0
42 (8)
40 (4)
0
0
33(1)
0(0)
0
0
31(5)
0(0)
0
0
38(3)
25(1)
"Mean of three replicates
b
3 day cocultures + hormone.
^P < 0.01
D = distal
much earlier than the other groups. At 24 h, distal caput
spermatozoa in 3 day cocultures + hormones had significantly
greater motility than controls (63% versus 22%; P < 0.001)
(Table I).
When distal caput spermatozoa were cocultured with 3 day
cauda epithelial cells + hormones, sperm motility increased
from 0 h through 24 h, unlike spermatozoa in 3 day cocultures hormones, 10 day cocultures + hormones and controls where
motility gradually dropped from 0 h through 24 h (Table 1).
At 24 h, distal caput sperm motility in 3-day cocultures +
hormones was significantly greater than controls (60% versus
20%; P < 0.001).
The motility of corpus spermatozoa decreased steadily with
time in 3-day corpus epithelial cocultures + hormones similar
to 3-day corpus cocultures - hormones, 10-day cocultures +
hormones and controls with no significant differences between
their values (Table II). However, sperm motility was retained
up to 48 h in 3-day cocultures and controls, unlike in 10-day
cocultures + hormones where motility lasted for only 12 h
(Table II).
The motility of corpus spermatozoa increased steadily up
1454
to 24 h and then dropped at 48 h when cocultured with 3-day
cauda epithelial cells supplemented with hormones, when
compared to the other coculture systems and controls where
motility reduced with time (Table II). At 24 h, the motility of
corpus spermatozoa in 3-day cauda cocultures + hormones
was significantly different from that of controls (corpus spermatozoa alone) (68 versus 30%; P < 0.001) (Table II).
When cauda spermatozoa were cocultured with cauda epidielial cells, motility decreased with time in all coculture groups,
similar to controls with no significant differences between
them. Cauda spermatozoa in 3-day cocultures and when
cultured in medium alone survived up to 72 h unlike 10-day
cauda cocultures and distal caput spermatozoa that survived
up to only 24 h (Table III).
Whilst the percentage of corpus spermatozoa showing forward progressive motility increased in the presence of cauda
cells with tune (Table II), at 24 h classical hyperactivated
motility patterns were observed.
Fertilization rates
There was no fertilization when zona-intact oocytes were
inseminated with distal caput spermatozoa in the presence of
distal caput, corpus or cauda epididymal cells (Table IV).
However, a low fertilization rate of 8% was observed with
zona-free oocytes inseminated with distal caput spermatozoa
in the presence of distal caput cells which was not significantly
different from die fertilization rate (6%) observed in controls
(Table IV). Fertilization rates of zona-free oocytes inseminated
with distal caput spermatozoa in the presence of corpus and
cauda cells were significantly higher man controls (20-22%
versus 6-10%; P < 0.01) (Table IV). When corpus spermatozoa
were used to inseminate zona intact and zona free oocytes, no
significant differences in fertilization rates were observed in
die presence of corpus cells and controls (Table V). However,
when corpus spermatozoa were used to inseminate zona intact
and zona free oocytes in the presence of cauda cells, fertilization
rates were significandy different from controls (50 versus 30%,
P < 0.01 and 45 versus 24%, P < 0.01 respectively) (Table
Sperm evaluation and epldidymal epithelium coculture
Table VI Fertilization and embryonic behaviour of oocytes inseminated
with cauda epididymal sperm cocultured with cauda epididymal epithelial
cells for 24 h
Groups
Oocytes* 2PN
Zona-intact
Cauda cellsb 100
Control
104
Zona-free
Cauda ceUsb 98
Control
100
2-cell
4-cell
Morulae Blastocyst
94 (94)
89(92)
98 (92)
97(89)
82 (75)
78(69)
78 (59)
73(50)
65 (38)
60(30)
86 (84)
84 (84)
95 (80)
91 (76)
80 (64)
74 (56)
70 (45)
68 (38)
-
'Mean of three replicates
••3 day cocultures + hormone
V). When cauda spermatozoa were used to inseminate zona
intact and zona free oocytes in the presence of corpus cells,
no significant differences in fertilization rates were observed
compared to controls (Table VI). Fertilization rates using cauda
spermatozoa were much higher than with corpus and caput
spermatozoa in all coculture and control groups (Tables IV, V
and VI).
Cleavage
The percentages of four-cell and compacted zona-free morulae
were generally greater in the coculture groups when compared
to controls in the distal caput spermatozoa-caput/corpus/cauda
cell combinations but these values were not significantly
different (Table IV). The percentages of two-cell, four-cell,
morula and blastocyst stage zona intact and zona free embryos
were also not significantly different between corpus cell
coculture and controls when corpus spermatozoa were used
for insemination (Table V). However, percentages for all zona
intact and zona free embryonic stages (two-cell to morula or
blastocyst) were significantly different between cauda cell
coculture and controls when corpus spermatozoa were used
for insemination (Table V). No significant differences in
embryonic behaviour were observed between cauda cell coculture and controls for zona intact and zona-free oocytes when
cauda spermatozoa was used for insemination (Table VI).
Discussion
Murine tubular fragments attached quite well to plastic dishes
in the presence of Chang's medium. After 3 days of culture,
epithelial outgrowths became established and microvilh
appeared on the apical surfaces of the everted tubular epithelium, similar to observations made in the hamster and human
(Moore et al, 1986, 1992). T6 medium with hormones did
not support growth in 3 days and hence Chang's medium
supplemented with hormones was used for initial primary
culture. Fragments of murine tubular epithelium when cut
open along their lengths and then plated in vitro everted into
balls of tissue similar to that described for the hamster (Moore
et al., 1986). The presence of hormones in the medium was
necessary to maintain in situ morphological characteristics
such as columnar epithelium with microvilh, epithelial confluency and mitotic activity. In the absence of these hormones,
attachment of tubular fragments and plating was slow, micro-
villi numbers were reduced and degenerative morphological
changes began to set in.
It appears that only half of the sperm population collected
fresh from the murine distal caput epididymis show vibratory
twitching motility while the other half are immotile. These
percentages are consistent with the observations of LachamKaplan and Trounson (1991). The percentage of spermatozoa
with this vibratory non-progressive flagellar motility increased
steadily and was sustained for 24 h when distal caput spermatozoa were cocultured with corpus and cauda epithelial cells
supplemented with hormones but not with caput cocultures.
This may suggest that as the caput spermatozoa traverse the
corpus and cauda epididymis in vivo the percentage of motile
caput spermatozoa may increase and flagellar non-progressive
forward motility is maintained. Lacham-Kaplan and Trounson
(1991) also demonstrated that about 40% of freshly collected
caput spermatozoa from the mouse have flagellar non-progressive motility. Mature epididymal spermatozoa are motile on
release from the epididymis (Lacham-Kaplan and Trounson,
1991; Soler et al, 1994). The same phenomenon appears
to hold true for corpus spermatozoa traversing the cauda
epididymis, because corpus sperm motility did not increase
with corpus epididymal cocultures but did increase with
cauda epididymal cells. Interestingly, both caput and corpus
spermatozoa could survive in 3 day coculture or cell-free
medium for a maximum period of only 24 h unlike the more
mature cauda spermatozoa which could survive up to 72 h.
The addition of hormones to day 10 cocultures reduced the
motile life of the spermatozoa, whereas day 3 cocultures with
and without hormones appeared to maintain sperm motility.
This suggests that the hormones alter the secretory nature of
day 10 epithelial cultures but may act in synergy with day 3
cultures to stimulate their secretory activity. In the absence of
a group with hormones alone, it is not possible to exclude a
direct effect of hormones. Slow and persistent flagellum
movement was also observed in hamster caput epididymal
spermatozoa after 8 and 24 h cocultures with 3-day old
corpus epididymal epithelial cells supplemented with hormones
(Moore et al., 1986).
The results of a study of fertilization rates demonstrate that
in general, the ability to penetrate the zona pellucida is not
improved by epididymal coculture of spermatozoa. However,
the ability of distal caput spermatozoa to fertilize zonafree ova was increased by coculture with corpus and cauda
epididymal cells. During coculture, maturation may occur of
sperm receptors for binding and fusion with the oolemma. It
has been reported that the sperm plasma membrane responsible
is already 'coated' with several macromolecules when spermatozoa leave the testis. During the passage through the epididymis these molecules are either lost or altered and new
macromolecules of epididymal origin are adsorbed or integrated
into the plasma membrane. Glycoproteins are the most prominent of these macromolecules. Also, the change in lecithinbinding ability of the sperm plasma membrane which occurs
during epididymal maturation indicates that certain terminal
saccharide residues of sperm membrane glycoproteins are
altered during maturation. Membrane lipids are also known to
undergo change during epididymal maturation (Yanagimachi,
1455
A.Bongso and A.Trounson
1988). The corpus epididymis has high cholesterol synthesis
activity (Hamilton and Fawcett, 1970) and this may suggest
that cholesterol is one of the lipid molecules integrated into
the sperm plasma membrane during sperm maturation. It has
been suggested that these substances, particularly glycoproteins, may be the sperm zona receptors involved in fertilization
(Yanagimachi, 1988). Bleil and Wassarman (1986) showed in
the mouse that the sperm's receptors for the zona protein ZP3
are on the plasma membrane over the entire acrosome of
capacitated spermatozoa, and ZP2 receptors can be detected
very clearly on the plasma membrane over the equatorial
segment as well on the inner acrosomal membrane. It is well
known that such receptors play an important role in spermoolemma interaction and subsequent fertilization. These receptors may be being acquired in coculture and thus improve
fertilization.
There was no effect of corpus epithelial cocultures on corpus
spermatozoa This would be expected but interestingly cauda
epithelial cocultures appeared to increase corpus sperm zona
penetration and oolemma binding and fusion as evident from
the increased fertilization seen in both zona-intact and zonafree ova. From the results of this study it is suggested that
zona penetration capabilities are not gained in the corpus but
rather in the cauda of the mouse, together with oolemma
binding and fusion capabilities.
The results demonstrate that embryos derived from distal
caput spermatozoa have a very reduced capability for development beyond two cells and that embryos derived from corpus
spermatozoa have reduced capabilities to compact and develop
beyond the four- to eight-cell stage. This capability for embryo
development improved for corpus spermatozoa by coculture
with cauda epithelial cells. Developmental incompetence after
the two-cell stage for non-cocultured murine caput spermatozoa
and blocks after the eight-cell stage for non-cocultured murine
corpus spermatozoa were also recently observed by LachamKaplan and Trounson (1994).
We propose that, in the mouse, sperm maturation is required
for embryonic developmental competence and this appears to
occur for spermatozoa that have traversed the epididymis and
are stored in the cauda. The results suggest that the epithelial
cells may be responsible for 'maturation' changes in the
spermatozoa. This maturation may be mediated via specific
secretions released by the epithelial cells.
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Received on February 5, 1996, accepted on April 16, 1996