2.7. Histological processing For half of the possums examined, the right side of the tract was fixed whole in
Bouin’s solution. Segments were immersed in Bouin’s fixative for 48 h, then washed in several changes of 70 ethanol and stored in 70 ethanol. Wax blocks were prepared
Ž .
and 7-mm sections were cut and stained with Harris’ haematoxylin and eosin HNE , or with one of the following mucopolysaccharide histochemical methods: 1 alcian blue,
Ž . Ž
. pH 2.5 or periodic acid-Schiff PAS
Pearse, 1968 . 2.8. Electron microscopy
For the remaining possums, the right side of the tract was fixed whole in Karnovsky’s Ž
. solution
Karnovsky, 1965 at pH 7.6 overnight at 48C before storage in 0.1 M
Ž .
phosphate buffer, pH 7.4 at room temperature Mate et al., 1992 . Post-fixation was in Ž
. 1 wrv OsO
in 0.1 M phosphate buffer for 1 h at 48C. This was followed by
4
washing in distilled water, dehydration and embedding in Spurr’s resin. Ultrathin sections were placed on nickel grids, stained with uranyl acetate and lead citrate and
examined on a Joel CX-100 electron microscope.
2.9. Statistical analysis Ovarian assessments are presented as mean values with range intervals. The numbers
of spermatozoa recovered from flushes of the tract are presented as means standard Ž
. error of the means S.E.M. . Comparison of treatment means was examined non-para-
metrically using a Kruskal–Wallis one way ANOVA on ranks. Dunns’ method was used for pairwise comparison to determine which treatment groups were different.
3. Results
Ž Histological processing revealed that the uterus and oviduct segments isthmus,
. middle segment and ampulla in T. Õulpecula were structurally distinct. Uterine sections
characteristically consisted of a series of small glands lined by tall columnar epithelial cells with small basal nuclei. All oviduct segments were lined with pseudo-stratified
Ž .
columnar epithelial cells Fig. 1 . The epithelium lining the isthmus region nearest the uterus consisted of highly-folded villi-type projections. Invaginations of this epithelium
or ‘folds’ were also abundant within this region. The middle oviduct region was characterised by a simpler less folded epithelial lining. The epithelium found within the
ampulla oviduct segment was the most simple in appearance. There were no deep epithelial folds in this region.
The columnar epithelium lining both the oviduct lumen and the epithelial fold regions Ž
. contained ciliated and mucus-secreting cells Fig. 2 , and TEM showed that these had
numerous electron-dense granules in their apical cytoplasm, together with many mito- Ž
. chondria and a considerable amount of rough endoplasmic reticulum Fig. 4 . In both the
Ž .
Ž . Ž .
Ž . Fig. 1. Longitudinal section LS through the ampulla a , middle segment b and isthmus segment c of the
Ž .
possum oviduct taken at 6 h after AI. Spermatozoa arrowhead ; E, epithelial fold.
oviduct lumen and deep within epithelial folds, light microscopy and TEM demonstrated that sperm tails were packed tightly together and aligned parallel to the length of the
lumen, while the heads of spermatozoa showed a variable orientation in relation to the
Ž . Ž
. Fig. 2. a LS through the ampulla region of the possum oviduct 6 h after AI 33 h after LH showing
Ž . secretion on the apical epithelial surface stained strongly with PAS. b LS through the ampulla at 1.5 h after
Ž .
AI 28.5 h after LH showing weak PAS staining of the apical epithelial surface.
Ž .
tails Figs. 3 and 4 . Appropriate sections through the sperm head also showed the Ž
. presence of an intact acrosome Fig. 4 .
3.1. 1.5 h after AI The ovarian response to PMSGrLH superovulation and AI is presented in Table 1.
At 1.5 h after AI none of the five animals had ovulated, but an average of 10–11
Ž .
Ž . Fig. 3. Higher magnification of spermatozoa arrowheads within an epithelial fold E .
Ž . Ž
. Fig. 4. TEM of an epithelial fold, showing ciliated C and mucus-secreting MU epithelial cells. Spherical
Ž . Ž
. apical granules G are apparent within secretory cells. Sperm tails ST are aligned parallel to the lumen,
Ž .
while sperm heads SH are randomly-oriented. Appropriate sections through the sperm head show an intact Ž .
acrosome A . c, Cilia.
follicles 2 mm per female were observed. Secretory activity was minimal in all regions of the genital tract at 1.5 h after AI, as indicated by low apical vesicle extrusion
from HNE-stained secretory cells and the weak staining of epithelial surfaces by PAS Ž
. and alcian blue Table 2 . The histological location of spermatozoa from unflushed
segments of the reproductive tract is presented in Table 2. At 1.5 h after AI, spermato- zoa were found in both the lumen and on the epithelial surfaces of all oviduct regions
with their heads opposed or in close proximity to the apical cells. No spermatozoa were
Table 1 The ovarian response of the brushtail possum following PMSGrLH superovulation and intrauterine artificial
insemination
a
Ž . Ž
. Time of analysis h
Proportion ovulating Mean number range per female
After LH After AI
Ovulation sites Follicles remaining 2 mm
Ž .
28.5 1.5
0r5 10.6 2–16
Ž .
30.0 3.0
1r5 16 from one female
9.8 1–14 Ž
. 33.0
6.0 1r5
3 from one female 10.4 3–22
Ž .
Ž .
36.0 9.0
4r5 4.4 0–12
4.8 0–14 Ž
. Ž
. 39.0
12.0 4r5
5.4 0–13 7.6 0–13
a
Animals that had G1 ovulation site were classed as having ovulated.
M.K. Jungnickel
et al.
r Animal
Reproduction
Science 59
2000 213
– 228
221 Table 2
The location of spermatozoa and secretory activity within the reproductive tract of the brushtail possum following PMSGrLH superovulation and intrauterine artificial insemination
Ž . Analysis time after AI h
Uterus Isthmus
Middle segment Ampulla
1.5 3
6 9
12 1.5
3 6
9 12
1.5 3
6 9
12 1.5
3 6
9 12
a
Secretory actiÕity Apical
y q
q q
q q
q qq
q q
q q
qq q
qq q
qq qqq
q qq
vesicle extrusion
Ž .
HNE Apical
q q
q q
q q
q qq
q q
q q
qq q
qq q
qq qqq
q qq
epithelial surface
ŽPASr alcian
b
. blue
c
Location of spermatozoa In lumenr
Few sperm q
qq qq
q qq
q q
q q
q q
q q
q q
epithelial found at
surface all sampling
periods in the lumen
of the uterine
glands
Within y
qq qq
q qq
y y
y y
y y
y y
y y
epithelial folds
a
Ž .
Ž .
Ž .
Ž .
Secretory activity: y absent, q weak, qq moderate, qqq strong.
b
No difference in staining pattern between PAS and alcian blue.
c
Ž .
Ž .
Ž .
Spermatozoa: y none, q few, qq many.
found within the epithelial folds. Within the uterus, sperm were located in the lumen of the uterine glands.
3
Ž .
Around 250 = 10 spermatozoa were flushed from the right uterus Table 3 . None of these displayed thumbtack head orientation. Approximately 16 = 10
3
spermatozoa were recovered from the isthmus, whereas 8 = 10
3
and 6 = 10
3
spermatozoa were flushed from the middle and ampulla, respectively. Thumbtack spermatozoa with a progressive
Ž .
motility rating of 4–5 on a 0–5 scale, Rodger et al., 1991 were recovered from the oviduct, with a progressive increase in the proportion of these spermatozoa recovered in
Ž .
Ž .
moving from the isthmus 20
to the middle 50
and finally to the ampulla Ž
. 90 . The relative proportion of thumbtack spermatozoa distributed within the
possum reproductive tract was similar at all sampling times after AI. 3.2. 3.0 h after AI
By 3 h after AI, one animal had ovulated in response to the PMSGrLH stimulation, Ž
. but there were still around 10 follicles 2 mm remaining per female Table 1 . HNE,
alcian blue and PAS staining indicated that secretory activity was greater at this Ž
. sampling period than at 1.5 h after AI Table 2 . Further, there was a progressive
increase in the amount of secretion from the uterus to the ampulla. Within all regions of the oviduct, spermatozoa were found in both the lumen and on the apical epithelial
Ž .
surface. Spermatozoa in the isthmus were also located within epithelial folds Table 2 . Sperm orientation within these regions was similar to that observed within the oviduct
lumen at 1.5 h. The numbers of spermatozoa recovered from the uterus and oviduct Ž
. regions at 3.0 h, were similar to the numbers recovered at 1.5 h after AI Table 3 .
3.3. 6.0 h after AI Even at 6 h after AI, only one out of five animals had ovulated and the ovarian
Ž .
response was similar to that reported at 3.0 h Table 1 . Secretory activity, was greater at Ž
. this sampling period than at 1.5 or 3.0 h Table 2 , and the amount of vesicle extrusion
Ž .
progressively increased from the uterus to the ampulla Fig. 2 . Spermatozoa were found in the lumen of the uterine glands. For the isthmus, middle and ampulla segments of the
Table 3 Ž
3
. Ž
. Total numbers of spermatozoa =10
recovered from the female reproductive tract meanS.E.M. after PMSGrLH superovulation and intrauterine artificial insemination
Ž .
Ž .
Within columns, means S.E.M. with different superscripts are significantly different P - 0.05 . Ž .
Time of analysis h Uterus
Oviduct After AI
After LH Isthmus
Middle Ampulla
ab a
a ab
1.5 28.5
254.9116.2 15.73.3
7.72.9 5.82.4
ab a
ab ab
3.0 30.0
248.885.1 34.612.0
4.31.4 11.35.2
a b
a a
6.0 33.0
274.9115.5 51.211.4
11.06.2 15.55.8
b ac
b b
9.0 36.0
52.432.9 8.54.0
0.70.2 1.10.4
ab b
ab b
12.0 39.0
109.617.6 103.127.8
2.00.6 0.90.1
oviduct, spermatozoa were located in both the lumen and at the apical surface of the epithelial cell lining. In the isthmus, spermatozoa were also observed within epithelial
Ž .
folds Table 2 . Ž
. Once again, similar numbers of spermatozoa were flushed from the uterus Table 3 .
The highest numbers of spermatozoa were recovered from all the oviduct segments compared with the 1.5- and 3.0-h sampling periods, but this was only significant for the
3
Ž .
isthmus where approximately 50 = 10 spermatozoa were recovered P - 0.05 . 3.4. 9.0 h after AI
By 9 h after AI, four of the five animals had ovulated, which resulted in a concomitant reduction in the mean number of remaining follicles 2 mm to around 5
Ž .
mm per female Table 1 . Secretory activity was lower at the 9-h sampling period than Ž
. at 6 h, and there was no increase in secretion from the uterus to the ampulla Table 2 .
Within all oviduct segments, spermatozoa were found in both the lumen and associated with the epithelial surfaces. Only few spermatozoa were found within the epithelial folds
at 9 h after AI. As in previous sampling periods, uterine spermatozoa were located within glandular regions.
Much lower numbers of spermatozoa were flushed from the uterus at this time than at Ž
. previous sampling periods P - 0.05 . A similar drop in sperm numbers occurred within
Ž .
all regions of the oviduct at 9 h relative to the 6-h sampling period P - 0.05, Table 3 . 3.5. 12.0 h after AI
The ovarian response at 12 h after AI was similar to the response at 9 h, with an Ž
. average of over five ovulation sites observed per female Table 1 . Secretory activity
Ž .
was greater at this sampling period than at 9 h after AI Table 2 and the pattern of secretion again showed a progressive increase from the uterus to the ampulla. Uterine
spermatozoa were located in glands at this sampling period. Within the ampulla, middle and isthmus regions of the oviduct, spermatozoa were found in the lumen and associated
with the apical surface of epithelial cells. Isthmic spermatozoa were also seen in the
Ž .
epithelial fold regions Table 2 . Twice as many spermatozoa were recovered from the uterus and a significantly
Ž .
greater number of spermatozoa P - 0.05 were recovered from the isthmus at 12 h
Ž
3
. compared with 9 h. Low numbers of spermatozoa 1–2 = 10
were flushed from the Ž
. middle and ampulla regions as for the 9-h sampling period Table 3 .
4. Discussion
