Content uploaded by Kathleen June Mullins
Author content
All content in this area was uploaded by Kathleen June Mullins on May 04, 2018
Content may be subject to copyright.
Content uploaded by Kathleen June Mullins
Author content
All content in this area was uploaded by Kathleen June Mullins on Mar 10, 2018
Content may be subject to copyright.
THE
ANATOMICAL RECORD 225:106-117 (1989)
Study
of
the Functional Anatomy of Bovine
Cervical Mucosa With Special Reference to
Mucus Secretion and -Sperm Transport
K.
JUNE
MULLINS
AND
R.G.
SAACKE
Department
of
Dairy Science, Virginia Polytechnic Institute and State University,
Blacksburg, Virginia
24061
ABSTRACT
The bovine cervical mucosa was investigated with respect to
structure, mucus secretory pattern, and sperm transport. Structural investigation
included stereomicroscopic examination of surface-stained tissue blocks and
graphic reconstruction of serial sections by using both computer-generated and
Plexiglas models. Histochemistry of the mucosa was evaluated in follicular- and
luteal-phase animals. Alcian blue, periodic acid Schiff, and high-iron diamine were
utilized to distinguish sialomucins, sulfomucins, and neutral mucins. Location and
orientation of cervical sperm in follicular phase animals were evaluated 12 h
postmating by using light and electron microscopy. Cervical mucosa was char-
acterized by longitudinal primary folds, most of which maintained continuity
throughout the cervix. Superimposed on these were secondary folds which varied
in length and depth. Abundant, shallow, uniformly spaced, and parallel longitu-
dinal “grooves” covered all surfaces. Grooves had greater continuity in regions
distal, as opposed to proximal, to the cervical canal. Blind-ending glands
or
crypts
were not apparent. Follicular-stage cervices exhibited a pronounced sialomucin
production in basal areas within grooves while neutral and sulfomucins were pre-
dominant in apical areas. In luteal-phase animals, basal sialomicin production was
markedly decreased while sulfated and neutral mucins remained abundant. Nu-
merous cranially oriented spermatozoa were observed within the shallow grooves
of cervical folds (sialomucin-rich areas) in mated animals and were unidirection-
ally opposed to ciliary beat. It appeared that privileged paths for transport of viable
spermatozoa may originate in the fornix vagina, extend through longitudinal pri-
mary folds at the external
os,
and progress to the uterus within continuous sialo-
mucin-rich channels which are associated with basal areas of the shallow grooves,
distal to the cervical canal.
A clearly defined three-dimensional model has not
been established for the bovine cervix. This precludes
development of concepts of sperm transport across this
organ in relation to the mucosa and pattern of mucous
secretion.
Historically, differences of opinion regarding cervi-
cal anatomy have centered on the presence
or
absence
of blind-ended tubular glands and/or crypts. Hammond
(1927)
first
reported the existence of simple sacculated
glands. This concept was substantiated by Cole (1930)
and Mattner (19661, whereas Hafez and Kanagawa
(1972) described a system of mucosal “crypts.” Alter-
natively, Herrick (1951) described the cervical mucosa
as being thrown up throughout its length into primary,
secondary, tertiary, and even quaternpry folds, but
without evidence of tubular glands
or
crypts.
The structure and composition of cervical mucus
are
complex, being permissive to sperm transport only dur-
ing the follicular phase of the estrous cycle (Litt
et
al.,
1976; Hafez, 1980). Alteration of cervical mucus com-
position in response to endocrine influence
or
stage
of
the estrous cycle has been examined for mucus-se-
creting ruminants (Wordinger
et
al., 1972; Hafez et aI.,
1971; Heydon and Adams, 1979). Wordinger et al.
(1972) found that in cows
at
metestrus (day
3
post-
estrus), cells lining the crypts of the cervix contained
both sulfated and nonsulfated (carboxylic acid, proba-
bly sialic acid) mucins, while cells in the base of the
crypts contained predominantly the nonsulfated acidic
mucins. Heydon and Adams (1979) also reported that
during estrus, sulfomucins were predominantly pro-
duced by regions nearest the cervical lumen, while
cells deepest within the crypts, away from the lumen,
stained primarily for sialomucins. They further dem-
onstrated in ovariectomized animals that sialomucin
production was in response to elevated levels of estra-
diol.
Sperm transport through the bovine cervix is un-
doubtedly dependent upon both the anatomy of the cer-
vical mucosa and the secretory pattern of cervical mu-
Received August
1,
1988; accepted February
15,
1989.
0
1989
ALAN
R.
LISS. INC.
107
FUNCTIONAL ANATOMY
OF
THE BOVINE CERVIX
cins in relation to that anatomy. On this basis the
following study was undertaken to clarify the struc-
ture of the bovine cervix and determine the nature of
mucin secretion in relation to that structure. In
addition, the relationship of spermatozoa coursing the
cervix to that anatomy and mucin secretory pattern
was examined.
MATERIALS AND METHODS
Structure
Three techniques were utilized to examine the ana-
tomical features of the bovine cervix. These included
stereomicroscopy following surface staining of the cer-
vical mucosa and graphic reconstruction of tracings
from
serial
sections by using both computer-generated
and Plexiglas models.
Intact cervices for this investigation were obtained
at an abattoir from
11
animals representing both nul-
liparous and multiparous females. In each case the cer-
vix was excised from the reproductive organs within
15
min following a stunning blow
at
the time of sacrifice,
and the cervical canal was exposed longitudinally.
Whole cervices were fixed in Bouin’s solution for
72
h
and rinsed with three changes of
70%
ethanol
at
24
h
intervals. Each fixed cervix was divided into halves by
cutting along the base of a major longitudinal fold. The
cervical epithelium of one-half of each cervix was sur-
face-stained by total immersion in Harris’s hematoxy-
lin (Pearse,
1985)
for
1-3
min followed by
a
tap-water
rinse and bluing in mild ammonia solution. Portions of
the other half-cervix were cut perpendicular to the lon-
gitudinal axis and similarly stained by immersion in
hematoxylin. Prepared tissue blocks from representa-
tive areas were photographed by using a Wild
M400
Photomakroskop.
A computer-aided reconstruction of
25
serial cross
sections
(5
pm) taken at
20
p,m intervals from
a
500
pm midcervical region between annular rings was
conducted on one animal to determine the presence
or
absence of blind-ending
or
tubular structures within
mucosal folds. Tracings of the epithelial conformation
were made from photo enlargements (approx.
40x1
onto clear acetate sheets which were then placed in
proper alignment with respect to the X and
Y
axes by
using
a
pair of fudicial points placed on each overlay in
the series. Aligned tracings were entered into
a
VAX
11/780
minicomputer with the aid of a VICOM digital
image processor. The computer was then instructed to
display the reconstructed image on a video display
system. Several stereopairs were produced by image
rotation as described in Dylewski
et
al.
(1984).
For additional clarity of mucosal anatomy, an alter-
nate method of model reconstruction was performed by
using
sets
of
15
serial sections
(5
pm) taken
at
20
pm
intervals from two additional animals. In these cases,
tissue
sections from
a
midcervical annular ring which
gave the macroscopic impression of possible glandu-
lar formation were selected. Mucosal tracings were
aligned with respect to paired fudicial points
(as
pre-
viously described) but between sheets of
3.18
mm Plex-
iglas (modified from Fluhmann,
1958).
Reconstructions
were viewed from various angles by using transmitted
and oblique lighting.
Histology
For histochemical localization of mucoid substances
and sperm distribution within the mucosa, eight cows
having
a
history of normal reproductive cycles were
utilized. Four of these were mated naturally to mature
bulls known to have normal semen qualities. Animals
were mated
at
first observation of estrus and sacrificed
8-12
h later. One additional animal was sacrificed in
early estrus without mating. These five animals were
considered to be in the follicular phase of the cycle.
Cervices from the remaining three animals were ob-
tained
at
diestrus
(10
days following first observation
of estrus) and represented the luteal phase of the es-
trous cycle.
The cervix of each animal was evaluated in four se-
quential segments of equal size from the posterior to
the anterior cervical
0s.
In the case of multiparous an-
imals having
a
relatively large cervix,
a
complete lon-
gitudinal strip approximately
1
cm wide was subdi-
vided into the four segments, while intact segments
from the smaller cervices of nulliparous animals were
utilized. Following
72
h fixation in Bouin’s solution,
the tissue was rinsed with three changes of
70%
etha-
nol
at
24
h intervals, dehydrated, cleared, and embed-
ded in paraffin. Replicate blocks from each quarter
were sectioned
at
4-5
pm.
Differentiation
of
mucins was accomplished using
the following staining techniques according to Pearse
(1985):
1)
Alcian blue 8GX (AB; Sigma Chem. Co.)
at
pH
1.0 (1%
in
0.1
N
HC1) with
a
30
min staining time;
2)
AB-8GX at pH
2.5 (1%
in
3%
acetic acid) with
a
30
min staining time;
3)
AB (pH
2.5)
for
60
min followed
by periodic acid Schiff (PAS; Sigma Chem. Co.),
10
min
oxidation in
1.0%
periodic acid, and
1
min staining
time in freshly prepared Schiff‘s reagent (stored not
longer than
1
month
at
5°C);
and
4)
high-iron diamine
(HID; Sigma Chem. Co.)
18
h stain time in freshly pre-
pared stain following
a
10
min oxidation in
1.0%
peri-
odic acid. Counterstain with AB (pH
2.5)
for
30
min.
Alcian blue was utilized
at
pH
1.0
to localize sulfomu-
cin and again
at
pH
2.5
to reveal
a
combination of
sialomucins and sulfomucins (Jones and Reid,
1973).
The addition of PAS to sections stained
first
with AB
at
pH 2.5
allowed differentiation of the neutral mucins
from the AB-positive acidic ones. Although glycogen
is
reportedly only present in very small amounts (Gib-
bons and Mattner,
1966)
the possibility of mucin stain-
ing interference from glycogen was examined by using
three pairs of adjacent sections which were randomly
obtained from one follicular animal. Digestion of one
section of each pair with Na amylase prior to staining
revealed no apparent difference in PAS intensity when
compared to the undigested section. On this basis, fur-
ther amylase digestion was not included in the exper-
imental protocol. High-iron diamine was utilized to
stain
a
combination of sulfomucins and neutral mucins
black, while an AB counterstain differentiated sialo-
mucins.
For each
cross
section, apical and basal areas of
grooves occurring within the deeper regions of primary
and secondary folds (peripheral) and apical and basal
areas of grooves occurring in regions lining the central
cervical canal (central) were evaluated. (See Fig.
1
for
diagrammatic identification of
areas
and regions con-
108
K.J.
MULLINS
AND
R.G.
SAACKE
REGION
h/2
n.
Fig.
1.
Schematic of
a
cervical cross section indicating the desig-
nated location of “central” and “peripheral” regions as well as “apical”
and “basal” areas (cross section
of
grooves) which occur within each
of
these regions. This terminology was established especially for the
histological evaluations presented in this study.
sidered in this study.) Sections stained with AB alone
(at pH 1.0 and at pH 2.5) were evaluated at 250~ by
scoring staining intensity within each area on a sub-
jective scale
of
1-5. In the case of PAS-AB (pH 2.5)-
stained slides, separate estimates were made for each
stain and a scale of 1-10 was used to estimate the per-
cent epithelial cells staining positive for each stain
within each region (1=10% to 10=100%). All esti-
mates (including both follicular and luteal) were made
by one individual using slides coded by another indi-
vidual.
High-iron-diamine-stained
tissue was not quan-
titated but was useful in distinguishing secreted mu-
cins held within the epithelial folds
of
both follicular
and luteal animals.
Results from these scored values were evaluated sta-
tistically by using a nonparametric analysis
of
vari-
ance procedure (SAS Institute, Inc., 1985)
to
test first
for significant differences in stain intensity based on
pH differences in the case of AB stained tissue and
second
to
determine differences in stain distribution for
AB vs. PAS in the PAS-AB-stained sections. Also in-
cluded in the model in both cases were differences
based on quarter, area, region, and cow. Interactions
considered in the model included stain by region, stain
by area, stain by quarter, region by quarter, and area
by quarter. Three-way interactions were not included.
Evaluations were carried out separately for follicular
and luteal data.
Sperm Transport
The general distribution of spermatozoa within cer-
vical tissue
of
mated animals was investigated in he-
matoxylin and eosin (H&E)-stained sections of par-
affin-embedded tissue. Plastic-embedded tissue was
utilized for a more detailed investigation of sperm ori-
entation using light and electron microscopy.
Tissue samples intended for plastic embedment were
fixed in Karnovsky’s fixative
for
1
1/2 h (Karnovsky,
1965), dehydrated in a graded alcohol series, and em-
bedded in either Historesin (LKB) for light microscopy
or
a mixture of Epon 812 and Araldite (Ladd In-
dustries). The Epon-Araldite-embedded samples were
thin-sectioned
for
ultrastructural examination, stained
in uranyl acetate and lead citrate according to Venable
and Coggeshall (1965), and examined with an RCA
EMU-3H electron microscope.
RESULTS
Structure
The stain-enhanced mucosal surface was found
to
consist of a complex system of longitudinally arranged
folds and grooves. Deep primary folds (Fig.
21,
some
originating within the fornix vagina, were found
to
traverse the external cervical
0s
and continue through
each additional annular ring, becoming shallow and
disappearing at the internal cervical
0s.
In the thick
annular rings of the cervix, primary folds were found
to
gain their greatest depth and complexity, which was
often expressed as bifurcation
or
merging
of
one longi-
tudinal fold with another. Secondary folds, produced by
longitudinal division within primary folds, were most
apparent at the annular rings and varied greatly in
length and depth. Numerous uniformly spaced shallow
grooves were evident along all surfaces of cervical
structure. These grooves were observed most fre-
quently as a longitudinal series of parallel furrows. In
central (Fig.
1)
regions lining the cervical canal and
specifically on annular ring surfaces, grooves were fre-
quently abrupt in their origin andlor termination (Fig.
3). Grooves occurring in peripheral regions, however,
maintained greater caudal-cranial continuity (Fig.
4).
Figure
5
presents a conceptulized illustration of a block
of cervical tissue from the fornix vagina through the
first annular ring, summarizing these observations.
Further evidence upholding these observations was
obtained from computer-aided reconstruction
of
seri-
ally sectioned tissue which was taken from a midcer-
vical region between annular rings. This technique re-
vealed a system of continual folds and offered positive
evidence that
no
blind-ending crypts, tubular glands,
or
tunnels are present within this system of folds and
grooves. Figure
6
represents one
of
the stereopairs pro-
duced by the computer and may be viewed with a ste-
reoscope for a three-dimensional perspective. Figure
7
is a graphic interpretation based on stereo images. In
addition, Plexiglas models produced from a series
of
sections which, individually, gave the impression
of
glandular formations within the annular rings, also
upheld the concept of a winding system
of
open ended
folds. Figure
8
diagrammatically illustrates one of
these models.
Histology
A histological study of the secretory epithelium com-
posing the anatomical model described in the Structure
section revealed significant differences in the staining
FUNCTIONAL ANATOMY
OF
THE BOVINE CERVIX
109
Fig.
2.
A
cross-cut of the cervical mucosa showing deep primary
(P)
folds. Note also branching of primary folds forming secondary
(S)
folds. This surface was cut at the base of an annular ring and shows
an increase in complexity as folds
on
the right side of the figure begin
to enter the annular ring.
x
6.5.
Fig.
3.
Luminal surface within the central cervical canal showing
discontinuous shallow longitudinal grooves (arrowheads). This pat-
tern was most frequently observed on surfaces nearest the cervical
canal.
~15.
Fig.
4.
Exposed wall of
a
primary fold showing shallow longitudinal
grooves which are predominantly continuous and parallel. This pat-
tern was most typical of surfaces within peripheral regions of primary
and secondary folds, i.e., distal to the cervical canal.
x
15.
Fig.
5.
An illustration depicting a block of tissue from the fornix
vagina through the cervical
0s
and
first
annular ring. The block has
been enlarged to show primary and secondary folds, subdivided by
numerous grooves. Note the fernlike discontinuous pattern of grooves
in isolated areas of luminal surface (arrow) as well
as
a
region of the
mucosa showing rows of parallel grooves within a peripheral region of
a primary fold which is exposed with forceps in lower right.
110
K.J.
MULLINS AND
R.G.
SAACKE
W
Fig.
6.
Stereopair from the computer-aided reconstruction of
25
se-
rial sections of midcervical tissue between annular rings. Boundary-
line image is displayed in diminishing shades from white to gray,
where white is the closest to the viewer and gray
is
the farthest.
Fig.
7.
Graphic illustration of the overall three-dimensional struc-
ture
as
drawn from the stereo image. Note primary folds
(P),
second-
ary branch
(S),
and lumen
(L)
grooves (G).
response
of
cells lining apical areas (between grooves)
vs. those lining basal areas (within the grooves) as they
occurred in central
or
peripheral regions (see Fig.
1).
For
tissue stained with alcian blue alone, differences in
staining due
to
quarter (caudal to cranial)
or
region
(central vs. peripheral) were found to be insignificant.
Tissue sections from animals in the follicular phase of
the estrous cycle stained with alcian blue
(AB)
at both
pH
1.0
and pH
2.5
revealed a significant
(P
<
.01)
variation in staining pattern in response to pH. Sec-
tions exposed to
AB
at pH
1.0
(specific for sulfomucins
only) revealed a stain intensity which was greatest in
the apical areas of grooves (Fig.
9).
This indicated an
apical sulfomucin concentration while sections stained
with
AB
at pH
2.5
(specific for sialomucins and sulfo-
mucins) revealed a stain intensity greatest in basal
Fig.
8.
Diagrammatic illustration based upon a Plexiglas recon-
struction of
15
serial sections from
a
midcervical region within an
annular ring. This figure demonstrates how glandular-appearing for-
mations in sectioned tissue may originate from
a
winding system of
open-ended folds and grooves.
Fig.
9.
Cervical tissue obtained from
a
cow in the follicular phase of
the estrous cycle. Section was stained with
AB
at
pH 1.0 (sulfomucins
only). Note denser stain intensity in apical
(A)
areas.
B,
basal.
x
230.
Fig.
10.
Tissue section adjacent
to
Figure
9
but stained with
AB
at
pH
2.5
(sulfomucins and sialomucins). Note increase in stain intensity
in basal (B) areas which may be attributed to sialomucin differenti-
ation with
AB
at pH
2.5.
A,
apical.
x
230.
FUNCTIONAL ANATOMY
OF
THE
BOVINE CERVIX
111
Fig.
8-10
112
K.J. MULLINS AND
R.G.
SAACKE
Alcian
Blue
pHI.0
vs
pH
2-5
4.0
2
3.5
_-
u)
5
3.0
-
2.5
2.0
1.5
t
c
c
--
v,
--
t
-
0
1.0
a
=
.5
Fol
I
i
cu
lar
*
Luteal
Apica
I
Ba
so
I
Apical Basal
Area
of
Groove
Fig.
11.
Comparison of differences in
AB
stain intensity
at
pH
1.0
(specific for sulfomucins) and
at
pH
2.5
(sialomucins and sulfomu-
cins). Intensity estimates for apical and basal areas
of
the grooves are
presented separately for both follicular
(left)-
and luteal (right)-phase
tissue. Values obtained separately for central vs. peripheral regions
(see
Fig.
1)
are pooled
as
they were not found to be different. In
follicular-phase tissue, note marked increase in intensity of basal
ar-
eas
at
pH
2.5,
compared
to
pH
1.0
("P<.02),
predominantly due to
sialomucin, while sulfomucins (pH
1.0)
actually decrease from apical
to basal areas (mean of five animals plus or minus standard error). By
contrast, basal areas
of
luteal-phase tissue revealed a small but in-
significant increase in intensity
at
pH
2.5
vs. pH
1.0
(reduced sialo-
mucins), although an increase in basal
vs.
apical intensity
at
pH
1.0
indicated an increase in basal sulfomucin during this phase (mean of
three animals plus or minus standard error).
areas
of
most grooves (Fig. 10) indicating a basal si-
alomucin concentration. This stain (pH 1.0 vs. pH 2.5)
by area (apical vs. basal) interaction was significant
(P
<
.02) for the follicular phase. On the contrary, for
luteal-phase animals there was no significant differ-
ence in stain estimates by area based upon pH shift,
indicating a dramatic reduction of sialomucin in basal
areas
of
luteal phase animals. These comparisons are
summarized in Figure
11.
Follicular-phase tissue stained with PAS in addition
to AB (pH 2.5) revealed a highly significant
(P
<
.01)
stain by area difference whereby cells within apical
areas stained predominantly with PAS, indicating the
presence of neutral mucins (in addition to the sulfo-
mucins noted above), while cells within basal areas
stained predominantly with AB, indicating a predom-
inance of acidic mucins (sulfomucins and sialomucins).
Regions were also found to be different
(P
<
.04)
due
primarily to a decrease in basal PAS in the peripheral
region (Fig. 12).
Luteal-phase tissue stained with PAS and AB also
revealed a significant
(P
<
.01)
predominance of PAS
in apical areas while AB remained the predominant
basal stain. However, in contrast to the follicular
phase, there was a reduction in the percent cells in
basal areas which contained AB-positive mucins, while
basal cells which contained PAS-positive mucins were
increased. A stain-by-region difference
(P
<
.01)
was
found in luteal-phase tissue due to an increased AB
stain response in both apical and basal areas
of
the
peripheral region (Fig.
12).
It was noted that individual cells often demonstrated
the presence of both PAS- and AB-positive mucin gran-
ules but in varying proportions depending on their lo-
cation within the groove. Cells in basal areas had pre-
dominantly, but not exclusively, AB-positive granules
and apical cells had predominantly, but not exclu-
sively, PAS-positive granules (Fig. 13).
High-iron diamine was utilized in a qualitative eval-
uation of both intracellular and luminal mucins. Lu-
minal mucins from follicular-phase samples revealed
the existence of a layered system of densely staining
HID-positive mucins (neutral and sulfomucins). Each
layer appeared to emerge from the apical area between
grooves especially within peripheral regions and ex-
tend toward the central lumen (Fig. 14). When consid-
ering the third dimension as described in the Structure
section, these neutral mucin layers could be envisioned
as parallel sheets (note Fig.
20).
These layers (or
sheets) were often observed
to
extend in close proxim-
ity to the epithelium in such a manner as to entrap the
less densely staining AB-positive mucins (sialomicins)
113
FUNCTIONAL ANATOMY
OF
THE BOVINE CERVIX
70
60
50
40
30
20
10
0
Luteal
Apical
AB
Basal
AB
Cent ra
I
Peripheral Central Peripheral
Region
of
Cervix
Fig.
12.
Comparison of the estimated percent cells staining positive
for PAS (neutral mucins) vs. those staining positive for AB (pH
2.5)
in
both follicular (left)- and luteal (right)-phase cervices. Estimates
were made for each stain in both apical and basal areas
of
the grooves.
Values for central and peripheral regions are shown separately
as
differences
(P<.04,
follicular;
P<.Ol,
luteal) were found to exist. In
follicular-phase tissue, note predominant PAS in apical areas and
predominant AB in basal areas with a reduction of basal PAS and
within grooves (Fig. 15). As layers of HID-positive mu-
cins originate along these apical areas, specifically
those deep within peripheral regions, and extend to-
ward the central cervical lumen, they appeared to lam-
inate into layers separated by mucins staining predom-
inantly with AB. In contrast to the follicular phase,
luminal mucins observed within cervices from luteal-
phase animals did not demonstrate
a
distinct layered
organization but rather appeared to be more tightly
interconnected, forming a dense meshwork (Fig.
16)
which stained predominantly
as
neutral mucins with
both HID and
PAS.
It
should be noted that while these
secreted luminal mucins could be subject to fixation
artifact, the differences noted between follicular and
luteal preparations were distinct and consistent.
Sperm
Transport
Paraffin-embedded tissues from animals which were
bred 8-12 h prior to sacrifice were sectioned
at
5
pm
and stained with hematoxylin and eosin for localiza-
tion of spermatozoa. Spermatozoa in these sections
were apparent in isolated regions of
all
sections but
varied greatly in number from an occasional single cell
to areas of relatively heavy concentration. Areas
heavily populated with spermatozoa were found in mu-
cus-filled luminal regions within primary and second-
ary folds. Cells observed within cross-sectioned cervical
slight increase in basal AB in peripheral regions (mean of five ani-
mals plus or minus standard error). In luteal-phase tissue PAS re-
mains the predominant apical stain but increases dramatically in
basal areas when compared
to
follicular tissue, while AB is reduced
but remains the predominant basal stain. Note increase in apical AB
in peripheral
vs.
central regions (mean
of
three animals plus or minus
standard error).
grooves were most often deep within basal
areas
(Fig.
17). Orientation of spermatozoa within the folds and
grooves of the cervix was evident in Historesin-em-
bedded tissue which was sectioned
at
2 pm and stained
with toluidine blue. In areas where longitudinal sec-
tions through
a
groove contained sagitally sectioned
spermatozoa, they were most frequently observed to be
unidirectionally aligned close to the epithelial surface
and traveling in opposition to the apparent direction
of
ciliary beat (Fig.
18).
Likewise, electron micrographs
from areas with good sperm retention also revealed
a
directional orientation of spermatozoa in opposition to
ciliary beat. This determination was based on the plane
of section through either an acrosomal ridge
or
a
por-
tion of the sperm flagellum (Fig.
19).
DISCUSSION
Due to the complexity of this winding but nearly
continual system of folds and grooves it
is
now clear
how cross sections through such
a
system could suggest
existence of blind-ending glands
or
“crypts” (a term
which implies slender pit
or
small glandular cavity).
The production of specific mucin types as prescribed by
location within the defined cervical structure would
suggest a complex pattern of mucoid structure also ex-
ists within the cervix and is determined by anatomical
features of the mucosa in addition to direction of flow.
114
K.J.
MULLINS
AND
R.G.
SAACKE
Fig. 13.
Historesin section stained with PAS-AB (no nuclear coun-
terstain) demonstrating mucin granules within secretory cells. Note
predominant PAS (lighter granules) in apical cells (A) and predomi-
nant AB (densely staining granules) in basal cells (B). Cells between
A and B contain a mixture of PAS and AB positive granules
as
noted
by difference in densities.
x
840.
Fig.
14.
A transverse section through a deep primary fold in follic-
ular tissue stained with HID-AB. Apparent are the densely stained
layers of HID-positive mucins (neutral and sulfomucin) emanating
from each apical area between grooves (arrows) and extending toward
the central canal. Lighter-staining AB-positive mucins (predomi-
nantly sialomucins) occur between denser HID mucin layers and
within grooves.
x40.
(Note AB intensity is reduced
to
a pale blue
when used in combination with HID.)
Fig.
15.
HID-AB-stained section showing a portion of a primary fold
(peripheral region) in follicular-phase cervical tissue where AB-posi-
tive sialomucins (Si) appear to be confined within basal areas of
grooves by denser-staining HID-positive neutral (N) and sulfomucin
layers as they are emitted from apical areas and flow in close prox-
imity to adjacent apical surfaces.
x
250.
Fig.
16.
An HID-AB-stained section through
a
fold taken from lu-
teal-phase cervical tissue revealing
a
loss in the layered organization
of luminal mucins evident in follicular tissue (Figs.
14,
15).
Note,
instead, a meshlike pattern of interconnected mucins which stain
predominantly for neutral mucins.
x
250.
FUNCTIONAL ANATOMY
OF
THE
BOVINE
CERVIX
115
Although
the
results of this study comparing tissue
from follicular and luteal phases of the cycle support
the concept reported by Heydon and Adams (1979) that
basal sialomucin production was
a
response to estrogen
stimulation, they do not support investigations utiliz-
ing PAS-AB staining techniques on cervical tissue
from follicular-phase animals (Wordinger et al., 1972;
Heydon and Adams, 1979). Wordinger
et
al. (1972)
found no difference in staining between apical and
basal areas (referred to as crypts) while Heydon and
Adams (1979) reported
a
predominance
of
bluish pur-
ple cells in apical areas with basal areas (referred to
as
secondary indentations) staining red and purple. This
pattern would indicate an apical production of acidic
mucins and basal production of neutral mucins which
is in sharp contrast to our results. Preliminary inves-
tigations in stain and fixation techniques could help
explain these differences. Overstaining with
PAS
was
found to mask AB, reducing the ability to obtain clear
localization, while tissue samples which were fixed in
formol-saline, the fixative utilized in the Heydon and
Adams investigation, revealed
a
reversal in the stain-
ing pattern (apical AB and basal PAS) in some sam-
ples.
Orientation of spermatozoa within this system of
mucus-filled channels was in opposition to the beat of
ciliated cells, suggesting that ciliated cells may func-
tion to
orient
spermatozoa toward the uterus by estab-
lishing gentle mucus flow toward the vagina. This
would be consistent with the observation that sperm
swim upstream (Zinner
et
al., 1982). The flow of mucus
toward the vagina was considered by Mattner (1966) in
his proposed concept whereby privileged paths were
established in the transport of spermatozoa toward the
mucosa via lines of strain. However, in Mattner’s
model, the lines of strain led through the central cer-
vical canal into blind-ending crypts/glands from which
continued sperm transport to the uterus was difficult to
envision
as
sperm would have to cross lines of strain to
leave the glands. A model which considers both the
three-dimensional cervical structure (a series of merg-
ing and diverging but continual grooves) and a super-
imposed pattern of mucus secretion (basal sialomucins
and apical neutral and sulfomucins) is presented in
Figure 20. It is conceivable that spermatozoa entering
the mucus structure within this model would be com-
pelled to follow the path of least resistance, which
would be within sialomucin-rich layers flowing in ba-
sal
areas of grooves with the aid of cilia
or
between
denser layers (sheets) of apically produced neutral mu-
cins.
The concept of sialomucin receptivity to sperm would
be consistent with the mechanism of mucus hydration
proposed by Daunter (1984) whereby uncomplexed
sialic acid associated with midcycle mucus in the hu-
man is able to bind water and thus undergo the swell-
ing that
is
associated with estrogen influence. Since
continual primarytsecondary cervical folds were ob-
served to originate within the fornix vagina, sperma-
tozoa naturally deposited in this area would have ac-
cess to direct entry into these folds. They could then
continue into and through peripheral regions of cervi-
cal structure where grooves were observed to be most
continuous
as
opposed to the central cervical canal
(Fig. 21). In a manner similar to that proposed by Matt-
ner (19661, privileged paths would still be established
and lead into basal areas within mucosal grooves. Lay-
ers of neutral mucin emitted from adjacent apical areas
could conceivably restrict lateral progression of sper-
matozoa
as
they are held on course within the more
hydrated sialomucin channels. In addition, wave cur-
rents established by cilia would encourage alignment
of spermatozoa along mucosal surfaces. Viable sperma-
tozoa entering peripheral grooves could thus continue
their progression toward the uterus within the confines
of continuous diverging and converging shallow folds
rather than being sequestered for storage as suggested
by Mattner (1966).
This concept of sperm transport is supported in the
present study by the distribution, orientation, and
membrane integrity of spermatozoa observed within
basal areas of grooves as well
as
a predominance of
sialomucins within grooves during the follicular phase.
It
is
uncertain whether populations of cells observed
within the denser mucin layers of some luminal areas
are indeed traversing the cervix within privileged
paths
or
simply are trapped within neutral mucins
which are being cleared to the vagina. Evidence for the
latter which would suggest their loss to the vagina is
presented in an investigation of the fate of sperm fol-
lowing uterine insemination (Mitchell
et
al., 1985).
These workers accounted for 73% of the inseminated
spermatozoa by 12 h postbreeding and 90% of those
were discharged in the mucus. Thus, a high porportion
of spermatozoa observed in luminal mucins may be in
the process of retrograde removal. In addition, Mattner
and Braden (1969) found that sperm deepest within the
cervical mucosa were more viable than those in the
luminal region, thus supporting the current concept
that such sperm are viable and most likely in transit to
the uterus.
Smith, Boland, and Gordon (unpublished data cited
in Gordon, 1977) studied conception
rate
in sheep after
shallow and deep cervical insemination. They found
that deep cervical insemination yielded lower fertility
than the more shallow deposition. This would be sur-
prising if sperm transport was primarily through the
cervical canal but not if access to privileged paths was
greatest in the fornix vagina with retrograde removal
taking place in the central canal. Other work by Fulk-
ersen et al. (1982) found that by using artificial insem-
ination in sheep, where semen deposition was within
the cervical canal, twice
as
many spermatozoa were
required to achieve
a
conception rate equivalent to nat-
Figs.
17-20
117
FUNCTIONAL ANATOMY
OF
THE BOVINE CERVIX
Fig.
21. Interpretative diagrammatic representation of the fornix
vagina and external cervical
0s
showing the potential mechanism of
penetration and subsequent sustained sperm transport whereby sper-
matozoa may enter sialomucin-rich layers extending from basal areas
of the peripheral cervix
at
the fornix vagina and progress to the
uterus within a system of folds and grooves. Retrograde flow of mu-
cins laden with leucocytes and disgarded spermatozoa occupy the cen-
tral cervical lumen (large arrow).
ural service using continually mated rams ejaculating
known numbers of sperm. This also suggests that sper-
matozoa deposited centrally within the cervical canal
are subject to removal with retrograde mucus flow and
may, therefore, be in
a
less favorable position for cer-
vical transport than those placed at
or
near the fornix
vagina where they may gain entry into basal areas of
folds and grooves.
ACKNOWLEDGMENTS
We are very grateful to
Dr.
Harry Steeves
for
direc-
tion in the histochemical investigation, to Dr. Daniel
Dylewski and Shiwen Gu for assistance with computer
graphics, and to
Dr.
Ronald Pearson for his help with
statistical design and analysis.
LITERATURE CITED
Cole, H.H.
1930
A study of the mucosa of the genital tract of the cow,
with special reference
to
cyclic changes. Am.
J.
Anat.,
46t261-302.
Daunter, B.
1984
Biochemical and functional-structural aspects of
human cervical mucus. Scanning Electron Microsc.,
1.343-358,
Dylewski, D.P., R.M. Haralick, and T.W. Keenan
1984
Three-dimen-
sional ultrastructure of the golgi apparatus in bovine mammary
epithelial cells during lactation. J. Ultrastruct. Res.,
87:75-85.
Fluhmann, C.F.
1958
The glandular structures of the cervix uteri.
Surg. Gynecol. Obstet.,
106t715-723.
Fulkersen, W.J., A.L. Synnott, and D.R. Lindsay
1982
Numbers of
spermatozoa required
to
effect
a
normal rate of conception in
naturally mated Merino ewes.
J.
Reprod. Fertil.,
66.129-132.
Gibbons, R.A., and P. Mattner
1966
Some aspects of the chemistry of
cervical mucus. Int. J. Fertil.,
11.366-372.
Gordon, J.
1977
Application of synchronization of estrus and ovula-
tion in sheep. In: Management of Reproduction in Sheep and
Goats Symposium. U. of Wisconsin, Madison, p.
23.
Hafez, E.S.E., A.A. El-Banna, and T. Yamashita
1971
Histochemical
characteristics of cervical epithelium of rabbits and cattle. Acta
Histochem. (Jena),
39:195-205.
Hafez, E.S.E., and Kanagawa
1972
Scanning electron microscopy of
the bovine reproductive tract in female. Cornell Vet.,
63:
469-482.
Hafez, E.S.E.
1980
Transport and survival of gametes. In: Reproduc-
tion in Farm Animals, 4th ed. E.S.E. Hafez, ed. Lea and Febiger,
Philadelphia, pp.
203-225.
Hammond
J.
1927
The Physiology of Reproduction in the Cow. Cam-
bridge University Press, London, pp.
54-55, 92-94.
Herrick, J.B.
1951
The cytological changes in the cervical mucosa of
the cow
(Bos
Taurus) throughout the estrous cycle. Am. J. Vet.
Res.,
12t276-283.
Heydon, R.A., and N.R. Adam
1979
Comparative morphology and
mucus histochemistry of the ruminant cervix: Differences be-
tween crypt and surface epithelium. Biol. Reprod.,
21.557-562.
Jones, R., and L. Reid
1973
The effect of pH on alcian blue staining of
epithelial acid glycoproteins.
1.
Sialomucins and sulphomucins
(singly
or
in simple combinations). Histochem. J.,
5:9-18.
Karnovsky, M.J.
1965
A formaldehyde gluteraldehyde fixative of
high osmolality for use in electron microscopy, J. Cell Biol.,
27:137A.
Litt, M., M.A. Khan, and D.P. Wolf
1976
Mucus rheology: Relation to
structure and function. Biorheology,
13:37-48.
Mattner, P.E.
1966
Formation and retention of the spermatozoan res-
ervoir in the cervix of the ruminant. Nature,
212:1479-1480.
Mattner, P.E., and A.W.H. Braden
1969
Comparison of the distribu-
tion of motile and immotile spermatozoa in the ovine cervix.
Aust.
J.
Biol. Sci.,
22.1069-1070,
Mitchell, J.R., P.L. Senger, and J.L. Rosenberger
1985
Distribution
and retention of spermatozoa with acrosomal and nuclear abnor-
malities in the cow genital tract. J. Anim. Sci.
61(41:956-967.
Pearse, A.G.E.
1985
Histochemistry, Theoretical and Applied. Vol.
2:
Analytical Technology, ed.
4.
Churchill Livingstone, Edinburgh.
SAS Institute, Inc.
1985
SAS
Users Guide: Basics, Version
5
Edition.
SAS Institute Inc., Cary, NC,
I290
pp.
Venable, J.H., and R. Coggeshall
1965
A simplified lead citrate stain
for use in electron microscopy.
J.
Cell Biol.,
25.407-408.
Wordinger, R.J., J.F. Dickey, and J.R. Hill
1972
Influence of
a
progestogen on the histology and carbohydrate histochemistry of
the bovine mucosa. J. Anim. Sci.,
352330435,
Zinner, N.R., A.M. Sterling, R.C. Ritter, and
J.
Ragter
1982
Human
sperm swim against the stream. J. Fertil. Steril.,
38276
(Abstract).
Fig.
17.
Hematoxylin-stained section of cervical tissue from
a
cow
which was mated naturally
at
estrus and slaughtered
8
h later. Note
spermatozoa deep within basal areas (arrows)
of
a groove.
x
300.
Fig.
18.
Spermatozoa within this narrow fold in a Historesin prep-
aration are observed oriented unidirectionally (note evidence of fla-
gella) in opposition to the beat pattern of ciliated cells.
x
1,060.
Fig.
19. Transmission electron micrograph
of
longitudinally
sectioned tissue showing spermatozoa traversing the cervix. Note
unified orientation of spermatozoa (left to right) in opposition to the
direction of ciliary beat (left) as evidenced by intact acrosomal
membranes (arrows) or section through the capitular region of the
flagellum.
x
7,100.
Fig.
20. An interpretive diagrammatic representation of
a
possible
secretory pattern based on structural and histological evidence
presented in this study. Apical secretion of neutral mucin in “sheets”
(N) extends toward the central lumen while less viscous sialomucins
(Si) produced in basal areas flow in a vaginal direction as influenced
by ciliary beat. Note also spermatozoa traversing in basal areas in
opposition to direction of ciliary beat.
