/ . Embryol. exp. Morph., Vol. 17, 2, pp. 433-440, April 1967
Printed in Great Britain
433
Studies on the cell
degeneration rate during the differentiation
of the epithelium in the uterine cervix and
Miillerian vagina of mouse
By JOHN-GUNNAR FORSBERG1
From the Department of Anatomy and the Tornblad Institute
for Comparative Embryology, University of Lund, Sweden
Glucksmann (1951) has summarized the results of earlier investigations on
cell degeneration processes during normal vertebrate development. To the few
investigations on the degeneration in the urogenital region made before the
publication of Gliicksmann's paper (.1951) can be added Bengmark's study
(1958) on degeneration processes during the separation between the Wolffian
duct and the ureteral anlage and Forsberg's study (1961) on the mechanism of
the division of the cloaca. Finally, Forsberg (1963) has given a description of
those regions where degeneration can be seen during the development of the
vaginal anlage in several species, including man.
In this investigation the author has made a quantitative investigation of cell
degeneration during the differentiation of the epithelium in the mouse Miillerian
vagina and the uterine cervix. Morphologically, this degeneration is characterized by the occurrence of degeneration granules, whose nature has earlier been
discussed by Bengmark & Forsberg (1959) and Forsberg (1963).
Concerning the differentiation of the Miillerian epithelium in the region
studied, it occurs as pseudostratified columnar epithelium at birth. It undergoes
an active proliferation (Forsberg, 1965) and 2-3 days after birth two epithelial
zones appear: one superficial with high columnar cells; one basal with smaller
cells. The latter zone is derived from cells migrating basally in the pseudostratified epithelium (Forsberg, 1965, 1966). The mitotic rate is higher in the
superficial zone than in the basal zone, the maximum being seen in the uterine
cervix (Forsberg, 1965). Not until 13 days after birth is the mitotic rate in the
vagina and the uterine cervix reduced to a similar low level.
1
Author's address: Department of Embryology, Biskopsgaten 7, Lund, Sweden.
434
J.-G. FORSBERG
MATERIAL AND METHODS
The material for this investigation was obtained from young albino mice from
a closed stock held at this Institute for several years and used by the author in
all earlier investigations on the differentiation of the vaginal epithelium. Newborn young and young aged 3, 5, 7, 10 and 13 days were studied. From each of
the first three stages, two animals were studied; from each of the latter three, one
animal. They were decapitated and immediately the vagina, uterine cervix and
the posterior part of the uterine horns were dissected out and fixed in Carnoy's
solution (Fig. 1). The preparations were then transferred to absolute alcohol,
paraffin embedded in the usual manner, and serially sectioned at 5 /i. The
sections were Feulgen stained; the time of hydrolysis was 8 min. Control
sections were incubated in DNase (Sigma Chemical Company, St Louis) at
pH 7 in the presence of magnesium sulphate (0-003 M).
Fig. 1. Schematic drawing of the region studied, u.h. uterine horn; u.c. uterine
cervix; / vaginal fornix; v. Miillerian (anterior) vagina. Only the epithelial part
is shown.
The section series were studied in an anterior posterior direction, beginning
somewhat anteriorly to the fusion between the two uterine horns. Thereafter,
every fifth section was studied in the posterior part of one of the uterine horns,
the uterine cervix, and the anterior part of the vagina (Fig. 1). Usually about
1000 epithelial cell nuclei, including mitotic nuclei, were counted in the region
studied in each of the sections. In the cervical canal the counts were made in one
of the lateral walls, always on the same side, as was the chosen uterine horn. Only
the surface epithelium was studied. In the vagina the calculations were also
restricted to the lateral wall and the bordering part of the ventral and dorsal
wall. When all cell nuclei had been counted, pycnotic nuclei and degeneration
435
Cell degeneration rate
Newborn
9 -
Newborn
M
t •
50//
F
t
V
m-s
Fig. 3
3 days
°o°o
5 3
x
Fig. 4
„ x
Fig. 5
Figs. 2-10. The degeneration rate distribution curves of each of the animals studied.
F indicates the fusion between the two uterine horns; V, the level of the opening of
the cervical canal into the vagina; m-s, the level of the border between the Mullerian
vagina and the sinus vagina; D.R. degeneration rate; M.R., mitotic rate. A cross
indicates the degeneration rate in the pseudostratified epithelium. After the epithelium has differentiated into a superficial zone and a basal zone, a cross indicates
the rate in the superficial zone, and a point, that in the basal zone. A triangle gives
the rate for the whole epithelial wall in the oldest stage where the two zones can
no longer be seen as separate entities. A circle denotes the rate in the fornix. In
Figs. 2, 6 and 9, both the degeneration-rate curve (D) and the mitotic-rate curve (M)
are shown. For details concerning the latter, the reader is referred to Forsberg
(1965). Note the close correspondence in appearance between the two curves. In
Fig. 2 the M curve represents a stage 6 h after birth.
436
J.-G. FORSBERG
5 days
5 days
as
S
7
a
5
«.
3
Q
2
3
\
D
X
X
xX
x
X
1
S
X
• •
XXX
IX
50//
Fig. 6
Fig. 7
10 days
7
OS
3
Q
w
5
et
S
r 3
C4
x
d
-
x"
D^x 2 ^^
SO ft
50//
Fig. 8
13 days
Figs. 6-10. For legend, see p. 435.
x*'X"
Cell degeneration rate
437
granules were counted in the same region. After the pseudostratified epithelium
had differentiated into the superficial zone and the basal zone, the counts were
made separately in the two zones. Granules situated on the borderline were disregarded. In the oldest stages where there is no longer a definite superficial zone
and basal zone, the counts were made on the whole epithelial wall. If two or
more distinctly separated granules lay close together, they were counted as
separate granules. All counts were made on a Zeiss photomicroscope, adjusted
for photographing, objective x 100, optovar x 1-25, ocular x 12-5. A green
filter was used.
The degeneration rate is expressed as a percentage, that is, as the number of
degeneration granules (each being considered to be derived from one nucleus;
see Discussion) and pycnotic nuclei in each 100 counted nuclei. These rates are
plotted on a co-ordinate system: the vertical axis represents the degeneration
rate as a percentage, the horizontal axis the extension of the region studied.
The degeneration rate curves thus constructed demonstrate the fluctuation of
the degeneration rate within the region studied.
RESULTS
The results of the calculations on the degeneration rate are shown in Figs.
2-10. From this, it can be seen that the degeneration rate is highest in the cervix
and the Miillerian part of the vaginal anlage at the newborn stage. The peak
of the curve corresponds approximately to the transitional region between the
vagina and the cervix. In one of the 3-day preparations studied, the peak is
situated in the most anterior part of the vagina and in the cervix; in the other
it is within the cervix. At 5 days after birth the peak of each curve is situated
within the cervix, and at 7 days and 10 days it has moved somewhat further
anteriorly in the cervix, now also including the posterior part of the uterine
horns. At 10 and 13 days the curve has become somewhat flattened compared
with the earlier stages but there is still a small peak in the cervix.
In those stages where the epithelium has differentiated into a basal zone and
a superficial zone, the degeneration rate in the region studied is considerably
lower in the former than in the latter. Some degeneration also occurs within the
uterine horns, at least within the posterior part studied.
A separate calculation was made to determine the degeneration rate in the
fornix. In the newborn stage, this is the same within the developing fornix
region as in the cervix and anterior part of the vaginal anlage. In one of the
3-day animals the degeneration rate in the fornix region does not reach the
level of the peak of the curve. However, in the other 3-day-old, the degeneration
rate in the fornix is higher than that corresponding to the peak of the curve.
In the same way, in one of the 5-day young studied the degeneration rate is
perhaps higher in the fornix than in the cervix and anterior part of the vagina,
whereas in the other it is approximately the same. The above-mentioned stages
438
J.-G. FORSBERG
show no trend for the degeneration rate to be lower or higher in the fornix
region than in the cervix and most anterior part of the vagina.
DNase treatment of the sections completely eliminated Feulgen staining
both of nuclei and degeneration granules, thus indicating the true nuclear
remnant character of the latter.
DISCUSSION
The peak of each of the degeneration rate curves moves in an anterior direction from the younger stages to the older. In the younger stages the peak is
localized in the most anterior part of the vaginal anlage and in the cervix;
thereafter, it moves into the cervix; finally, it involves also the posterior part of
the uterine horns. A similar behaviour is also shown by the mitotic rate distribution curves from the same regions (JForsberg, 1965). When the pseudostratified
epithelium has differentiated into two zones, the degeneration rate is considerably lower in the basal zone than in the superficial. The mitotic rate is also
lower in the basal zone than in the superficial zone (Forsberg, 1965) although
the difference is not so pronounced as it is for the degeneration rate. This has
been interpreted as being due to the basal zone consisting of more differentiated
cells than the superficial zone, which is mainly cell-producing during the period
studied (Forsberg, 1965). This circumstance may well explain the difference in
degeneration rate between the zones. Part of the degeneration seen in the basal
zone may be due to cells damaged in the superficial zone or to degeneration
granules moving basally in the epithelium.
At 10 days the degeneration rate curve is flattened compared with the earlier
stages. This applies also to the mitotic rate curve from the same age stage.
At 13 days after birth the mitotic rate in both the cervix and the vagina is the
same and low, but the degeneration rate curve shows a small peak in the cervix.
Obviously, counting degeneration granules might introduce some methodological error. Thus, for instance, one degenerating nucleus may give rise to one,
two, or even more condensed chromatin particles, somewhat separated from
one another. This will result in too high a degeneration rate, However, there
are no reasons for believing that there is a greater tendency for the nuclei to
give rise to more particles in one region than in another. An error may thus be
introduced into the measurement of the absolute degeneration rate, but not of the
relative distribution of degeneration. Nuclei can naturally also be seen in earlier
phases of degeneration, and it is remarkable that the chromatin in these cases
is often condensed in two or three regions along the nuclear membrane, thereby
suggesting the later formation of two or more separate degeneration granules.
Crucial for the estimation of the true degeneration rate is the relation between
the formation of new granules and the speed of their disappearance. The shape
of the curves, compared at different times after birth, does not indicate any
obvious accumulation of granules. Instead the 'input' seems to balance the
'output' (cf. Gliicksmann, 1951).
Cell degeneration rate
439
It may be argued that the degeneration process studied here is not related to
epithelial differentiation, but might instead result from a hormone withdrawal
effect at the time of birth. Opposed to this view is the fact that a hormone
withdrawal effect would probably be reflected in a decreased mitotic rate in the
neonatal period. This, however, does not occur; the mitotic rate does not
decrease in the cervical region until about 10 days after birth (Forsberg, 1965).
Instead, there is a remarkable correspondence between the curves for the
mitotic rate distribution in the vagina, cervix, and posterior part of the uterine
horns and those for the degeneration rate distribution in the same regions. The
peak of each curve appears in the same region; the curves also follow a very
similar course. In one experiment (Forsberg, unpublished results) colchicine was
injected into a pregnant mouse on the 18th day of gestation, and the foetuses
were fixed 6 h later. An accumulation of mitoses in the vaginal anlage, cervix,
and uterine horns resulted. A study of the mitotic rate distribution in the Mlillerian part of the vaginal anlage and in the cervical region in two foetuses
revealed no signs that the mitotic rate in these regions was higher than that seen
in newborn; the rate seems to be lower before birth than after. It would thus
appear that the change in hormonal environment does not lead to any atrophic
changes in the epithelium under study.
Is the degeneration studied related to the fusion of the two Miillerian ducts
(uterine horns) to form a common cervical canal? If so, it could be argued that
the high degeneration rate in the cervix might depend on persistent degeneration
granules originating from the disappearance of the common wall formed at the
fusion. To avoid this possible source of error, the author studied the degeneration in only the lateral cervical walls. It is not probable that eventual degeneration remnants should 'flow' in the epithelium from the medial part of the ventral
and dorsal wall into the lateral walls. Nor are there any indications that the
development of the fornix is associated with any morphological degeneration.
The hypothesis that the degeneration described here results from an anterior
growing sinus epithelium replacing the Miillerian epithelium is not consonant
with the author's earlier autoradiographic investigations on the derivation of
the epithelium in the anterior part of the vagina in mouse, neither in normal nor
in oestradiol-treated animals (Forsberg, 1965, 1966).
The most conspicuous result from this study is the great resemblance between
the mitotic-rate curves (Forsberg, 1965) and the degeneration-rate curves. This
indicates that some error in the mitotic mechanism might, at least to some extent,
explain the degeneration in the region under study (cf. Kallen, 1965). This
statement is supported by the finding of irregular epithelial anaphases, e.g.
chromosome bridges, in squash preparations from the region studied.
440
J.-G. FORSBERG
SUMMARY
The degeneration rate during the differentiation of the epithelium in the Miillerian anterior part of the vaginal anlage and cervix in mice was studied. A peak in
the degeneration-rate distribution curves was noted in approximately the cervical
region; earlier stages included also the anterior part of the vaginal anlage, later
stages the posterior part of the uterine horns. The peak of each of these curves
largely coincides with that seen in the mitotic-rate distribution curves from the
same region. Possible errors in the method used are discussed. The correspondence between the mitotic-rate and degeneration-rate curves indicates that
irregular mitoses might, at least to some extent, explain this degeneration.
RESUME
Etudes sur le taux de degenerescence pendant la differentiation de Vepithelium
dans les ebauches du col uterin et du vagin de la souris
Chez la souris, le taux de degenerescence au niveau de la partie anterieure
de l'ebauche vaginale et du col a ete etudie pendant la differentiation de
I'epithelium. Un pic dans les courbes de distribution du taux de degenerescence
a ete note approximativement dans la region du col; les stades plus precoces
comportaient aussi la partie anterieure de l'ebauche vaginale; les stades plus
tardifs, la partie posterieure des cornes uterines. Le pic de chacune de ces
courbes coincide largement avec celui observe pour la meme region dans les
courbes de distribution du taux de mitoses. Les erreurs possibles de la methode
utilisee sont discutees. La correspondance entre les courbes de taux mitotique
et de taux de degenerescence montre que des mitoses irregulieres pourraient,
au moins dans une certaine mesure, expliquer cette degenerescence.
This investigation was supported by grants from the Swedish Cancer Society (no. 65:107)
and from the Swedish Medical Research Council (no. 12X-579-02).
REFERENCES
S. (1958). The Prostatic Urethra andProstatic Glands. Lund: C. W. K. Gleerup.
S. & FORSBERG, J.-G. (1959). Some remarks on degeneration granules in the
genital sphere. Z. Zellforsch. 49, 694-8.
FORSBERG, J.-G. (1961). On the development of the cloaca and the perineum and the formation
of the urethral plate in female rat embryos. /. Anat. 95, 423-36.
FORSBERG, J.-G. (1963). Derivation and differentiation of the vaginal epithelium. Thesis,
University of Lund.
FORSBERG, J.-G. (1965). Mitotic rate and autoradiographic studies on the derivation and
differentiation of the epithelium in mouse vaginal anlage. Ada anat. 62, 266-82.
FORSBERG, J.-G. (1966). The effect of estradiol-17/? on the epithelium in the mouse vaginal
anlage. Acta anat. 63, 71-88.
GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86.
KALL£N, B. (1965). Degeneration and regeneration in the vertebrate central nervous system
during embryogenesis. In Progress in Brain Research, vol. xrv, pp. 77-96. Ed. M. Singer
and J. P. Schade. Amsterdam: Elsevier Publishing Company.
BENGMARK,
BENGMARK,
{Manuscript received 28 October 1966)