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Histochemical studies on cervical glands in ewes with clover disease
.J. COMP.
PATH.
1977.
87.
VOL.
HISTOCHEMICAL
STUDIES ON CERVICAL WITH CLOVER DISEASE
EWES
R. Diraision
353
A.
of Animal
HEYDON Health,
CSIRO,
and PIivale
N.
R.
GLANDS
IN
ADAMS
Bag, ll’embly,
lVe.ctern :htralia
INTRODUCTION
Ewes which have grazed oestrogenic subterranean clover for prolonged periods develop clover disease and become permanently infertile (Schinckel, 1948). The infertility is thought to be due primarily to abnormal cervical secretions which impair the establishment of spermatozoa in the cervix after mating (Smith, 1971). C onvoluted tubular glands form in the cervix of the infertile ewes and have been shown to react differently to stains for mucus when compared to the normal cervical epithelium (Adams, 1976). These cervical glands therefore appear likely to play a role in the abnormal cervical secretion and the infertility. Consequently, the nature of the cervical glands in ewes with clover disease is worthy of further study. In the female mouse treated with oestrogen at birth, glands with a uterine type of epithelium develop in the cervix (Forsberg, 1969). In women, on the other hand, gland-like clefts occur as part of the normal cervical architecture (Fluhmann, 1958). The present study compares the histology and histochemistry of cervical glands in clover-diseased ewes with both uterine glands and the cervical epithelium of normal ewes in order to better define theit nature. MATERIALS
AND
METHODS
Biological material. The reproductive tracts were removed from 25 mature, nonpregnant South Australian type Merino ewes at the time of slaughter. The ewes were in early metoestrus, as shown by the presence of a fresh corpus luteum, less than 3 mm in diameter, in the ovary. Seventeen of the ewes were from a flock that had grazed strongly oestrogenic Yarloop subterranean clover pasture for 3 years. Only 43 per cent of the ewes in this flock had produced a lamb from the previous mating. The other 8 ewes grazed non-oestrogenic pastures and all had lambed after the previous mating. Each cervix was split longitudinally. From one half, transverse segments 0.5 cm thick were taken from the rostral, middle and caudal portions of the cervix and were immediately immersed in isopentane and frozen in a liquid nitrogen bath. The other half was fixed whole in cold 10 per cent buffered neutral formol-saline. Segments were also cut from the body of the unopened uterus for both freezing and formol fixation. Staining. (a) Para@ sections. The formalin-fixed tissues were routinely processed, paraffin embedded and cut at 7 pm. Sections were stained using: (1) (2)
Haematoxylin and eosin. Various methods for the identification (1968), including the Schmorl method
of lipopigments suggested by Pearse for lipofuscin, Gomori’s chrome alum
354
13 (5) (6)
(7)
R.
A.
HEYDON
AND
N.
R.
ADAMS
haematoxylin method for lipofuscins, the Long-Ziehl-Neelsen method for acid fast lipofuscins, Sudan Black B and the periodic acid-Schiff (PAS) reaction. Gomori’s trichrome, as described by Luna (1968). PAS-Alcian blue method for mucosubstances at pH 2.5 (Luna, 1968). The Gallocyanin-chromalum method for ribonucleic acid of Berube, Powers, Kerkay and Clark (1966). Heidenhain’s iron haematoxylin, as prepared by Lillie (1965) for distinguishing ciliated cells. For counts of ciliated cells 100 cells were counted from the midregion of each cervix in three random fields, as described by Restall and Lightfoot ( 1970). The procedures outlined in Pearse (1968) to show proteins containing tyrosine, arginine and -SH groups, including Baker’s modifications of the Millon reaction and the Sakaguchi reaction, and the Dihydroxy-dinaphthyldisulphide method.
(b) Frozen sections. The frozen sections for those used in the NADH-diaphorase at 4 “C in 4 per cent formaldehyde in slides were washed in running tap water The enzymes studied were:
were cut at 10 pm in a cryostat and, except reaction, were fixed immediately for 20 min Holt’s gum sucrose. Prior to incubation the for 5 min.
(1)
NADH-d’ za@horuse, stained by the Nitro-BT method of Pearse (1968) and subsequently fixed for 15 min in buffered formalin. (2) Acid phosphatase, using both the Naphthol AS and hexazotized pararosanilin method of Barka and Anderson as described by Pearse (1968), and Lake’s (1965) modification of Gomori’s lead acetate method. (3) Alkaline phosjhtuse, by the calcium-cobalt method of Gomori, as in Pearse (1968). (4) Jlion-spec$cesteruse, by the hexazotized pararosanilin and cc-naphthyl acetate method, as described in Pearse (1968). Control slides were prepared by incubation in a portion of the test solution from which the substrate had been omitted. No nuclear counterstains were used to avoid introducing a second stain which could obscure the final reaction product. Lipid droplets were demonstrated by impregnation with Sudan Black B in propylene glycol (Luna, 1968). RESULTS
The intensity of staining of each cell type was uniform throughout the cervix with each stain used. No differences were observed between ewes exposed to phyto-oestrogen and control ewes in the distribution or intensity of staining of the epithelium of the crypts or surface of the cervix. The staining reactions of the cervical glands, which occurred only in the oestrogen-affected ewes, could therefore be compared with those of the surface epithelium in the one section, allowing a more accurate assessmentof variations in staining intensity. Where possible, the staining reactions have been summarized in Table 1. (A) Para$in Sections (1) General histology. The architectural pattern of the normal sheep cervix was similar to that described by Restall (1966). In contrast to the thin villi found in control ewes [Fig. l(a)], villi in ewes with clover disease were
CERVICAL
GLANDS
IN CLOVER TABLE
SOME STAINING
REACTIONS
OF CERVICAL
AND
I
UTERINE EPITHELIUM EWES
OF CLOVER-AFFECTED
Source
Cervicalsurface, affected and control ewes
Stain
C;allocyanin-chromalum .Millon reaction Proportion of ciliated NADH-diaphorase &id phosphatase: (a) Naphthol A.S. (b) Gomori .Alkaline phosphatase Non-specific esterase Sudan Black B
0: no stain;
f , very
faint
+.
glands. ewes
l’terine glandJ affected and contrai ewe5
42 per cent +
* +
-c + &. ./.
:+ 0
i’ 0
slight
CONTROL
+ 0
42 per cent ++
stain;
AND
of epithelium
Cervical a&ted on&
++ +
cells
355
DISEASE
stain:
$- +,
moderate
stain;
-i- $ --.
strong
stain.
flattened, and appeared in places to have fused [Fig. 1 (b)]. Tubular convoluted glands similar to those previously described (Adams, 1976), were found in the cervix of the phyto-oestrogen-exposed ewes. The glands could easily be distinguished from crypts by their coiled tubular structure, narrow dimensions, and position in the lamina propria. The cervical lamina propria of affected ewes took on a more uterine appearance and frequently contained increased numbers of plasmacytes and neutrophils.
l:ig.
1. Cervix from normal (a) and clover-diseased (b) ewes, showing the blunting and fusion of villi in clover disease. The darkly staining mucus material is prominent in the surface epithelium of the control ewe, and almost absent from the glands of the (‘we with clover disease. Alcian blue-PAS stain, ‘/ 2.5.
(2) Ciliated cells. Ciliated cells were found, in variable numbers, in all parts of the cervical and uterine glands. Both glandular and surface epithelium of the cervix had an average of 42 per cent of ciliated cells, whereas the uterine glands had only 13 per cent. (3) Glandular epithelial pigment. An accumulation of pale yellow refractile
356
R.
A.
HEYDON
AND
N.
R.
ADAMS
pigment was seen in the epithelial cells of shallow glands. This pigment was stained with Sudan Black B and was present in about one-quarter of the frozen sections of cervix from ewes with clover disease (Fig. 2), but not in control ewes. Sudan Black B impregnated paraffin sections revealed that about one-third of the granules of this pigment were soluble in fat solvents.
Fig. 2. Shallow: cervical gland Black B. frozen section,
from a ewe with x 800.
clover
disease,
showing
lipid
pigment
masses.
Sudan
Only a small amount of the original pigment stained with either Schmorl’s method or the chrome alum haematoxylin procedure for oxidized lipofuscins. However, all the lipopigment was found to be acid fast and PAS positive. Granules also had strong acid phosphatase activity. This evidence suggests that lipofuscins of the ceroid type were common in the epithelium of the cervical glands of the clover-affected ewes. There was only a small amount of true lipofuscin and autoxidizing lipid. No lipopigments were found in the uterine glands. (4) Gomori’s trichrome connective tissue stain. In ewes which had been grazing oestrogenic clover the lamina propria of the cervix was very cellular with few strands of collagen. The stromal cells had large, ovoid nuclei, and the appearance was strikingly similar to that observed in the uterus. The lamina propria of control sheep was not very cellular and consisted of a dense mat of collagen fibres.
CERVICAL
GLANDS
IN
CLOVER
DISEASE
357
(5) Alcian blue and periodic acid-Scfzi$ at PH 2.5. In both affected and control ewes, cervical mucus stained purple. The surface and crypt epithelium of the cervix of clover-affected ewes contained considerably less mucus than that of the control ewes. Even so, the cervical epithelium of the ewes affected with clover disease was stained much more intensely than that of the cervical glands. Many glands were unstained, particularly in their deepest parts (Fig. 1). The uterine glands were unstained, apart from a few fine PAS-positive droplets throughout the cells. (6) Gallocyanin-chromalum. The surface and crypt epithelial cells of the cervix were moderately stained. The epithelial cells of the cervical glands, particularly those deep in the glands, generally contained little diffuse stain in the cytoplasma. In contrast, the uterine glands were rather more intensely stained. particularly in their more distal parts (Table 1).
Fig.
3. cervix (a) and uterus (b) from distribution of NADH-diaphorase than in the glands. x 140.
a ewe with clover staining. In both
disease, showing the surface cases. staining is more intense
and glandular on the surface
(7) Protein stains. The Millon reaction for tyrosine showed that the epithelium of both the cervical and uterine glands was unstained, but the cervical surface epithelium showed a slight reaction. A similar result was obtained when staining for proteins containing -SH groups. The whole cervical and uterine tissue gave a negative reaction with the Sakaguchi reaction for arginine.
358
R. A. HEYDON
AND
N. R. ADAMS
(B) Frozen Sections (1) JVADH-d’ laph orase. The epithelium of both the surface and crypts of the cervix was consistently well stained (Fig. 3). Both cervical and uterine glands stained similarly, in that they were as dark as the surface epithelium in their shallowest parts, but were paler in the deeper parts. The cervical glands were slightly paler than those of the uterus (Table 1). (2) Acid phosphatase. (a) Naphthol-AS method. Surface and crypt epithelium of the cervix contained relatively little stain. It was consistently found that the cervical glands, particularly in their deepest parts, contained much more demonstrable enzyme than the surface and crypt epithelium.
Fig. 4. Cervix froman ewe with clover disease, showing the more intense staining compared with the surface. Gomori’s acid phosphatase stain. % 50.
ofglandular
epithelium,
Although not every gland stained with equal intensity, the cervical and uterine glands appeared to stain similarly-both in regard to intensity and to the diffuse, apical distribution of the stain. (b) Gomori’s method. Again the cervical and uterine glands were found to stain very similarly and strongly. Both stained moderately well in the superficial parts of the glands and became progressively more intensely stained until they were almost completely black in their deepest parts (Fig. 4). However, the uterine glands were intensely stained from the mid-gland region distally, whereas only the bases of the cervical glands were so strongly stained.
CERVICAL
GLANDS
IN CLOVER
DISEASE
359
In contrast, the epithelium of the surface and crypts of the cervix was almost unstained (Table 1). The lipofuscin in the glandular epithelium of the cervix stained very intensely with both methods for acid phosphatase. (3) Alkaline phosphatase. No consistent difference was detected between the surface and glandular epithelium in the cervix. Both contained little of this enzyme and there was no progressive alteration in staining towards the distal end of the glands. Although not all the uterine glands contained equal amounts of this enzyme, they were more intensely stained than the cervical glands (Table 1). was well stained. (4) Non-specijc esterase. The cervical surface epithelium Cells of the cervical glands, particularly in their distal regions, stained less intensely. The uterine glands for most of their length contained a similar amount 01‘ enzyme to the cervical glands. However, in the more distal parts of the utcrinr glands, the cells were more strongly stained. 1’5) Lipids. Apart from lipofuscin pigments, there was no demonstrable lipid in the epithelium of either cervix or uterus. DISCUSSION
The staining reactions of the cervical glands were intermediate in intensity between those of the cervical surface epithelium and the uterine glands. With rvery stain used, the cervical glands, at least in their most distal parts, reacted differently from the epithelium of the crypts and surface. The lamina propria also became more like that of the uterus. Uterine and cervical glands were uniformly stained for acid phosphatase by the Naphthol-AS method, and for the various amino-acid radicals. However, staining with NADH-diaphorasc, alkaline phosphatase, Gomori’s acid phosphatase and the gallocyaninchromalum stain was in each case less intense in the cervical than in the utcrim glands. Furthermore, in contrast to the uterine glands, cervical glands contained many more ciliated cells. From these results it appears that chronic exposure to plant ocstrogcns stimulates the cervix to transform towards a uterine-type structure. The transition does not appear to be abrupt. At the mouth of the cervical glands thr cbpithelium appears to be still of cervical nature. Lower down the glands the cpithelium becomes progressively less like cervical cells and more like those of the uterine endometrial glands. In the embryological development of man, oestrogen is hclieved to cause the cervix to develop in size and complexity (Mossman, 1973). Protrusion and fusion of villi cause branching crypts to develop, resulting in a “glandular“ appearance (Fluhmann, 1958). Although within the cervix of the ewes treated with plant oestrogens there appeared to be some fusion of villi and cellular multiplication in the stoma, it w-as obvious that the glands arose by downgrowth of epithelium from the crypts. The histochemical results also support the contention that the epithelium of the cervical glands formed in clover disease is distinct from that of villi and thus is not analogous to the gland-likt
360
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HEYDON
AND
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R.
ADAMS
clefts found in the normal human cervix (Fluhmann, 1958; Tack and Shilkin, 1970). In the cervix of mice neonatally treated with oestradiol, the surface epithelium permanently assumes a uterine appearance, and glands which form in the stroma show the same distribution of acid and alkaline phosphatase as uterine epithelium (Forsberg, 1969). Although this is a far more radical change than occurs in the ewe, the two processes appear to be qualitatively similar, and it is possible that a similar process is involved in both phenomena. Lipofuscin pigments are not uncommon in reproductive tissues. Cervicitis is common in ewes with clover disease (Adams, 1976), and this may have contributed to the relatively high incidence of pigment in affected ewes. It is worth noting, however, that most of the pigments were in the relatively unoxidized, “ceroid” form, and that prolonged treatment with oestrogen has been reported to cause the appearance of similar ceroid pigment in the adrenal of the mouse (Alpert, 1953). It is interesting to speculate on the possible functions of the cervical glands. They contained less RNA, a lower concentration of certain amino-acid radicals and lower activity of the enzyme NADH-diaphorase compared with normal cervical epithelium and are therefore probably not as active as the rest of the cervix. Since these glands resemble those of the uterus, rather than the cervix, in their morphology and staining characteristics, it is possible that they do also in function. Analysis of the cervico-vaginal fluid from affected ewes for components normally found in uterine secretions may, therefore, prove valuable. SUMMARY
Histological and histochemical observations were made on the cervices and uteri from 17 ewes with clover disease, and on 8 controls. The coiled, tubular cervical glands found in clover disease contained very little mucin demonstrable by alcian blue or PAS, but had a similar proportion of ciliated cells to normal cervical epithelium. In other aspects, including staining for acid and alkaline phosphatase, NADH-diaphorase, amino acid radicals, and with gallocyaninchromalum, the cervical glands were similar to uterine glands, although staining in the cervical glands was generally less intense. It is concluded that prolonged exposure of ewes to plant oestrogens induces a transformation of the cervix towards a uterine type of structure while still retaining some characteristics of cervix. REFERENCES
Adams, N. R. (1976). Pathological changes in the tissues of infertile ewes with clover disease. Journal of Comparative Pathology, 86, 29-35. Alpert, M. (1953). Hormonal induction of deposition of ceroid pigment in the mouse. The Anatomical Record, 116, 469-493. Berube, G. R., Powers, M. M., Kerkay, J., and Clark, G. (1966). The Gallocyaninchrome alum stain: influence of methods of preparation on its activity and separation of active staining compound. Stain Technology, 41, 73-81. Fluhmann, C. F. (1958). The glandular structures of the cervix uteri. Surgery, Gynecology and Obstetrics, 166, 715-723.
CERVICAL
GLANDS
IN
CLOVER
DISEASE
361
Forsberg, J. G. (1969). The development of atypical epithelium in the mouse uterine cervix and vaginal fornix after neonatal oestradiol treatment. British Journal qf Experimental Pathology, 50, 187-195. Lake, B. D. (1965). The histochemistry of phosphatases: the use of lead acetatr instead of lead nitrate. Journal of the Royal Microstopical Society, 85, 73-75. Lillie. R. D. (1965). Histopathologic Technic and Practical Histochemis+~, 3rd edit.. McGraw-Hill Book Co., New York. Luna, L. G. (1968). Manual of Histological Staining Methods of the Armed Forces Znsfitufu qf Pathology, 3rd edit., McGraw-Hill Book Co., New York. Mossman, H. W. (1973). The embryology of the cervix. In The Biology of the C’ervi.\, Blandon, R. J. and Moghissi, K., Eds, The University of Chicago Press, Chicago. Pcarse, A. G. E. (1968). Histochemistry : Theoretical and Applied, 3rd edit., J. and A. Churchill Ltd, London. Restall, B. J. (1966). Histological observations on the reproductive tract of the cwt’. Australian Journal of Biological Sciences, 19, 673-686. Kestall, B. J., and Lightfoot, R. J. (1970). Ciliated cells in cervix of the cwt. Justralian Journal of Biological Sciences, 23, 1265-l 269. Schinckel, P. G. (1948). Infertility in ewes grazing subterranean clover pastures. Observations on breeding behaviour following transfer to ‘isound” country. Australian Veterinary Journal, 24, 289-294. Smith, J, F. (1971). Studies on ovine infertility in agricultural regions of Western Australia: cervical mucus production by fertile and infertile ewes. *4ustralian Journal of Agricultural Research, 22, 513-519. Tack, E. P. C., and Shilkin, K. B. (1970). Histochemical study of mucosubstances and glycogen of the postmenopausal human cervix uteri. American .yournal uJ’ Obstetrics and Gynecology, 107, 194-20 1. [Received for publication,
August 3 lst, 19761
PATH.
1977.
87.
VOL.
HISTOCHEMICAL
STUDIES ON CERVICAL WITH CLOVER DISEASE
EWES
R. Diraision
353
A.
of Animal
HEYDON Health,
CSIRO,
and PIivale
N.
R.
GLANDS
IN
ADAMS
Bag, ll’embly,
lVe.ctern :htralia
INTRODUCTION
Ewes which have grazed oestrogenic subterranean clover for prolonged periods develop clover disease and become permanently infertile (Schinckel, 1948). The infertility is thought to be due primarily to abnormal cervical secretions which impair the establishment of spermatozoa in the cervix after mating (Smith, 1971). C onvoluted tubular glands form in the cervix of the infertile ewes and have been shown to react differently to stains for mucus when compared to the normal cervical epithelium (Adams, 1976). These cervical glands therefore appear likely to play a role in the abnormal cervical secretion and the infertility. Consequently, the nature of the cervical glands in ewes with clover disease is worthy of further study. In the female mouse treated with oestrogen at birth, glands with a uterine type of epithelium develop in the cervix (Forsberg, 1969). In women, on the other hand, gland-like clefts occur as part of the normal cervical architecture (Fluhmann, 1958). The present study compares the histology and histochemistry of cervical glands in clover-diseased ewes with both uterine glands and the cervical epithelium of normal ewes in order to better define theit nature. MATERIALS
AND
METHODS
Biological material. The reproductive tracts were removed from 25 mature, nonpregnant South Australian type Merino ewes at the time of slaughter. The ewes were in early metoestrus, as shown by the presence of a fresh corpus luteum, less than 3 mm in diameter, in the ovary. Seventeen of the ewes were from a flock that had grazed strongly oestrogenic Yarloop subterranean clover pasture for 3 years. Only 43 per cent of the ewes in this flock had produced a lamb from the previous mating. The other 8 ewes grazed non-oestrogenic pastures and all had lambed after the previous mating. Each cervix was split longitudinally. From one half, transverse segments 0.5 cm thick were taken from the rostral, middle and caudal portions of the cervix and were immediately immersed in isopentane and frozen in a liquid nitrogen bath. The other half was fixed whole in cold 10 per cent buffered neutral formol-saline. Segments were also cut from the body of the unopened uterus for both freezing and formol fixation. Staining. (a) Para@ sections. The formalin-fixed tissues were routinely processed, paraffin embedded and cut at 7 pm. Sections were stained using: (1) (2)
Haematoxylin and eosin. Various methods for the identification (1968), including the Schmorl method
of lipopigments suggested by Pearse for lipofuscin, Gomori’s chrome alum
354
13 (5) (6)
(7)
R.
A.
HEYDON
AND
N.
R.
ADAMS
haematoxylin method for lipofuscins, the Long-Ziehl-Neelsen method for acid fast lipofuscins, Sudan Black B and the periodic acid-Schiff (PAS) reaction. Gomori’s trichrome, as described by Luna (1968). PAS-Alcian blue method for mucosubstances at pH 2.5 (Luna, 1968). The Gallocyanin-chromalum method for ribonucleic acid of Berube, Powers, Kerkay and Clark (1966). Heidenhain’s iron haematoxylin, as prepared by Lillie (1965) for distinguishing ciliated cells. For counts of ciliated cells 100 cells were counted from the midregion of each cervix in three random fields, as described by Restall and Lightfoot ( 1970). The procedures outlined in Pearse (1968) to show proteins containing tyrosine, arginine and -SH groups, including Baker’s modifications of the Millon reaction and the Sakaguchi reaction, and the Dihydroxy-dinaphthyldisulphide method.
(b) Frozen sections. The frozen sections for those used in the NADH-diaphorase at 4 “C in 4 per cent formaldehyde in slides were washed in running tap water The enzymes studied were:
were cut at 10 pm in a cryostat and, except reaction, were fixed immediately for 20 min Holt’s gum sucrose. Prior to incubation the for 5 min.
(1)
NADH-d’ za@horuse, stained by the Nitro-BT method of Pearse (1968) and subsequently fixed for 15 min in buffered formalin. (2) Acid phosphatase, using both the Naphthol AS and hexazotized pararosanilin method of Barka and Anderson as described by Pearse (1968), and Lake’s (1965) modification of Gomori’s lead acetate method. (3) Alkaline phosjhtuse, by the calcium-cobalt method of Gomori, as in Pearse (1968). (4) Jlion-spec$cesteruse, by the hexazotized pararosanilin and cc-naphthyl acetate method, as described in Pearse (1968). Control slides were prepared by incubation in a portion of the test solution from which the substrate had been omitted. No nuclear counterstains were used to avoid introducing a second stain which could obscure the final reaction product. Lipid droplets were demonstrated by impregnation with Sudan Black B in propylene glycol (Luna, 1968). RESULTS
The intensity of staining of each cell type was uniform throughout the cervix with each stain used. No differences were observed between ewes exposed to phyto-oestrogen and control ewes in the distribution or intensity of staining of the epithelium of the crypts or surface of the cervix. The staining reactions of the cervical glands, which occurred only in the oestrogen-affected ewes, could therefore be compared with those of the surface epithelium in the one section, allowing a more accurate assessmentof variations in staining intensity. Where possible, the staining reactions have been summarized in Table 1. (A) Para$in Sections (1) General histology. The architectural pattern of the normal sheep cervix was similar to that described by Restall (1966). In contrast to the thin villi found in control ewes [Fig. l(a)], villi in ewes with clover disease were
CERVICAL
GLANDS
IN CLOVER TABLE
SOME STAINING
REACTIONS
OF CERVICAL
AND
I
UTERINE EPITHELIUM EWES
OF CLOVER-AFFECTED
Source
Cervicalsurface, affected and control ewes
Stain
C;allocyanin-chromalum .Millon reaction Proportion of ciliated NADH-diaphorase &id phosphatase: (a) Naphthol A.S. (b) Gomori .Alkaline phosphatase Non-specific esterase Sudan Black B
0: no stain;
f , very
faint
+.
glands. ewes
l’terine glandJ affected and contrai ewe5
42 per cent +
* +
-c + &. ./.
:+ 0
i’ 0
slight
CONTROL
+ 0
42 per cent ++
stain;
AND
of epithelium
Cervical a&ted on&
++ +
cells
355
DISEASE
stain:
$- +,
moderate
stain;
-i- $ --.
strong
stain.
flattened, and appeared in places to have fused [Fig. 1 (b)]. Tubular convoluted glands similar to those previously described (Adams, 1976), were found in the cervix of the phyto-oestrogen-exposed ewes. The glands could easily be distinguished from crypts by their coiled tubular structure, narrow dimensions, and position in the lamina propria. The cervical lamina propria of affected ewes took on a more uterine appearance and frequently contained increased numbers of plasmacytes and neutrophils.
l:ig.
1. Cervix from normal (a) and clover-diseased (b) ewes, showing the blunting and fusion of villi in clover disease. The darkly staining mucus material is prominent in the surface epithelium of the control ewe, and almost absent from the glands of the (‘we with clover disease. Alcian blue-PAS stain, ‘/ 2.5.
(2) Ciliated cells. Ciliated cells were found, in variable numbers, in all parts of the cervical and uterine glands. Both glandular and surface epithelium of the cervix had an average of 42 per cent of ciliated cells, whereas the uterine glands had only 13 per cent. (3) Glandular epithelial pigment. An accumulation of pale yellow refractile
356
R.
A.
HEYDON
AND
N.
R.
ADAMS
pigment was seen in the epithelial cells of shallow glands. This pigment was stained with Sudan Black B and was present in about one-quarter of the frozen sections of cervix from ewes with clover disease (Fig. 2), but not in control ewes. Sudan Black B impregnated paraffin sections revealed that about one-third of the granules of this pigment were soluble in fat solvents.
Fig. 2. Shallow: cervical gland Black B. frozen section,
from a ewe with x 800.
clover
disease,
showing
lipid
pigment
masses.
Sudan
Only a small amount of the original pigment stained with either Schmorl’s method or the chrome alum haematoxylin procedure for oxidized lipofuscins. However, all the lipopigment was found to be acid fast and PAS positive. Granules also had strong acid phosphatase activity. This evidence suggests that lipofuscins of the ceroid type were common in the epithelium of the cervical glands of the clover-affected ewes. There was only a small amount of true lipofuscin and autoxidizing lipid. No lipopigments were found in the uterine glands. (4) Gomori’s trichrome connective tissue stain. In ewes which had been grazing oestrogenic clover the lamina propria of the cervix was very cellular with few strands of collagen. The stromal cells had large, ovoid nuclei, and the appearance was strikingly similar to that observed in the uterus. The lamina propria of control sheep was not very cellular and consisted of a dense mat of collagen fibres.
CERVICAL
GLANDS
IN
CLOVER
DISEASE
357
(5) Alcian blue and periodic acid-Scfzi$ at PH 2.5. In both affected and control ewes, cervical mucus stained purple. The surface and crypt epithelium of the cervix of clover-affected ewes contained considerably less mucus than that of the control ewes. Even so, the cervical epithelium of the ewes affected with clover disease was stained much more intensely than that of the cervical glands. Many glands were unstained, particularly in their deepest parts (Fig. 1). The uterine glands were unstained, apart from a few fine PAS-positive droplets throughout the cells. (6) Gallocyanin-chromalum. The surface and crypt epithelial cells of the cervix were moderately stained. The epithelial cells of the cervical glands, particularly those deep in the glands, generally contained little diffuse stain in the cytoplasma. In contrast, the uterine glands were rather more intensely stained. particularly in their more distal parts (Table 1).
Fig.
3. cervix (a) and uterus (b) from distribution of NADH-diaphorase than in the glands. x 140.
a ewe with clover staining. In both
disease, showing the surface cases. staining is more intense
and glandular on the surface
(7) Protein stains. The Millon reaction for tyrosine showed that the epithelium of both the cervical and uterine glands was unstained, but the cervical surface epithelium showed a slight reaction. A similar result was obtained when staining for proteins containing -SH groups. The whole cervical and uterine tissue gave a negative reaction with the Sakaguchi reaction for arginine.
358
R. A. HEYDON
AND
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(B) Frozen Sections (1) JVADH-d’ laph orase. The epithelium of both the surface and crypts of the cervix was consistently well stained (Fig. 3). Both cervical and uterine glands stained similarly, in that they were as dark as the surface epithelium in their shallowest parts, but were paler in the deeper parts. The cervical glands were slightly paler than those of the uterus (Table 1). (2) Acid phosphatase. (a) Naphthol-AS method. Surface and crypt epithelium of the cervix contained relatively little stain. It was consistently found that the cervical glands, particularly in their deepest parts, contained much more demonstrable enzyme than the surface and crypt epithelium.
Fig. 4. Cervix froman ewe with clover disease, showing the more intense staining compared with the surface. Gomori’s acid phosphatase stain. % 50.
ofglandular
epithelium,
Although not every gland stained with equal intensity, the cervical and uterine glands appeared to stain similarly-both in regard to intensity and to the diffuse, apical distribution of the stain. (b) Gomori’s method. Again the cervical and uterine glands were found to stain very similarly and strongly. Both stained moderately well in the superficial parts of the glands and became progressively more intensely stained until they were almost completely black in their deepest parts (Fig. 4). However, the uterine glands were intensely stained from the mid-gland region distally, whereas only the bases of the cervical glands were so strongly stained.
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In contrast, the epithelium of the surface and crypts of the cervix was almost unstained (Table 1). The lipofuscin in the glandular epithelium of the cervix stained very intensely with both methods for acid phosphatase. (3) Alkaline phosphatase. No consistent difference was detected between the surface and glandular epithelium in the cervix. Both contained little of this enzyme and there was no progressive alteration in staining towards the distal end of the glands. Although not all the uterine glands contained equal amounts of this enzyme, they were more intensely stained than the cervical glands (Table 1). was well stained. (4) Non-specijc esterase. The cervical surface epithelium Cells of the cervical glands, particularly in their distal regions, stained less intensely. The uterine glands for most of their length contained a similar amount 01‘ enzyme to the cervical glands. However, in the more distal parts of the utcrinr glands, the cells were more strongly stained. 1’5) Lipids. Apart from lipofuscin pigments, there was no demonstrable lipid in the epithelium of either cervix or uterus. DISCUSSION
The staining reactions of the cervical glands were intermediate in intensity between those of the cervical surface epithelium and the uterine glands. With rvery stain used, the cervical glands, at least in their most distal parts, reacted differently from the epithelium of the crypts and surface. The lamina propria also became more like that of the uterus. Uterine and cervical glands were uniformly stained for acid phosphatase by the Naphthol-AS method, and for the various amino-acid radicals. However, staining with NADH-diaphorasc, alkaline phosphatase, Gomori’s acid phosphatase and the gallocyaninchromalum stain was in each case less intense in the cervical than in the utcrim glands. Furthermore, in contrast to the uterine glands, cervical glands contained many more ciliated cells. From these results it appears that chronic exposure to plant ocstrogcns stimulates the cervix to transform towards a uterine-type structure. The transition does not appear to be abrupt. At the mouth of the cervical glands thr cbpithelium appears to be still of cervical nature. Lower down the glands the cpithelium becomes progressively less like cervical cells and more like those of the uterine endometrial glands. In the embryological development of man, oestrogen is hclieved to cause the cervix to develop in size and complexity (Mossman, 1973). Protrusion and fusion of villi cause branching crypts to develop, resulting in a “glandular“ appearance (Fluhmann, 1958). Although within the cervix of the ewes treated with plant oestrogens there appeared to be some fusion of villi and cellular multiplication in the stoma, it w-as obvious that the glands arose by downgrowth of epithelium from the crypts. The histochemical results also support the contention that the epithelium of the cervical glands formed in clover disease is distinct from that of villi and thus is not analogous to the gland-likt
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clefts found in the normal human cervix (Fluhmann, 1958; Tack and Shilkin, 1970). In the cervix of mice neonatally treated with oestradiol, the surface epithelium permanently assumes a uterine appearance, and glands which form in the stroma show the same distribution of acid and alkaline phosphatase as uterine epithelium (Forsberg, 1969). Although this is a far more radical change than occurs in the ewe, the two processes appear to be qualitatively similar, and it is possible that a similar process is involved in both phenomena. Lipofuscin pigments are not uncommon in reproductive tissues. Cervicitis is common in ewes with clover disease (Adams, 1976), and this may have contributed to the relatively high incidence of pigment in affected ewes. It is worth noting, however, that most of the pigments were in the relatively unoxidized, “ceroid” form, and that prolonged treatment with oestrogen has been reported to cause the appearance of similar ceroid pigment in the adrenal of the mouse (Alpert, 1953). It is interesting to speculate on the possible functions of the cervical glands. They contained less RNA, a lower concentration of certain amino-acid radicals and lower activity of the enzyme NADH-diaphorase compared with normal cervical epithelium and are therefore probably not as active as the rest of the cervix. Since these glands resemble those of the uterus, rather than the cervix, in their morphology and staining characteristics, it is possible that they do also in function. Analysis of the cervico-vaginal fluid from affected ewes for components normally found in uterine secretions may, therefore, prove valuable. SUMMARY
Histological and histochemical observations were made on the cervices and uteri from 17 ewes with clover disease, and on 8 controls. The coiled, tubular cervical glands found in clover disease contained very little mucin demonstrable by alcian blue or PAS, but had a similar proportion of ciliated cells to normal cervical epithelium. In other aspects, including staining for acid and alkaline phosphatase, NADH-diaphorase, amino acid radicals, and with gallocyaninchromalum, the cervical glands were similar to uterine glands, although staining in the cervical glands was generally less intense. It is concluded that prolonged exposure of ewes to plant oestrogens induces a transformation of the cervix towards a uterine type of structure while still retaining some characteristics of cervix. REFERENCES
Adams, N. R. (1976). Pathological changes in the tissues of infertile ewes with clover disease. Journal of Comparative Pathology, 86, 29-35. Alpert, M. (1953). Hormonal induction of deposition of ceroid pigment in the mouse. The Anatomical Record, 116, 469-493. Berube, G. R., Powers, M. M., Kerkay, J., and Clark, G. (1966). The Gallocyaninchrome alum stain: influence of methods of preparation on its activity and separation of active staining compound. Stain Technology, 41, 73-81. Fluhmann, C. F. (1958). The glandular structures of the cervix uteri. Surgery, Gynecology and Obstetrics, 166, 715-723.
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Forsberg, J. G. (1969). The development of atypical epithelium in the mouse uterine cervix and vaginal fornix after neonatal oestradiol treatment. British Journal qf Experimental Pathology, 50, 187-195. Lake, B. D. (1965). The histochemistry of phosphatases: the use of lead acetatr instead of lead nitrate. Journal of the Royal Microstopical Society, 85, 73-75. Lillie. R. D. (1965). Histopathologic Technic and Practical Histochemis+~, 3rd edit.. McGraw-Hill Book Co., New York. Luna, L. G. (1968). Manual of Histological Staining Methods of the Armed Forces Znsfitufu qf Pathology, 3rd edit., McGraw-Hill Book Co., New York. Mossman, H. W. (1973). The embryology of the cervix. In The Biology of the C’ervi.\, Blandon, R. J. and Moghissi, K., Eds, The University of Chicago Press, Chicago. Pcarse, A. G. E. (1968). Histochemistry : Theoretical and Applied, 3rd edit., J. and A. Churchill Ltd, London. Restall, B. J. (1966). Histological observations on the reproductive tract of the cwt’. Australian Journal of Biological Sciences, 19, 673-686. Kestall, B. J., and Lightfoot, R. J. (1970). Ciliated cells in cervix of the cwt. Justralian Journal of Biological Sciences, 23, 1265-l 269. Schinckel, P. G. (1948). Infertility in ewes grazing subterranean clover pastures. Observations on breeding behaviour following transfer to ‘isound” country. Australian Veterinary Journal, 24, 289-294. Smith, J, F. (1971). Studies on ovine infertility in agricultural regions of Western Australia: cervical mucus production by fertile and infertile ewes. *4ustralian Journal of Agricultural Research, 22, 513-519. Tack, E. P. C., and Shilkin, K. B. (1970). Histochemical study of mucosubstances and glycogen of the postmenopausal human cervix uteri. American .yournal uJ’ Obstetrics and Gynecology, 107, 194-20 1. [Received for publication,
August 3 lst, 19761