Changes in cervical histology in ewes following prolonged grazing on oestrogenic subterranean clover

J. COMP.

PATH.

1979.

CHANGES FOLLOWING

VOL.

89.

367

IN CERVICAL PROLONGED SUBTERRANEAN

HISTOLOGY GRAZING ON CLOVER

IN EWES OESTROGENIC

BY

R. J.

LIGHTFOOT

Department of .4griculture, South Perth, Western Australia, 6151 and

N. R. ADAMS DiGsion of Animal Health, CSIRO. C’nioersi!vof Wz4, Nedlands, Western Australia, 6009 IBTRODUCTION

The permanent infertility seen in ewes following prolonged grazing on oestrogenic subterranean clover pastures is largely due to reduced ovum fertilization, associated with a failure of sperm transport. Lightfoot, Croker and Neil (1967) presented evidence indicating that the primary breakdown in the sperm transport process occurs in the cervix. It is likely that this breakdown is associated with an abnormal pattern of cervical mucus production observed by Smith (1971) and Lightfoot, Smith, Cumming, Marshall, Wroth and Hearnshaw (1974) in affected ewes. Histological studies suggest that this process is accompanied by a transformation of the cervix towards a more uterine appearance (Adams, 1976; Heydon and Adams, 1977). Opportunity has been taken in the present study to carry out further histological observations on the cervices of clover-affected ewes drawn from 3 separate experiments. The large number of samples available allowed the results to be analysed in a semi-quantitative fashion, so that previously unreported changes could be detected. In particular, the quantitative distribution of glandular tissue was studied because of possible associations with mucus production and the mechanism of failure in sperm transport. MATERIALS

AND

METHODS

Sheep. Aged Merino ewes were obtained from 3 separate studies which had been designed to compare the fertility of ewes grazing oestrogenic or non-oestrogenic pastures. Details of the ewes and their grazing histories are given in Table 1. In each experiment the ewes subjected to prolonged grazing on oestrogenic pasture suffered a progressive decline in fertility. More complete descriptions of the grazing trials have already been published (Davies and Maller, 1970; Lightfoot et al., 1974; Neil, Lightfoot and Fells, 1974). After the development of infertility, ewes were grazed on non-oestrogenic pasture for at least 7 months (Table 1) to ensure that the changes observed were of a permanent nature, and were not confounded with changes due to concurrent phyto-oestrogenic intake. Collection and treatment of tissues. In Experiments 1 and 3, the eweswere run on nonoestrogenic pasture with vasectomized rams fitted with harnesses and crayons, and the ewes were killed within 24 h of the onset of oestrus. In Experiment 2, all ewes were slaughtered at a common time, regardless of the stage of the oestrous cycle. oozl-9975/79/030367+07

$02.00/O

0

1979

Academic

Press

Inc.

(London)

Limited

368

R.

J.

LIGHTFOOT

AND

N.

R.

ADAMS

At slaughter the genital tract was removed and the cervix was severed from the uterus. In Experiment 1, the cervix was frozen in liquid nitrogen and stored at - 15 “C until frozen sections were prepared from cranial, middle and caudal segments. In Experiment 2 the 3 cervical segments were placed immediately in buffered formol-saline. In Experiment 3 the cervix was flushed with saline through the cervical canal for sperm recovery before division into cranial, middle and caudal segments and then fixed in buffered formol-saline. Paraffin-embedded sections were cut from cranial, middle and caudal segments of the cervix in Experiments 2 and 3. All sections were stained with haematoxylin and eosin. Examination of sections. Sections were examined without knowledge of the treatment groups to which they belonged. Histological features were quantified as follows: Glandular development. Tubular, convoluted glands were identified by their position in the lamina propria and by their circular or elliptical shape on cross-section. The total number of gland intersections was counted on each slide. StratiJiedsquamousepithelium. Score: 0 = absent; 1 = stratified squamous epithelium present in some cervical crypts; 2 = extensive areas of stratified squamous epithelium covering both cervical crypts and villi. Proportion of columnar epithelial cells identiJable as mucus cells. Score : 0 = (6 per cent mucus cells; 1 = 6 to 25 per cent mucus cells; 2 = >25 per cent mucus cells. Acute cervicitis. Score: 0 = absent; 1 = large numbers of neutrophils and mononuclear cells passing through he epithelium and lamina propria, usually associated with degenerating epithelial cells. Chronic cervicitis. Score: 0 = absent; 1 = most high power ( Y 400) microscopic fields of the lamina propria contained less than 5 plasma cells; 2 = many plasma cells, often aggregated in groups and accompanied by mononuclear cells and lymphoid follicles. Statistical analyses. The data were analysed by Chi Square according to the method of Claringbold (1961). For the purpose of analysis previous grazing history (control v oestrogenic) was regarded as a treatment, the 3 experiments as replicates, and region of the cervix (caudal v mid v cranial) as a split plot factor. RESULTS

Gross pathological changes were consistent with those described previously for ewes with clover disease (Turnbull, Braden and George, 1966; Fels and Neil, 1968). There was a high incidence of macroscopic cysts in the uterus, as shown in Table 1. Hydrops uteri, pyometra, or massive adhesions over the genital tract were common findings in oestrogenic treatment ewes from all 3 experiments. Glandular

Development

in the Lamina

Propria

Intersections of glands were least numerous in the caudal region of the cervix, a progressive increase towards the middle and cranial regions (P < O-001). Overall, the mean (&s.e.) numbers of intersections observed in the caudal, middle, and cranial cervix were 117 f12, 166f20, and 282&42 respectively. In each of the 3 experiments, ewes subjected to prolonged grazing on oestrogenic pasture showed far greater glandular development than control animals (Table 2 ; P < O*OOl), although the magnitude of this difference varied between experiments. The control ewes in Experiment 2, which had grazed the slightly oestrogenic Mt Barker cultivar of subterranean clover showed greater development of glands (mean number: 571) than control ewes in Experiments 1 (265) and 3 (276) which had grazed less oestrogenic pasture. with

CERVICAL

HISTOLOGY

AND

CLOVER

369

DISEASE

Furthermore, within the oestrogenic treatments, ewes from Experiment 1 which had grazed oestrogenic pasture for only 4 years had fewer glands than ewes in Experiments 2 and 3, each of which spent 6. years on oestrogenic pasture (means 554, 1051 and 1254, respectively). TABLE1 DETAILS

OF PREVIOUS

TREATMENT

Experiment

Number of ewes : oestrogenic control Experimental pasture oestrogenic

1N THE

PRESENT

I

L’

26 19

15 17

STUDY

3

grazed: 100 per cent Dwalganup* Grass

control

Duration

OF EWES

Yarloop* Mt

Dwalganup* or Dinninup* Grass, Serradella, Kondinin, Rose Blackwood*

Barker*

or

of grazing:

(i)

on experimental

pasture

(years)

(ii)

on non-oestrogenic pasture before slaughter (months) Age of ewes at slaughter (years) Mean number of macroscopic cysts in the uterus: oestrogenic control Mean number of lambs born per ewe in last 3 years of experimental grazing: oestrogenic control

6

6

17

33

7

7

10

8

19 1

13 1

68 2

3.0 3.65

0.23 2.27

2.61 3.68

Reference

Davies and Maller (1970)

* Cultivars Mt Barker

of Subterranean and Blackwood

CX.ASS,F,CATION

clover (Trrifolium are relatively low

OF EWES FROM OESTROGENIC NITMBER OF GLANDS OBSERVED

Total number qf glands seen in cross-section*

O-100 101-200 201-400 401-800 >800

Experiment Control

5 6 4 0”

Total number ewes

* Total

4

1 Oestrogen

et al.

Lightfoot (1974)

Neil

el al.

(1974)

subterraneum). in phytooestrogens.

AN” CONTROL. TREATMENTS RCCORDlKC IN SECTIONS OF THE CERVICAI. MlTCOSA

Experiment Control

0 2 1;

; i

4

4

26

17

2 Oestrogen

T O THE

Experiment Control

0

3 Oestrogen

0

i 5 10

ii 9 4 0

: 3 11

1.5

20

17

of

of 3 cervical

19

sections

(caudal

+ mid+

cranial)

per

ewe.

370

R. J. LIGHTFOOT

Stratijed

AND

N. R. ADAMS

Squamous Epithelium

Overall, the amount of stratified squamous epithelium was greater in the caudal region than in the middle or cranial regions of the cervix (Table 3; P < 0.001). Oestrogenic and control ewes did not differ in this respect, but oestrogenic treatment ewes tended to have less stratified squamous epithelium than control ewes (P < O-001), the difference being least marked in Experiment 1 (treatment x experiment P < 0.05). TABLE

CLASSIFICATION TREATMENTS

3

OF CERVICAL SECTIONS (3 PER EWb) FROM OESTKOGENIC ACCORDING

T O THEIR

SCORE

FOR STRATIFIED

SQUAMGUS

AND CONTROL EPITHELIUM

Treatment

Score for stratz$ed squamous epithelium

0

Section of cervix cranial middle caudal Exoeriment number

Oe.rtrogeni~ 1

56 39 22

”

1 1;

A 21

0

Cbntrol I

2

48 28 4

6 17 15

2 11 37

: 3

‘I‘ABLE 4 CLASSIFICATION OF CERVICAL SECTIONS PROPORTION OF MUCOUS CELLS

Mucous

cell xore

Section of cervix: cranial middle caudal Treatment: oestrogenic control

0

Experiment I

34 10 5 30 19

(3

PER EWE) IN CERVICAL

1

ACCORDING COLUMNAR

L’

0

Experiment I

7 10 10

4 25 29*

30 12 5

2 15 7

21 6

27 3**

24 23

12 12

* In Experiments 1 and 3, the epithelium of stratified squamous cells.

of the caudal

section

T O THEIR

SCORE

2

from

FOR

EPITED2LIU.M

2

0

Experirnen I

0 2;

30 ‘1 14

4 9 a

3 7 14*

9 16

47 18

4 17

!I 24”

1 control

ewe consisted

t 3 2

entirely

Mucus Cells In both oestrogenic and control ewes, the frequency of mucus cells increased from the cranial to the caudal region of the cervix (Table 4; P < 0.001). Within each cervical region, mucus cells tended to be less common in the epithelium of oestrogenic treatment than control ewes (P < 0.001). There was considerable variation in this regard, however, between experiments. Particularly high values were recorded in Experiment 1, possibly due to difficulty

CERVICAL

HISTOLOGY

AND

CLOVER

371

DISEASE

in reading mucus cells from the frozen sections used in this experiment. The most marked suppression of mucus cells was seen in oestrogenic treatment ewes from Experiment 3, which also suffered the most severe decline in fertility. TABLE5 CLASSlFICATION TREATMENTS

OF CERVICAL SECTIONS (3 PER EWE) FROM OESTROGENIC AND CONTROL ACCORDING T O THEIR SCORE FOR THE DEGREE OF CHRONIC CERVICITIS

Treatment Chronic

ceroicitis score

Experiment 1

0

Control I

-?

0

Oestrogenic I

?

number

Ceraicitis Acute cervicitis tended to be more common in sections from oestrogenic treatment than control ewes (66/174 vs 47/168; P = O-05). This effect was most marked in Experiments 2 and 3. The mean score for chronic cervicitis tended to be higher in caudal sections compared with those from the mid and cranial regions of the cervix (O-73, 0.64 and 0.58 respectively; P < O-05). There was a marked difference between experiments in the amount of chronic cervicitis recorded (Table 5, P < 0.001). Chronic cervicitis was both more common and more severe in the oestrogenic treatment ewes (P < O*OOl), although again this was not as apparent in Experiment 1. DISCUSSION

The results of all three experiments demonstrate increased glandular development in the cervical lamina propria of ewes after prolonged exposure to oestrogenic pasture. The proliferation of tubular convoluted glands is apparently a permanent change, because it persisted for a comparatively long interval between oestrogenic grazing and slaughter (up to 33 months). The extensive glandular development most likely reflects the transformation of the cervix towards a uterine appearance (Adams, 1976; Heydon and Adams, 1976) and possibly relates to the abnormal pattern of cervical mucus production observed in clover-affected ewes (Smith, 1971; Lightfoot et al., 1974). Enumeration of glandular transections may offer an objective means of assessing the degree of change in the cervix since the number varied both between treatments and experiments in proportion to the extent of oestrogenic exposure. The amount of stratified squamous epithelium in the cervix of control ewes was similar to that reported previously (Cole and Miller, 1935; Raynaud, 1973). Oestrogenic treatment resulted in a decrease in the amount of both stratified squamous epithelial cells and mucous cells in the cervix. This finding was unexpected, because exposure to oestrogen normally causes an increase in mucus

372

R.

J. LIGHTFOOT

AND

N.

R.

ADAMS

secretion (Smith, 1971) and an extension of the stratified squamous epithelium towards the uterus (Zuckerman, 1940; Adams, 1977), even in ewes which have been exposed to oestrogenic pasture for several years (Hearnshaw, Brown, Gumming, Goding and Nairn, 1974; Adams, 1976). It is suggested that the differences between previous studies and those reported here relate to the period between oestrogenic exposure and slaughter. In previous studies, ewes have been slaughtered either while still exposed to plant oestrogens, or not more than 10 weeks after removal from oestrogenic pasture. Appropriately, the changes reported have been consistent with those normally seen in adult animals treated with oestrogen (Zuckerman, 1940). In the present study, ewes had been held off oestrogenic pasture for at least 28 weeks, and it is suggested that all temporary effects of oestrogen had regressed, so that only the more persistent changes caused by oestrogen were expressed. These changes in the epithelium are consistent with the general change towards a more uterine-like structure in the cervix, shown by the development of glands and changes in the lamina propria (Adams, 1976). A change of epithelium in the cervix and the cranial part of the vagina to a more uterine type has been caused by oestrogen in neonatal mice (Forsberg, 1975) and prenatal humans (Hart, Townsend, Aldrich, Henderson, Roy and Benton, 1976), so that the type of change caused by oestrogen in the present study is not without precedent. However, such a change has not been produced previously by treating adult animals with oestrogen. The change caused by oestrogen in cervical differentiation in the neonatal mouse is believed to be due to a modification of embryological development (Forsberg, 1970; Iguchi, Ohta and Takasuji, 1976). Such a mechanism cannot be invoked in the adult ewe, so that a study of the pathogenesis of the changes described in this paper may well prove of interest. SUMMARY

The cervical histology of ewes affected with clover disease (caused by prolonged grazing on oestrogenic subterranean clover pastures) was compared with that of control animals. The cervix from diseased ewes showed much an increased incidence of cervicitis, fewer greater glandular development, goblet cells and a lower incidence of stratified squamous epithelium than since they were observed in ewes controls. These changes were “permanent” slaughtered up to 2 years after removal from oestrogenic pastures. The changes are consistent with the hypothesis that the cervix of affected ewes assumes a more uterine-like nature. REFERENCES

Adams, N. R. (1976). Pathological changes in the tissues of infertile ewes with clover disease. Journal of Coqbarative Pathology, 86, 29-35. Adams, N. R. (1977). Morphological changes in the organs ofewes grazing oestrogenic subterranean clover. Research in Veterinary Science, 22, 2 16-221. Claringbold, P. J. (1961). The use of orthogonal polynomials in the partition of Chi-square. Australian Journal of Statistics, 3, 48-59.

CERVICAL

HISTOLOGY

AND

CLOVER

DISEASE

Cole, H. H., and Miller,

373

R. F. (1935). Changes in the reproductive organs of the ewe with some data bearing on their control. American Journal of Anatomy, 57, 39-87. Davies, H. L., and Maller, R. A. (1970). The fertility of ewes grazing pastures containing different proportions of subterranean clover in south western Australia. Proceedings of the Australian Society of Animal Production, 8, 394399. Fels, H. E., and Neil, H. G. (1968). Effects on reproduction of prolonged grazing of oestrogenic pastures by ewes. Australian Journal of Agricultural Research, 19, 1059-1068. Forsberg, J. G. (1970). An estradiol mitotic rate inhibiting effect in the Mullerian epithelium in neonatal mice. Journal of Experimental zoology, 175, 369-374. Forsberg, J. G. (1975). Late effects in the vaginal and cervical epithelia after injections of diethylstilbestrol into neonatal mice. American Journal of Obstetrics and Gynecology, 121, 102-l 04. Hart, W. R., Townsend, D. E., Aldrich, J. O., Henderson, B. E., Roy, M., and Benton, B. (1976). Histopathologic spectrum of vaginal adenosis and related changes in stilbestrol-exposed females. Cancer, 37, 763-775. Hearnshaw, H., Brown, J. M., Cumming, I. A., Goding, J. R., and Nairn, M. E. (1972). Endocrinological and histopathological aspects of the infertility in the ewe caused by oestrogenic clover. Journal of Reproduction and Fertility, 28, 160-l 6 1. Heydon, R. A., and Adams, N. R. (1977). Histochemical studies on cervical glands in ewes with clover disease. Journal of Comparative Pathology, 87, 353-361. Iguchi, T., Ohta, Y., and Takasugi, N. (1976). Mitotic activity of vaginal epithelial cells following neonatal injections of different doses of estrogen in mice. Development, Growth and Daxerentiation, 18, 69-78. Lightfoot, R. J., Croker, K. P., and Neil, H. G. (1967). Failure of sperm transport in relation to ewe infertility following prolonged grazing on oestrogenic pastures. Australian Journal of Agricultural Research, 18, 755-765. Lightfoot, R. J., Smith, J. F., Cumming, I. A., Marshall, T., Wroth, R. H., and Hearnshaw, H. (1974). Infertility in ewes caused by prolonged grazing on oestrogenic pastures : oestrus, fertilization and cervical mucus. Australian Journal of Biological Sciences, 27, 409-414. Neil, H. G., Lightfoot, R. J., and Fels, H. G. (1974). Effect of legume species on ewe fertility in south western Australia. I. Badgingarra. Proceedings of the Australian Society of Animal Production, 10, 136. Raynaud, F. (1973). Physiologie du co1 de l’uterus de la brebis. Effet d’un progestagene de synthese: l’acetate de flurogesterone. Annales de Biologie Animale Biochimie Biophysique, 13, 335-346. Smith, J. F. (1971). Studies on ovine infertility in agricultural regions of Western Australia: cervical mucus production by fertile and infertile ewes. Australian Journal of Agricultural Research, 22, 5 13-5 19. Turnbull, K. E., Braden, A. W. H., and George, J. M. (1966). Fertilization and earl) embryonic losses in ewes that had grazed oestrogenic pastures for six years. Australian Journal of Agricultural Research, 17, 90779 17. Zuckerman, S. (1940). The histogenesis of tissues sensitive to oestrogens. Biological Reviews, 15, 231-271. [Receivedfor

publication,

October 2nd, 19781