- Email: [email protected]
Pituitary prolactin levels in canine mammary cancer
Europ. J. Cancer Vol. 10, pp. 63-66. Pergamon Press 1974. Printed in Great Britain
Pituitary Prolactin Levels in Canine Mammary Cancer P. G. SALUJA*, J. M. HAMILTON, M. GRONOW* and W. MISDORP t *Department of Experimental Pathology and Cancer Research, The University of Leeds, Leeds, England ~Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Abstract--Canine pituitary prolactin concentration was estimated densitometrically
after isoeleetric focusing in polyaerylamide gel columns and staining by Coomassie brilliant blue. In bitches with mammary carcinoma, prolactin levels were significantly higher than in normal animals of comparable endocrine state. This finding indicated that prolactin imbalance may be involved in canine mammary neoplasia.
The reproductive state of all bitches (tumorous and control) was ascertained by external inspection combined with gross and histological examination of the reproductive organs. The dogs were destroyed by intravenous injection of sodium pentobarbitone. All control bitches and over half the tumorous cases were killed between 11.00-12.00 hours and the remainder at various times throughout the day.
INTRODUCTION PROLACTIN is of prime importance in the genesis of mammary neoplasia in rodents [1, 2]. It is unknown, however, whether, or not, the hormone is similarly implicated in other species. There is some evidence in man that serum prolactin levels may be elevated in patients with breast cancer [3, 4], but much remains to be investigated, particularly the problem regarding the role of prolactin in the early stages of tumour induction. In Leeds, the bitch is the subject of study of the association between prolactin and mammary neoplasia and the following report describes an investigation into the prolactin content of the adenohypophysis of bitches bearing mammary tumours.
Pituitaries The adenohypophyses were removed within 30-60 min after death and either analysed for prolactin concentration immediately or stored at --20°C for intervals not exceeding 2 months (stainable canine pituitary prolactin has been shown to be durable for at least 3 months [6]). Prolactin estimations were performed by isoelectric focusing in polyacrylamide gel columns containing Ampholine carrier ampholytes of pH range 5-7 (LKB-Produkter). After completion of electrofocusing, gels were fixed in 12 % trichloroacetic acid, stained with Coomassie brilliant blue and scanned densitometrically in a Joyce-Loebl Chromoscan M K II. The details of the method have been described in an earlier publication [6]. A quantity of extract equivalent to 1 mg adenohypophysis per gel was sufficient for clear detection and densitometric measurement of the prolactin band and for each gland the mean optical density of the prolactin band was calculated from the scans of eight gels and expressed as equivalent gg of ovine prolactin standard (regression analysis
MATERIALS AND M E T H O D S
Animals A series of 19 bitches, mostly mongrels, bearing spontaneous mammary turnouts was used. None of these animals had received hormonal therapy. Post-mortem and histopathological examination were performed on all cases.
A series of 39 normal, sexually mature, nonpregnant bitches, again mostly mongrels, in various endocrine states, used in a previous study [5], served as a control group in this investigation. Accepted 4 December 1973.
63
64
P. G. Saluja, d. M . Hamilton, M . Gronow and W. Misdorp
had shown that ovine prolactin displayed the same dye-binding properties as canine prolactin in adenohypophysial extracts). Statistical analysis
Previous studies on normal bitches [5] had shown that pituitary prolactin concentration varied with the endocrine state and, accordingly, the latter was taken into account when assessing levels in the tumorous bitches. Low pituitary prolactin concentrations occurred in dioestrous females while significantly higher concentrations were found in oestrous, metoestrous (luteal) and ovariectomized animals. Significant differences did not occur amongst the oestrous, luteal or spayed categories. Accordingly, in the present study, the results for tumorous animals in oestrous, luteal and spayed conditions were pooled and compared with the mean level for normal animals in these conditions. Prolactin levels in tumorous animals in dioestrous were compared with the dioestrous level in normal animals. Tests for differences in prolactin levels were carried out by Student's t-test [7] using an Olivetti Programma 101 desk-top computer. In assessing estimations obtained from individual pituitaries, prolactin concentration was considered to be elevated when it was above the 95% confidence limit of the upper end of the normal range and to be depressed when it was below the 95% confidence limit of the lower end of the normal range for a given endocrine state.
Table 1. Description of mammary tumour cases in which pituitary prolactin concentration was measured
Number of animals 3 12 1 I I 1
Pituitary prolactin level
Diagnosis Localized infiltrating carcinoma Recurrent and/or metastatic carcinoma Recurrent, metastatic malignant mixed turnout Recurrent, metastatic osteosarcoma Recurrent, metastatic fibrosarcoma Multiple pleomorphic adenomata
2N; 1E 3N; 9E N N N D
Abbreviations used: N = Normal, E = Elevated, D = Depressed.
~'
5
28
a4
P
} 20
P<0.025
&
iiii!iiiii! ..... . ....,.,.~ . . . : : ..
. .... . ....
....,,,... ,.,...,::
::.. ,:. . ....
.'.v:.'
, ..,:::
v.'.'.'. ,,.....,..
. . ,...., .:..::,
.::::.
.....
~...,.,,..
8
RESULTS
. . v ....,
t.J
The distribution of the cases according to t u m o u r type, stage of disease and pituitary prolactin level is shown in Table 1. Statistical analysis showed that, as a group, bitches with m a m m a r y tumours had significantly higher pituitary prolactin concentration than normal females of comparable endocrine state (Fig. 1). This difference was somewhat more significant among dioestrous bitches ( 0 . 0 2 > P > 0 . 0 1 ) than among oestrous-lutealspayed animals (0.025 > P > 0 . 0 2 ) . Elevated prolactin levels were confined to those animals with carcinomas (Table 1). DISCUSSION
The dog, a species which closely shares man's environment, suffers from spontaneous malignant m a m m a r y neoplasms at least as frequently as man [8] and, furthermore, canine
4
~
, ,.....
o
ORMAL BREAST TUMOUR
NORMAL BREAST TUMOUR
I
(IZ) (7) DIOEST"RU$
I
I
(27) 02)
.I
OESTRUS-LUTEAL-SPAYED
Mean pituitary prolactin concentration in normal dogs and dogs with mammary tumours. The vertical bars represent S.E.M. and the numerals in brackets indicate the number of dogs examined.
F~g.
1.
and h u m a n breast cancer are remarkably similar with regard to age of onset, age specific incidence and, in m a n y cases, histological appearance and clinical course [8, 9]. In the dog, as in the woman, there is circumstantial evidence that endocrine factors influence the incidence and course of m a m m a r y tumours
Pituitary Prolactin Levels in Canine Mammary Cancer although the specific hormones involved have not been identified as yet in either species. In the bitch a protective effect is conveyed by ovariectomy early in life [10] and also, possibly by frequent pregnancies [10, 11]. Oestrogens have been incriminated [12, 13, 14] but experimental support for this hypothesis has not been forthcoming [ 15, 16, 17]. Involvement of the pituitary gland is suggested by reports of enlarged adenohypophysis [18] and increased numbers of acidophils [19] in bitches bearing m a m m a r y tumours. Using differential pituitary staining, Barboni and Romanelli [20] found marked hyperplasia and hypertrophy of the prolactin cells in bitches with fibrocystic mastopathy with and without concomitant m a m m a r y cancer, as compared to normal bitches. It is not too surprising, therefore, that the above investigation has shown that bitches with m a m m a r y tumours had significantly higher pituitary prolactin concentration than normal females, although the elevation was actually confined to animals with carcinomas. Although diurnal secretory cycles for prolactin have been described in the rat [21] and the h u m a n [22], in the present study the time of death did not appear to influence the levels observed. The mean age of the tumorous bitches was 11.8 years (4-S.D. 2.7 years) with no significant age difference between oestrous-lutealspayed bitches and dioestrous bitches ( P > 0.50). Since precise information was not available for all control bitches, the age distribution of this series could not be presented. However, the following evidence indicated that age did not influence pituitary prolactin *r = product moment correlation coefficient [23].
levels in the present study: (1) Amongst the m a m m a r y tumour bitches, there was no significant correlation between increasing age and elevation of pituitary prolactin, i.e. for oestrousluteal-spayed bitches, r* = 0.07, P > 0 . 4 0 and for dioestrous bitches, r = 0.22, P>0.35. (2) When the control animals were classed into the broad age groups of young, intermediate and old, analysis o ~ variance did not disclose any significant differences attributable to age. For these reasons, it was felt that the control and tumorous groups were comparable despite possible differences in age-distribution. All animals, tumorous and normal, were subject to the same degree of acute stress with regard to handling and destruction. However the m a m m a r y tumour animals had suffered the additional stress of a serious illness. In this regard it is recognized that pituitary prolactin should be examined in dogs with serious illnesses other than m a m m a r y malignancy, although such cases were not available during the course of the present investigation. The findings of this study suggest that prolactin m a y be of importance in m a m m a r y neoplasia in the dog. However, information regarding its mode of action awaits elucidation and, towards this end, methods are being developed for the measurement of serum prolactin. This, combined with assessment of pituitary content, would enable further investigation into the role of prolactin in m a m m a r y tumorigenesis in the bitch. A c k n o w l e d g e m e n t s - - T h e authors wish to thank Mr. J. Wright for technical assistance. The ovine prolactin standard was kindly supplied by the Endocrinology Study Section of the N.I.H. The financial support of the Yorkshire Cancer Research Campaign is gratefully acknowledged.
REFERENCES
I.
2. 3. 4.
5. 6.
7.
65
O . H . PEARSON, O. LLERENA, L. LLERENA, A. MOLINA and T. BUTLER, Prolactin-dependent rat mammary cancer: a model for man ? Trans. Ass. Am. Physns. L X X X I I , 225 (1969). L . M . BOOT, Prolactin and mammary gland carcinogenesis. The problem of human prolactin. Int. J. Cancer 5, 167 (1970). P. BERLE and K. D. VOIOT, Plasma-Prolaktinkonzentrafionen beim mammakarzinom der frau. Acta endocr. Copenh. suppl. 159, 38 (1972). R . M . L . MURRAY, G. MOZAFFARIANand O. H. PEARSON, Prolactin levels with L-Dopa treatment in metastatic breast carcinoma, in Prolactin and Carcinogenesis (Edited by A. R. BOYNS and K. GRIFFITHS), p. 158. Alpha Omega Alpha, Cardiff (1972). P . G . SALUJA, J. M. HAMILTON and M. GRONOW, Prolacfin content of canine pituitary gland. J. Endocr. 57, 495 (1973). P . G . SALUJA, M. CTRONOW and J. M. HAMILTON, Measurement of canine pituitary prolacfin. J. Endocr. 56, 245 (1973). J. G. PEATMAN, Introduction to Applied Statistics, p. 209. Harper & Row, Evanston and London.
66
P. G. Saluja, 3". M . Hamilton, M. Gronow and W. Misdorp
8.
R. Sell,raIDER, Comparison of age, sex and incidence rates in human and canine breast cancer. Cancer (Philad.) 26, 419 (1970). 9. W. MISDORP, Malignant mammary tumours in the dog and the cat compared with the same in the woman. Doctoral thesis, Veterinary Faculty, University of Utrecht (1964). 10. R. SCHN~.IDER,C. R. DORN and D. O. N. TAYLOR, Factors influencing canine mammary cancer development and post.surgical survival. J. nat. Cancer Inst. 43, 1249 (1969). 11. A . C . ANDERSON, Parameters of mammary gland tumors in aging beagles. or. Amer. vet. med. Assoc. 147, 1653 (1965). 12. J. DE VITTA, Mammary adenofibroma of the female dog. N. Am. Vet. 20, 53 (1939). 13. G. DOZZA and G. CoLuzzI, Biological assay of urinary oestrogens in bitches with tumours of the mammary gland. Atti Soc. ital. Sci. vet. 17~ 354 (1964). 14. G. DozzA, M. L. CALANDRAand A. S. LI, Measurement by gas chromatography of urinary oestrogens in bitches with mammary dysplasia of neoplasia. Nuova Vet. 47~ 71 (1971). 15. R . M . MULLIOAN, Some effects of chronic doses of stilbestrol in female dogs. Exp. ivied. Surg. 5, 196 (1947). 16. A. JABARA, Canine mixed tumours. Aust. vet. or. 36, 212 (1960). 17. A. J~ARA, Canine mammary carcinomata. Aust. vet. or. 36~ 389 (1960). 18. C. HUOGINSand P. V. MOULDER, Studies on the mammary tumors of dogs. l Lactation and the influence of ovariectomy and suprarenalectomy thereon. J. exp. Med. 80~ 441. (1944). 19. R.M. MULUOAN,In Neoplasms of the Dog.Williams & Wilkins, Baltimore (1949). 20. E. BARBONIand V. RO~ANF.LLI,Remarks and considerations about fibrocystic mastopathy and about "cancer ex fibrocystic mastopathy" of the dog. In International Symposium on Mammary Cancer (Edited by L. SEVERI), p. 547. Division of Cancer Research, Perugia (1958). 21. J . D . DUNN, A. AmMURA and L. E. SCHEVING, Effect of stress on circadian periodicity in serum LH and prolactin concentration. Endocrinology 90~ 29 (1972). 22. J. NoKIN, M. VEKEMANS,M. L'H~.RMITE and C. ROBYN, Circadian periodicity of serum prolactin concentration. Brit. med. or. 3~ 561 (I 972). 23. M.J. MORONEY.In Factsfrom Figures, p. 286, Penguin Books, Harmondsworth, Middlesex.
Pituitary Prolactin Levels in Canine Mammary Cancer P. G. SALUJA*, J. M. HAMILTON, M. GRONOW* and W. MISDORP t *Department of Experimental Pathology and Cancer Research, The University of Leeds, Leeds, England ~Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands Abstract--Canine pituitary prolactin concentration was estimated densitometrically
after isoeleetric focusing in polyaerylamide gel columns and staining by Coomassie brilliant blue. In bitches with mammary carcinoma, prolactin levels were significantly higher than in normal animals of comparable endocrine state. This finding indicated that prolactin imbalance may be involved in canine mammary neoplasia.
The reproductive state of all bitches (tumorous and control) was ascertained by external inspection combined with gross and histological examination of the reproductive organs. The dogs were destroyed by intravenous injection of sodium pentobarbitone. All control bitches and over half the tumorous cases were killed between 11.00-12.00 hours and the remainder at various times throughout the day.
INTRODUCTION PROLACTIN is of prime importance in the genesis of mammary neoplasia in rodents [1, 2]. It is unknown, however, whether, or not, the hormone is similarly implicated in other species. There is some evidence in man that serum prolactin levels may be elevated in patients with breast cancer [3, 4], but much remains to be investigated, particularly the problem regarding the role of prolactin in the early stages of tumour induction. In Leeds, the bitch is the subject of study of the association between prolactin and mammary neoplasia and the following report describes an investigation into the prolactin content of the adenohypophysis of bitches bearing mammary tumours.
Pituitaries The adenohypophyses were removed within 30-60 min after death and either analysed for prolactin concentration immediately or stored at --20°C for intervals not exceeding 2 months (stainable canine pituitary prolactin has been shown to be durable for at least 3 months [6]). Prolactin estimations were performed by isoelectric focusing in polyacrylamide gel columns containing Ampholine carrier ampholytes of pH range 5-7 (LKB-Produkter). After completion of electrofocusing, gels were fixed in 12 % trichloroacetic acid, stained with Coomassie brilliant blue and scanned densitometrically in a Joyce-Loebl Chromoscan M K II. The details of the method have been described in an earlier publication [6]. A quantity of extract equivalent to 1 mg adenohypophysis per gel was sufficient for clear detection and densitometric measurement of the prolactin band and for each gland the mean optical density of the prolactin band was calculated from the scans of eight gels and expressed as equivalent gg of ovine prolactin standard (regression analysis
MATERIALS AND M E T H O D S
Animals A series of 19 bitches, mostly mongrels, bearing spontaneous mammary turnouts was used. None of these animals had received hormonal therapy. Post-mortem and histopathological examination were performed on all cases.
A series of 39 normal, sexually mature, nonpregnant bitches, again mostly mongrels, in various endocrine states, used in a previous study [5], served as a control group in this investigation. Accepted 4 December 1973.
63
64
P. G. Saluja, d. M . Hamilton, M . Gronow and W. Misdorp
had shown that ovine prolactin displayed the same dye-binding properties as canine prolactin in adenohypophysial extracts). Statistical analysis
Previous studies on normal bitches [5] had shown that pituitary prolactin concentration varied with the endocrine state and, accordingly, the latter was taken into account when assessing levels in the tumorous bitches. Low pituitary prolactin concentrations occurred in dioestrous females while significantly higher concentrations were found in oestrous, metoestrous (luteal) and ovariectomized animals. Significant differences did not occur amongst the oestrous, luteal or spayed categories. Accordingly, in the present study, the results for tumorous animals in oestrous, luteal and spayed conditions were pooled and compared with the mean level for normal animals in these conditions. Prolactin levels in tumorous animals in dioestrous were compared with the dioestrous level in normal animals. Tests for differences in prolactin levels were carried out by Student's t-test [7] using an Olivetti Programma 101 desk-top computer. In assessing estimations obtained from individual pituitaries, prolactin concentration was considered to be elevated when it was above the 95% confidence limit of the upper end of the normal range and to be depressed when it was below the 95% confidence limit of the lower end of the normal range for a given endocrine state.
Table 1. Description of mammary tumour cases in which pituitary prolactin concentration was measured
Number of animals 3 12 1 I I 1
Pituitary prolactin level
Diagnosis Localized infiltrating carcinoma Recurrent and/or metastatic carcinoma Recurrent, metastatic malignant mixed turnout Recurrent, metastatic osteosarcoma Recurrent, metastatic fibrosarcoma Multiple pleomorphic adenomata
2N; 1E 3N; 9E N N N D
Abbreviations used: N = Normal, E = Elevated, D = Depressed.
~'
5
28
a4
P
} 20
P<0.025
&
iiii!iiiii! ..... . ....,.,.~ . . . : : ..
. .... . ....
....,,,... ,.,...,::
::.. ,:. . ....
.'.v:.'
, ..,:::
v.'.'.'. ,,.....,..
. . ,...., .:..::,
.::::.
.....
~...,.,,..
8
RESULTS
. . v ....,
t.J
The distribution of the cases according to t u m o u r type, stage of disease and pituitary prolactin level is shown in Table 1. Statistical analysis showed that, as a group, bitches with m a m m a r y tumours had significantly higher pituitary prolactin concentration than normal females of comparable endocrine state (Fig. 1). This difference was somewhat more significant among dioestrous bitches ( 0 . 0 2 > P > 0 . 0 1 ) than among oestrous-lutealspayed animals (0.025 > P > 0 . 0 2 ) . Elevated prolactin levels were confined to those animals with carcinomas (Table 1). DISCUSSION
The dog, a species which closely shares man's environment, suffers from spontaneous malignant m a m m a r y neoplasms at least as frequently as man [8] and, furthermore, canine
4
~
, ,.....
o
ORMAL BREAST TUMOUR
NORMAL BREAST TUMOUR
I
(IZ) (7) DIOEST"RU$
I
I
(27) 02)
.I
OESTRUS-LUTEAL-SPAYED
Mean pituitary prolactin concentration in normal dogs and dogs with mammary tumours. The vertical bars represent S.E.M. and the numerals in brackets indicate the number of dogs examined.
F~g.
1.
and h u m a n breast cancer are remarkably similar with regard to age of onset, age specific incidence and, in m a n y cases, histological appearance and clinical course [8, 9]. In the dog, as in the woman, there is circumstantial evidence that endocrine factors influence the incidence and course of m a m m a r y tumours
Pituitary Prolactin Levels in Canine Mammary Cancer although the specific hormones involved have not been identified as yet in either species. In the bitch a protective effect is conveyed by ovariectomy early in life [10] and also, possibly by frequent pregnancies [10, 11]. Oestrogens have been incriminated [12, 13, 14] but experimental support for this hypothesis has not been forthcoming [ 15, 16, 17]. Involvement of the pituitary gland is suggested by reports of enlarged adenohypophysis [18] and increased numbers of acidophils [19] in bitches bearing m a m m a r y tumours. Using differential pituitary staining, Barboni and Romanelli [20] found marked hyperplasia and hypertrophy of the prolactin cells in bitches with fibrocystic mastopathy with and without concomitant m a m m a r y cancer, as compared to normal bitches. It is not too surprising, therefore, that the above investigation has shown that bitches with m a m m a r y tumours had significantly higher pituitary prolactin concentration than normal females, although the elevation was actually confined to animals with carcinomas. Although diurnal secretory cycles for prolactin have been described in the rat [21] and the h u m a n [22], in the present study the time of death did not appear to influence the levels observed. The mean age of the tumorous bitches was 11.8 years (4-S.D. 2.7 years) with no significant age difference between oestrous-lutealspayed bitches and dioestrous bitches ( P > 0.50). Since precise information was not available for all control bitches, the age distribution of this series could not be presented. However, the following evidence indicated that age did not influence pituitary prolactin *r = product moment correlation coefficient [23].
levels in the present study: (1) Amongst the m a m m a r y tumour bitches, there was no significant correlation between increasing age and elevation of pituitary prolactin, i.e. for oestrousluteal-spayed bitches, r* = 0.07, P > 0 . 4 0 and for dioestrous bitches, r = 0.22, P>0.35. (2) When the control animals were classed into the broad age groups of young, intermediate and old, analysis o ~ variance did not disclose any significant differences attributable to age. For these reasons, it was felt that the control and tumorous groups were comparable despite possible differences in age-distribution. All animals, tumorous and normal, were subject to the same degree of acute stress with regard to handling and destruction. However the m a m m a r y tumour animals had suffered the additional stress of a serious illness. In this regard it is recognized that pituitary prolactin should be examined in dogs with serious illnesses other than m a m m a r y malignancy, although such cases were not available during the course of the present investigation. The findings of this study suggest that prolactin m a y be of importance in m a m m a r y neoplasia in the dog. However, information regarding its mode of action awaits elucidation and, towards this end, methods are being developed for the measurement of serum prolactin. This, combined with assessment of pituitary content, would enable further investigation into the role of prolactin in m a m m a r y tumorigenesis in the bitch. A c k n o w l e d g e m e n t s - - T h e authors wish to thank Mr. J. Wright for technical assistance. The ovine prolactin standard was kindly supplied by the Endocrinology Study Section of the N.I.H. The financial support of the Yorkshire Cancer Research Campaign is gratefully acknowledged.
REFERENCES
I.
2. 3. 4.
5. 6.
7.
65
O . H . PEARSON, O. LLERENA, L. LLERENA, A. MOLINA and T. BUTLER, Prolactin-dependent rat mammary cancer: a model for man ? Trans. Ass. Am. Physns. L X X X I I , 225 (1969). L . M . BOOT, Prolactin and mammary gland carcinogenesis. The problem of human prolactin. Int. J. Cancer 5, 167 (1970). P. BERLE and K. D. VOIOT, Plasma-Prolaktinkonzentrafionen beim mammakarzinom der frau. Acta endocr. Copenh. suppl. 159, 38 (1972). R . M . L . MURRAY, G. MOZAFFARIANand O. H. PEARSON, Prolactin levels with L-Dopa treatment in metastatic breast carcinoma, in Prolactin and Carcinogenesis (Edited by A. R. BOYNS and K. GRIFFITHS), p. 158. Alpha Omega Alpha, Cardiff (1972). P . G . SALUJA, J. M. HAMILTON and M. GRONOW, Prolacfin content of canine pituitary gland. J. Endocr. 57, 495 (1973). P . G . SALUJA, M. CTRONOW and J. M. HAMILTON, Measurement of canine pituitary prolacfin. J. Endocr. 56, 245 (1973). J. G. PEATMAN, Introduction to Applied Statistics, p. 209. Harper & Row, Evanston and London.
66
P. G. Saluja, 3". M . Hamilton, M. Gronow and W. Misdorp
8.
R. Sell,raIDER, Comparison of age, sex and incidence rates in human and canine breast cancer. Cancer (Philad.) 26, 419 (1970). 9. W. MISDORP, Malignant mammary tumours in the dog and the cat compared with the same in the woman. Doctoral thesis, Veterinary Faculty, University of Utrecht (1964). 10. R. SCHN~.IDER,C. R. DORN and D. O. N. TAYLOR, Factors influencing canine mammary cancer development and post.surgical survival. J. nat. Cancer Inst. 43, 1249 (1969). 11. A . C . ANDERSON, Parameters of mammary gland tumors in aging beagles. or. Amer. vet. med. Assoc. 147, 1653 (1965). 12. J. DE VITTA, Mammary adenofibroma of the female dog. N. Am. Vet. 20, 53 (1939). 13. G. DOZZA and G. CoLuzzI, Biological assay of urinary oestrogens in bitches with tumours of the mammary gland. Atti Soc. ital. Sci. vet. 17~ 354 (1964). 14. G. DozzA, M. L. CALANDRAand A. S. LI, Measurement by gas chromatography of urinary oestrogens in bitches with mammary dysplasia of neoplasia. Nuova Vet. 47~ 71 (1971). 15. R . M . MULLIOAN, Some effects of chronic doses of stilbestrol in female dogs. Exp. ivied. Surg. 5, 196 (1947). 16. A. JABARA, Canine mixed tumours. Aust. vet. or. 36, 212 (1960). 17. A. J~ARA, Canine mammary carcinomata. Aust. vet. or. 36~ 389 (1960). 18. C. HUOGINSand P. V. MOULDER, Studies on the mammary tumors of dogs. l Lactation and the influence of ovariectomy and suprarenalectomy thereon. J. exp. Med. 80~ 441. (1944). 19. R.M. MULUOAN,In Neoplasms of the Dog.Williams & Wilkins, Baltimore (1949). 20. E. BARBONIand V. RO~ANF.LLI,Remarks and considerations about fibrocystic mastopathy and about "cancer ex fibrocystic mastopathy" of the dog. In International Symposium on Mammary Cancer (Edited by L. SEVERI), p. 547. Division of Cancer Research, Perugia (1958). 21. J . D . DUNN, A. AmMURA and L. E. SCHEVING, Effect of stress on circadian periodicity in serum LH and prolactin concentration. Endocrinology 90~ 29 (1972). 22. J. NoKIN, M. VEKEMANS,M. L'H~.RMITE and C. ROBYN, Circadian periodicity of serum prolactin concentration. Brit. med. or. 3~ 561 (I 972). 23. M.J. MORONEY.In Factsfrom Figures, p. 286, Penguin Books, Harmondsworth, Middlesex.