SEXUAL DIFFERENTIATION OF THE BRAIN: ENDOCRINE AND BEHAVIOURAL RESPONSES OF ANDROGENIZED EWES TO OESTROGEN
CLARKE, R. J. SCARAMUZZI AND R. V. SHORT M.R. C. Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EHI 2QW I. J.
(Received
11 June
1976)
Testosterone treatment of female mammals during a critical period of foetal or neonatal life affects their postpubertal endocrine and behavioural activity. For example, it prevents the occurrence of regular ovulatory cycles in adult rats (Barraclough & Gorski, 1961), guinea-pigs (Brown-Grant & Sherwood, 1971), hamsters (Swanson & Brayshaw, 1973) and sheep (Short, 1974), which is apparently due to a failure of oestrogen to facilitate the release of luteinizing hormone (positive feedback) (Brown-Grant, 1974; Short, 1974). Positive feedback is a sexually dimorphic character in sheep and is only shown by ewes (Short, 1974; Karsch & Foster, 1975). Female sheep foetuses exposed to testosterone from days 20 or 60 of gestation until birth not only failed to show positive feedback (Short, 1974), but were incapable of displaying behavioural oestrus, even when given 800 \g=m\goestradiol benzoate (OB) (I. J. Clarke, guinea-pigs, rats, unpublished results). It has also been shown that androgenization of female and hamsters and dogs alters their sexual behaviour by suppressing female display accentuating male-like patterns (Whalen, 1968; Beach, 1975). It has even been suggested that the presence of positive feedback in men is correlated with homosexual behaviour (Dorner, Rohde, Stahl, Krell, & Masius, 1975). The purpose of the present investigation was to see how closely endocrine and behavioural events were correlated with one another in experimentally androgenized female
sheep.
Dorset Horn sheep foetuses were exposed to androgen for 50 day periods by implanting their mothers with 1 g testosterone (Clarke, Scaramuzzi & Short, 1976). Those females that were treated between days 30 and 80 (D30-80; n 6) had male-type external genitalia, whereas those treated between days 50 and 100 (D50-100; 7) only showed hyper¬ trophy of the clitoris (Clarke et al. 1976). The androgenized ewes were run with a contemporary group of normal ewe lambs (n 8), and a raddled vasectomized ram was introduced during the Finn
=
=
=
first mating season. All of the control ewes showed regular cycles of 16-7 ±0-6 (S.E.M.) days throughout their first mating season with a total of 44 oestrous periods. None of the D30-80 animals showed regular cycles, and only eight heats were recorded. Similarly, the D50-100 ewes failed to show regular cycles, and only nine heats were recorded. At the end of the first mating season after all the control ewes had become anoestrous (May), the positive feedback effect of OB on luteinizing hormone (LH) discharge was evaluated.a All the ewes were primed with 12 daily injections of 10 mg progesterone, and then given single i.m. injection of 50 Mg OB in oil 24 h after the last progesterone injection. Jugular venous blood was collected at 2-4 h intervals for 36 h, and plasma LH concentrations were measured by radioimmunoassay (Martensz, Baird, Scaramuzzi & Van Look, 1976). All of the control ewes discharged LH within 22-36 h of the OB injection (Table 1), whereas only two out of the six D30-80 ewes and one out of the seven D50-100 ewes showed a dis¬ charge. Following the OB injection, the ewes were checked for oestrus at 4h intervals using a vasectomized ram. The sheep were also paired with two oestrous ewes, and in this way it was possible to score male sexual behaviour. Four out of the eight control ewes displayed oestrus * Present address: C.S.I.R.O. Division of Animal Production, Prospect, P.O. Box 239, Blacktown, New South Wales, Australia.
Table 1. Behavioural responses and LH concentrations in androgenized and normal ewes given an injection of 50 pg oestradiol benzoate during summer anoestrus Number of ewes
Androgen treatment during gestation Days 30-80
showing: Positive
Oestrus
Mounting
feedback
(ng/ml)
2 1
78-2 17-6 65-5 ± 10-6*
Days 50-100 No treatment
(control) *Means
±
Peak plasma LH concentrations
Time from to LH
injection
peak (h)
22-0 18-0 27-7 ±2-5*
S.E.M.
none of the D30-80 or D50-100 ewes came into heat (Table 1). The failure of half the ewes to show oestrus in response to an oestrogen injection confirmed earlier work showing that ewes become relatively refractory to the behavioural effects of oestrogen during anoestrus (Gibson & Robinson, 1971; Beck & Reeves, 1973), and suggests that the endocrine and behavioural centres of the brain may operate independently of one another. This conclusion was also borne out by the androgenized animals. Male-type mounting behaviour, which was never seen in controls, was shown by two of the D30-80 ewes and one D50-100 ewe and yet these behaviourally masculinized animals also happened to be the only ones to show a female-type positive feedback response to oestrogen. Since the endocrine and behavioural responses to oestrogen in sheep vary independently of one another during the year, it is impossible to predict the type of sexual behaviour from the presence or absence of positive feedback.
whereas control
The authors thank the Farm Staff of the Animal Breeding Research Organization for the of the animals. Mr W. G. Davidson provided technical assistance. One of us (U.C.) is in receipt of a William Georgetti Scholarship.
care
REFERENCES
Gorski, (1961). Endocrinology 68,68-79. Barraclough, Beach, F. A. (1975). Psychoneuroendocrinology 1, 3-23. Beck, T. W. & Reeves, J. J. (1973). Journal of Animal Science 36,566-570. Brown-Grant, K. (1974). Journal of Endocrinology 62, 319-332. Brown-Grant, K. & Sherwood, M. R. (1971). Journal of Endocrinology 49, 277-291. Clarke, I. J., Scaramuzzi, R. J. & Short, R. V. (1976). Journal of Embryology and Experimental. Morphology (In Press.) Dorner, G., Rohde, W., Stahl, F., KreU, L. & Masius, W. (1975). Archives of Sexual Behaviour 4, 1-8. Gibson, W. R. & Robinson, T. J. (1971). Journal of Reproduction and Fertility 24, 9-18. Karsch, F. J. & Foster, D. L. (1975). Endocrinology 85, 133-142. Martensz, N. D., Baird, D. T., Scaramuzzi, R. J. & Van Look, P. F. A. (197'6). Journal of Endocrinology 69, C. A. &
R. A.
227-237.
R. V. (1974). In Sexual endocrinology of the perinatal period. Colloque International, pp. 121-142. Eds M. G. Forest & J. Bertrand. Lyon: INSERM. Swanson, H. H. & Brayshaw, J. S. (1973). Advances in the Biosciences 13, 119-136. Whalen, R. E. (1968). In Reproduction and sexual behaviour, pp. 303-340. Ed. M. Diamond. Indiana University Press.
Short,
CLARKE, R. J. SCARAMUZZI AND R. V. SHORT M.R. C. Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EHI 2QW I. J.
(Received
11 June
1976)
Testosterone treatment of female mammals during a critical period of foetal or neonatal life affects their postpubertal endocrine and behavioural activity. For example, it prevents the occurrence of regular ovulatory cycles in adult rats (Barraclough & Gorski, 1961), guinea-pigs (Brown-Grant & Sherwood, 1971), hamsters (Swanson & Brayshaw, 1973) and sheep (Short, 1974), which is apparently due to a failure of oestrogen to facilitate the release of luteinizing hormone (positive feedback) (Brown-Grant, 1974; Short, 1974). Positive feedback is a sexually dimorphic character in sheep and is only shown by ewes (Short, 1974; Karsch & Foster, 1975). Female sheep foetuses exposed to testosterone from days 20 or 60 of gestation until birth not only failed to show positive feedback (Short, 1974), but were incapable of displaying behavioural oestrus, even when given 800 \g=m\goestradiol benzoate (OB) (I. J. Clarke, guinea-pigs, rats, unpublished results). It has also been shown that androgenization of female and hamsters and dogs alters their sexual behaviour by suppressing female display accentuating male-like patterns (Whalen, 1968; Beach, 1975). It has even been suggested that the presence of positive feedback in men is correlated with homosexual behaviour (Dorner, Rohde, Stahl, Krell, & Masius, 1975). The purpose of the present investigation was to see how closely endocrine and behavioural events were correlated with one another in experimentally androgenized female
sheep.
Dorset Horn sheep foetuses were exposed to androgen for 50 day periods by implanting their mothers with 1 g testosterone (Clarke, Scaramuzzi & Short, 1976). Those females that were treated between days 30 and 80 (D30-80; n 6) had male-type external genitalia, whereas those treated between days 50 and 100 (D50-100; 7) only showed hyper¬ trophy of the clitoris (Clarke et al. 1976). The androgenized ewes were run with a contemporary group of normal ewe lambs (n 8), and a raddled vasectomized ram was introduced during the Finn
=
=
=
first mating season. All of the control ewes showed regular cycles of 16-7 ±0-6 (S.E.M.) days throughout their first mating season with a total of 44 oestrous periods. None of the D30-80 animals showed regular cycles, and only eight heats were recorded. Similarly, the D50-100 ewes failed to show regular cycles, and only nine heats were recorded. At the end of the first mating season after all the control ewes had become anoestrous (May), the positive feedback effect of OB on luteinizing hormone (LH) discharge was evaluated.a All the ewes were primed with 12 daily injections of 10 mg progesterone, and then given single i.m. injection of 50 Mg OB in oil 24 h after the last progesterone injection. Jugular venous blood was collected at 2-4 h intervals for 36 h, and plasma LH concentrations were measured by radioimmunoassay (Martensz, Baird, Scaramuzzi & Van Look, 1976). All of the control ewes discharged LH within 22-36 h of the OB injection (Table 1), whereas only two out of the six D30-80 ewes and one out of the seven D50-100 ewes showed a dis¬ charge. Following the OB injection, the ewes were checked for oestrus at 4h intervals using a vasectomized ram. The sheep were also paired with two oestrous ewes, and in this way it was possible to score male sexual behaviour. Four out of the eight control ewes displayed oestrus * Present address: C.S.I.R.O. Division of Animal Production, Prospect, P.O. Box 239, Blacktown, New South Wales, Australia.
Table 1. Behavioural responses and LH concentrations in androgenized and normal ewes given an injection of 50 pg oestradiol benzoate during summer anoestrus Number of ewes
Androgen treatment during gestation Days 30-80
showing: Positive
Oestrus
Mounting
feedback
(ng/ml)
2 1
78-2 17-6 65-5 ± 10-6*
Days 50-100 No treatment
(control) *Means
±
Peak plasma LH concentrations
Time from to LH
injection
peak (h)
22-0 18-0 27-7 ±2-5*
S.E.M.
none of the D30-80 or D50-100 ewes came into heat (Table 1). The failure of half the ewes to show oestrus in response to an oestrogen injection confirmed earlier work showing that ewes become relatively refractory to the behavioural effects of oestrogen during anoestrus (Gibson & Robinson, 1971; Beck & Reeves, 1973), and suggests that the endocrine and behavioural centres of the brain may operate independently of one another. This conclusion was also borne out by the androgenized animals. Male-type mounting behaviour, which was never seen in controls, was shown by two of the D30-80 ewes and one D50-100 ewe and yet these behaviourally masculinized animals also happened to be the only ones to show a female-type positive feedback response to oestrogen. Since the endocrine and behavioural responses to oestrogen in sheep vary independently of one another during the year, it is impossible to predict the type of sexual behaviour from the presence or absence of positive feedback.
whereas control
The authors thank the Farm Staff of the Animal Breeding Research Organization for the of the animals. Mr W. G. Davidson provided technical assistance. One of us (U.C.) is in receipt of a William Georgetti Scholarship.
care
REFERENCES
Gorski, (1961). Endocrinology 68,68-79. Barraclough, Beach, F. A. (1975). Psychoneuroendocrinology 1, 3-23. Beck, T. W. & Reeves, J. J. (1973). Journal of Animal Science 36,566-570. Brown-Grant, K. (1974). Journal of Endocrinology 62, 319-332. Brown-Grant, K. & Sherwood, M. R. (1971). Journal of Endocrinology 49, 277-291. Clarke, I. J., Scaramuzzi, R. J. & Short, R. V. (1976). Journal of Embryology and Experimental. Morphology (In Press.) Dorner, G., Rohde, W., Stahl, F., KreU, L. & Masius, W. (1975). Archives of Sexual Behaviour 4, 1-8. Gibson, W. R. & Robinson, T. J. (1971). Journal of Reproduction and Fertility 24, 9-18. Karsch, F. J. & Foster, D. L. (1975). Endocrinology 85, 133-142. Martensz, N. D., Baird, D. T., Scaramuzzi, R. J. & Van Look, P. F. A. (197'6). Journal of Endocrinology 69, C. A. &
R. A.
227-237.
R. V. (1974). In Sexual endocrinology of the perinatal period. Colloque International, pp. 121-142. Eds M. G. Forest & J. Bertrand. Lyon: INSERM. Swanson, H. H. & Brayshaw, J. S. (1973). Advances in the Biosciences 13, 119-136. Whalen, R. E. (1968). In Reproduction and sexual behaviour, pp. 303-340. Ed. M. Diamond. Indiana University Press.
Short,
