Commonwealth Scientificand IndustrialOrganization
Journal
of Sreroid Biochrmisfr): Vol. Il. pp. 951 to %I 0 PergamonPressLtd 1979.Printedin Great Britain
29. Biological effects of Steroid Antibodies ANTIBODIES TO ANDROGENS, AND OVULATION IN THE EWE R. J. C.S.I.R.O.,
SCARAMUZZI
Division of Animal Production, P.O. Box 239, Blacktown, N.S.W. 2148. Australia SUMMARY
Ewes were actively immunized against androstenedione-1 I-BSA, androstenedione-7-HSA and testosterone-3-BSA. Antibody titre was monitored and, when necessary, maintained by booster immunizations. The effects of these immunizations on ovulation and oestrous cycles were investigated. Immunization against androstenedione-1 I-BSA or androstenedione-7-HSA increased ovulation rate (2.2 and 1.6, respectively) compared to control ewes (1.2). Immunization against testosterone-3-BSA produced an anovulatory state in most ewes, but increased ovulation rate in those ewes still ovulating. The oestrous cycle was not significantly altered by immunization against androstenedione, but was abolished in 4/5 ewes immunized against testosterone. Gonadotrophins were measured in jugular venous plasma taken from ewes immunized against androstenedione-1 l-BSA and testosterone-3-BSA. The circulating levels of LH were elevated in all immunized ewes, while FSH levels were either reduced (androstenedione-immunized) or unchanged (testosterone-immunized). These results suggest that neutralization of most of the biological activity of androgens produces alterations in hypothalamic-pituitaryovarian relationships, and/or in the incidence of follicular atresia. These changes can increase ovulation rate, but in their more extreme form they can also cause anovulation. It appears that ovarian androgens, acting either directly or indirectly as oestrogen precursors, are important components of the hypothalamic-pituitary-ovarian axis in the ewe.
INTRODUCDON The ovary of many mammals is able to secrete androgens, principally androstenedione and testosterone [l]. The presence of androgens in ovarian venous blood might simply represent nonfunctional by-products formed during the biosynthesis of oestrogens. Alternatively it could be that these androgens are secreted for a purpose and that they serve necessary biological function(s). The available evidence would generally support this latter view [2-4]. In the ewe, for example, testosterone administered exogenously can induce normal oestrous behaviour [S], LH release [6,7] and ovulation [8] and exogenous androstenedione can induce oestrus in the ovariectomized ewe [9]. Recent literature also indicates that testosterone might also have local effects on ovulation [lo] and follicular atresia [ll]. In recent years a number of studies have used active immunization against steroidal haptens to neutralize the biological activity of endogenous androgens [4,12]. This paper reviews the use of this technique as a method of investigating the role of ovarian androgens in the regulation of ovarian function and ovulation. ANDROGEN
ewes are presented (Table 1) and confirm that the sheep ovary is capable of secreting appreciable quantities of androstenedione and to a lesser extent of testosterone [lS]. The secretion rate of androstenedione was significantly correlated with the secretion rate of oestradiol-17/J (r = 0.514, P < 0.01, n = 33; r =0.756, P 3 mm diameter. The ovaries appeared otherwise normal although occasionally an abnormally large (> 10 mm diameter) unruptured follicle was observed. Figure 1 illustrates the frequency distribution for the numbers of corpora lutea (i.e. ovulation rate) from a group of Welsh Mountain ewes. It is apparent that the ovulation rate has increased in ewes immunized against androstenedione-1 l-BSA. Furthermore the excessive superovulation which commonly occurs when pregnant mare serum gonadotrophin is used to increase ovulation rate has been largely avoided. Permanently anoestrous ewes which had been immunized against testosterone-3-BSA had ovaries which were grossly enlarged. These ovaries contain
and ovulation
Immunization against androstenedione-1 I-BSA or androstenedione-‘I-HSA had very little effect on the length of the oestrous cycle in sheep (Table 2). Immunized ewes continued to show oestrus at intervals of 16-19 days and the duration of oestrus also appeared to be normal [4]. Immunization against testosterone3-BSA resulted in the rapid development of an anoestrus state in 4/S treated ewes (Table 2), which persisted for the 13-month duration of the experiment. The ovaries of treated ewes were examined at laparotomy and compared to ovaries from control
Table 2. The effect of immunization against androgens on the oestrous cycle, ovulation rate and follicular development in the ewe (mean + SE. of the mean) (data adapted from Scaramuzzi et 01.[4]; Van Look et a/.[18] and R. J. Scaramuzzi, R. 1. Cox and R. M. Hoskinson, (unpublished data)
Breed of ewe
Antigen
No. of ewes
Length of oestrous cycle (days)
Ovulation rate of ewes ovulating
No. follicles 33 mm dia.
Welsh Mountain Welsh Mountain Welsh Mountain
Androstenedione-1 I-BSA Testosterone-3-BSA BSA; control
23 5 12
16.7 f 0.2 Anoestrus 17.5 f 0.8
2.38 f 0.13 2.80 f 0.25 1.26 f 0.09
4.2 f 0.8 6.7 f 0.9 1.6 + 0.2
Merino Merino Merino
Androstenedione-l I-HSA Androstenedione-7-HSA HSA : control
5 12 6
17.7 f 0.6 17.1 f 0.2 17.3 f 0.4
2.20 f 0.20 1.64 f 0.20 1.23 f 0.12
3.4 f 0.5 3.0 f 0.5 2.4 f 0.6
HSA: human Serum albumin; BSA: bovine serum albumin.
Androgens and ovulatian
959
very large (18-29 mm diameter) follicle8 some of which were thin-walled and fluid-filled while others had thickened asymmetrical walls which appeared to be partially luteinized. Twa of the 4 permanently anoestrous ewes had solid structures reminiscent of old or modified corpora lutea present on their ovaries suggesting the sporadic occurrence numerous
of’silent o+uktion in t&e animals. One treated animal continued to show reguiar ctesfrusand had an
increased ovulation rate, her ovaries were devoid of abnormally large follicles as seen in the anoestrous ewes described above. These data show that i~u~za~on against androgens leads to ovarian stimulation. This stimulation may be beneficial if, as with immunization against androstenedione, it increases ovulation rate. On the other hand the excessive ovarian stimulation seen in ewes immunized against testosterone-3-BSA is obviously detrimental to the animals’ reproductive potential. The fertility of sheep immunized against androgens has not been extensively tested. Some preliminary information [18f shows no improvement in fertility despite an increased ovulation rate in a group of ewes immunized against androstenedione-i I-BSA. A significant alteration in the sex ratio of lambs born to ewes immunized against androstenedione-1 I-BSA has also been reported [18], The fertility of ewes immunized against testosterone-3-BSA has not been tested The problem of translating increases in ovulation rate to increases in fertility is a function of the control that can be brought to the antisteroid antibody response so as to prevent overstimulation of the ovaries. l.H (ng I ml)
22 I 16 t
to-
as4-
,“tmtrt;r;l CONTROL
Fig. 2. The cor~ntration (mean f SE. of the mean) of LH in jugular venous p&ma on days 2, 7, 12 and 16 of the aestroua cycle in ewes immunized against BSA (control) or androstenedione-11-BSA (AAJ and in anoestrous ewes immunized against testosterono3BSA (T). Each bar represents the comzntration of LH in at least 4 animals bkd every 15 min for a period of 6 h. Data adapted from Martensz and Scaramuzzi[iCf7Jand Martensz[Zl].
Fig. 3. The concentration (mean * SE. of the mean) of FSH in iuaular venous plasma on days 2. 7, 12 and 16 of the oe’stks cycle in ekes immunized ag&nst BSA (control), or androstenedior~ll-BSA (AA+) and in anocstrous ewes immunized against t~t~~~~~A fl). Each k represents the concentration of FSH in at least 4 animals bled every 15 min for a period of 6 h. Data adapted from Martensz and !karamuzzi[lfl and Martensz[Zl].
Indwelling catheters were placed in the jugular veins of treated and control ewes, Blood samples were then collected every 15 tin over a tch period on selected days of the oestrous cycle. The concentrations of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were measured by ra~oi~uno~ys [19,2Q The overall mean cclncentrations of LH and FSH for controt and androgen inimunized groups of ewes are shown in Figs 2 and 3. Immunization again& androstenedione-1 l-MA was followed by increased LI-I and decreased FSH levels. The -immunization of sheep against testosterone+ BSA was followed by a rise in LH levels but with no change in FSH levels. The very high levels of LH in ewes immunized against testosterone-3-BSA probably explains the gross o~rstirnu~~on of the ovaries of these animals. Examination of the inditidual patterns of LH and FSH levels suggest that the frequency and amplitude of pulsatile LH release has been increased marginally by j~u~~on agailrst androstenedione-1 l-BSA and substantiaiiy increased by immunization against testosterone9-BSA C173. Gonadotrophin levels in ewes immunized against ~~~ten~i~~7-Ha have not been mea&red. 4. Possible mechanisms Immunization against androstenedione-ll-BSA leads neither to formation of antibodies able to bind oestradiol-17p nor to reduced oestradiol~17fl secretion [22]. On the contrary oestradiol-l’tg sacretion rises [22]. Despite the latter LH levels also increase (Fig, 2) indicating that the usual negative feedback effect of oestradioM7j3 01t LH secretion is considerably reduced. Such a reduction could conceivably
R. J.
960
SCARAMUZZI
occur if androstenedione were a normal modulator of this feedback effect of oestradiol-178 [4]. Neutralization of part or all of the biological effect of androstenedione by its antibodies would therefore interfere with the control system and lead to elevated LH levels. Similarly, elevated LH levels would also result from any reduction in peripheral or CNS formation of oestradiol-178 from circulating androstenedione. In ewes immunized against testosterone-3-BSA a more complex situation exists for here antibodies which can bind to both testosterone, and oestradiol-178 are produced [23]. It is, however, perhaps easier to understand why LH levels are high in these animals because biologically active oestradiol-178 could be neutralized by antibodies able to bind this steroid [23,24]. Why it is in both situations that high LH levels lead to increases in ovulation rate is even now not f&y understood. An alternative mechanism to explain the increase in ovulation rate could relate to the local effect of androgens. It is known that androgens can act locally to induce atresia[ll) and that antibodies can enter follicular fluid [21,25]. A~ordingfy in ewes immunized against androgens follicular atresia could be reduced and more follicles made available for ovulation. The levels of FSH observed in response to immu-
nization are difficult to understand and even more difficult ta relate to any proposed function of FSH in the regulation of follicular development and ovulation rate. Sheep immunized against androstenedione-11-B!% show a doubling of ovulation rate in the face of decreased FSH levels. These data suggest that the ovarian feedback control of LH and FSH can be separate and that any function of FSH in relation to ovulation rate is permissive rather than regulatory.
CONCLUSIONS The data summarized in this paper leads to two conclusions. Firstly, the ovary of the ewe is able to secrete appreciable quantities of both androstenedione and testosterone. Secondly, neutralization of part or all of the biological activity of these androgens leads to ovarian stimulation and increased ovulation rate. These two conclusions provide strong support for a functional role for androstenedione and testosterone in the regulation of ovarian activity in the ewe. The mechanisms by which these androgens influence ovarian function are still ,speculative, and several possibiiities are proposed. The two main possibilities are either an interference with negative feedback of ovarian steroids on the hypothalamopituitary axis and/or a reduction in local androgeninduoed follicular atresia. These results also suggest a method for increasing fertility in sheep flocks, especially since low ovulation rates are a normal factor limiting fertility in some
breeds of sheep. While initial tests have not given increases in fertilitytl8-J further study is warranted
Acknowledgements-I
wish to acknowledge the contributions of my research collaborators, Prof. D. T. Baird, Drs. I. J. Clarke, N. D. Martensz and P. F. A. Van Look and Mr. W. G. Davidson from the M.R.C. Unit of Reproductive Biology, Edinburgh, Scotland, and Drs. R. I. Cox and R. M. Hoskinson from C.S.I.R.O., Division of Animal Production, Prospect, N.S.W. Australia.
REFERENCES 1. Baird D. T.: Synthesis and secretion of steroid hormones by the ovary in viva. In The Ovary (Edited by
2. 3. 4.
5. 6.
7.
Sir S. Zuckerman and B. Weir). Academic Press, New York and London, 2nd ed., Vol. 3 (1978) pp. 305-357. Carter A. C., Cohen E. J. and Shorr E.: The use of androgens in women. Virams Horm. 5 (1947) 317-391. Everitt B. J. and Herbert J.: The effects of dexamethasone and androgens on sexual receptivity of female rhesus monkevs. J. Endocr. 51 (19711575-588. Scaramuzzi RI J., Davidson W: G. and Van Look P. F. A.: increasing the ovulation rate of sheep by active immunization against an ovarian steroid androstenedione. Nature, Land. 269 (1977) 817818. Lindsay D. R. and Robinson T. J.: Oestrus-inducing activity of testosterone in the ewe. Nurure, Lmd. 192 (1961) 761-762. Pant H. C.: Effect of androgens on concentration of LH and FSH in the peripheral plasma of anoestrous ewes. J. Reprod. Fertif. 50 (1977) 133-136. Clarke I. J. and Scaramuzzi R. 1.: Sexual behaviour
and LH secretion in spayed androgenized ewes after a single iniection of testosterone Repr;;d
or oestradiol-178. J.
Fertil. 52 (1978) 313-320.
8. Radford H. M. and Wallace A. L. C.: The effect of testosterone propionate on ovarian activity in sheep. 1. Reprod. Ferril. 24 (1971) 439440. 9. Lindsay D. R. and Scaramuzzi R. J.: Oestrogenic and anti-oestrogenic activities of a number of steroids in behavioural oestrus and vaginal smear assays in the ewe. f. Endocr. 45 (1969) 549-555. 10. Mori T., Suzuki A., Nishimura T. and Kambegawa A.: Evidence for androgen participation in induced ovulation in immature rats. Endocrinology 101 (1977) 623-626. 11. Louvet J. P., Harman S. M., Schreiber J. R. and Ross G. T.: Evidence for a role of androgens in follicular maturation. Endocrinology 97 (1975) 366-372. 12. Hillier S. G., Groom G. V., Boyns A. R. and Cameron E. H. D.: Development of polycystic ovaries in rats actively immunized against T-bBSA Nnture, Land. 250 (1974) 233434. 13. Baird D. T.. Land R. B.. Scaramuzzi R. J. and Wheeler A. G.: Endocrine changes associated with luteal regression in the ewe; secretion of ovarian oestradiol, progesterone and androstenedione and uterine prostaglandin Fz. throughout the oestrous cycle. J. Endocr. 69 (1976) 275-286. 14. Corker C. S. and Davidson D. W.: A radioimmunoassay for testosterone in various biological fluids without chromatozraohv. J. steroid. Biochem. 9 (1978) 373-3?4. 1.5. Baird D. f., &ling J. R.. Ichikawa Y. and McCracken J. A.: The secretion of steroids from the autotransplanted ovary in the ewe spontaneously and in response to systemic gonadotrophin. J. Endocr. 42 (1968) 282-299.
Androgens and ovulation 16. Scaramuzzi R. J., Corker C. S., Young G. and Baird D. T.: Production of antisera to steroid hormones. In Steroid Immunoassay (Edited by E. H. D. Cameron, S. G. Hillier and K. Griffiths). Alpha Omega Alpha Publications, Cardiff (1975). Proc. Vth Tenovus Workshop CarditT, April, 1974, pp. 111-122. 17. Martensz N. D. and Scaramuzzi R. J.: The plasma concentrations of luteinizing hormone, follicle:stimulating hormone and progesterone during the breeding season in ewes immunized against androstenedione or testosterone. J. Endocr. (1979). In press. 18. Van Look, P. F. A., Clarke I. J., Davidson W. G. and Scaramuzzi R. J.: Ovulation and lambing rate in ewes actively immunized against androstenedione. J. Reprod. Fert. 53 (1978) 129-130. 19. Martensz N. D., Baird D. T.. Scaramuzzi R. J. and Van Look P. F. A.: Androstenedione and the control of luteinizing hormone in the ewe during anoestrus. J. Endocr. 69 (1976) 227-237. 20. Lincoln G. A., Peet M. J. and Cunningham R. A.: Seasonal and circadian changes in the episodic release of
961
follicle-stimulating hormone, luteinizing hormone and testosterone in rams exposed to artificial photoperiods. J. Endocr.
12 (1977) 337-349.
21. Martensz, N. D.: The experimental manipulation of gonadotrophins in nonpregnant sheep by immunization against steroid hormones. Ph.D. thesis. Uniuersiry of Edinburgh
(I 977).
22. Scaramuzzi R. J., Baird D. T., Clarke I. J., Martensz N. D. and Van Look P. F. A.: Ovarian morphology and the concentration of steroids during the oestrous cycle of sheep actively immunized against androstenedione. J. Reprod. Fertil. (1979). In press. 23. Scaramuzzi R. J., Baird D. T., Martensz N. D.. Turnbull K. E. and Van Look P. F. A.: Ovarian function in the ewe following active immunization against testosterone. In preparation. 24. Rawlings N. C., Kennedy S. W. and Hendricks D. M.: EfFect of active immunization of the cyclic ewe against oestradiol-17g. J. Endocr. 76 (1978) 11-19. 25. Edwards R. G.: Follicular fluid. J. Reprod. Fertil. 37 (1974)
189-219.
Journal
of Sreroid Biochrmisfr): Vol. Il. pp. 951 to %I 0 PergamonPressLtd 1979.Printedin Great Britain
29. Biological effects of Steroid Antibodies ANTIBODIES TO ANDROGENS, AND OVULATION IN THE EWE R. J. C.S.I.R.O.,
SCARAMUZZI
Division of Animal Production, P.O. Box 239, Blacktown, N.S.W. 2148. Australia SUMMARY
Ewes were actively immunized against androstenedione-1 I-BSA, androstenedione-7-HSA and testosterone-3-BSA. Antibody titre was monitored and, when necessary, maintained by booster immunizations. The effects of these immunizations on ovulation and oestrous cycles were investigated. Immunization against androstenedione-1 I-BSA or androstenedione-7-HSA increased ovulation rate (2.2 and 1.6, respectively) compared to control ewes (1.2). Immunization against testosterone-3-BSA produced an anovulatory state in most ewes, but increased ovulation rate in those ewes still ovulating. The oestrous cycle was not significantly altered by immunization against androstenedione, but was abolished in 4/5 ewes immunized against testosterone. Gonadotrophins were measured in jugular venous plasma taken from ewes immunized against androstenedione-1 l-BSA and testosterone-3-BSA. The circulating levels of LH were elevated in all immunized ewes, while FSH levels were either reduced (androstenedione-immunized) or unchanged (testosterone-immunized). These results suggest that neutralization of most of the biological activity of androgens produces alterations in hypothalamic-pituitaryovarian relationships, and/or in the incidence of follicular atresia. These changes can increase ovulation rate, but in their more extreme form they can also cause anovulation. It appears that ovarian androgens, acting either directly or indirectly as oestrogen precursors, are important components of the hypothalamic-pituitary-ovarian axis in the ewe.
INTRODUCDON The ovary of many mammals is able to secrete androgens, principally androstenedione and testosterone [l]. The presence of androgens in ovarian venous blood might simply represent nonfunctional by-products formed during the biosynthesis of oestrogens. Alternatively it could be that these androgens are secreted for a purpose and that they serve necessary biological function(s). The available evidence would generally support this latter view [2-4]. In the ewe, for example, testosterone administered exogenously can induce normal oestrous behaviour [S], LH release [6,7] and ovulation [8] and exogenous androstenedione can induce oestrus in the ovariectomized ewe [9]. Recent literature also indicates that testosterone might also have local effects on ovulation [lo] and follicular atresia [ll]. In recent years a number of studies have used active immunization against steroidal haptens to neutralize the biological activity of endogenous androgens [4,12]. This paper reviews the use of this technique as a method of investigating the role of ovarian androgens in the regulation of ovarian function and ovulation. ANDROGEN
ewes are presented (Table 1) and confirm that the sheep ovary is capable of secreting appreciable quantities of androstenedione and to a lesser extent of testosterone [lS]. The secretion rate of androstenedione was significantly correlated with the secretion rate of oestradiol-17/J (r = 0.514, P < 0.01, n = 33; r =0.756, P 3 mm diameter. The ovaries appeared otherwise normal although occasionally an abnormally large (> 10 mm diameter) unruptured follicle was observed. Figure 1 illustrates the frequency distribution for the numbers of corpora lutea (i.e. ovulation rate) from a group of Welsh Mountain ewes. It is apparent that the ovulation rate has increased in ewes immunized against androstenedione-1 l-BSA. Furthermore the excessive superovulation which commonly occurs when pregnant mare serum gonadotrophin is used to increase ovulation rate has been largely avoided. Permanently anoestrous ewes which had been immunized against testosterone-3-BSA had ovaries which were grossly enlarged. These ovaries contain
and ovulation
Immunization against androstenedione-1 I-BSA or androstenedione-‘I-HSA had very little effect on the length of the oestrous cycle in sheep (Table 2). Immunized ewes continued to show oestrus at intervals of 16-19 days and the duration of oestrus also appeared to be normal [4]. Immunization against testosterone3-BSA resulted in the rapid development of an anoestrus state in 4/S treated ewes (Table 2), which persisted for the 13-month duration of the experiment. The ovaries of treated ewes were examined at laparotomy and compared to ovaries from control
Table 2. The effect of immunization against androgens on the oestrous cycle, ovulation rate and follicular development in the ewe (mean + SE. of the mean) (data adapted from Scaramuzzi et 01.[4]; Van Look et a/.[18] and R. J. Scaramuzzi, R. 1. Cox and R. M. Hoskinson, (unpublished data)
Breed of ewe
Antigen
No. of ewes
Length of oestrous cycle (days)
Ovulation rate of ewes ovulating
No. follicles 33 mm dia.
Welsh Mountain Welsh Mountain Welsh Mountain
Androstenedione-1 I-BSA Testosterone-3-BSA BSA; control
23 5 12
16.7 f 0.2 Anoestrus 17.5 f 0.8
2.38 f 0.13 2.80 f 0.25 1.26 f 0.09
4.2 f 0.8 6.7 f 0.9 1.6 + 0.2
Merino Merino Merino
Androstenedione-l I-HSA Androstenedione-7-HSA HSA : control
5 12 6
17.7 f 0.6 17.1 f 0.2 17.3 f 0.4
2.20 f 0.20 1.64 f 0.20 1.23 f 0.12
3.4 f 0.5 3.0 f 0.5 2.4 f 0.6
HSA: human Serum albumin; BSA: bovine serum albumin.
Androgens and ovulatian
959
very large (18-29 mm diameter) follicle8 some of which were thin-walled and fluid-filled while others had thickened asymmetrical walls which appeared to be partially luteinized. Twa of the 4 permanently anoestrous ewes had solid structures reminiscent of old or modified corpora lutea present on their ovaries suggesting the sporadic occurrence numerous
of’silent o+uktion in t&e animals. One treated animal continued to show reguiar ctesfrusand had an
increased ovulation rate, her ovaries were devoid of abnormally large follicles as seen in the anoestrous ewes described above. These data show that i~u~za~on against androgens leads to ovarian stimulation. This stimulation may be beneficial if, as with immunization against androstenedione, it increases ovulation rate. On the other hand the excessive ovarian stimulation seen in ewes immunized against testosterone-3-BSA is obviously detrimental to the animals’ reproductive potential. The fertility of sheep immunized against androgens has not been extensively tested. Some preliminary information [18f shows no improvement in fertility despite an increased ovulation rate in a group of ewes immunized against androstenedione-i I-BSA. A significant alteration in the sex ratio of lambs born to ewes immunized against androstenedione-1 I-BSA has also been reported [18], The fertility of ewes immunized against testosterone-3-BSA has not been tested The problem of translating increases in ovulation rate to increases in fertility is a function of the control that can be brought to the antisteroid antibody response so as to prevent overstimulation of the ovaries. l.H (ng I ml)
22 I 16 t
to-
as4-
,“tmtrt;r;l CONTROL
Fig. 2. The cor~ntration (mean f SE. of the mean) of LH in jugular venous p&ma on days 2, 7, 12 and 16 of the aestroua cycle in ewes immunized against BSA (control) or androstenedione-11-BSA (AAJ and in anoestrous ewes immunized against testosterono3BSA (T). Each bar represents the comzntration of LH in at least 4 animals bkd every 15 min for a period of 6 h. Data adapted from Martensz and Scaramuzzi[iCf7Jand Martensz[Zl].
Fig. 3. The concentration (mean * SE. of the mean) of FSH in iuaular venous plasma on days 2. 7, 12 and 16 of the oe’stks cycle in ekes immunized ag&nst BSA (control), or androstenedior~ll-BSA (AA+) and in anocstrous ewes immunized against t~t~~~~~A fl). Each k represents the concentration of FSH in at least 4 animals bled every 15 min for a period of 6 h. Data adapted from Martensz and !karamuzzi[lfl and Martensz[Zl].
Indwelling catheters were placed in the jugular veins of treated and control ewes, Blood samples were then collected every 15 tin over a tch period on selected days of the oestrous cycle. The concentrations of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were measured by ra~oi~uno~ys [19,2Q The overall mean cclncentrations of LH and FSH for controt and androgen inimunized groups of ewes are shown in Figs 2 and 3. Immunization again& androstenedione-1 l-MA was followed by increased LI-I and decreased FSH levels. The -immunization of sheep against testosterone+ BSA was followed by a rise in LH levels but with no change in FSH levels. The very high levels of LH in ewes immunized against testosterone-3-BSA probably explains the gross o~rstirnu~~on of the ovaries of these animals. Examination of the inditidual patterns of LH and FSH levels suggest that the frequency and amplitude of pulsatile LH release has been increased marginally by j~u~~on agailrst androstenedione-1 l-BSA and substantiaiiy increased by immunization against testosterone9-BSA C173. Gonadotrophin levels in ewes immunized against ~~~ten~i~~7-Ha have not been mea&red. 4. Possible mechanisms Immunization against androstenedione-ll-BSA leads neither to formation of antibodies able to bind oestradiol-17p nor to reduced oestradiol~17fl secretion [22]. On the contrary oestradiol-l’tg sacretion rises [22]. Despite the latter LH levels also increase (Fig, 2) indicating that the usual negative feedback effect of oestradioM7j3 01t LH secretion is considerably reduced. Such a reduction could conceivably
R. J.
960
SCARAMUZZI
occur if androstenedione were a normal modulator of this feedback effect of oestradiol-178 [4]. Neutralization of part or all of the biological effect of androstenedione by its antibodies would therefore interfere with the control system and lead to elevated LH levels. Similarly, elevated LH levels would also result from any reduction in peripheral or CNS formation of oestradiol-178 from circulating androstenedione. In ewes immunized against testosterone-3-BSA a more complex situation exists for here antibodies which can bind to both testosterone, and oestradiol-178 are produced [23]. It is, however, perhaps easier to understand why LH levels are high in these animals because biologically active oestradiol-178 could be neutralized by antibodies able to bind this steroid [23,24]. Why it is in both situations that high LH levels lead to increases in ovulation rate is even now not f&y understood. An alternative mechanism to explain the increase in ovulation rate could relate to the local effect of androgens. It is known that androgens can act locally to induce atresia[ll) and that antibodies can enter follicular fluid [21,25]. A~ordingfy in ewes immunized against androgens follicular atresia could be reduced and more follicles made available for ovulation. The levels of FSH observed in response to immu-
nization are difficult to understand and even more difficult ta relate to any proposed function of FSH in the regulation of follicular development and ovulation rate. Sheep immunized against androstenedione-11-B!% show a doubling of ovulation rate in the face of decreased FSH levels. These data suggest that the ovarian feedback control of LH and FSH can be separate and that any function of FSH in relation to ovulation rate is permissive rather than regulatory.
CONCLUSIONS The data summarized in this paper leads to two conclusions. Firstly, the ovary of the ewe is able to secrete appreciable quantities of both androstenedione and testosterone. Secondly, neutralization of part or all of the biological activity of these androgens leads to ovarian stimulation and increased ovulation rate. These two conclusions provide strong support for a functional role for androstenedione and testosterone in the regulation of ovarian activity in the ewe. The mechanisms by which these androgens influence ovarian function are still ,speculative, and several possibiiities are proposed. The two main possibilities are either an interference with negative feedback of ovarian steroids on the hypothalamopituitary axis and/or a reduction in local androgeninduoed follicular atresia. These results also suggest a method for increasing fertility in sheep flocks, especially since low ovulation rates are a normal factor limiting fertility in some
breeds of sheep. While initial tests have not given increases in fertilitytl8-J further study is warranted
Acknowledgements-I
wish to acknowledge the contributions of my research collaborators, Prof. D. T. Baird, Drs. I. J. Clarke, N. D. Martensz and P. F. A. Van Look and Mr. W. G. Davidson from the M.R.C. Unit of Reproductive Biology, Edinburgh, Scotland, and Drs. R. I. Cox and R. M. Hoskinson from C.S.I.R.O., Division of Animal Production, Prospect, N.S.W. Australia.
REFERENCES 1. Baird D. T.: Synthesis and secretion of steroid hormones by the ovary in viva. In The Ovary (Edited by
2. 3. 4.
5. 6.
7.
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