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Partial deferminization by administration of androstenedione to neonatal female rats
Pergamon PTeae
Vol. 8, Part 1, pp. 1003-1008, 1989. Life 3cienc a Printed in Great Britain.
PARTIAL DEFEMINI2ATION BY ADMINISTRATION OF ANDROSTENEDIONE TO NEONATAL FEMALE RATS William G . Luttge and Richard E . Whalen Department of Psychobiology University of California, Irvine Irvine, California 92664
(received 4 June 1989; in final form 7 July 1989) bURING the past decade the administration of both homotypical and heterotypical hormones to prenatal and neonatal animals has been used by investigators to elucidate the role of gonadal hormones in the differentiation of those neural mechanisms which control gonadotropin secretion and sexual behavior .
For
example, it has been shown that the administration of testosterone propionate (TP) to neonatal female rata can permanently alter the cyclic pattern of pitunary gonadotropin secretion and the display of female-like mating behavior (1-3) .
Testicular, but not ovarian, grafts can mimic moat of these effects,
which include induction of persistent vaginal cornification, anovulatory sterility, and inhibition of female-like mating behavior (3) .
In addition,
studies employing the castration of neonatal male rata (4), or the repeated administration of high doses of the antiandrogen cyproterone acetate (6-chloro17-hydroxy-1n,2~-methylenepregna-4,6-thane-3,20-diene acetate) (5), have shown that one can produce a male with the female cyclic pattern of gonadotropin secretion .
Unlike normal melee, these demasculinized males can readily be
induced to display the female-like mating pattern when exogenous estrogen ie administered (4) .
Both of these experimental approaches (i .e ., surgical or
chemical gonadectomy of the male and the treatment of the female with male hormoms) have increased our understanding of the sexual differentiation process . Ia particular these approaches have contributed to the hypothesis that under normal developmental c~nditione the male differentiates from an essentially
1003
1004
DEFENIINIZATION WITH ANDROBTENEDIONE Vol . 8, Pb. 17
female condition to the one characteristic of the male under the inflaeace of testicular hormones (4) . In the past it has been generally assumed that the critical testicular hormone involved in the differentiation process is testosterone .
However, the
critical periods and least effective doses for defeminization have been ex plored for the moat part using only the synthetic steroid testosterone propionote, rather than the naturally occurring testicular steroids teatoaterone and androatenedioae .
In contrast to these studies, Goraki (3) screened a variety
of synthetic and natural steroidal and aonateroidal hormones for their ability to produce anovulatory sterility .
In thiB study androatenedione was found to
be ineffective ; however, its effectiveness was evaluated by only a single dose and time of administration .
Therefore, the complete ineffectiveness of the
single 1000 4+g done of androstenedione administered by Gorski (3) to five day old rats may be due to its peculiar dose and duration of action characteristics . Recent eaperimenta have shown that several androgens other Chan teatoaterone, including androetenedione and dihydrotestoaterone, may be the physiologically active androgens in some systems (6-9) .
Therefore the potential sterilizing
capacity of androatenedione should not be diamisaed .
The . preaent experiment
wan designed to reevaluate the possibility of neonatal defeminization by androatenedione using a relatively high dose repeatedly administered to neonatal female rata . MBTHODS Eighteen Sprague-Dawley female rata, born and raised in our vivarium, rare divided into two groups and treated as follws .
At approximately 48,
72, 96, and 120 hours after birth group I (n ~ 13) received repeated sc lnjectlons of 500 Ng of androsteaedione in 0 .05 cc peamit-oil :benzyl-benzoate carrier (90 :10, v :v) ; group II (n ~ 5) received the carrier only .
The purity
of the androstenedione ran checked by thin layer chrooatography and fowd to be approxir taly 99 .92 testosterone free .
Frog 65 through 75 days of age and
Vol . 8~, No . 17
DEÏ~EMIHIZATION WITH AI~ROBTENEDIONE
1005
88 through 94 days of age daily vaginal smears were obtained Eton all animals . At approximately 96 days of age all animals were ovariectamised and the ovaries examined for the presence of corpora lutes .
1t~o weeks following ovariectomy
all animals received 1 Ng estradiol benzoate ac in 0 .1 cc peanut oil daily for two days .
On the third day, 3~ hours prior to the behavioral tests, all ani-
male received 500 4+g progesterone ac in 0 .1 cc peanut oil .
The behavioral
teats were conducted at approximately the midpoint of the dark period of a reversed light-dark cycle .
Each behavioral teat consisted of placing the
Effects of androatenedione or vehicle administered to neonatal female rata on vaginal smear pattern and mating behavior in adulthood .
Smears were
taken at 65-75 and 88-94 days of age and classified as estrus (S), proeatrus (P), or diestrua (D) .
Sexual receptivity following estrogen-progesterone
treatment was assessed at 110 days of age end expressed as the ratio of lordoeis responses to mounts by the male (R .Q .) .
1008
DEFENIINIZATION WITH AI~ROBTENEDIONE
Vol . 8, No. 17
experimental female into a large glass cylinder with a sexually experienced male, which was permitted to mount the female at least ten times .
The presence
or absence of a lordotic response to each of these mounts was recorded using an event recorder . RESULTS The repeated injection of 500 Wg androstenedione to the group I neonatal females at 48, 72, 96, and 120 hours after birth resulted in persistent vaginal cornification in adulthood (Fig . 1) .
The first period of vaginal smearing,
conducted when the animals were between 65 and 75 days of age, revealed that the group I females averaged 62 .4 13 .6x dieatrue smears .
estrus smears, 24 .Ox proeatrus smears and
During the same period the group II females (carrier
only as neonates) averaged 26 .Ox eatrua smears, 20 .OX prcestrus smears and 54 .OX dieetrus smears .
1ûe second period of vaginal smearing, conducted when
the animals were between 88 and 94 days of age, produced essentially the same record :
Group I averaged 75 .6
6 .4% dieetrus smears .
estrus smears, 17 .9% proeatrua smears, and
Group II females averaged 24 .1X eatrua smears, 24 .1
prcestrua smears, and 51 .77 dieetrus smears .
1ûe distribution of smears
between the two groups was found to be significantly different during both periods of vaginal smearing :
Period I p< .001 (X2 ~ 32 .5, df ~ 2) .
Period 2
p< .001 (R2 ~ 32 .7, df ~ 2) . At approximately 96 days of age examination of the ovaries revealed that 24 of 26 ovaries from the group I females lacked corpora lutes while none of the ovaries from the group II females lacked them (p< .001) .
At this age the
average weight of the group I females was 253 grams, while the group II females averaged 241 grams . During the behavioral teats, conducted when the animals were approximately 110 days of age, both groups exhibited high receptivity quotients (RQ total member lordotic resaanaes X 1000 . total member mounts with thrusting
The medn RQ for the group I
Vol . 8, No. 17
females was 88 .7
DEFENIINIZATION WITH ANDROSTENEDIONE
1007
and for the group II females it was 85 .3 . DISCUSSION
Thus, in contrast to the results reported by Gorski (3), our results indicate that androatenedione can inhibit the formation of corpora lutes as well as the cyclic cornification of the vaginal epithelium .
However, its failure to
inhibit female-like mating indicates that even multiple injections of 500 Ng each cannot produce complete neonatal defeminization .
These data suggest a
novel interpretation of the effects of early androgen treatment upon the development of functional hypothalamic mechaaiama, namely that the hypothalamic mechanisms which control the cyclic pattern of gonadotropin release are sensitive to androatenedione, while those which control the display of aexual .behavior are sensitive only to testosterone .
In this light it is interesting to
note the similarity of our results with those recently reported by Goldfoot, Feder, and Goy (10) who administered androatenedione every other day for the first 19 days after birth to male rats castrated on the day after birth .
This
procedure failed to inhibit the potential for the display of female-like mating behavior, but it did stimulate the penis, seminal vesicles, and prostate auch that they could respond to TP replacement therapy when the neonatal castrates were adults .
TP treatment during the same period would both stimulate peri
pheral structures and inhibit the potential to display female-like sexual behavior (1-3) . An alternative interpretation is that the hypothalamic mechanisms which control gonadotropin secretion and sexual behavior are differentially sensitive to the same androgenic stimulation .
Barraclough and Gorski (1) reported that
administration of 10 Wg TP to five day old female rata inhibits the potential for ovulation and normal vaginal cyclicity, but it does not inhibit the potential for the display of female-like mating .
The similarity of ttte Barraclough
and Gorski results, with those obtained here using androatenedione, suggests the argument that the defeminization obtained with androatenedione is a result of
DEFENIINIZATION WITH ANDROrSTENEDIONE
1008
its metabolic conversion to testosterone .
Vol . 8;, No. 17
Pearlman and Pearlman (11) have
shown that tritiated androatenedione can be reduced to form teatoaterone in intact male rats .
The fact that only partial defeminization of both neonatal
fessle and neonatal castrate male rate has been demonstrated using androatenedione could be explained by the relatively incomplete conversion of androatenedione to teatoaterone, and of the relatively short half-life of androatenedione and teatoaterone (11) . SUMMARY The repeated administration of 500 N,g androstenedlone to neonatal female rata at 48, 72, 96, and 120 hours after birth results is the inhibition of corpora lutes production and cyclic vaginal cornification .
However, unlike
the defeminization produced by high doses of teatoaterone propionate these females have essentially normal female-like sexual behavior .
REF ERENCES and NOTES 1.
C .A . Barraclough, Endocrinology 68, 62 (1961) .
2.
C .A . Barraclough and R .A . Goraki, J . Endocrinol . 2~5 , 175 (1962) .
3.
R .A, Goraki, J . Reprod . Fertil . Suaal .
4.
R .E . Whalen and D,A . Edwarda, Anat . Rec . 157, 173 (1967) .
5.
F . Neumann, W . Elger, and M . Rramer, Endocrinology 78, 628 (1966) .
6.
K .M . Anderson and S . Liao, Nature ~, 277 (1968) .
7.
N . Bruchovaky and J .D . Wilsoa, J . Biol . Chew . 243, 2012 (1968) .
8.
B .W . Harding and L,T . Samuels, Endocrinology 70, 109 (1962) .
9.
R .E . Whalen, W .G . Luttge and R . Green, Endocrinology 84, 217 (1969) .
1, 67 (1966) .
10 .
D .A . Goldfoot, R .H . Feder, and R .W . Goy, J . camp . phyaiol . Psychol . 67, 41 (1969) .
11 .
W .R . Pearlmsn and M.R .J . Pearlman, J . Biol . Chem . 236, 1321 (1961) .
12 .
This research vae supported by Grant HD-00893 to R .E . Whalen from NICHD of the Pational Institutes of Health and by a NDEA Title IV Fellowship to W .G . Luttge .
Vol. 8, Part 1, pp. 1003-1008, 1989. Life 3cienc a Printed in Great Britain.
PARTIAL DEFEMINI2ATION BY ADMINISTRATION OF ANDROSTENEDIONE TO NEONATAL FEMALE RATS William G . Luttge and Richard E . Whalen Department of Psychobiology University of California, Irvine Irvine, California 92664
(received 4 June 1989; in final form 7 July 1989) bURING the past decade the administration of both homotypical and heterotypical hormones to prenatal and neonatal animals has been used by investigators to elucidate the role of gonadal hormones in the differentiation of those neural mechanisms which control gonadotropin secretion and sexual behavior .
For
example, it has been shown that the administration of testosterone propionate (TP) to neonatal female rata can permanently alter the cyclic pattern of pitunary gonadotropin secretion and the display of female-like mating behavior (1-3) .
Testicular, but not ovarian, grafts can mimic moat of these effects,
which include induction of persistent vaginal cornification, anovulatory sterility, and inhibition of female-like mating behavior (3) .
In addition,
studies employing the castration of neonatal male rata (4), or the repeated administration of high doses of the antiandrogen cyproterone acetate (6-chloro17-hydroxy-1n,2~-methylenepregna-4,6-thane-3,20-diene acetate) (5), have shown that one can produce a male with the female cyclic pattern of gonadotropin secretion .
Unlike normal melee, these demasculinized males can readily be
induced to display the female-like mating pattern when exogenous estrogen ie administered (4) .
Both of these experimental approaches (i .e ., surgical or
chemical gonadectomy of the male and the treatment of the female with male hormoms) have increased our understanding of the sexual differentiation process . Ia particular these approaches have contributed to the hypothesis that under normal developmental c~nditione the male differentiates from an essentially
1003
1004
DEFENIINIZATION WITH ANDROBTENEDIONE Vol . 8, Pb. 17
female condition to the one characteristic of the male under the inflaeace of testicular hormones (4) . In the past it has been generally assumed that the critical testicular hormone involved in the differentiation process is testosterone .
However, the
critical periods and least effective doses for defeminization have been ex plored for the moat part using only the synthetic steroid testosterone propionote, rather than the naturally occurring testicular steroids teatoaterone and androatenedioae .
In contrast to these studies, Goraki (3) screened a variety
of synthetic and natural steroidal and aonateroidal hormones for their ability to produce anovulatory sterility .
In thiB study androatenedione was found to
be ineffective ; however, its effectiveness was evaluated by only a single dose and time of administration .
Therefore, the complete ineffectiveness of the
single 1000 4+g done of androstenedione administered by Gorski (3) to five day old rats may be due to its peculiar dose and duration of action characteristics . Recent eaperimenta have shown that several androgens other Chan teatoaterone, including androetenedione and dihydrotestoaterone, may be the physiologically active androgens in some systems (6-9) .
Therefore the potential sterilizing
capacity of androatenedione should not be diamisaed .
The . preaent experiment
wan designed to reevaluate the possibility of neonatal defeminization by androatenedione using a relatively high dose repeatedly administered to neonatal female rata . MBTHODS Eighteen Sprague-Dawley female rata, born and raised in our vivarium, rare divided into two groups and treated as follws .
At approximately 48,
72, 96, and 120 hours after birth group I (n ~ 13) received repeated sc lnjectlons of 500 Ng of androsteaedione in 0 .05 cc peamit-oil :benzyl-benzoate carrier (90 :10, v :v) ; group II (n ~ 5) received the carrier only .
The purity
of the androstenedione ran checked by thin layer chrooatography and fowd to be approxir taly 99 .92 testosterone free .
Frog 65 through 75 days of age and
Vol . 8~, No . 17
DEÏ~EMIHIZATION WITH AI~ROBTENEDIONE
1005
88 through 94 days of age daily vaginal smears were obtained Eton all animals . At approximately 96 days of age all animals were ovariectamised and the ovaries examined for the presence of corpora lutes .
1t~o weeks following ovariectomy
all animals received 1 Ng estradiol benzoate ac in 0 .1 cc peanut oil daily for two days .
On the third day, 3~ hours prior to the behavioral tests, all ani-
male received 500 4+g progesterone ac in 0 .1 cc peanut oil .
The behavioral
teats were conducted at approximately the midpoint of the dark period of a reversed light-dark cycle .
Each behavioral teat consisted of placing the
Effects of androatenedione or vehicle administered to neonatal female rata on vaginal smear pattern and mating behavior in adulthood .
Smears were
taken at 65-75 and 88-94 days of age and classified as estrus (S), proeatrus (P), or diestrua (D) .
Sexual receptivity following estrogen-progesterone
treatment was assessed at 110 days of age end expressed as the ratio of lordoeis responses to mounts by the male (R .Q .) .
1008
DEFENIINIZATION WITH AI~ROBTENEDIONE
Vol . 8, No. 17
experimental female into a large glass cylinder with a sexually experienced male, which was permitted to mount the female at least ten times .
The presence
or absence of a lordotic response to each of these mounts was recorded using an event recorder . RESULTS The repeated injection of 500 Wg androstenedione to the group I neonatal females at 48, 72, 96, and 120 hours after birth resulted in persistent vaginal cornification in adulthood (Fig . 1) .
The first period of vaginal smearing,
conducted when the animals were between 65 and 75 days of age, revealed that the group I females averaged 62 .4 13 .6x dieatrue smears .
estrus smears, 24 .Ox proeatrus smears and
During the same period the group II females (carrier
only as neonates) averaged 26 .Ox eatrua smears, 20 .OX prcestrus smears and 54 .OX dieetrus smears .
1ûe second period of vaginal smearing, conducted when
the animals were between 88 and 94 days of age, produced essentially the same record :
Group I averaged 75 .6
6 .4% dieetrus smears .
estrus smears, 17 .9% proeatrua smears, and
Group II females averaged 24 .1X eatrua smears, 24 .1
prcestrua smears, and 51 .77 dieetrus smears .
1ûe distribution of smears
between the two groups was found to be significantly different during both periods of vaginal smearing :
Period I p< .001 (X2 ~ 32 .5, df ~ 2) .
Period 2
p< .001 (R2 ~ 32 .7, df ~ 2) . At approximately 96 days of age examination of the ovaries revealed that 24 of 26 ovaries from the group I females lacked corpora lutes while none of the ovaries from the group II females lacked them (p< .001) .
At this age the
average weight of the group I females was 253 grams, while the group II females averaged 241 grams . During the behavioral teats, conducted when the animals were approximately 110 days of age, both groups exhibited high receptivity quotients (RQ total member lordotic resaanaes X 1000 . total member mounts with thrusting
The medn RQ for the group I
Vol . 8, No. 17
females was 88 .7
DEFENIINIZATION WITH ANDROSTENEDIONE
1007
and for the group II females it was 85 .3 . DISCUSSION
Thus, in contrast to the results reported by Gorski (3), our results indicate that androatenedione can inhibit the formation of corpora lutes as well as the cyclic cornification of the vaginal epithelium .
However, its failure to
inhibit female-like mating indicates that even multiple injections of 500 Ng each cannot produce complete neonatal defeminization .
These data suggest a
novel interpretation of the effects of early androgen treatment upon the development of functional hypothalamic mechaaiama, namely that the hypothalamic mechanisms which control the cyclic pattern of gonadotropin release are sensitive to androatenedione, while those which control the display of aexual .behavior are sensitive only to testosterone .
In this light it is interesting to
note the similarity of our results with those recently reported by Goldfoot, Feder, and Goy (10) who administered androatenedione every other day for the first 19 days after birth to male rats castrated on the day after birth .
This
procedure failed to inhibit the potential for the display of female-like mating behavior, but it did stimulate the penis, seminal vesicles, and prostate auch that they could respond to TP replacement therapy when the neonatal castrates were adults .
TP treatment during the same period would both stimulate peri
pheral structures and inhibit the potential to display female-like sexual behavior (1-3) . An alternative interpretation is that the hypothalamic mechanisms which control gonadotropin secretion and sexual behavior are differentially sensitive to the same androgenic stimulation .
Barraclough and Gorski (1) reported that
administration of 10 Wg TP to five day old female rata inhibits the potential for ovulation and normal vaginal cyclicity, but it does not inhibit the potential for the display of female-like mating .
The similarity of ttte Barraclough
and Gorski results, with those obtained here using androatenedione, suggests the argument that the defeminization obtained with androatenedione is a result of
DEFENIINIZATION WITH ANDROrSTENEDIONE
1008
its metabolic conversion to testosterone .
Vol . 8;, No. 17
Pearlman and Pearlman (11) have
shown that tritiated androatenedione can be reduced to form teatoaterone in intact male rats .
The fact that only partial defeminization of both neonatal
fessle and neonatal castrate male rate has been demonstrated using androatenedione could be explained by the relatively incomplete conversion of androatenedione to teatoaterone, and of the relatively short half-life of androatenedione and teatoaterone (11) . SUMMARY The repeated administration of 500 N,g androstenedlone to neonatal female rata at 48, 72, 96, and 120 hours after birth results is the inhibition of corpora lutes production and cyclic vaginal cornification .
However, unlike
the defeminization produced by high doses of teatoaterone propionate these females have essentially normal female-like sexual behavior .
REF ERENCES and NOTES 1.
C .A . Barraclough, Endocrinology 68, 62 (1961) .
2.
C .A . Barraclough and R .A . Goraki, J . Endocrinol . 2~5 , 175 (1962) .
3.
R .A, Goraki, J . Reprod . Fertil . Suaal .
4.
R .E . Whalen and D,A . Edwarda, Anat . Rec . 157, 173 (1967) .
5.
F . Neumann, W . Elger, and M . Rramer, Endocrinology 78, 628 (1966) .
6.
K .M . Anderson and S . Liao, Nature ~, 277 (1968) .
7.
N . Bruchovaky and J .D . Wilsoa, J . Biol . Chew . 243, 2012 (1968) .
8.
B .W . Harding and L,T . Samuels, Endocrinology 70, 109 (1962) .
9.
R .E . Whalen, W .G . Luttge and R . Green, Endocrinology 84, 217 (1969) .
1, 67 (1966) .
10 .
D .A . Goldfoot, R .H . Feder, and R .W . Goy, J . camp . phyaiol . Psychol . 67, 41 (1969) .
11 .
W .R . Pearlmsn and M.R .J . Pearlman, J . Biol . Chem . 236, 1321 (1961) .
12 .
This research vae supported by Grant HD-00893 to R .E . Whalen from NICHD of the Pational Institutes of Health and by a NDEA Title IV Fellowship to W .G . Luttge .