성홀몬(Estrogen 및 Testosterone)이 자성백서(雌性白鼠)의 생식선급(生殖腺及) 그와 관련된 타내분비선(他內分泌腺)에 미치는 조직학적 변화에 관한 실험적 연구

Abstract

[한글]

Experimental Studies on the Histologic Alteration of the Gonad and other Related

Endocrine Organs induced by the Administration of Estrogen and Testosterone In

Female Rats

Hi Chul Hahn, M. D.

(Directed by: Prof. Deng Sik Kim)

Department of Pathology

College of Medicine, Yonsei University Seoul, Korea

In general the sex hormones are divided into two major groups, namely, estrogenic

and androgenic according to their actions, However, this division is not an

absolute one, and Korenchevsky in 1937 already stressed more or less bisexual

activities of each hormones through his animal experiments with various derivatives

of both estrogenic and androgenic hormones. This fact is further proved by blotter

effect of combined treatment with both groups of hormones in many clinical

conditions (Newman 1952,Katzman 1952) than the treatment with single hormone. These

evidences suggest that latestrogenic and androgenic hormones act not only

antagonistically but also in synergetic way.

The actions of the sex hormones in living body can be divided into two ways. The

first mode of action is a feed-back chain action between higher center and gonads,

that is, hypothalamus, hypophysis, and gonads. And the second type of action is

direct one way action to the external genital organs and secondary sex

characteristics, The sex hormones are normally produced and liberated from gonads,

namely, female hormones from the ovaries and male hormones from the testes. But

soma portion is also produced in the other organs, such as adrenals and the gonads

of the opposite sex. Therefore, both groups of sex hormones are naturally

coexisting in the body of every single individuals, usually keeping a certain

balance. When this balance is disturbed, various degree of abnormal phenomena are

manifested, such as pseudohermaphroditism.

Wells and van Wegenen (1954) and others experimentally induced this phenomena by

the administration of sex hormones during fetal life of mammalian animals, and some

of their results showed opposite sex directed structural alteration of gonads,

suggesting the possibility of experimental sex exchange.

The influence of sex hormones on the gonads of young and mature animals also have

been studied by many investigators. But the most of them dealt with changes on

follicles and corpus luteum of ovaries, and the mechanism of ovulation. Very little

has been investigated on detail histologic alteration, particularly about intersex

structures of gonads, and many of their report contradicted each ocher depending on

the dose and duration of hormone administration.

Therefore, the present investigation is intended to study a detail morphologic

alteration of ovaries, with special reference to possible male directed structural

changes, and at the same time the histologic alteration of other related endocrine

organs, following along term administration of both estrogen and testosterone,

singly and combined, in female rats.

Materials and Methods

Fifty one female albino n7, around 250 Ems. were divided into eleven groups.

Group I consisted of 3 animals as normal untreated control group; group If of 3

animals as the castration group: group Ⅲ, Ⅳ and V of 5 animals each as estrogen

treated groups with doses of 0.016 mg.,0.03 mg. and 0.066 mg. of estrogen to each

animal, recpectively; groups Ⅵ, Ⅶ and Ⅷ of 5 animals each as testosterone

treated groups with doses of 0.016 mg., 0.033 mg. and 0.066 mg. of testosterone,

respectively; and groups If, X and Xl of ? animals each as combined estrogen and

testosterone treated groups with doses of 0.066 mg., 0.033 mg. and 0.066 mg. of

each hormones, respectively. The hormones were administered intramuscularly at

thigh once every other day for one month period. When both hormones were given

combined, each hormone was injected separately at each thigh.

The hormones used in this experiment were products of Merck Co., prepared inform

of estradiol benzoate and testosterone propionate. The dose of hormones is

calculated from human therapeutic dome based on the weight of animal for the lowest

dose. Then double and quadriple doses were given to produce the effect of overdose.

All animals were killed 3 days after the last injection of hormones. The weight

of the ovaries, hypophysis, thyroid and adrenals were measured. All tissue were

fixed in10 per cent neutral formal in solution after macroscopic examination of

each organ. Sections were prepared after paraffin embedding and stained by

hematoxylin-eosin, van Gieson, Verhoff's and periodic acid Schiff's methods for the

microscopic examination. The body weight was weighed three times, first at the

beginning, 2nd at 10 days later, and the 3rd at the 30th day of the experiment. The

weight of each organs was expressed in mg. per 100 gm. of the body weight.

Results and Summary

The body weight increased from 16 to 30 per cent of original weight at the end of

experiment in all groups including normal control and castration groups. However,

no definite pattern of tendency according to the dose and kind of the hormones

administered is noted.

The weight of ovaries decreased in all groups treated with hormones in comparison

with the normal control group. The decrease was most marked in groups treated with

both estrogen and testosterone combined, and least in the groups treated with

estrogen alone. The degree of decrease in each groups was more marked as the dose

of hormones increased. The weight of hypophysis also decreased in all groups in

comparison with the normal control group. But no definite difference by the kind or

dose of hormones was noted. The thyroid glands showed most irregular pattern of

weight changes, increase in some groups and decrease in other groups, irrespective

to the kind and dose of the hormones. The adrenals, on the other hand, showed an

increase of weight in castration and estrogen treated groups, and slight decrease

in testosterone treated groups. The groups treated with estrogen and testosterone

combined showed increase of weight when treated with small dose and decrease of the

weight when treated with large dose.

The ovaries in groups treated with estrogen showed increased height of germinal

epithelium without actual proliferation, mild atrophy of stromal tissue, and

moderate hyperplasia of interstitial cells with decreased numbers of interstitial

glands. The follicles showed increased numbers of medium sized and vesicular form

of follicles with over-expansion of antral lumen, increased numbers of degenerating

follicles, appearance of anovular follicles in few animals, and marked degree of

degenerating changes of corpus luteum. These changes showed increased severity by

the increasing the dose of hormone, but no qualitative change was noted by the

difference of the dose of hormone.

The ovaries in groups treated with testosterone alone showed mild hypertrophy and

slight hyperplasia of germinal epithelial cells, slight hyperplasia of stromal

tissue, and moderate atrophy of interstitial cells. The follicles showed marked

decrease of normal mature form, and severe degenerative changes of follicles with

frequent appearance of anovular follicles in practically all animals. The corpus

luteum also showed marked retrogressive changes. The most interesting findings in

this group was the appearance of peculiar structures similar to developing

seminiferous tubules in 1/3 of animals. These structures seemed to be transformed

from stromal tissue by the stimulation of testosterone.

The ovaries of animals treated with estrogen and testosterone together showed

mild or no notable histologic alteration in groups treated with small and medium

doses, but in the group treated with large dose showed changes similar to the

animals treated with testosterone alone, but in milder degree.

The histologic alteration of the hypophysis consisted of marked increase of

basophiles in the castration group, decrease of basophiles and hypertrophy of

chromophobe cells in all hormone treated groups.

The thyroid gland showed no notable histologic alteration except mild follicular

haperplasia in groups treated with testosterone alone and combined groups. The most

significant changes in adrenal glands were the population of PAS positive cells in

reticular zone. These cells were scattered in small numbers in normal animals, but

markedly decreased in estrogen treated animals, while moderate to marked increase

was noted in testosterone treated groups. The combined groups showed mild increase

of these cells.

In summary, the prolonged administration of estrogen and testosterone, either

alone or combined, produced toxic effect and structural modification on the

ovaries, and certain morphologic alterations in the hypophysis and adrenals. Some

of changes are com-mon to both hormones while others are qualitatively different.

When they are given together, androgenic action seems to be dominated.

The most interesting and new observation in this experiment is the appearance of

structures resembling developing immature seminiferous tubules in the ovaries of

am-mats treated with testosterone. This phenomena may provide morphologic back-up

for the possibility of the experimental production of intersexuality, and further

investigation in this direction would be rewarding.

[영문]

In general the sex hormones are divided into two major groups, namely, estrogenic and androgenic according to their actions, However, this division is not an absolute one, and Korenchevsky in 1937 already stressed more or less bisexual activities of each hormones through his animal experiments with various derivatives

of both estrogenic and androgenic hormones. This fact is further proved by blotter effect of combined treatment with both groups of hormones in many clinical conditions (Newman 1952,Katzman 1952) than the treatment with single hormone. These evidences suggest that latestrogenic and androgenic hormones act not only

antagonistically but also in synergetic way.

The actions of the sex hormones in living body can be divided into two ways. The first mode of action is a feed-back chain action between higher center and gonads, that is, hypothalamus, hypophysis, and gonads. And the second type of action is

direct one way action to the external genital organs and secondary sex characteristics, The sex hormones are normally produced and liberated from gonads, namely, female hormones from the ovaries and male hormones from the testes. But soma portion is also produced in the other organs, such as adrenals and the gonads of the opposite sex. Therefore, both groups of sex hormones are naturally coexisting in the body of every single individuals, usually keeping a certain balance. When this balance is disturbed, various degree of abnormal phenomena are manifested, such as pseudohermaphroditism.

Wells and van Wegenen (1954) and others experimentally induced this phenomena by the administration of sex hormones during fetal life of mammalian animals, and some of their results showed opposite sex directed structural alteration of gonads, suggesting the possibility of experimental sex exchange.

The influence of sex hormones on the gonads of young and mature animals also have been studied by many investigators. But the most of them dealt with changes on follicles and corpus luteum of ovaries, and the mechanism of ovulation. Very little has been investigated on detail histologic alteration, particularly about intersex structures of gonads, and many of their report contradicted each ocher depending on the dose and duration of hormone administration.

Therefore, the present investigation is intended to study a detail morphologic alteration of ovaries, with special reference to possible male directed structural changes, and at the same time the histologic alteration of other related endocrine organs, following along term administration of both estrogen and testosterone, singly and combined, in female rats.

Materials and Methods

Fifty one female albino n7, around 250 Ems. were divided into eleven groups.

Group I consisted of 3 animals as normal untreated control group; group If of 3 animals as the castration group: group Ⅲ, Ⅳ and V of 5 animals each as estrogen treated groups with doses of 0.016 mg.,0.03 mg. and 0.066 mg. of estrogen to each animal, recpectively; groups Ⅵ, Ⅶ and Ⅷ of 5 animals each as testosterone treated groups with doses of 0.016 mg., 0.033 mg. and 0.066 mg. of testosterone, respectively; and groups If, X and Xl of ? animals each as combined estrogen and testosterone treated groups with doses of 0.066 mg., 0.033 mg. and 0.066 mg. of each hormones, respectively. The hormones were administered intramuscularly at thigh once every other day for one month period. When both hormones were given combined, each hormone was injected separately at each thigh.

The hormones used in this experiment were products of Merck Co., prepared inform of estradiol benzoate and testosterone propionate. The dose of hormones is calculated from human therapeutic dome based on the weight of animal for the lowest

dose. Then double and quadriple doses were given to produce the effect of overdose.

All animals were killed 3 days after the last injection of hormones. The weight of the ovaries, hypophysis, thyroid and adrenals were measured. All tissue were fixed in10 per cent neutral formal in solution after macroscopic examination of

each organ. Sections were prepared after paraffin embedding and stained by hematoxylin-eosin, van Gieson, Verhoff's and periodic acid Schiff's methods for the microscopic examination. The body weight was weighed three times, first at the beginning, 2nd at 10 days later, and the 3rd at the 30th day of the experiment. The

weight of each organs was expressed in mg. per 100 gm. of the body weight.

Results and Summary

The body weight increased from 16 to 30 per cent of original weight at the end of experiment in all groups including normal control and castration groups. However, no definite pattern of tendency according to the dose and kind of the hormones administered is noted.

The weight of ovaries decreased in all groups treated with hormones in comparison with the normal control group. The decrease was most marked in groups treated with both estrogen and testosterone combined, and least in the groups treated with

estrogen alone. The degree of decrease in each groups was more marked as the dose of hormones increased. The weight of hypophysis also decreased in all groups in comparison with the normal control group. But no definite difference by the kind or

dose of hormones was noted. The thyroid glands showed most irregular pattern of weight changes, increase in some groups and decrease in other groups, irrespective to the kind and dose of the hormones. The adrenals, on the other hand, showed an increase of weight in castration and estrogen treated groups, and slight decrease in testosterone treated groups. The groups treated with estrogen and testosterone combined showed increase of weight when treated with small dose and decrease of the weight when treated with large dose.

The ovaries in groups treated with estrogen showed increased height of germinal epithelium without actual proliferation, mild atrophy of stromal tissue, and moderate hyperplasia of interstitial cells with decreased numbers of interstitial glands. The follicles showed increased numbers of medium sized and vesicular form of follicles with over-expansion of antral lumen, increased numbers of degenerating follicles, appearance of anovular follicles in few animals, and marked degree of

degenerating changes of corpus luteum. These changes showed increased severity by the increasing the dose of hormone, but no qualitative change was noted by the difference of the dose of hormone.

The ovaries in groups treated with testosterone alone showed mild hypertrophy and slight hyperplasia of germinal epithelial cells, slight hyperplasia of stromal tissue, and moderate atrophy of interstitial cells. The follicles showed marked decrease of normal mature form, and severe degenerative changes of follicles with frequent appearance of anovular follicles in practically all animals. The corpus luteum also showed marked retrogressive changes. The most interesting findings in this group was the appearance of peculiar structures similar to developing seminiferous tubules in 1/3 of animals. These structures seemed to be transformed from stromal tissue by the stimulation of testosterone.

The ovaries of animals treated with estrogen and testosterone together showed mild or no notable histologic alteration in groups treated with small and medium doses, but in the group treated with large dose showed changes similar to the animals treated with testosterone alone, but in milder degree.

The histologic alteration of the hypophysis consisted of marked increase of basophiles in the castration group, decrease of basophiles and hypertrophy of chromophobe cells in all hormone treated groups.

The thyroid gland showed no notable histologic alteration except mild follicular haperplasia in groups treated with testosterone alone and combined groups. The most significant changes in adrenal glands were the population of PAS positive cells in reticular zone. These cells were scattered in small numbers in normal animals, but markedly decreased in estrogen treated animals, while moderate to marked increase was noted in testosterone treated groups. The combined groups showed mild increase of these cells.

In summary, the prolonged administration of estrogen and testosterone, either alone or combined, produced toxic effect and structural modification on the ovaries, and certain morphologic alterations in the hypophysis and adrenals. Some of changes are com-mon to both hormones while others are qualitatively different.

When they are given together, androgenic action seems to be dominated.

The most interesting and new observation in this experiment is the appearance of structures resembling developing immature seminiferous tubules in the ovaries of am-mats treated with testosterone. This phenomena may provide morphologic back-up

for the possibility of the experimental production of intersexuality, and further investigation in this direction would be rewarding.