Studies on the effects of the ovary on testicular differentiation in heterosexual transplantation of embryonic gonads in the rats
The "freemartion" phenomenon, the situation in which twin calves are of opposite sex, was first described by Lille)1916) and by Keller and Tandler(1916). This foundation for generally accepted notion that very early in embryonic development sex glands of the vertebrates secrete sex hormones which are responsible for the differentiation of two sexes from an indeterminate type prevailing in the embryo has received a considerable attention from numerous investigators.
However this sex hormone theory of gonadal differentiation from an indeterminate type of gonad was not entirely proved at the mammalian level.
Willer(1939)described th early embryonal gonad as a bisexual premordium capable differentiation to either sex. Male of female potentialities are represented by specific histological elements, medulla and cortex, which have alternative roles in gonadogenesis.
In his amphibian experiments, Witschi(1934) obtained information that formed a portion of his theory concerning the so-called corticomedullary inductors, cortexin and medullarin, in the sex differentiation of amphibia.
The corticomedullary inductor theory was deduced concerning the sex differentiation and was then proven by others. Witschi(1934) concluded that the inductors produce their effects by the release of morphogenic substances called medullarin and cortexin each of which may be the antagonist of the other. The theory was proven by others
Macintyre(1956) and Macintyre et al.(1959) obtained results interrupted as substantiating at the placental mammalian level that portion of the corticomedullary inductor theory of sex differentiation which deals with the secretion of and inductor by the medullary tissue and by the cortical tissue of a developing gonadal anlage.
The author has attempted the present experiment in order to study the effect of each inductor(corticomedullary), especially the inductor secreted by the cortical tissue of a developing gonadal anlage, to the opposite gonadal tissue in rats(Placental mammalian level vertebrates).
Materials and methods
Both testes and ovaries of the 16-day old rat fetus(post coitem or estimated by crown-rump length of the albino rat as presented by Kupfer and Koeller(1951) and by Christie(1964) were obtained from pregnant albino rats. The excised gonads from the fetuses at this stage may be surgically separated from adjacent tubular structures and cleaned by the aid of the stereomicroscope under the magnification of 15×1.33 diameters.
Grafts of both gonads were made to a subcapsular portion in the kidneys of adult male rat hosts which have been castrated at least 3 week prior to their use.
The author adopted the transplantation period of three weeks in the kidney site recommended by Macintyre(1956). In the first experimental group, the male and female gonads were transplanted side by side to the subcapsular site of the host.
For the control group the fetal testis or ovary of the 16-day old rat fetus, as described above, was transplanted to the subcapsular site of the host kidney in the absence of gonadal tissue of opposite sex. The developed testis or ovary, grown in
the site for 3 weeks, was compared with the grown or grafted gonads of the experimental groups.
For the experimental group, A) the male and female gonads were transplanted to the subcapsular site of the host side by side and 15 grafts removed from the hosts were histologically checked. B) one fetal testis and multiple ovaries (4 to 14 ovaries) were, in manual contact, grafted in one subcapsular site of the host kidney before removal. These graft were tested in the same manner.
The grown grafts were removed from the host after 3 weeks, fixed in Bouin's fluid, embedded in paraffin, sectioned serially at 6μ and stained with hematoxylin and eosin.
1. Transplanted testis without ovary:
The fetal testis, developed in the subcapsular site of the host kidney for 3 weeks' grew into a large mass measuring about 5mm. in length. Two or three layers of spermatogonia-like cells were arranged around the growth of seminiferous tubules. The cells showed fairly active proliferation. Relatively well developed
interstitial cells, whose cytoplasma was well stained with eosin dye, were found in the stroma. It was evident that 3 week old grafts of fetal male gonads showed some precocity of growth and differentiation as compaired to normal testis of equivalent
2. Transplanted ovary of the certain developmental stage grew into a relatively small mass of about 3mm. in length and contained several follicles showing different stages of the primary follicle, the growing follicle, and the young Graafian follicle. The stroma showed a fairly well-developed picture, The ovary also showed certain precocity of growth and differentiation as compared with the normal one of equivalent age.
A. Transplanted testis with ovary in close contact.
In the testis, the growth and differentiation were not different from those of the control group without the ovary. However, the growth and differentiation of the ovary which developed with the testis in close contact was inhibited and depressed to one side probably due to a more rapid growth and differentiation of the testis compared with the ovary as well as to the inhibition of the growth and differentiation of the ovary. In certain instance, the sex cell cord of the ovary developed into a tubular structure which contained degenerating oocytes. In this group the definite effect or an inhibitory action of the testis on the ovary was easily demonstrated by the phenomena of recessive growth and inhibited differentiation of the ovary transplanted with the testis in mutual contact.
B. Transplanted testis with multiple ovaries in mutual contact:
I) Transplanted testis with 4 to 9 ovaries in close contact:
In these 6 hosts the growth and development of the testis was not different from those of the transplanted control testis without ovary. However, the multiple ovarian grafts grown in close contact with the transplanted testis were suppressed in growth and differentiation. These showed degenerating follicles and the appearances of seminiferous-like tubules in the ovarian medulla and rate ovarian grafts grown in close contact but the latter were depressed by the former.
ii) Transplanted testis with 10 to 14 ovaries in close contact:
In these 13 hosts, the growth and development of the transplanted testis, grown in multiple ovarian grafts in mutual contact, was somewhat inhibited and most of the seminiferous tubules showed a degeneration characterized by a single layer of
spermatogonia-like cells, separation and scattering of the cells, and degenerating spermatogonia-like cells. However, the multiple ovarian grafts showed some abortive ovarian structures as the experimental group with transplanted testis and ovary in close contact. These results indicated that testicular graft was moderately suppressed by multiple ovarian grafts(more than ovaies).
Summary and Conclusion
The author studied the "corticomedullay inductor" of sex differentiation presented by Sitschi(1934) in respect to the effect of inductor substance and the possibility of the substance and the possibility of the substance acting as an inhibitor to each other at the mammalian level by the use of heterosexual grafts of the feral gonads from albino rats in very close proximity to each other.
The heterosexual grafts of the 16 day old fetal gonads were performed by the method of Macintyre(1956). The hosts were castrated at least 3 weeks prior to their use and the grafts were allowed to grow in the host's subcapsular renal site.
The ovarian explant, grown with testis in close contact was inhibited and depressed to one side. This was probably due to a more rapid growth and differentiation of the testis as compared with the ovary as well as to predominant testicular effect upon the nearby ovary by the activity of the diffused inductor substance originating from the testis.
The transplanted testis with 4 to 0 ovaries in close contact was not different from those of the trasnplanted testis without the ovary and was not effected by such numbers of fetal ovaries, probably due to a recessive ovarian effect of the diffused inductor substance originating from the multiple ovaries.
The transplanted testis with more than 10 ovaries in mutual contact was somewhat inhibited in growth and development. It showed an abortive growth pattern and a degenerating picture of seminiferous tubules associated with a single layer of spermatogonia-like cells, separation of the spesumed a predominant effect of the multple (more than 10 ovaried) ovarian graft upon the testis to be due to the activity of the diffused inductor substance originating from the multiple ovaries.
By these observations the autnor concluded that the fetal testis supressed the growth and differentiation of the fetal ovary and vice versa, and proved the existence and biological activity of the "corticomedulary inductor" as indirectly
presented by Witschi(1934).