Untersuchungen zur Wirkung von östrogenwirksamen Stoffen auf die Gonadenentwicklung bei Fröschen (Rana temporaria und Xenopus laevis) sowie ultrastrukturelle und immunhistochemische Untersuchungen an Froschgonaden (Rana temporaria)

The influence of estrogen-like substances on the gonadal
development in frogs (Rana temporaria and Xenopus laevis).
Ultrastructural and immunohistochemical investiga-tions on the
gonads of frogs (Rana temporaria). The aim of the present thesis
was the investigation and assessment of the estrogen pollu-tion of
a river in South-Germany above and below a sewage plant outlet and
its possible effects on the gonadal development in amphibians. In
an exposure experiment with differ-ent sewage dilutions the effect
on the indigenous gras frog (Rana temporaria) were com-pared with
the effects on the African claw frog (Xenopus laevis). Because of
the lack of relevant data in current literature it was necessary to
first collect some basic data on light microscopy, ultrastructure
and immunohistochemistry with reference to frogs. Furthermore the
sexual differentiation of two populations of different origin were
compared histologically. Characterisation of the gonads The ovaries
of Xenopus laevis displayed a garland-like structure in contrast to
the compact ovaries of Rana temporaria. The ovaries of both frog
species were surrounded by an epi-thelium and filled with germ
cells which enclosed an ovarian cavity. The oogonies were mainly
found in the peripheral parts of the organ. The oocytes were
characterised by a big and irregularly shaped cell nucleolus with
peripherally located nucleoli. They were sur-rounded by a thin
one-layered follicular epithelium. Electron microscopical
examination of the ovaries of Rana temporaria showed elongated
tubular mitochondria in the cytoplasm which were found exclusively
in oocytes. Some of the mitochondria from subadult frogs contained
yolk crystals. Additionally an accumulation of electron-tight
Granule was found just below the plasma membrane, which could be a
preliminary stage of the cortical granule. The testis of the frogs
were enveloped by an epithelium and a tunica albuginea which
al-ready showed a tubular structure. The testis from Rana
temporaria and the Xenopus laevis differed in their developmental
stages. The testis of juvenile Rana temporaria contained only germ
cells whereas the testis of some Xenopus laevis already contained
spermato-cytes and spermatozoa. The spermatocysts, characteristic
of frog testis, could be seen in conjunction with the appearance of
spermatocytes only. By using light microscopy two different types
of germ cells in both frog species could be distinguished which
probably were primary and secondary germ cells. By electron
microscopical investigation of the testis of Rana temporaria the
somatic cells could, on the basis of location and morphology, be
differentiated in two types. The somatic cells of the first type
were located inside the Tubuli seminiferi and have probably the
same function as the Sertoli cells of mammals. The cells of the
second type, the Leydig cells or interstitial cells, were located
outside the Tubuli and were characterized by granular vesicle in
the cytoplasm. With view to the sexual differentiation of Rana
temporaria a comparative histological inves-tigation of the gonads
of juvenile and subadult frogs of a native midland population and a
high alpine population was performed. The midland population proved
to be a sexually semi-differentiated species since apart from
clearly male or female animals it also com-prises intersex
individuals in different stages of the transformation process. The
genotypi-cally male animals developed female gonads in the first
place which secondly converted into testis during a hermaphrodite
stage. The transformation process from female into male gonads was,
on the basis of morphological criteria, classified into three
stages. The highal-pin population on the other hand proved to be a
sexually differentiated race. By using the
Avidin-Biotin-Complex-Technique five different antibodies were
tested on the gonadal tissue of juvenile and subadult Rana
temporaria. The test for Laminin, a non-collagen glycoprotein,
which is part of the basal membrane in mammals, resulted in a
posi-tive reaction. It seems therefore that similar to Laminin in
mammals a glycoprotein plays an important part in the basal
membrane of frogs. α-Actin, a fibrous protein of the smooth
muscles, which was detected in the Theca externa of the layer of
follicle cells in different species, could be established in the
blood vessel wall only and not in the layer of follicle cells.
Furthermore the occurrence of a Zona pellucida by using antibodies
against the por-cine glycoprotein ZP3 was investigated. The ovum
including the surrounding layer of follicle cells showed a negative
reaction which however did not implicitly exclude the occurrence of
a glycoprotein layer with a different antigenicity. Until now the
existence of a glycoprotein layer in anurans, in connection with
microscopical anatomy of ovaries in Rana temporaria or other
species of frogs has not been mentioned in references. The
examination of the go-nadal tissue as to the occurrence of ACE
(Angiotensin Converting Enzymes) turned out negative as well.
Example of application With the exception of a temporarily higher
concentration of alkylphenol in the beginning of the exposure, the
chemical analyses revealed a relatively low degree of pollution
with alcyl-phenols and steroids. The results were comparable to
other results of German rivers and stayed well below other European
comparative data. According to the results of this study the
current level of pollution of the experimental water with estrogens
does not endanger the amphibian population. The examination did not
reveal any influence of the sewage on the embryonal and larval
development. Furthermore, the histological investigation of the
gonads in exposed and unexposed frogs with reference to the gonadal
sexual differentia-tion as well as the sex ratios did not reveal
significant changes. There was no correlation as to the frequency
of the occurrence of intersex in the groups of exposed and
unexposed frogs, neither of Xenopus laevis nor of Rana temporaria.
Only the transformation process from ovaries to testis of the
sexually semi-differentiated species of Rana temporaria was slowed
down in the group of exposed animals in contrast to unexposed
animals. The rea-son for this phenomenon could be the inhibitive
influence of the low-level but more continu-ous estrogen pollution
in sewage than in river water. Conversely, a link between the
accel-eration of the transformation process within the group of
unexposed animals and the tem-porarily higher alkylphenol level in
the beginning of the exposure cannot completely be ruled out.
According to references alkylphenol can cause an increase of
testosterone. The semiquantitative RT-PCR detecting
Vitellogenin-mRNA carried out by the Institute of Freshwater
Ecology and Inland Fisheries in Berlin showed a minor increase in
females of Xenopus laevis which were exposed to sewage in the
ration of a 2:1 dilution in contrast to the unexposed animals.
Taking into account the synergistic effects of estrogens, the
in-crease could be attributed to the higher estrogen pollution of
the sewage. The histopa-thological analysis for the detection of
toxical effects of the sewage as well as other poten-tial
influential factors provided no hints as to a possibly toxical
influence of the sewage.