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Ultrastructural events during early gonadal development in Rana pipiens and Xenopus laevis.

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THE ANATOMICAL RECORD 199~349-360(1981)
Ultrastructural Events During Early Gonadal
Development in Rana pipiens and Xenopus laevis
HORACIO MERCHANT-LARIOS AND IRMA VILLALPANDO
Departamento de Biologia del Desarrollo, Instituto de Inuestigaciones
Bioddicas, Uniuersidad Nacional Autdnoma de Mexico,
Apartado Postal 70228, Mexico 20 D P . , Mhico
ABSTRACT
The establishment of the undifferentiated gonad was studied in
Xenopus laevis and Rana pipiens using high resolution techniques. It was found
that the cells of the so-called “mesonephric blastema” had no structural resemblance to the cells of the gonadal medulla in both species. Furthermore, there was
no morphological evidence that would suggest a migration of the former cells
towards the incipient gonad at the time of its appearance. However, the basal
lamina of the coelomic epithelium was interrupted in the region of the genital crest,
and there was a definite ultrastructural similarity between the cells of this
epithelium and those that first form the medulla. These observations suggest that,
in amphibians, the cells of the gonadal medulla come from a cellular line arising
from the coelomic epithelium and not from the “mesonephric blastema,” as has
been proposed.
The embryological origin of the somatic cells
of the vertebrate gonad has long been a subject
of considerable controversy. The most durable
hypothesis formulated to explain the establishment and sexual differentiation of the
gonad has been that which postulates the
existence of two primordia, cortical and medullary, with different embryonic origins
(Witschi, ’67). This has mainly been based on
observations and experiments carried out in
different amphibian species in which the differentiation of the cortical and medullary regions, and thus their potential for feminization
and masculinization, respectively, are evident
from very early stages of development
(Merchant-Larios, ’78). However, in other vertebrates, such as mammals, in which the segregation of the two gonadal primordia takes place
at a later time, the participation of the
mesonephros in the formation of the gonad has
again come to occupy the attention of various
investigators. As a result of the use of high
resolution techniques, the old debate has been
reopened (Byscov, ’78; Merchant-Larios and
Centeno-Urruiza, ’79; Upadhyay et al., ’79;
Zamboni et al., ’79).
Success in obtaining total reversion of the
gonadal sex and therefore of the somatic sex
using steroid hormones has been demonstrated
in Xenopus laevis (Gallien, ’53; Chang and
Witschi, ’56) and in R a m pipiens (Foote, ’38).
We believe that it is important to reconsider the
problem of the initiation of gonadal develop0003-276W81/19934349$03.500 1981 ALAN R. LISS.INC.
ment in these species using high resolution
techniques, with reference to the embryological
origin of the cells. Such a study is a necessary
antecedent to the investigation of the effects of
steroid hormones on the establishment,
morphogenesis, and differentiation of the
gonad.
MATERIALS AND METHODS
Tadpoles of R a m pipiens and Xenopus laevis
were raised from fertilized eggs obtained by
induced ovulation according to the methods of
Rugh (’62) and Gurdon (quoted by New, ’66),
respectively. They were given declorinated tap
water (21°C t 1°C)and fed with lettuce leaves
(R.pipiens) or powdered alfalfa leaf (X. laevis) .
Nine animals from each group were killed at
each of the following developmental stages: R .
pipiens, stages I-VII (Taylor and Kollros, ’46);
X . laevis, stages 4 9 5 6 (Nieuwkoop and Faber,
’56). The gonads, including the mesonephros,
were removed and immediately placed in the
fixative described by Kalt and Tandler (’71),
omitting the acrolein. They were postfixed in
1% OsO, in 0.1114 cacodylate buffer, dehydrated
in acetone, and embedded in Epon 812.
Semithin (1pm) and thin sections were cut
with a n LKB microtome and stained with toluidine blue or lead citrate for light and electron microscopy, respectively.
Received December 19, 1979; accepted July 9, 1980
350
HORACIO MERCHANTLARIOS AND IRMA VILLALPANDO
RESULTS
Xenopus laeuis
The undifferentiated gonad is present during
stages 4%54 and is characterized by the establishment of the two primordia, medulla and
cortex. The genital crest first appears in stage
49 a s two evaginations of the coelomic
epithelium which run parallel to the sides of
the intestinal mesentery in the ventral medial
region of the mesonephros. Some primordial
germ cells (PGCs) are already present in the
interior of the crests, where cells of the coelomic
epithelium are gradually changing from a flat
to a cuboidal shape (Figs. 1, 2).
The genital crests become elongated between
stages 50 and 51; the first PGCs remain in the
distal part of the original evaginations. It is a t
this time that the movement of some cells from
the coelomic epithelium towards the interior of
the genital crest can be detected. While it is
possible to find some evidence of this process in
semithin sections observed under the light
microscope (Fig. 2), it can be definitely corroborated with electron microscopy (Fig. 3). Initially, the first cells that form the gonadal
medulla appear in the intermediate zone of the
gonad, next to the distal region containing the
PGCs that arrived earliest at the crest. In the
former area, the basal lamina that originally
covers the coelomic epithelium is interrupted,
through which pass the cells migrating towards
the interior of the gonad (Fig. 3). It should be
noted that the inter-epithelial space in the
proximal zone of the genital crests (next to the
mesonephric region) is rather narrow, has
abundant collagenousfibrils, and, most importantly, does not contain cells that would indicate a migration from the mesonephric region
towards the gonad (Figs. 2, 3).
From these initial stages of gonadal development onwards, it can be seen that the cells of
the so-called “mesonephric blastema” form a
heterogeneous population with respect to their
ultrastructural characteristics. The most differentiated cells at the onset of gonadal development (stage 49) are those which compose the
interrenal gland (Figs. 1,4).They appear to be
steroidogenic cells that contain large mitochondria with tubular cristae and big lipid inclusions (Fig. 4). The rest of the cells of the
“mesonephric blastema” are found distributed
between the mesonephric tubules and the blood
vessels. Their form and size vary, although
they are basically of three types during stages
49 and 50: cells with numerous small, very electron-dense inclusions, identified as chromaffin
cells (Fig. 5 ) ; amoeboid-like cells with irregu-
larly shaped inclusions of medium density
(Figs. 5 , 6); and cells with relatively small
amounts of poorly differentiated cytoplasm and
a very large nucleus containing abundant
heterochromatin (Fig. 5 ) . There are also other
poorly differentiated mesenchymal cells, but
they are few in number. Finally, highly pigmented cells are always to be found lining the
coelomic epithelium (Figs. 1, 2, 5 ) .
The next phase of gonadal development,
characterized by a great proliferation of medullary cells and a compact gonadal structure (Fig.
7),takes place between stages 52 and 53. Under
the electron microscope, it can be observed that
there is a definite structural similarity between the cells of the gonadal epithelium and
those of the medulla. Furthermore, there is direct contact between the cells since no basal
lamina is present (Fig. 8).It is also important to
note that the cells of the medulla display none
of the ultrastructural characteristics typical of
the three most abundant cellular types in the
“mesonephric blastema” (compare Figs. 9 and
10).
The last phase in the establishment of the
undifferentiated gonad occurs during stages 54
and 55. There is an invasion of connective tissue appearing to come from the mesonephric
region that separates the cells of the medulla
from those of the cortex (Fig. 11). This tissue is
not distributed evenly over the length of the
gonad, but rather is found in separate masses
as can be seen in serial sections (Figs. 11, 12).
The gradual appearance of a basal lamina
which together with the connective tissue functions to segregate the two primordia can be
observed with the electron microscope. However, the basal lamina of the cortex is still continuous a t some points with that of the medulla
which has barely begun to form (Fig. 13). Finally, it should be pointed out that even during
this last phase prior to the sexual differentiation of the gonad, it is impossible to distinguish the cells of the medulla from those of the
cortex by means of their ultrastructure. In contrast, there is no structural similarity whatever between the cells of these two primordia
and those of the “mesonephric blastema” which
are even more differentiated in stage 55.
R a m pipiens
The appearance of the genital crest as a small
evagination in the coelomic epithelium occurs
during stage I (“limb bud,” 13 mm). Although
the morphogenetic events in this speciesclosely
resemble those described in Xenopus, there are
several important differences that should be
mentioned.
Fig. 1. Stage 49, Xempus. Two genital crests (arrows) are visible in this light micrograph; the right one contains a
primordial germ cell. Cells of the “mesonephricblastema” (MB), interrenal gland (IR),and a growing mesonephric tubule
(MT)are present. x 380.
Fig. 2. Stage 51,Xenopus. Light micrograph showing two genital crests separated by intestinal mesentery (IM). Some
epithelial cells have moved toward the interior in the left gonad (arrow). Note the narrowness ofthe upper region of the gonads
in the vicinity of the mesonephros (MI. x 500.
Fig. 3. Stage 51, Xennpus. Electron micrograph of a genital crest a t the same developmental stage. The basal lamina
(small arrows) of the epithelium is interrupted (big arrows) by cells (*) which appear to be migrating toward the interior of the
gonad. Part of a primordial germ cell (PGC) is visible in the lower portion of the figure. x 7,200.
352
HORACIO MERCHANT-LARIOS AND IRMA VLLALPANDO
The PGCs arrive as a group and not gradually as in the previous case, such that most of
the volume of the gonad is occupied by these
cells a t the beginning of development (Fig. 14).
Another interesting distinction is that the
mesonephros develops much more slowly with
respect to the gonad in Rana. As in Xenopus,
the cells that form the “mesonephric blastema”
are numerous and morphologically varied
(Figs. 14, 15). The most abundant are those
with steroidogenic characteristics that form
the interrenal gland; they are arranged in isolated groups along the two mesonephric ducts
(Fig. 16). Another cell type has large granules
with a homogeneous highly electron-dense appearance (Fig. 15). There are also cells with
amoeboid characteristics similar to those in
Xenopus containing small irregular moderately electron-dense granules, as well as others
that are poorly differentiated and have a
mesenchymal appearance; in this. species, the
latter are more numerous (Fig. 15).
The medullary primordium appears in stage
I11 (22 mm) (Fig. 16). Again, it is not uniformly
distributed throughout the genital crests; in
fact, i t is rare that the primordium can be
simultaneously seen in both gonads in transverse serial sections. Although the structural
resemblance and continuity of the first medullary cells with those of the gonadal epithelium
(cortex) are very clear initially (Fig. 171, the
former soon segregate (Stage V) and form a
separate entity with staining characteristics
similar to the cells of the “mesonephric
blastema.” At the level of the intermediate region connecting the gonad with the mesonephros, interruptions of the basal lamina are frequently found where epithelial cells appear to
be moving towards the interior of the gonad
(Figs. 18, 19). It is possible that there they
might be incorporated into the growing medulla..
In R. pipiens as in Xenopus, there is an early
invasion of mesenchymal tissue and blood vessels, doubtlessly arising from the mesonephric
region, which separates the cortex from the
medulla. This morphogenetic process marks
the last stage in the establishment of the undifferentiated gonad, since sexual differentiation
takes place from this moment on through the
greater development of one or the other of the
two gonadal primordia.
epithelium does not participate in the formation of the medulla in the amphibian gonad.
Discussion has primarily been held between
proponents of the theory that the medulla is
formed by the migration of cells from the
“mesonephric blastema” (Witschi, ’67)and others who maintain that it arises from the “interrenal blastema” (Vannini, ’52;Nieuwkoop and
Faber, ’56).However, the results of the present
study appear to be opposed to these earlier
interpretations based on observations made on
material that was processed using classichistological techniques.
As can be seen in several of the light micrographs presented in this work, many of the cells
of the “mesonephric blastema” appear similar
to those of the growing gonadal medulla, owing
to their staining characteristics and topographic relationship. This is so, even taking
into account that the preservation of material
embedded in Epon and the resolution obtained
with the light microscope using semithin sections are much better than those obtained by
previous workers. Under these circumstances,
it is logical that they should have concluded
that the cells of the “mesonephric blastema” or
“interrenal blastema” which appear prior to
the medulla must emigrate more or less massively towards the gonad (posteriorly), giving
rise to the latter tissue. Nevertheless, there
exists some evidence from experiments with
anurans that suggests that the gonad is capable
of developing independently of the mesonephros.
Humphrey (‘33) unilaterally extirpated the
intermediate mesodermal region of the wood
frog (Rana syluatica), which contained the
“mesonephricblastema.” He interpreted his results in the following manner: “Although the
rete cords in particular are commonly regarded
as derivatives of the mesonephric blastema,
they are nevertheless frequently developed on
the operated side in the entire absence of distinguishable mesonephric tubules” (p. 255).
In the present study, there are three main
morphological findings that suggest an active
participation of the gonadal epithelium in the
formation of the medulla: 1)The basal lamina
of the coelomic epithelium is interrupted in the
region of the genital crest where the first cells
of the medulla appear; close contact is a t once
established between these cells. 2) There is a
marked ultrastructural resemblance between
DISCUSSION
the cells of the medulla a t the beginning of its
The great majority of investigators, both in development and those of the gonadal epthe classical literature and in recent pub- ithelium. Moreover, there are structural diflications, are of the opinion that the coelomic ferences found during every developmental
Fig. 4. Stage 49, Xempus. Electron micrograph of a cell of the interrenal gland containing mitochondria with tubular
cristae and big lipid inclusions (LD). x 20,000.
Fig. 5. Stage 50, Xempus. Electron micrograph showing the three main types of cells in the “mesonephric blastema”:
chromaffin cells (CF), amoeboid-like cells (A), and cells with poorly differentiated cytoplasm and a large nucleus containing
heterochromatin (H).Highly pigmented cells next to the coelomic epithelium are also present. x 19,000.
Fig. 6. Stage 50, Xempus. Electron micrograph of part of an amoeboid-like cell with irregularly shaped granules of
medium density (arrows). x 18,000,
354
HORACIO MERCHANT-LARIOSAND IRMA V I L W P A N D O
Fig. 7. Stage 52,Xenopus. Two gonads with an apparent asymmetrical developmentare shown in this light micrograph. "he
left one has a compact appearance. Two mitotic figures are present, suggesting active growth (arrows). x 380.
Fig. 8. Stage 52,Xenopus. Electron micrographofthe proximal part of the gonad near the mesonephros. The upper epithelium
is formed by a single layer of cells (arrow).The lower region, however, has grown toward the interior, forming the initial gonadal
medulla (m). x 8,OOO.
Fig. 9. Stage 52, Xenopus. Electron micrograph of the mesonephric region showing its main cellular components: a mesonephric tubule (MT),
part of the interrenal gland (IG),and the so-called “mesonephric blastema”situated between the two former
structures, formed by various types of cells.Note the differencesin the cytological characteristics of these cells and those in Figure
10. x 4.500.
Fig. 10. Stage 52,Xempus. Small gonad taken from the same tadpole as that in Figure 9. The arrow indicates what appears to
be an ingrowth of the gonadal epithelium, giving rise to the first medullary cells. ~ 4 , 5 0 0 .
Fig. 11. Stage 54,Xenopus. The upper gonad has a compact medulla separated from the cortex by connective tissue (arrows)in
this light micrograph, while the lower gonad appears to contain only connective tissue. X 380.
Fig. 12. Stage 54,Xenopus. The reverse situation prevails in this serial section taken from the same tissue. The medulla is
evident only in the lower gonad, where a small cavity can be seen (arrow). x 380.
Fig. 13. Stage 54,Xenopus. Electron micrograph showing a continuous basal lamina (arrows) between the cells of the cortex
(c) and medulla (m).The latter have begun to organize as an epithelium as suggested by the presence ofseveral desmosomes (D).
x 7,200.
Fig. 14. Stage I (14mm), Rana. The genital crest is almost completely filled by primordial germ cells with abundant yolk
inclusions. Cells of the “mesonephricblasterna” are also present (M) in this light micrograph. x 500.
Fig. 15. Stage I (14 mm),Rana. Part of a mesonephric tubule can be seen in the upper right corner of this electron micrograph.
Two main types of cellspresent in the “mesonephricblasterna” can be observed: amoeboid-likecells with small granules (A)and
cells with large spheroidal electron-dense granules of a homogeneous appearance (B). x 6,300.
358
HORACIO MERCHANT-LARIOS AND IRMA VILLALPANDO
-
Fig 16 Stage 111 (22 mm),Rana Light micrograph showng the medulla (m) a s well a s the interrenal gland (IR)near a
mesonephric duct x 380
Fig 17 Stage 111 (22 mm), Rana In this electron micrograph, the basal lamina is interrupted and cortical (c) and
medullary (m) cells are in direct contact (arrows) Note the structural similarity of these cells x 7,200.
Fig. 18. Stage IV (35 mm),Rana. Low magnificationelectron micrograph of the intermediateregion connecting the gonad
on the left where part of a primordial germ cell (pgc)can be Seen and the mesonephros (M) on the right. Two cells (*I appear to
be emerging from the epithelium. x 2,500.
Fig. 19. Stage IV (35 mm),Rana. High magnificationof the same two starred cells shown in Figure 18.The basal lamina
(arrows) is interrupted where the two cells appear to be emerging from the epithelium (*, arrows). x 18,000.
360
HORACIO MERCHANT-LPLRIOSAND IRMA VILLALPANDO
stage between the cells of the medulla and
ACKNOWLEDGMENTS
those of the “mesonephric blastema.” 3) The
We
would
like
to thank Mr. Felipe Olvera for
later formation of a basal lamina which sepahis
excellent
photographic
work and Ms. Marrates the cells of the medulla in the proximal
zone from the mesonephros, as well as the ex- cella Vogt for her skillful editorial assistance.
tensive mitotic activity observed in the former
LITERATURE CITED
cells, suggests that the growth of the medullary Bryant, S.V. l1978)Pattern regulation and cell commitment
primordium takes place by the proliferation of
in amphibian limbs. In: The Clonal Basis of Development.
S. Subtelny and I.M. Sussex, eds. Academic Press, New
cells originating in the epithelium, and not by
York, pp. 63-82.
the aggregation of cells from the mesonephric Byscov,
G.A. (1978) The anatomy and ultrastructure of the
region.
rete system in the fetal mouse ovary. Biol. Reprod.,
Cells from the mesonephric zone do invade
19:72@735.
the gonad in both X . laevis and R . pipiens dur- Chang, C.Y., and E. Witschi (1956) Genic control and hormonal reversal of sexdifferentiation inxenopus. Proc. Soc.
ing the final stage of the establishment of the
Exp. Biol. Med., 93:140-144.
undifferentiated gonad. However, the incur- Foote, C.L. 11938) Influence of hormones on sex difsion is carried out by the connective tissue and
ferentiation in amphibia (Rana pipiens). Anat. Rec.,
Suppl. 72: 12CL-121.
blood vessels which function to segregate the
L.. (1953) Inversion totale du sexe chez Xempus
two gonadal primordia. It is probable that this Gallien,
laeuis Daud. a la suite d’un traitement gynogene par le
event also contributed to the formulation of the
benzoate d’oestradiol administre pendant la vie larvaire.
theory of the mesonephric origin of the gonadal
C.R. Acad. Sci. [Dl (Paris), 237:1565-1566.
Humphrey, R.R. (1933) The development and sex difmedulla.
ferentiation of the gonad in the wood frogfRana syluaticd
Traditionally, the term “blastema” has been
following extirpation or orthotopic implantation of the
used in embryology to refer to a group of pluriintermediate segment and adjacent mesoderm. J. Exp.
Zool., 65:243-269.
potent cells that are capable of giving rise to
one or several organs, to a limb (in the case of Johnson, M.H., A.H. Handyside, and P.R. Braude (1977)
Control mechanisms in early development. In: Developregeneration), or to a complete organism (in
ment in Mammals. M.H. Johnson, ed. NorthHolland Pubasexual reproduction). The observations in this
lishing Co., Amsterdam, pp, 67-97.
study made with high resolution techniques Kalt, R.M., and B. Tandler (1971)A study of fixation of early
amphibian embryos for electron microscopy. J. Ultrasuggest that the mesonephric blastema construct. Res., 41r635-645.
tains cells from which originate the different Merchant-Larios,
H. (1978) Ovarian differentiation. In: The
cellular types that form the mesonephros as
Vertebrate Ovary. R.E. Jones, ed. Plenum Press, New
well as the interrenal gland. Although it can be
York, pp. 47-81.
argued that in a morphological study such as Merchant-Larios, H., and B. Centeno-Urruiza (1979) Origin
of the somatic cells in the rat gonad An autoradiographic
the present one it is not possible to definitely
approach. Ann. Biol. Anim. Biochim. Biophys., 19:1219ascertain the origin of the medullary cells, it is
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even more difficult, in light of the biological New, D.A.T. (1966) The Culture of Vertebrate Embryos.
Academic Press, London.
principle of economy, to accept the possibility
P.D., and J. Faber (1956) Normal Table of
that the cells of the “mesonephric blastema” Nieuwkoop,
Xenopus lamis (Daudin).A Systemical and Chronological
ultrastructurally de-differentiate while deSurvey of the Development from the Fertilized Egg Till the
End of Metamorphosis. North-Holland Publishing Co.,
scending from the gonadal primordium and
Amsterdam.
transform into cells similar to those of the
Rugh, R. (1962) Experimental Embryology. Techniques and
coelomic epithelium.
Procedures, 3rd Ed. Burgess Publishing Co., Minneapolis,
These observations imply that the estabMinnesota.
lishment of the undifferentiated gonad and its Taylor, A.C., and J.K. Kollros (1946) Stages in the normal
development of Rana pipiens larvae. Anat. Rec., 94:7-24.
subsequent sexual differentiation in amS., J.M. Luciani, and L. Zamboni (1979)The role
phibians do not differ fundamentally from that Upadhyay,
of the mesonephros in the development of indifferent gowhich occurs in other vertebrates (Merchantnads and ovaries of the mouse. AM. Biol. Anim. Biochim.
Larios, ’78). The early commitment of two cellBiophys., 19:117%1196.
ular lines from a common origin owing to the Vannini, E. (1952) Organogenese des gonades et determinism du sexe chez les amphibians e t les amniotes. Arch.
different location of cells in the tissue has been
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experimentally demonstrated in other develop- Witschi, E. (1967) Biochemistry of sex differentiation in
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postulate different embryonic origins for the Zamboni,
L., P. Mauleon, and J. Bezard (1979)The role of the
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has previously been done.
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