Ultrastructural events during early gonadal development in Rana pipiens and Xenopus laevis.код для вставкиСкачать
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 1229. 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 Anat. Microsc. Morphol. Exp., 39t295-313. experimentally demonstrated in other develop- Witschi, E. (1967) Biochemistry of sex differentiation in ing systems (Johnson et al., ’77; Bryant, ’78). vertebrate embryos. In.The Biochemistry of Animal Development. R. Weber, ed. Academic Press, New York, pp. Thus, it does not appear to be necessary to 19S225. postulate different embryonic origins for the Zamboni, L., P. Mauleon, and J. Bezard (1979)The role of the two gonadal primordia (cortex and medulla) as mesonephros in the development of the sheep fetal ovary. Ann. Biol. Anim. Biochim. Biophys., 19:1155-1178. has previously been done.