I N T E R N A T 1-0 N A L J 0 U R N A L 0 F A N D R 0 L 0 G Y

7 (1984) 529-541

Department of Expm'mentul Mgbhology, Faculty of Medicine, University of Chile,

Santiago, Chile

Foetal meiosis in the testis of the rodent Octodon degus

BY

M. A. Rojas, B. Morales and P. Esponda*

Different stages of meiotic prophase have been studied in foetal testes of the rodent, Octodon degus, using the light and electron microscope. Special attention was focused on the ultrastructural morphology of these meiotic cells in comparison to pre-spermatogonia of foetal testes and meiotic spermatocytes of the adult male testis. Meiosis occurs in only a few cells located among fibroblasts of the tunica albuginea or in the region of the gonadal blastema. The foetal meiotic process resembles adult meiosis in its ultrastructural characteristics; typical pachytene synaptonemal complexes and leptotene or diplotene axial elements appear associated to the chromatin. This process occurs at the same foetal age that meiosis commences in the ovary, thus reinforcing the idea that both meiosis-inducing and meiosis-preventing sub- stances are secreted in both sexes. The intra-or extracordonal localization of the germ cells would be an important factor in determining the cells' response to these substances.

Key words: foetal testis - meiotic germ cells - ultrastructure.

Gonadal differentiation in mammals has been studied from many different points of view (Witschi 1970; Jost et al. 1973; Gondos 1977; Wartenberg 1981a, 1982). The control mechanisms by which germinal cells commence meiosis in the male and female gonad is one of the most interesting aspects of this process. Testicular cords are normally differentiated during the male's foetal life. Gonocytes and sustenta-

Received on March 5th, 1984. * Instituto de BioloGa Celular (Consejo Superior de Investigaciones Cientificas, Madrid,

Espaiia).

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cular (pre-Sertoli) cells are the only differentiated cells in some species (Gondos 1977), whilst in others,.pre-spermatogonia can be observed and, in a few cases, foetal spermatogonia have been identified (Gondos & Hoebel 1971). It is well known that male germ cells do not enter meiotic prophase until early puberty, probably because they are enclosed by the foetal Sertoli cells Uost et al. 1974) which may secrete a meiosis-preventing substance (MPS) (Byskov & Saxen 1976; Luciani et al. 1977) On the other hand, in the female the onset of meiosis occurs in foetal life in response to the secretion of a Meiosis-Inducing Substance (MIS) which is secreted by the rete ovarii (Byskov 1978; Grinsted & Byskov 1981). Nevertheless, in some cases stages of meiotic prophase have been observed under the light microscope in normal (Ohno et al. 1962; Ozdzenski 1972) and experimental conditions (Byskov & Saxen 1976; Mc Laren et al. 1972) in the male. However, the ultrastructure of these meiotic cells in the gonads of mammalian foetuses has not been studied in detail.

The rodent degu (Octodon degus. Fam. Octodontidae; Suborder Caviomorpha; Order Rodentia) has a longer period of gestation (range 87-93 days, see Weir 1974) than that of other rodents, and this permits an accurate study of the different phases of development. The embryonal development of this species has been analysed recently (Rojas et al. 1982), and in 41-day post-coitum embryos a testis could be recognized.

In this report, we present some ultrastructural aspects of the differentiated foetal testis of Octodon degus, with emphasis on the meiotic cells found in the testis.

Materials and Methods

Octodon degus were maintained in the laboratory at 20-22°C under natural light and with free access to food and water. Foetuses were obtained according to the previously described method (Rojas et al. 1982), as follows: females presenting a perforated vaginal membrane were joined with selected males; the initial day of pregnancy (day 0) was determined by the presence of spermatozoa in vaginal smears which were taken daily. Females were isolated and embryos obtained by laparatomy. In the present study, foetuses of 65,66 and 67 days from three females and of 72 days from two females were analysed. Foetuses were dissected in saline and examined macroscopically to evaluate their external morphology. Gonads were then processed for electron microscopy: fixation was carried out in 6% glutaralde- hyde in 0.1 M phosphate buffer followed by post-fwation in 2 % Os04 for 30 min. After dehydration in alcohols, samples were embedded in Epon. Thick sections (1-2 pm) obtained with an LBK Ultramicrotome were stained with toluidine blue (2%) and used for histological study and foetal sex determination. Ultra-thin sections

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were stained with uranyl acetate and lead citrate and examined with a Philips 300 and a Jeol 100 B electron microscope. Testes from three adult males which were in their sexuaily active period were processed in a similar way to that described above.

Results

In foetuses, testes could be distinguished from ovaries by the presence of a tunica albuginea, testicular cords and Leydig cells (Figs. 1 and 2), in addition to the presence of well developed Wolffian ducts and degenerated Mullerian ducts (Rojas et al. 1982).

In the testicular cords three different and distinct cells types were evident-: gonocytes in the center of the cords, and pre-spermatogonia and pre-Sertoli cells in the peripheral area (Fig. 2). Gonocytes were characterized by their regular round shape with a high nucleo-cytoplasmatic ratio, a large centrally located spherical nucleus with evenly distributed chromatin and prominent reticular nucleoli. Pre-spermatogonia or foetal spermatogonia were arranged in pairs at the tubular periphery. Except for their peripheral location, the general appearance of sperma-

Fig. 1. Semithin section of the testis of a 66-day-old foetus showing a superficial epithelium (se), tunica albuginea (ta), testicular cords (cord) and Leydig cells (leydig). A small gonadal blastema is indicated by the dotted white line and the black circle shows ectopic germinal cells.

x 93.

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Fig. 2. A testicular cord in cross section showing gonocytes (g) in the center and pre-spermatogonia (p) and sustentacular cells (s) in the peripheral area. Thick section stained with Toluidine blue

from a 66-day-old foetus. X 460.

FIX. 3. Electron micrograph of an ultra-thin section of the testis of a 65day-old foetus. Two pre-spermatogonia (pre-esp) can be seen; the nucleus shows diffuse chromatin and a reticular nucleolus (N-ret). Pre-Sertoli cells appear in between the pre-spermatogonia: the nucleus shows dense chromatin adhered to the nuclear membrane, and the cytoplasm has an

appreciable quantity of organelles and membranes. x 6200.

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togonia was similar to that of gonocytes under the light microscope. Pre-Sertoli cells in the peripheral area were distinguished by their columnar shape and indented irregular nucleus.

Testicular cords were surrounded by mesenchymal cells and between them precursors of Leydig cells could be observed. In all foetuses analysed under the electron microscope the testicular cords showed two principal cell types: pre- spermatogonia and sustentacular or pre-Sertoli cells, which could be distinguished clearly (Fig. 3). In the nucleus of pre-spermatogonia the chromatin was usually dispersed, and a prominent nucleolus formed by nucleolar strands could be observed. Cytoplasmic organization was simple : mitochondria often appeared associated to a dense ‘cementing’ substance. Golgi apparatus and elongated cisternae of the endoplasmic reticulum, membranes and ribosomes were scattered. Vacuolated bodies, electron dense structure or ‘nuages’ (see Eddy 1975) and intercellular bridges were also commonly seen. Pre-Sertoli cells appeared among pre-spermatogonia, and their cytoplasm was related to the basal lamina, whereas their nuclei were situated towards the centre of the cord.

Fig. 4. Semi-thin section from the testis of a 65-day-old foetus. A group of cells which appear in the small gonadal blastema can be seen. In some cells the chromatin appears as strands (arrow).

X 630.

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Fig. 5. A differentiated germinal cell (Meiotic G.C.) in the small gonadal blastema of a 66day-old foetus. The cytoplasm shows an appreciable quantity of organelles and a well developed Golgi (G) and vesicles (v). In the nucleus, dense chromatin masses appear associated to axial

structures (arrows). The surrounding cell can also be seen. X 5700.

The nuclei of pre-Sertoli cells showed dense chromatin masses and rounded nucleoli. The cytoplasm was similar to that studied in other species (Gondos 1977), with a high density of ribosomes, polyribosomes and glycogen, many mitochondria, a moderate granular reticulum and a Golgi zone composed of flattened cisternae and vesicles.

Germinal cells situated outside of the testicular cords were found in all samples analysed. These cells were located mainly between fibroblasts or mesenchymal-like cells corresponding to the tunica albuginea or in the central gonadal blastema (Figs. 6-7 and 4), near to the area of the future rete testis. In the tunica albuginea, germinal cells could be seen alone or in groups of 4 or 6 cells in a linear or rounded disposition. In the central gonadal blastema the cells formed rounded groups sometimes surrounded by undifferentiated blastema cells. A notable difference between the germinal cells situated outside and those located within the testicular cords could be observed, especially concerning the nucleus. In the former, dense

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chromatin strands and an augmented nuclear volume were evident compared to gonocytes within the cord and pre-spermatogonia (Fig. 4). The nucleus measured 1 1.6 pm in diameter in comparison to 7.6 pm in the pre-spermatogonial nucleus. In these bigger nuclei, axial elements associated to dense chromatin, resembling those forming the meiotic synaptonemal complex could be observed (Fig. 5). These synaptonemal complexes, described in pachytene cells of many species (Moses 1969; Gillies 1975) showed lateral elements of an approximate diameter of 40 nm associated to the chromatin with a space of 120 nm between them. In this space a parallel central elements and some transverse filaments could be seen. Nucleoli were conspicuous in these cells, and the cytoplasm was denser than in pre- spermatogonia. The most remarkable cytoplasmic characteristics were the .abundant ribosomes and membranes, as well as a well-developed Golgi apparatus (Fig. 5).

In other testes, similar cells appeared among the fibroblasts of the tunica albuginea (Figs. 8 and 9). The cells showed different stages of meiotic prophase, and both synaptonemal complexes (Fig. 9) and single axial elements (Fig. 8) were evident, and which were typical of the pre-pachytene stage. In the gonadal blastema of some samples the foetal meiotic cells had nuclear axial elements associated with very dense chromatin masses which were close to a dense nucleolus. The nucleolus was in a central position and was associated with dense chromatin; within the nucleolus, fibers and granules were observed.

Figs. 6 and 7. Fig. 6. Semithin section of the showing testis of a 65-day-old foetus a cell of the superficial epithelium (s.e.), the tunica albuginea, testicular cord (cord), and ectopic germinal cells X 5 10. In Fig. 7. Meiotic germinal cells (MGC) are shown close to the tunica albuginea (ta) in a

65-day-old foetus. Mesenchymal like cells or fibroblasts (F) are also shown. x 3500.

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Figs. 8 and 9. Two germinal cells close to the tunica albuginea of a 65-day-old foetus. In Fig. 8 an axial core (arrow) appears, x 7700, whilst in Fig. 9 a typical synaptonemal complex (arrow) is observed

associated to dense chromatin masses. x 9700.

No typical pre-Sertoli cells were observed in association with the foetal meiotic cells. Stromal cells in both the central blastema and tunica albuginea function as supporting cells. The meiotic cells situated in the tunica albuginea were found in close relation to mesenchymal-like cells. The meiotic cells situated in the gonadal blastema were closely surrounded by the blastema cells which had a dense cytoplasm and nucleus.

No meiotic stages were observed in older foetuses.

Discussion

Despite studies on the biochemical control of the meiotic process (Stern & Hotta 1977) the main mechanisms concerning the commencement and control of this phenomenon remains unknown. In mammals, the problem is even more difficult due to the fact -that meiosis begins a: different stages of development in the male and the female. In this respect the existence of regulatory substances such as MIS

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and MPS (Byskov & Saxen 1976; Grinsted & Byskov 1981), the role of other cells such as the pre-Sertoli cells Uost et al. 1974) and the location of gonocytes in different regions of the male gonad (Wartenberg 1978) could be factors involved in initiating or inhibiting meiosis. Such factors may also be responsible for the normal presence of meiotic cells in different stages of foetal life in the female. Despite this, studies carried out under the light microscope have show in that, ocassionally, some gonocytes undergo meiosis in the foetal testis. In cats, Ohno et al. (1962) observed that a few cells in pachytene and diplotene normally occurred whilst a similar case was reported in mice by Ozdzenski (1972). Preleptotene stages have also been reported in the neonatal rabbit (Gondos & Byskov 1981) and human (Luciani et al. 1977). Moreover Upadhyay & Zamboni (1982) noted the presence of meiotic germ .cells in the adrenal glands of mice of both sexes. On the other hand, pre-meiotic stages have also been observed in the foetal mouse testis cultured close to ovaries (Byskov & Saxen 1976) and in foetal testes of chimaeras, which probably have an XX chromosome constitution (McLaren et al. 1972).

Some authors (Byskov 1978) claim that the gonads of both sexes have the capacity to secrete MIS, via cells derived from the mesonephros. The gonocytes inside the testicular cords would be unable to respond to this substance, but those present in other areas of the gonad could undergo meiosis (Byskov & Grinsted 1981). Gondos & Byskov (1981) point out that germinal cells in both the male and female are responsive to MIS in early stages of development, but that different environmental factors (i.e. hormones) would stop the process. Wartenberg (1978, 1981b) has proposed that meiotic initiation would depend on the numerical relationship between two different populations of supporting cells. In this respect it has been suggested that two kinds of Sertoli cells could be involved , as there is evidence that the dark and light supporting cells of the foetal testis exercise either a stimulatory or an inhibitory effect on the germ cells via direct cellular contact.

The observations carried out in 0.degus suggest that true meiotic prophase stages occur in a few cells of the foetal testis and these show similar ultrastructural characteristics to primary spermatocytes of the adult male degu (Fernindez- Donoso 1982). The presence of synaptonemal complexes is a universal feature during the zygotene and pachytene stages of meiotic prophase in plants and animals (Moses 1969; Westergaard & von Wettstein 1972; Gillies 1975). One of the characteristics of the synaptonemal complex is its construction, being composed of two lateral and one central element with a distance of 120 nm between laterals (The pairing space which separates the pair of homologous chromosomes). Attachment of the synaptonemal complex to the inner nuclear envelope is another characteristic of this structure which is commonly observed in mammalian spermatocytes (Esponda & Gimenez-Martin 1972). During leptotene and early zygotene stages, single axial cores are usually associated to the nuclear envelope, representing the future lateral element of the synaptonemal complex; during the diplotene stage, single axes have been described as remnants of the elements of the synaptonemal

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complex (Solari 1970). With respect to its role in riieiosis, the synaptonemal complex is undoubtly related to the pairing of hornlogous chromosomes and possibly to the crossing-over phenomenon. Oocytes from mammalian foetuses analysed under the electron microscope show typical synaptonemal complexes and single axes during the different stages of meiotic prophase in the female (Baker & Franchi 1967; Condos et al. 1971a).

The ultrastructural analysis of meiotic germ cells in foetal testes of 0.degus demostrates that they are similar to spermatocytes present in the adult. The nucleolus is in a central position and is associated with dense chromatin. On the other hand, the cytoplasmatic organization of the foetal meiotic cells resembles that of the adult meiotic spermatocytes although there are a few similarities to pre-spermatogonia. In this respect, evidence exists that several changes take place in the cytoplasm of pre-meiotic cells, for example an increase of organelles leading to the image of a typical auxocyte (Burgos et al. 1970; Eddy 1975). These characteristics and those of the nucleus support the suggestion that the foetal meiotic cells are similar to those of the adult. However, it is premature to conclude that they correspond exactly to true primary spermatocytes.

The fact that no stages of meiotic metaphase I or spermatids have been observed and the absence of meiotic cells in older foetuses may indicate degeneration of these early meiotic cells. It has been noted by other authors that a notable increase in germ cell degeneration occurs coincident with meiotic initiation (Gondos & Byskov 1981 ; Byskov & Grinsted 1981). Again Upadhyay & Zamboni (1982) observed that no ectopic germ cells in meiosis were seen in the adrenal glands after day 12 of life as the result of degeneration.

The initiation of meiosis in female and male gonads seems to be controlled by two opposing substances a meiosis-inducing substance (MIS) and a meiosis preventing substance (MPS), which are secreted by the mesonephros and mesonephric -derived tissues in both sexes. Using cultures of foetal mouse gonads (Nikitin & Byskov 198 l), indicated that this mesonephric influence varies with age triggering meiosis of germinal cells during certain stages of development but later inhibiting meiosis. These observations probably also explains the absence of meiotic germ cells in older male foetuses.

It is interesting to note that meiosis in the foetal testis starts at the same time as it does in the ovary of 0. degus (Rojas et al. 1982), a fact also observed in other species (Ohno et al. 1962; Luciani et al. 1977; Gondos & Byskov 1981; Upadhyay & Zamboni 1982). According to Hilscher et a1 (1974) and Wartemberg (1981a) pre-meiotic mitosis of oogonia is terminated at a time when male germ cells have gone through a similar sequence of mitotic divisions. In the foetal testis, however, the ‘pre-meiotic’ mitotic proliferation is halted within the testicular cords, and it is likely that ectopic germ cells run through an identical sequence of mitotic divisions and are not preyented from entering meiosis due to the different local cellular composition.

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Upadhyay & Zamboni (1982) noted the presence of ectopic germ cells in the adrenal glands of animals of both sexes, and claimed that mammalian germ cells are capable of undergoing sustained differentiation outside of the gonads and that, in ectopic sites, all ‘germ cells’ differentiate into oocytes, even in males. These ectopic germ cells were closely surrounded by adrenal cortical cells or by the chromaffin cells of the medulla. These cells occasionally displayed a crescent shape similar to that of the granulosa cells lining the walls of unilaminar ovarian follicles. In the present study, no typical pre-Sertoli were observed adjacent to the meiotic cells. Instead the meiotic cells situated in the tunica albuginea were found in close relation to mesenchymal cells and the meiotic cells situated in the gonadal blastema were closely surrounded by the blastema cells. These stromal cells had a dense -cytoplasm and nucleus and cellular prolongations.

Uphadhyay & Zamboni (1982) have suggested that germinal cells differentiate along a male line only in the testis and that is only in this organ that they are prevented from entering meiosis during foetal life and differentiating into oocytes. However, our present observations indicate that meiosis also occurs normally in the mammalian testis during foetalife, and these meiotic germ cells show similar ultrastructural characteristics to those of primary spermatocytes. The fact that meiosis occurs outside, but not inside, of the testicular cords suggests that meiosis may be prevented if germ cells are located within the cords, and perhaps it is this difference in location which determines the response of the cells to MIS and MPS.

Acknowledgments

We thank Dr. Raul Fernandez-Donoso for his scientific assistance and Mrs. Elena Zanelli for typing the manuscript. This study was supported in part by Grants B-1497-8433 DDI (Univesity of Chile) and No. 131 from C.A.I.C.Y.T. (Spain). The author’s work at C.S.I.C. in Spain was supported by a Fellowship from Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.

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Author’s address: Mariana A. Rojas, Departamento Morfologia Experimental, Facultad de Medicina, Div. Cs. Medicas Norte. Universidad de Chile. Casilla 2988, Correo Central, Santiago, Chile.

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