Ultrastructure of the Early Ovary and Testis in Pig Embryos LAURI J. PELLINIEMI Department of A n a t o m y and Laboratory of Electron Microscopy, University of T u r k u , SF-20520 T u r k u 52, Finland

ABSTRACT Pig embryos aged 26-27 days were used for an ultrastructural study of the early ovary and testis. Sex was identified by both chromosomal analysis and gonadal histology, with consistent results. The gonads occupied their original site in the medial coelomic angles in both sexes. The female gonad was composed of three tissues: the surface epithelium, the gonadal blastema and the mesenchyme. The gonadal structure was similar to that seen earlier at the age of 24 days. At 26 days the testis had distinctly differentiated into four tissues. The new components were the testicular cords and the interstitium, both derived from the gonadal blastema. The testicular cords resembled anastornosing sheets more than cords. The ultrastructure of the tissues and their cell types are described and compared to the previous indifferent stage at the age of 24 days. The cells of the surface epithelium, of the primitive cords, of the mesenchyme, and the primordial germ cells had an ultrastructure that was similar in both sexes. The sustentacular cells of the testicular cords resembled the primitive cord cells and the spermatogonia were similar to the primordial germ cells. No Leydig cells were present yet. The process of testicular differentiation is described on the basis of the present and a previous study, and a new hypothesis, based on the vascular organization, is presented.

The mammalian gonad remains morpho- monkey (Wagenen and Simpson, '65), the logically at a sexually indifferent stage pig (Allen, '04; Whitehead, '04; Grunwald, until the testicular cords and tunica albu- '34, '42; Black and Erickson, '65; Moon ginea develop in the male gonad (Gier and and Raeside, '72; Moon and Hardy, '73; Marion, '70; Jirasek, '71; Jost et al., '73). Moon et al., '73), the rabbit (Allen, '04), At the time when these characteristic the rat (Jost et al., '73) and the mouse changes occur in the testis, the morphol- (Peters, '70). Ultrastructural investigations ogy of corresponding ovaries remains un- of the fetal human testis (Pelliniemi and changed (Jost et al., '73). The morphologi- Niemi, '69; Wartenberg et al., '71; Holstein cal differentiation of the testis is thought et al., '71), the rat testis and ovary (Eddy, to be regulated by the sex-determining '74), the rabbit testis (Gondos and Conner, chromosomal genes, acting in the somatic '73; Bjerregaard et al., '74), the mouse cells of the gonadal blastema (Ohno, '67; ovary (Odor and Blandau, '69a,b), the Hamerton, '68; Boczkowski, '71, '73; Short, guinea pig testis (Black and Christensen, '72). '69) and the hamster testis (Gondos et al., The main emphasis in previous studies '74) are available. has been on the development of the ovary The present investigation was underand testis during the fetal period, and less taken because there is a lack of systematic attention has been paid to the stages of ultrastructural studies of the process of early differentiation. The fetal gonads have sexual differentiation i n the gonads of embeen studied at the light microscope level bryos whose chromosomal sex has been in man (Gillman, '48; McKay et al., '53; identified. The pig was chosen because it Pinkerton et al., '61; Wagenen and Simpson, '65; Jirasek, '71; Vossmeyer, '71), the Accepted March 1, '75. AM. J. ANAT., 144: 89-112.

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is suitable for correlation with studies of human development (Book and Bustad, ’74). In this paper the ultrastructure of the early ovary and testis in the pig is described and compared with the preceding indifferent stage (Pelliniemi, ’75b). MATERIALS AND METHODS

Pregnant sows of Nordic landrace do-mestic pigs ( S u s scrofa) were slaughtered 26 and 27 days after artificial insemination. The crown-rump lengths of the embryos were 16 m m and 22 mm, respectively. The chromosomal sex of the 26-day embryos was identified by chromosome analysis (Pelliniemi and Salonius, ’75). The gonadal sex was also identifiable by light microscopy of epoxy-embedded sections, and these results were in agreement with the chromosomal studies. Therefore the sex of the 27-day old embryos was identified solely on the basis of the gonadal histology. A total of eight male and six female embryos was examined by light and electron microscopy. Specimen preparation has been described in detail in a previous paper (Pelliniemi, ’75a). Various fixatives were tested and the best results were obtained with a solution containing 0.24 mol/l glutaraldehyde (Glutaraldehyde EM, TAAB Laboratories, England), 4.1 mmol/l CaC1, and 0.086 mol/l sodium cacodylate-HC1 buffer a t pH 7.4. The postfixative contained 39 mmol/l osmium tetroxide in 0.1 mol/l sodium cacodylate-HCl buffer at pH 7.4. After dehydration in ethanol the specimens were embedded in Epon (Luft, ’61). Thin sections were cut on a n LKB-Huxley Ultramicrotome with a diamond knife and stained with aqueous (Watson, ’58) or methanolic (Stempak and Ward, ’64) uranyl acetate and lead citrate (Venable and Coggeshall, ’65). The specimens were examined in a JEOL JEM-T8 electron microscope. The terms “male” and “female” refer here to chromosomal as well as to gonadal sex. “Gonadal blastema” (Ohno, ’67; Jirasek, ’71) is used to denote the tissue compartment between the surface epithelium and the mesenchyme which occupies the lateral part of the gonad. The male germ cells in the testicular cords are called spermatogonia and the somatic testicular

cord cells are called sustentacular cells (Wagenen and Simpson, ’65). OBSERVATIONS

At the age of 26-27 days gestation the gonads appeared as longitudinal protrusions along the medial mesonephric surfaces on each side of the mesenteric root (fig. 1 ) . They were Fttached by a thick mesogonadium to the mesonephros (figs. 3 , 4). I n transverse sections the anteroposterior axis measured 0.5 mm and the transverse axis 0.4 mm. Topographically there were no differences between the male and female gonad. Tissue types. The female gonad consisted of three different tissues (fig. 3 ) . The first was the surface epithelium. Below the epithelium the second tissue, the undifferentiated gonadal blastema, occupied the main area of the gonadal cross section. The cortical layer of the blastema contained primitive cords and the medullary part was the blastema proper. There was no clear boundary between these two areas and the cells were similar to each other. For these reasons the primitive cords were included in the gonadal blastema. The third tissue, the mesenchyme, was located i n the basal part of the ovary. Even by 27 days this organization remained similar to that of the earlier indifferent stage. In contrast to the ovary, the early testis contained four different tissues (figs. 3 , 4 ) . The structure and location of the testicular surface epithelium and mesenchyme were similar to those of the ovary. In contrast with the compact and undifferentiated gonadal blastema of the ovary (fig. 3), by 26 days the testicular blastema proper had differentiated into testicular cords and interstitium (figs. 4, 5 ) . This was the earliest stage at which the male gonad in the pig could be morphologically identified as a testis on the basis of the testicular cords. Accordingly, the female gonad was characterized by the absence of similar cords. Surface epithelium. The ovary and testis were similarly covered by surface epithelial cells. The cell shape and the number of cell layers varied on the anterior, medial and posterior surfaces, and the cells were arranged irregularly (figs. 3, 4, 5). On the lateral surfaces, anterior

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and posterior to the mesogonadal insertion, the cells of the surface epithelium had a columnar shape and were arranged regularly in a single layer, with a continuous basal lamina (figs. 4, 7 ) . The tunica albuginea had not differentiated yet. The location of the nucleus in the surface epithelial cells varied from basal to apical (figs. 6, 7). The mitochondria were rod-shaped with lamellar cristae. The Golgi complex was small, and the granular endoplasmic reticulum was composed of a few solitary cisternae. Numerous polysomes, some lipid droplets and coated vesicles were seen i n the cytoplasm. These organelles were similar in all the various forms of surface epithelial cells. Cytoplasmic processes and folds were seen on the apical cell membrane (fig. 6 ) . At the coelomic border the superficial cells were connected with each other by junctional complexes. They were composed of zonulae occludens and zonulae adherens with a n occasional desmosome located basal to these structures. The cell membranes of adjacent epithelial cells ran smoothly parallel and some desmosomes and patches resembling zonulae adherens were encountered. Ultrastructurally there were no sex differences. Primitive cords. The peripheral layer of the gonadal blastema was structurally similar in both sexes by 27 days. It was a compact tissue composed of cord cells as the principal cell type, and of singly occurring primordial germ cells (fig. 5 ) . The primitive cords were continuous with the surface epithelium in many places on the anterior, medial and posterior surfaces (figs. 4, 6). On the central side the primitive cords were continuous with the testicular cords in the male and with the blastema proper i n the female. I n this way the structural connections from the surface epithelium to the ovarian blastema proper and testicular cords were still maintained. The border between testicular and primitive cords was characterized by large capillaries located between the peripheral ends of the testicular cords (fig. 4 ) . No capillaries were seen in the area of the primitive cords. A similar organization of the capillaries was noted in the ovary (fig. 3 ) , and therefore these vessels were also regarded there as an indicator of the border between the primitive cords and the blas-

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tema proper. The cord cells were organized in r p d o m l y oriented short cords (fig. 5 ) . There were also places where no specific pattern could be seen. The shape and size of the cord cells and their nuclei varied considerably. The testicular cells directly under the epithelium were sometimes elongated in the plane of the gonadal surface, possibly representing the earliest signs of the differentiation of the tunica albuginea (fig. 6 ) . At the ultrastructural level the cord cells showed no sex differences and resembled both the surface epithelial cells and the sustentacular cells, with the exception that they did not have a free coelomic surface. Single primordial germ cells were located randomly in the primitive cords among the cord cells. They were seen directly under the surface epithelium (fig. 6 ) and also at all levels down to and including the blastema proper in the ovary and the testicular cords in the testis. The primordial germ cells of both sexes found in the mesenchyme and in the aforementioned locations, including testicular spermatogonia, were ultrastructurally similar (figs. 6, 8, 11, 12, 13, 17). Therefore, they could be distinguished by location only. The nucleus was round and the chromatin finely dispersed. One to three large nucleoli were seen. The mitochondria were round and distributed randomly i n the cytoplasm. The cristae were lamellar or tubular, and the matrix was denser in mitochondria containing tubular cristae. One or two small Golgi complexes were usually found in the vicinity of the nucleus, i n the same region as the centrioles. Granular endoplasmic reticulum occurred as long solitary cisternae or closely associated shorter pairs (figs. 6, 8, 11). They were distributed randomly in the cytoplasm and differed from the sustentacular cells in their lack of association with mitochondria. Numerous free polysomes were scattered in the cytoplasm. A cluster of dense bodies of varying shape was noticed i n some cells. Lipid droplets and coated vesicles were also encountered. An aggregate of chromatin-like material was occasionally present in the vicinity of the nucleus, as shown in figure 12. The cell membrane was smooth, containing only a few isolated processes. In a few places on the cell membrane patches of zonulae

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adherens were seen joining adjacent cells. Blastema proper. The histological pattern of the central region of the gonadal blastema, bounded by the peripheral capillaries, was undifferentiated only in the female gonad. This lack of differentiation was found in both developmental stages studied (fig. 3). By 27 days the blastema was denser and had grown in size in comparison to that of the 26-day specimen. The closely packed blastema cells were oriented randomly as single cells or small groups, at times showing a cord-like arrangement (figs. 8, 9 ) . The cells and their nuclei had varying shapes. One or two large nucleoli were usually present. The mitochondria were elongate with lamellar transverse cristae. The cisternae of the granular endoplasmic reticulum were often closely associated with mitochondria. Solitary rolls of agranular membranes were sometimes noted, but no conventional agranular endoplasmic reticulum was observed (fig. 8). The cytoplasm contained numerous free polysomes, coated vesicles, lipid droplets and fine filaments. Occasional cells contained large numbers of filaments. Some blastema cells and cord cells had a large inclusion body which contained remnants of a nucleus and cytoplasm with some organelles (fig. 10). The columnar blastema cells arranged a s cords had a basal lamina and the nuclei were basal or apical. The Golgi complex was either on the basal or apical side of the nucleus. The cell membranes of adjacent blastema cells were smoothly parallel and sometimes attached by a patch of zonula adherens. Some cells had long and narrow cytoplasmic processes that continued around a n adjacent blastema cell or around a primordial germ cell (fig. 9 ) . The second cell type i n the ovarian blastema, primordial germ cells, occurred singly and no organization into oogonial clusters or primordial follicles was encountered. They were completely bound by the surrounding blastema cells (fig. 8). The ultrastructure of the primordial germ cells has already been described in connection with the primitive cords. Testicular cords. By 26 days the edge of the central region of the male gonadal blastema was demarcated by the most peripheral capillaries and showed sex-specific

differentiation into testicular cords (figs. 4, 5). Peripherally they were continuous with the primitive cords, and centrally with the often, as yet undifferentiated, most central region of the gonadal blastema. This small area of undifferentiated blastema in the very center of the testis was often seen at the age of 26 days (fig. 4 ) , and by the 27th day it had totally differentiated into testicular structures. The thickness of the testicular cords varied from one to approximately eight cell layers. The broadest cords were located in the periphery and the narrowest in the central part. I n order to elucidate the threedimensional structure and relations of the early testicular cords, the gonads were also cut in the sagittal plane of the embryo. This plane of section showed a longitudinal profile of the testis in a plane parallel with the posterior portion of the mesentery (figs. 1, 2, 4 ) . I n the sagittal as well as transverse planes the testicular cords were irregularly shaped and anastomosing (figs. 2, 4 ) . Isolated roundish cords were seen very infrequently, whereas roundish interstitial areas of that shape were frequent (fig. 13). This indicates that, in a literal sense, the cords had not yet formed, and that the sustentacular cells were actually organized as an irregular, three-dimensional network of anastomosing sheets or walls of varying thickness around the interstitial space. The great majority of cells in the testicular cords were sustentacular cells, also known in the mature testis as Sertoli cells (figs. 13, 14). The sustentacular cells resembled the surface epithelial cells in that the size and shape of the cells and their nuclei varied. One, or sometimes two, large nucleoli were present. The mitochondria were elongate with transverse, lamellar cristae. Cisternae of granular endoplasmic reticulum were often observed in close association with mitochondria (fig. 14). The varying location of the Golgi complex indicated that the cells were not yet polarized. Agranular endoplasmic reticulum was not noted. Numerous polysomes were scattered evenly in the cytoplasm. Some coated vesicles, a few lipid droplets and a variable amount of fine filaments, approximately 8 n m in diameter, were oriented randomly in the cytoplasm. Occasionally a large in-

ULTRASTRUCTURE O F EARLY OVARY AND TESTIS

clusion body was noted, like that seen in figure 10, which resembled a degenerating cell in its texture. The cell membranes of adjacent cells ran parallel with a n interspace of about 40 nm. Patches of zonulae adherens were occasionally seen between the cells. The cell membrane facing the interstitium was covered by a discontinuous basal lamina (fig. 14). Its thickness varied from 40 n m to 90 n m and it was composed of loose fibrous material. Ultrastructurally, sustentacular cells were similar to the blastema cells of the ovary, exclusive of the differences caused by the different orientation of the cells. The germ cells, called spermatogonia within the cords, constituted the second cell type and appeared as large, roundish cells with a diameter of 12-20 pm. They were located singly among the sustentacular cells, which completely surrounded them within the cords (figs. 11, 13). Their ultrastructure was similar to that of the primordial germ cells in the primitive cords. Interstitium. The second derivative of the testicular blastema proper is the interstitial tissue (figs. 5, 13). At the age of 26 days it was separated from the testicular cords by a large intercellular space and by the, as yet incomplete, basal lamina of the testicular cords. However, there were places where there was no basal lamina, and in which interstitial cells were in direct contact with sustentacular cells by cell membrane apposition and patches of zonulae adherens (fig. 16). On the basis of examination of transverse and sagittal sections (figs. 2, 4 ) , and the fact that the testicular cords did not contain blood vessels, the interstitium was concluded to be a continuous tissue compartment that occupied the space surrounding the anastomosing sheets of the testicular cords. Hence the interstitium was more cord-like than the testicular cords themselves at this stage of development. The interstitium was composed of small, irregularly shaped, undifferentiated interstitial cells and blood capillaries (figs. 2, 4, 5, 13, 15). Neither primordial germ cells nor other cell types were seen. Both cell shape and nuclear shape of the undifferentiated interstitial cells vaned greatly (fig. 15). The chromatin was finely dispersed and the nucleoli, usually one or two

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per cell, were small and often attached to the nuclear envelope. The mitochondria were rod-shaped with lamellar transverse or tubular cristae. The Golgi complex was usually small. The cisternae of the granular endoplasmic reticulum occurred singly and often associated with mitochondria. Free polysomes were present abundantly i n the cytoplasm. Filaments of 8 n m in diameter and coated vesicles, similar to those in the sustentacular cells, were also noted in the interstitial cells. The intercellular space was generally large and the cells contacted each other by parallel apposition of the cell membranes and by short contacts of the tips of cytoplasmic processes (fig. 15). The interstitial cells were similar to each other ultrastructurally and no agranular endoplasmic reticulum, a sign of the early differentiation of future Leydig cells, could be seen. Mesenchyme. The mesenchymal tissue was located in the basal part of the gonad, lateral to the testicular cords or ovarian blastema proper, respectively, and i n the mesogonadium it extended to the medial border of the capsules of the mesonephric glomeruli (figs. 1, 4 ) . The shapes of the mesenchymal cells varied and the intercellular space was often large (fig. 17). The mesenchyme was denser than the testicular interstitium. Ultrastructurally, the mesenchymal cells were similar in both sexes and resembled the undifferentiated interstitial cells. The presence of single primordial germ cells in the ovarian and testicular mesenchyme was a striking difference when compared to the interstitium (fig. 17). Even though there were direct connections between the mesenchymal and interstitial compartments, germ cells had not entered the interstitial spaces between the testicular cords. The primordial germ cells were ultrastructurally similar to those in the primitive cords, and no sexual differences or signs of degeneration of the primordial germ cells were noted. Areas of membrane apposition and cytoplasmic processes connected the mesenchymal cells to the primordial germ cells. Patches of zonulae adherens were also seen. Blood vessels. The vasculature of the ovary and testis consisted of capillaries of varying diameter. They were located in the mesenchyme and blastema proper in the

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ovary, and i n the mesenchyme and interstitium in the testis (figs. 3, 4). Surface epithelium, primitive cords and testicular cords were devoid of vasculature. I n both sexes the most peripheral capillaries were located about 80 pm below the gonadal surface. In the testis they marked the border of the blastema proper that had differentiated into testicular cords and interstitium. At times the capillary endothelial cells had basal lamina-like material on their outer surface (fig. 15). The ovarian blastema cells were usually in close contact with the endothelial cells, whereas in the testis the interstitial cells were usually separated from the capillaries by a n extracellular space of varying width (figs. 5, 15). The mesenchymal capillaries of both sexes were similar to those in the testicular interstitium. DISCUSSION

I n the present study the testis could be distinguished at a n earlier stage of embryonic development (26-days and 16 m m ) than reported by other investigators. According to Allen ('04) differentiation is not yet clearly established at the length of 18 mm, but in 25 m m pig embryos the difference is striking. Whitehead ('04) described a testis in a 22 m m pig embryo, and in later papers the earliest stages of gonadal sex differentiation by age and length were 20 mm (Griinwald, '42), 27 days (Black and Erickson, '65), 35 days (Gier and Marion, '70), 30 days and 25 m m (Moon and Hardy, '73). I n most of the aforementioned papers, the tunica albuginea was the sex specific feature, whereas in the present study the testicular cords were found to differentiate earlier than the tunica albuginea. This difference is apparently explained by better fixation in glutaraldehyde and osmium, and by Epon embedding, both of which permit a section thickness of only 1 El.mor less for light microscopy. Therefore, the contorted testicular cords are seen clearly because they do not overlap each other, as in thick conventional sections. The tunica albuginea can be seen even in thick sections because it consists of a continuous layer of distinctively shaped cells. The tissue and cell types of the early ovary and testis are essentially identical in the pig and

other mammals, including man (Gier and Marion, '70). The description by Allen ('04) of the gonadal surface epithelium is the only one available for comparison with the present results. His drawings show basically the same morphology as described here in both sexes. The elongation of occasional epithelial cells in a deeper layer parallel with the gonadal surface in the testes of the present embryos is regarded as a sign of early development of the tunica albuginea. The subepithelial layer of the embryonic gonad in both sexes, as presented by M e n ('04), is comparable to the area of the primitive cords as described i n the present study. Contradictory views about the relationships between the surface epithelium and the primitive cords have been presented in earlier studies, as reviewed by Jost et al. ('73). The direct continuity of these two tissues and their basal laminae was demonstrated ultrastructurally in the present study. In this respect the pig ovary is different from that of the mouse, where no cord-like formations were observed (Odor and Blandau, '69a). In the pig a striking feature of this peripheral layer of gonadal blastema is its invariable structure from the early stages of gonadal ridge to the stage of the early ovary and testis (Pelliniemi, '75a,b). In contrast to the testis, the central region of the ovarian gonadal blastema has been studied less. It has been described as cords in the pig (Allen, '04), as an intermixture of germ and somatic cells in the mouse (Odor and Blandau, '69a) and as a blastema sending strands towards the periphery in cattle (Ohno, '67). Medullary cords resembling distinct testicular cords (Allen, '04) were not seen in the ovaries of the present study. This difference may be explained by the fact that the 25 mm embryos employed in Allen's study were at a later developmental stage than the 22 mm embryos in the present material. The same explanation is probably also applicable to cattle, although no exact stages were given. I n the present study the cordlike cell groups in the ovarian blastema could be a sign of early cord formation. In contrast to the mouse (Odor and Blandau, '69a) and the rat (Eddy, '74), germ cells in the pig ovary were not arranged in

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groups at the time when testicular differ- clear outline, numerous polysomes, rodentiation was noted. The ultrastructure of like mitochondria and solitary cisternae of both germ cells and somatic cells in the granular endoplasmic reticulum. The presmouse is essentially similar to that i n the ent study reveals the same features in pig pig. The cellular organization of gonadal sustentacular cells. These structures are blastema i n the 27-day pig ovary was already present in columnar blastema cells similar to that of the 24-day indifferent in the cord-like portions of the indifferent gonad i n both sexes; hence, it is still called pig gonad at the age of 24 days (Pelliniemi, by the same name (Pelliniemi, '75b). The '75b). Also the contacts with adjacent cells results indicate that in the pig sex-specific and the basal laminae are similar at both ovarian organogenesis of the somatic as ages. This indicates that the organization well as of the germinal components had of cell groups, but not the internal strucnot yet begun by 27 days. This is in agree- ture of individual cells, is altered during ment with earlier studies (Peters, '70), sexual differentiation of the testis. where the beginning of oogenesis in the The material from which basal lamina pig occurred at 30 days. is formed is thought to be synthesized in Pig testicular cords have been described the granular endoplasmic reticulum and in by earlier light microscopists (Allen, '04; the Golgi complex of the epithelial cells Whitehead, '04; Griinwald, '42; Moon and (Pierce, '70). These structural componHardy, '73) and i n man their structure ents are present in the sustentacular cells has proved to be essentially similar (Gill- and also in other cell types i n the present man, '48; Wagenen and Simpson, '65; study. The basal lamina of the testicular Pelliniemi and Niemi, '69; Jirasek, '71; cords is not yet continuous, indicating that Vossmeyer, '71 ). A stereological examina- its formation has not yet been completed. tion of the early human testis has shown This view is supported by a similar, gradthat in reality the cords are sheets (Elias, ual appearance of the basal lamina at var'71). This interpretation is in agreement ious sites in the differentiation of the renal with the present results obtained from metanephric vesicles (Jokelainen, '63) and transverse and sagittal sections of the 26- in tubulogenesis from metanephric mesenday pig embryos. The mechanism by which chyme (Wartiovaara,'66). the cords are formed from sheets has not Spermatogonia in the rabbit had nuclei been studied. The small area of the indif- and nucleoli similar to those of pig cells, ferent blastema in the center of the testis and the cytoplasm contained the same at the age of 26 days apparently repre- types of organelles, with the exception of sented the region of the future rete testis some elongate mitochondria (Gondos and (Grunwald, '34). The sustentacular cells Conner, '73). Phagocytosis of degenerating seem to be derivatives of blastema cells cells was also noted in the rabbit. The from the previous stage (Pelliniemi, '75b). aggregates of chromatin-like material obIn man it is not possible to distinguish served in some germ cells of both sexes in whether the blastemal cells have differen- the present study resemble those described tiated from mesenchymal or epithelial in germ cells of the rat (Eddy, '74). Their components (Jirasek, '71) and this is also reported association with mitochondria, true in the pig. Therefore, the problem of was, however, not seen. determining whether the origin of the susThe structure of the interstitium in the tentacular cells is epithelial or mesenchy- pig testis has been very accurately dema1 cannot be solved by the present study, scribed by Whitehead ('04) in 22 mm emeven though ultrastructurally the susten- bryos. The tissue was similar to the mesentacular cells resemble the surface epithe- chyme, as was also evident from the lial cells more than the mesenchymal cells. present study. His interpretation of it as a Characteristic features of sustentacular syncytial structure is a n understandable cells in the testicular cords in the guinea error owing to the low resolution of the pig (Black and Christensen, '69), in the light microscope. In his material the Leydig rabbit (Gondos and Conner, '73; Bjerre- cells first appeared in 24 mm embryos and gaard et al., '74) and in the hamster hence their absence i n 22 m m embryos (Gondos et al., '74) are a n irregular nu- matches well the present results with em-

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bryos of the same length. The undifferentiated interstitial cells in the human ( Pelliniemi and Niemi, '69 ) , the guinea pig (Black and Christensen, '69) and the hamster (Gondos et al., '74) show an ultrastructure similar to the corresponding pig cells, With the electron microscope the interstitial cells were not distinguishable from the mesenchymal cells located in the basal part of the gonad. The interstitial cells, which have differentiated from blastema cells, may have either a mesenchyma1 or an epithelial origin as previously mentioned in regard to sustentacular cells. Usually they are regarded as derivatives of mesenchymal cells (Black and Christensen, '69) and their structural resemblance is a strong argument for this. However, the direct contacts of the interstitial cells and the sustentacular cells in the present study are consistent with a common origin of the sustentacular and interstitial cells. It might also be interpreted a s resulting from a migration of cord cells into the interstitium, or vice versa, providing a new possibility regarding the origin of Leydig cells. The absence of primordial germ cells in the interstitium indicates that there is some selective mechanism by which the primordial germ cells accept only cord cells, sustentacular cells and mesenchyma1 cells a s their environment, because histologically there are open channels for migration into the interstitium as well. The topographic organization of the mesenchyme had changed greatly from the situation in 21-24 mm embryos, through morphogenetic changes in the gonads in both the male and female (Pelliniemi, '75a,b). The mesenchymal cells had, however, remained structurally unaltered. A very low activity of A5-3p-hydroxysteroid dehydrogenase was shown histochemically by Moon and Raeside ('72) in 20 mm pig fetuses of undetermined sex, and a high activity was found in the testes of 25 mm fetuses. In both cases the reactions appeared to be confined to the cords. The activity of 17p-hydroxysteroid dehydrogenase was very low at the former and not detectable at the latter stage. The activities of these enzymes in the pig were localized to the cords and not the interstitium. In the present study there was no agranular endoplasmic reticulum in the susten-

tacular cells in the pig a t 27 days. This organelle appears in the interstitial and Sertoli cells of fetal guinea pigs at a slightly more advanced stage (Black and Christensen, '69). The cordal localization of hydroxysteroid dehydrogenases in the pig and the occurrence of agranular endoplasmic reticulum in the guinea pig are consistent with the aforementioned assumption that Leydig cells derive from cord cells, based on the close contacts between interstitial and sustentacular cells seen in the present study. The histochemical results suggest that there is a possibility of androgen synthesis in the developing gonad well before agranular endoplasmic reticulum, the ultrastructural hallmark of steroid-synthesizing cells (Christensen, '74), is observed i n gonadal cells. Organ culture of fetal pig gonads at the age of 26 and 30 days indicated secretion of androgens from the testis but not from the ovary (Moon and Hardy, '73; Moon et al., '73). From previous histochemical and culture studies, and from the present ultrastructural studies, it can be concluded that the early pig testis is capable of androgen synthesis even if no typical steroidsecreting cells can be observed with the electron microscope. Whether these reactions also occur in vivo remains to be clarified by other methods. The studies of gonadal sex differentiation available at present are at the light microscope level. In the human male gonad the process is started by the formation of testicular cords, with subsequent differentiation of the tunica albuginea and appearance of Leydig cells in the interstitium (Gillman, '48; Wagenen and Simpson, '65; Jirasek, '71). In the pig the earliest sign of testicular differentiation was the tunica albuginea (Black and Erickson, '65; Moon and Hardy, '73). The cords, however, appeared before the Leydig cells (Whitehead, '04). A general scheme for the differentiation process based on cattle has been presented by Ohno ('67). The indifferent gonad consists of a central somatic blastema and peripheral primordial germ cells. Differentiation is achieved by rearrangement and growth of these basic components. The earliest phases of testicular differentiation in the pig can now be described on the basis of the present and

ULTRASTRUCTURE OF EARLY OVARY AND TESTIS

previous study (Pelliniemi, '75b). The common indifferent blastema (Ohno, '67; Jirasek, '71 ), containing scattered primordial germ cells, is present in the pig gonad at the age of 24 days. Differentiation begins with the formation of testicular cords and the interstitium from the central region of the blastemal tissue. The primordial germ cells become enclosed in the cords, and blood capillaries appear in the interstitium by the age of 26 days. The newly formed testicular cords are gradually bound by a basal lamina. The peripheral portion of the gonadal blastema remains unchanged as primitive cords. The first signs of differentiation of the tunica albuginea in the surface epithelium also become evident by the twenty-seventh day. The interstitial cells of Leydig do not appear at this earliest stage of the morphologically identifiable testis. During this period (24-27 days) the female gonad remains devoid of any morphological differentiation. Attempts have been made to unite the genetic regulatory factors of the histological differentiation of gonadal sex, their location and mode of function in the chromosomes, and the time and the place of their action into a general model (Ohno, '67; Hamerton, '68; Boczkowski, '71, '73; Short, '72). It seems evident that the primordial germ cells do not play an active role in the differentiation, as normal testicular structures can develop even in the absence of primordial germ cells (Ohno, '67). The distribution of the primordial germ cells in all compartments of the gonad in the pig further supports their passive role. In the male the sex-determining factors are apparently first activated in the somatic blastemal cells of the indifferent gonad, as the morphological changes, including establishment of specific cell contacts and simultaneous formation of a basal lamina, are occurring in these cells. The present results indicate that the differentiation of an indifferent gonad into an identifiable testis proceeds without any cells which resemble ultrastructurally mature, differentiated Leydig cells (Belt and Cavazos, '67). This supports the view that Leydig cells and androgens (Elger et al., '71) have no role in the early formation of testicular cords. The formation of the basal

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lamina is not yet completed when the testicular cords have already separated from the interstitium. Therefore, the beginning of synthesis of the basal lamina does not seem to be an initiator of cord formation. Furthermore, small areas of cords with a basal lamina were already present in the indifferent gonadal blastemas of both sexes. An interesting feature that has not been described previously is the overlapping of the peripheral borders of the vascular bed and the area of the testicular cords. Differentiation begins in the vascular periphery and the central cords are formed last. In other words, the interstitium develops only in the area which also contains capillaries. The histological differentiation of the testicular cords can now be presented on the basis of a new hypothesis in which the interstitial tissue is regarded as the active part. The blastemal cells adjacent to the capillaries are presumed to differentiate into interstitial cells and anastomosing channels of loose interstitial tissue form along the capillaries. The rest of the blastema is thought to remain as anastomosing sheets of compact tissue, known thereafter as the testicular cords. It is not yet possible to decide which of the two tissues, the interstitium or the testicular cords, is the primary organizer in testicular morphogenesis. The present observations, however, indicate that the role of the vasculature in the regulation of the gonadal sex differentiation might deserve more attention. ACKNOWLEDGMENTS

I am indebted to professor M. Niemi, M.D., for the initiation of this study and to associate professors P. T. Jokelainen, M.D., and T. Nevalainen, M.D., for their valuable suggestions in the preparation of the manuscript. The helpful cooperation of Anna-Liisa Salonius, M.Sc., in the cytogenetic part of the study is sincerely acknowledged. I thank Ms. M. Aaltonen, Mrs. S. From, Mrs. A. Ketola, Ms. U. Mantyla, Mr. M. Lehtimaki and Mr. U. Reunanen for skillful technical assistance in the laboratory and office work. This study was supported by grants from the University of Turku and from the Finnish Medical Foundation.

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LITERATURE CITIED Allen, B. M. 1904 The embryonic development of the ovary and testis of the mammals. Am. J. Anat., 3: 89-146. Belt, W. D., and L. F. Cavazos 1967 Fine structure of the interstitial cells of Leydig in the boar. Anat. Rec., 158: 333-350. Bjerregaard, P., F. Bro-Rasmussen and T. Reumert 1974 Ultrastructural development of fetal rabbit testis. 2. Zellforsch., 147: 401-413. Black, J. L., and B. H. Erickson 1965 Oogenesis and ovarian development in the prenatal pig. Anat. Rec., 161: 45-56. Black, V. H., and A. K. Christensen 1969 Differentiation of interstitial cells and sertoli cells in fetal guinea pig testes. Am. J. Anat., 124: 2 11-238. Boczkowski, K. 1971 Sex determination and gonadal differentiation in man. A unifying concept of normal and abnormal sex development. Clin. Genetics, 2: 379-386. 1973 Male and female differentiation of the human gonad. Clin. Genetics, 4: 213-219. Book, S. A,, and L. K. Bustad 1974 The fetal and neonatal pig in biomedical research. J. Anim. Sci., 38: 997-1002. Christensen, A. K. 1974 Leydig cells. In: Handbook of Physiology. Sect. 7, Vol. 4. s. R. Geiger, ed. Amer. Physiol. SOC., Washington, D. C., in press. Eddy, E. M. 1974 Fine structural observations on the form and distribution of nuage in germ cells of the rat. Anat. Rec., 178: 731-757. Elger, W., F. Neuman and R. von BerswordtWallrabe 1971 The influence of androgen anatagonists and progestogens on the sex differentiation of different mammalian species. In: Hormones in Development. M. Hamburgh, and E. J. W. Barrington, eds. Appleton-CenturyCrofts, New York, pp. 651-667. Elias, H. 1971 Development of the human testis, stereologically examined. Anat. Rec., 169: 310. Gier, H. T., and G. B. Marion 1970 Development of the mammalian testis. In: The Testis. A. D. Johnson, W. R. Gomes and N. L. Vandemark, eds. Academic Press, New York, pp. 1 4 5 . Gildman, J. 1948 The development of the gonads in man; with a consideration of the role of the fetal endocrines and the histogenesis of ovarian tumors. Contr. Embryol. Carneg. Instn., 32: 81-131. Gondos, B., and L. A. Conner 1973 Ultrastructure of developing germ cells in the fetal rabbit testis. Am. J. Anat., 136: 23-42. Gondos, B., D. C. Paup, J. Ross and R. A. Gorski 1974 Ultrastructural differentiation of Leydig cells in the fetal and postnatal hamster testis. Anat. Rec., 178: 551-565. Griinwald, P. 1934 Uber Form und Verlauf der Keimstrkge bei Embryonen der Saugetiere und des Menschen. I Die Keimstrange des Hodens. Z. Anat. Entwick1.-Gesch., 103: 1-19. Griinwald, P. 1942 The development of the sex cords i n the gonads of man and mammals. Am. J. Anat., 70: 359-397. Hanerton, J. L. 1968 Significance of sex chro-

mosome derived heterochromatin in mammals. Nature (London), 219: 910-914. Holstein, A. F., H. Wartenberg and J. Vossmeyer 1971 Zur Cytologie der pranatalen Gonadenentwicklung beim Menschen. 111. Die Entwicklung der Leydigzellen im Hoden von Embryonen und Feten. Z. Anat. Entwick1.-Gesch., 135: 43-66. Jirasek, J. E. 1971 Development of the Genital System and Male Pseudohermaphroditism. The Johns Hopkins Press, Baltimore and London. Jokelainen, P. T. 1963 A n electron microscope study of the early development of rat metanephric nephron. Acta anat. (Basel), Suppl. 47: 1-71 + 17 plates. Jost, A., B. Vigier, J. Prbpin and J. P. Perchellet 1973 Studies on sex differentiation in mammals. Recent Progr. Hormone Res., 29: 1-41. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol., 9: 409-414. McKay, D. G . , A. T. Hertig, E. C. Adams and S. Danziger 1953 Histochemical observations on the germ cells of human embryos. Anat. Rec., 117: 201-219. Moon, Y. S., and M. H. Hardy 1973 The early differentiation of testis and interstitial cells i n the fetal pig and its duplication in organ culture. Am. J. Anat., 138: 253-268. Moon, Y. S., M. H. Hardy and J. I. Raeside 1973 Biological evidence for androgen secretion by the early fetal pig testes i n organ culture. Biol. Reprod., 9: 330-337. Moon, Y. S., and J. I. Raeside 1972 Histochemical studies on hydroxysteroid dehydrogenase activity of fetal pig testes. Biol. Reprod., 7: 278287. Odor, D. L., and R. J. Blandau 1969a Ultrastructural studies on fetal and early postnatal mouse ovaries. I. Histogenesis and organogenesis. Am. J. Anat., 124: 163-186. Odor, D. L., and R. J. Blandau 1969b Ultrastructural studies o n fetal and early postnatal mouse ovaries. 11. Cytodifferentiation. Am. J . Anat., 125: 177-216. Ohno, S. 1967 Sex Chromosomes and Sexlinked Genes. Springer-Verlag, Berlin. Pelliniemi, L. J. 1975a Ultrastructure of gonadal ridge in male and female pig embryos. Anat. Embryol., 147: 19-34. 197513 Ultrastructure of indifferent gonad in male and female pig embryos. To be published. Pelliniemi, L. J., and M. Niemi 1969 Fine structure of the human foetal testis. I. The interstitial tissue. Z. Zellforsch., 99: 507-522. Pelliniemi, L. J., and A-L. Salonius 1975 Identification of the chromosomal sex in pig embryos at indifferent gonadal stages. To be published. Peters, H. 1970 Migration of gonocytes into the mammalian gonad and their differentiation. Phil. Trans. B, 259: 91-101. Pierce, G. B. 1970 Epithelial basement membrane: Origin, development and role in disease. In: Chemistry and Molecular Biology of the Intercellular Matrix. E. A. Balazs, ed. Academic Press, London and New York, pp. 471-506. Pinkerton, J. H. M., D. C. McKay, E. C. Adams

ULTKASTRUCTURE OF EARLY OVARY AND TESTIS and A. T. Hertig 1961 Development of the human ovary. A study using histochemical technics. Obst. and Gynec., 18: 152-181. Short, R. V. 1972 Germ cell sex: In: Proc. Int. Symp. the Genetics of the Spermatozoon. R. A. Beatty and S. Gluecksohn-Waelsch, eds. Departments of Genetics of the University of Edinburgh and of the Albert Einstein College of Medicine, pp. 325-345. Stempak, J. G., and R. T. Ward 1964 A n improved staining method for electron microscopy. J. Cell Biol., 22: 697-701. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Vossmeyer, J. 1971 Zur Cytologie der priinatalen Gonaden-Entwicklung beim Menschen. I. Histogenese des Hodens, an Eponschnitten untersucht. Z . Anat. Entwick1.-Gesch., 134: 146-164. Wagenen, G. van., and M. E. Simpson 1965

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Embryology of the Ovary and Testis, Homo sapiens and Macaca mulatta. Yale University Press, New Haven. Warterberg, H., A-F. Holstein and J. Vossmeyer 1971 Zur Cytologie der pranatalen GonadenEntwicklung beim Menschen. 11. Elektronenmikroskopische Untersuchunger iiber die Cytogenese von Gonocyten und fetalen Spermatogonien im Hoden. 2. Anat. Entwickl.-Gesch., 134: 165-185. Wartiovaara, J. 1966 Cell contacts in relation to cytodifferentiation in metanephrogenic mesenchyme in vitro. Ann. Med. exp. Fenn., 44: 1-35.

Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol., 4: 475478. Whitehead, R. H. 1904 The embryonic development of the interstitial cells of Leydig. Am. J. Anat., 3 : 167-182.

PLATE 1 EXPLANATION OF FIGURES

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1

Age 27 days. A survey light micrograph of a transverse paraffin section of a pig embryo at the abdominal levels. The arrows point to the outer surface of the embryo. A, aorta; DM, mesonephric duct; GD, gonad; GL, glomerulus; H, liver; ME, mesentery and gut; MN, mesonephros; V, gall bladder primordium. X 17.5.

2

Testis, age 27 days. A light micrograph of a sagittal Epon section. The longit u d k a l axis of the testis is vertical in the picture. The section plane is perpendicular to the plane in figure 4, where the direction is indicated by the arrow. IS, interstitium; L, coelomic cavity; PC, primitive cords; SE, surface epithelium; TC, testicular cords. x 120.

3

Ovary, age 27 days. A light micrograph of a transverse section. The whole area of the gonadal blastema (GB) is still undifferentiated. Note the cross sections of large capillaries (arrowheads) at a relatively constant distance from the surface. The mark for the mesenchyme (MS) is in the mesovarium. GA, glomerular capsule; L, coelomic cavity; PC, primitive cords; SE, surface epithelium; U , urinary space. x 120.

4

Testis, age 26 days. A light micrograph of a transverse section. The characteristic testicular cords (TC) and interstitium ( I S ) are seen in the central region. Note the cross sections of large capillaries (arrowheads) at a relatively constant distance from the surface. The arrow indicates the direction of the sagittal section i n figure 2. The mark for the mesenchyme (MS) is in the mesotestis. A n area of undifferentiated gonadal blastema (GB) is seen in the center of the testis. GA, glomerular capsule; GL, glomerulus; L, coelomic cavity; PC, primitive cords; SE, surface epithelium. x 120.

5

Testis, age 26 days. A light micrograph of a transverse section showing the surface epithelium (SE), primitive cords (PC), testicular cords (TC) and interstitium ( I S ) . CA, capillary; L, coelomic cavity; PG, primordial germ cell. x 600.

ULTRASTRUCTURE OF EARLY OVARY AND TESTIS Lauri J. Pelliniemi

PLATE 1

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PLATE 2 EXPLANATION OF FIGURES

102

6

Testis, age 27 days. A n electron micrograph of surface epithelium. A primitive cord with a primordial germ cell (PG) is continuous with the epithelium between the asterisks. The inset shows a junctional complex at the luminal borders of two surface epthelial cells. B, basal lamina; D, desmosome; E, surface epithelial cell; F, cytoplasmic filaments; G, Golgi complex; GR, granular endoplasmic reticulum; J, junctional complex; L, coelomic cavity; M, mitochondrion; S, surface folds; ZA, zonula adherens. x 4,275; inset x 49,400.

7

Testis, age 27 days. The surface epithelium in an area adjacent to the mesotestis consists of single layer of columnar cells ( E ) . B, basal lamina; G, Golgi complex; GR, granular endoplasmic reticulum; J. junctional complex; L, coelomic cavity; LI, lipid droplet; M, mitochondrion. X 6,080.

ULTRASTRUCTURE OF EARLY OVARY AND TESTIS Lauri J. Pelliniemi

PLATE 2

PLATE 3 EXPLANATION O F FIGURES

8

104

Ovary, age 27 days. Portions of two primordial germ cells ( P G ) in the gonadal blastema. The inset shows endoplasmic membranes arranged as concentric circular layers (arrow) in a blastemal cell. BC, blastemal cell; G, Golgi complex; GR, granular endoplasmic reticulum; LI, lipid droplets; M, mitochondrion; Z, zonula adherens. x 10,450; inset x 10,450.

ULTRASTRUCTURE OF EARLY OVARY AND TESTIS Lauri J. Pelliniemi

PLATE 3

PLATE 4 EXPLANATION O F FlGURES

9

Ovary, age 27 days. Two cells of the gonadal blastema ( B C ) in a cord. B, basal lamina; CP, cytoplasmic processes; G, Golgi complex; GR, granular endoplasmic reticulum; M, mitochondrion; Z, zonula adherens. x 6,080.

10

Ovary, age 27 days. Two cord cells containing phagosomes ( F S ) and located below the base of a columnar surface epithelial cell ( E ) . CP, cytoplasm; GR, granular endoplasmic reticulum; LI, lipid droplet; M, mitochondrion; N, nucleus. x 5,225.

11 Testis, age 27 days. A spermatogonium (PG) in a testicular cord. DB, dense bodies; G, Golgi complex; GR, granular endoplasmic reticulum; M, mitochondrion; S, surface folds; SC, sustentacular cell; Z zonula adherens. X 7,410.

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ULTRASTRUCTURE OF EARLY OVARY AND TESTIS

Lauri J. Pelliniemi

PLATE 4

PLATE 5 EXPLANATION OF FIGURES

12 Testis, age 27 days. A n aggregate of chromatin-like material (arrow) in the vicinity of the nucleus in a spermatogonium. M, mitochondrion; N, nucleus. x 22,800. 13 Testis, age 27 days. A survey picture of testicular cords (TC) surrounding interstitial areas ( I S ) . B, basal lamina; GR, granular endoplasmic reticulum; IC, interstitial cell; M, mitochondrion; PG, spermatogonium; SC, sustentacular cell. X 2,660. 14 Testis, age 27 days. A testicular cord between interstitial areas ( I S ) . B, basal lamina; G, Golgi complex; GR, granular endoplasmic reticulum; M, mitochondrion. X 5,700.

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ULTRASTRUCTURE OF EARLY OVARY AND TESTIS Lauri J. Pelliniemi

PLATE 5

PLATE 6 EXPLANATION OF FIGURES

15 Testis, age 27 days. T w o interstitial cells ( I C ) on the left and one capillary endothelial cell on the right. B, basal lamina; CA, capillary lumen; CV, coated vesicles; F, cytoplasmic filaments; G, Golgi complex; GR, granular endoplasmic reticulum; M, mitochondrion. x 7,600. 16

Testis, age 27 days. A direct contact between an interstitial cell ( I C ) and a sustentacular cell (SC). B, basal lamina; F, cytoplasmic filaments; M, mitochondrion; TC, testicular cord; Z, zonula adherens. X 12,350.

17 Testis, age 27 days. Mesenchymal tissue in the mesotestis with a primordial germ cell (PG). MC, mesenchymal cell. X 3,040.

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ULTRASTRUCTURE OF EARLY OVARY A N D TESTIS Lauri J. Pelliniemi

PLATE 6