embryonic development of the porcine indifferent gonad and testis

J. Vet. Med. A 38, 594-607 (1991) 0 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1 - 184X

From the Department of Anatomy and Histology and of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences,

Uppsala, Sweden

Embryonic Development of the Porcine Indifferent Gonad and Testis

M. HuRsT>, A.-M. DALIN and H. RODRIGUEZ-MARTINEZ

Address of authors: Dr. M. HURST, Department of Pathology, Dr. A,-M. DALIN, Department of Obstetrics and Gynaecology

and Dr. H. Rodriguez-Martinez, Department of Anatomy and Histology, Swedish University of Agricultural Sciences, P. 0. Box 7028, S-750 07 Uppsala Sweden

With 8figures

(Received for publication September IS, 1990)

Summary The early gonadal development in the pig from day 18 to day 36 post conception, when distinct

testes were present in male embryos, has been studied. The development of the porcine gonad followed the general mammalian pattern. During testicular differentiation, the germ cells and the relevant somatic cells, the Sertoli cells, became enclosed in testicular cords, thus creating an intracordal germ cell compartment and an extracordal compartment. The development of conspicuous cell junctions and the production of a basal lamina were evident in the Sertoli cells during the early testis development. Their origin in this species remains unclear.

Introduction The mammalian embryonic gonads develop on the ventro-medial surface of the

mesonephric ridges (Rev. by BYSKOV, 1986). The gonads are formed as a result of very specific interactions between cells of different origins. The primordial germ cells arise outside the presumptive gonads (WITSCHI, 1948) and migrate to the mesonephric ridges via the mesentery (CHIQUOINE, 1953). The embryonic origin of the gonadal somatic cells is not entirely clear, but three different origins have been suggested, i. e. the mesonephros, the coelomic epithelium covering the mesonephric ridge and rnesenchymal cells resident in the ridge (NIEMI et a]., 1967; WARTENBERG, 1978; UPADHYAY et al., 1981).

Fig. 1 A-E. Light micrographs of the developing gonadal ridges of pig embryos at 18-24 days post conception (p. c.). In A (10 X) a transverse section of a pig embryo at 18 days p. c. showing the position of the gonadal ridges on the ventromedial surface of the mesonephri (long arrows) (G: mesonephric glomerulus, T: mesonephric tubuli, Me: mesentery, NT: neurtal tube, A: aorta, M: mesenchyme). In

::- To whom correspondence should be addressed.

U.S. Copyright Clraranrc Crnter Code Statement: 093 1 - 184X/91/3808 - 0594$02.50/0

Early development of pig gonads 595

Figures B--E (100 x), transverse sections of the developing gonadal ridge at 18,21,22 and 23 days p. c. respectively. Note the gradually increasing size of the ridge caused by the growing gonadal blastema (GB). The irregular surface epithelium (SE) gives rise to increasing numbers of cellular cords (C), which intermingle with the subepithelial tissue. Some epithelial cells form radiate patterns (short

arrows). Note also the presence of germ cells (arrowheads). HE

596 HURST. DALIN and RODRIGUEZ-MARTINEZ

The exat r61e of the mesonephros in gonadal development is yet unclear. Some authors claim that mesonephric epithelial cells leave the mesonephros, migrate to the presumtive gonadal ridge and there differentiate into gonadal somatic cells (ZAMBONI et al., 1979; UPADHYAY et al., 1981; WARTENBERG, 1981). MERCHANT-LARIOS (1979), however, claims that no conclusive evidence for a mesonephric origin of the somatic cells has yet been provided. The somatic cells interact very early with the primordial germ cells, presumably as a part of gonadal differentiation. There are, however, reports suggesting that the presence of germ cells is not necessary for the early development of a morphologically normal gonad (MERCHANT-LARIOS, 1976).

The development of the porcine gonads seems to follow the general mammalian pattern. The mesonephros of the pig is very large and functional throughout embryonic life (TIEDEMANN, 1979), reaching maximal size (9 mm) at a fetal length of 50 mm. In the pig, the pre- and postnatal Sertoli cells have a clear cytoplasmic carbonic anhydrase activity, which makes it possible to trace them by histochemical methods ( R I D D E R S T ~ L E et a]., 1985; RODRIGUEZ-MARTINEZ et al., 1990). PELLINIEMI (1975) described the ultrastructure of gonads from pig embryos aged between 21 and 27 days (post conception), but no evidence of the origin of the somatic cells was provided. Here, we studied the morphology of the gonads from pig embryos aged between 18 and 36 days (post conception), with special attention paid to the somatic-germ cell relationships in the differentiating testes.

Material and Methods A total of eleven Landrace x Yorkshire multiparous sows were mated mice during a spontane-

ous oestrus and slaughtered at 18, 21, 22, 23, 24, 25, 27, 28 or 36 days after the last mating. After slaughter and removal of the genital organs, the uterine horns were longitudinally opened

and the embryos with their extra-embryonic membranes were collected. The embryos were dissected free from the membranes and fixed by immersion in Karnowsky's fixative (KARNOWSKY, 1965). Thereafter, the gonadal ridgedgonads and the neighbouring mesonephri of embryos selected from each sow were dissected under a stereomicroscope.

The trimmed tissues were dehydrated and embedded in water soluble methylmetacrylate (Historesin@ LKB, Sweden). Semithin sections (1 pm) were obtained with a microtome (Historange@ LKB, Sweden), and stained with haematoxylin-eosin (HE). After confirmation of proper dissection, embryonic gonadal tissue was removed in the same way for electron microscopy. Small pieces of tissue were washed with buffer, poststained with a 2 % Os04 soluton for one hour, dehydrated and embedded in Agar-lOO@ plastic resin. Semithin (1 pm) sections were cut with an Ultratome@ (LKB, Sweden) and stained with buffered toluidine blue for selection of further thin sectioning. Thin sections were then cut, picked upon formvar film-coated copper grids, counterstained with uranyl acetate and lead citrate, and examined in a Philips 201 EM electron microscope at 60 kV.

Results Development of the gonadal ridge

In embryos at 18 days post conception (p. c.) the gonadal ridges were just beginning to form on the ventromedial surfaces of the mesonephri (fig. 1 A). the surface epithelium in these parts was taller and more irregular than the coelomic epithelium covering neighbouring structures. It consisted of a mixture of cylindrical cells in a single layer, frequently forming radiate patterns, and cuboidal cells in 1-3 layers. Cellular cords, seemingly formed by the epithelial cells, were intermingled with the underlying mesenchyme (Figs. 1 B and 2 A). Scattered germ cells were found in the epithelium and in the mesenchyme.

The gonadal ridges gradually developed in size, so that at 23 days p.c. they were clearly protruding into the coelomic cavity (Figs. 1 C-E). During this period the gonadal ridges consisted of the surface epithelium and a cellular mass, the gonadal blastema, located between the surface epithelium and the mesenchyme at the base. The gonadal blastema was composed mainly of irregular cellular cords, cells that were morphologically similar to the cord cells, but seemingly not organized in cord like structures, and mesenchymal cells. Via the basal mesenchyme the gonadal ridges were connected with the mesonephri. The surface


A

Fig.2A-D. Electron micrographs of the porcine gonadal ridge at 18 (A, 1 2 0 0 ~ ) , 21 (B, 2800x), 22 (C, 1200 X) and 23 (D, 1500 X) days p. c. showing the surface epithelium (SE) (Figs. A and C; bl: basal lamina) and germ cells (Fig. A, C: short arrows) just under the epithelium. Note the cellular cord formation (C) adjoining the germ cells. In B, a germ cell in detail with its typical rounded shape, a central nucleus (n) containing two prominent nucleoli (nu); (m: mitochondria). Surface epithelial cells are during this period seen migrating into the underlying tissue (Fig. C: arrows, *: coelomic cavity) forming a cellular cord. At 23 days cellular cords are found deep in the gonadal blastema (Fig. D) and

are often, like this one, devoid of germ cells

epithelium became more irregular with a more marked tendency to form radiate patterns, and the formation of cellular cords increased (Figs.2A, C). A basal lamina was present underneath the surface epithelium, but it was interrupted in the places where the cords appeared to be formed. At both light microscopic and ultrastructural levels, the surface


epithelial cells and the cord cells were similar. They had lightly stained nuclei with somewhat varying shape, usually oval or elongated with one or two nucleoli. Prominent features of the cytoplasm were rodshaped mitochondria, some cisternae of rough endoplasmic reticulum and polysomes. Germ cells were present in the surface epithelium as well as in the blastema and in the underlying mesenchyme. The irregular cords were usually not incorporating germ cells (Fig. 2 D), although cord cells sometimes appeared to, at least partially, surround germ cells. The germ cells were large rounded cells with round nuclei containing one or two prominent nucleoli (Fig. 2 B). The organelles included round mitochondria, some granulated endoplasmic reticulum and an abundance of polysomes.

The gonadal ridges at 24 days p. c. had increased further in size, but the wide base connecting them with the mesonephri yet remained (Fig. 3 A). The surface epithelium was still apparently forming cellular cords, which intermingled with the underlying tissue (Figs. 3 A and 4 A). The gonadal blastema contained irregular cellular cords, mesenchymal cells and many capillaries. Germ cells were found throughout the gonadal ridge i. e. in and just under the surface epithelium, in the blastema and in the mesenchyme at the base. During the formation of the gonadal ridges, the neighbouring mesonephric glomeruli appeared intact and signs of degenerative changes were not found anywhere in the mesonephri.

The indifferent gonad At 25 days p. c. indifferent gonads had formed (Fig. 3 B). These gonads had a narrow

basal connection with the mesonephri and an oval shape in transverse sections. The surface epithelium consisted of low cylindrical or cuboidal cells in one or two layers. Irregular cellular cords were still being formed by the epithelium (Fig. 3 C). These cords appeared continuous with the cords located more centrally in the blastema. The blastema was morphologically similar to that at 24 days. Numerous germ cells were present (Figs. 3 C-D, arrowheads) sometimes surrounded by cord cells, and germ cells in different stages of mitosis were frequently seen. The germ cells still showed a very primitive ultrastructure, with few organelles, such as rounded mitochondria, rough endoplasmic reticulum and polysomes (Fig. 4 B). At the base of the gonad there were several layers of mesenchymal cells, a few germ cells and a large number of blood vessels (Fig. 3 D).

The newly differentiated testis At 27 days the undifferentiated gonad had developed into a morphologically distin-

guishable testis (Fig. 5 A-D). A primitive tunica albuginea, consisting of several layers of regularly arranged mesenchymal cells, had began to form just underneath the now cuboidal surface epithelium. It appeared to be continuous with the basal mesenchyme and was best developed near the base. At the most apical (or ventromedial) surface of the testis the tunica albuginea had not yet formed and the epithelium was similar to that at 25 days i. e. low cylindrical or cuboidal in one or two layers, still apparently forming cellular cords. Most of the former gonadal blastema had become organized into distinct testicular cords and interstitial tissue, although some of it remained undifferentiated in the central part of the testis at this stage.

The testicular cords were branched irregular structures consisting of cord cells, now called sustentacular or early Sertoli cells and germ cells, now termed prespermatogonia. The sustentacular cells had a varying morphology, but tended to have elongated or oval shaped nuclei, often with one or more prominent nucleoli. Their cytoplasm stained more intensely than that of the germ cells. Ultrastructural features included elongated mitochondria, rough endoplasmic reticulum and numerous polysomes. Desmosomes were often seen where the sustentacular cells were in contact with prespermatogonia (Fig. 6, arrows). A basal lamina had began to form, but was incomplete. The prespermatogonia appeared to be completely surrounded by the sustentacular cells and were often located centrally in the cords (Fig. 5 B). No germ cells were found outside the cords, except in the most ventro-

600 HURST, DALIN and RODRIGUEZ-MARTINEZ

Fig.4A-B. Ultrastrucutre of the indifferent gonad at 24 (A, 2 3 0 0 ~ ) and (B, 5 0 0 0 ~ ) days p.c. showing the surface epithelium (SE) and the formation of a cellular cord ( C ) (Fig. A). Figure B shows a contact zone between a germ cell and some cord cells (C). Note the rounded nucleus (11) and round mitochondria (m) of the germ cell (rer: rough endoplasmic reticulum; short arrows: polysomes; long

arrows: desmosomes)

medial area, where the tunica albuginea had not yet formed. Several prespermatogonia were undergoing mitosis at this stage of testicular development (Fig. 5 B, D arrows).

The dominating cells in the interstitium were undifferentiated interstitial cells, with varying morphology. The nuclei were often oval shaped or elongated and contained one or

Early development of pig gonads 60 1

Fig. 5 A-D. Light microscopy of the newly differentiated testis in pig embryos at 27 days p. c., showing at low magnification (Fig. A, 40 X) the primitive tunica albuginea (TA) under the surface epithelium (SE), the distinct testicular cords (TC) and the loose interstitial tissue (IT) (": coelomic cavity, M: mesentery). Some undifferentiated gonadal blastema (GB) still remains in the centre of the testis. Figures B-D (500x), show a higher magnification of the testis in Fig. A. In B, the tunica albuginea (TA) consists of several layers of regularly arranged mesenchymal cells and many capillaries (ca). As seen in Figs. B-D; the testicular cords (TC), consisting mainly of sustentacular cells (S),


Fig. 7 A-C. Light micrographs of the testis in pig embryos at 28 (Fig. A) and 36 (Figs. B-C) days p. c., showing in Figs. A (100 X) and B (80 X) the tunica albuginea (TA), testicular cords (TC) and interstitial tissue (IT), SE: surface epithelium (": coelomic cavity). In B, the interstitium (IT) is now dominated by recently differentiated interstitial cells (Leydig cells). Degenerating germ cells (arrows) are frequently observed in the testicular cords. A detail of the testis in Fig. B, is shown in Fig. C (500 X) with a testicular

cord (TC) with germ (G) and sustentacular cells (S), L: Leydig cell, ca: capillaries, H E

604 HURST, DALIN and RODRIGUEZ-MARTINEZ

* v- , , , . .- .. .

Fig. 8 A-B. Electron micrograph from the pig testis showing in Fig. A (28 days p. c., 5400 X) a germ cell surrounded by sustentacular cells (S). Note the typical round nucleus (n) with its prominent nucleolus (nu). In the cytoplasm round mitochondria (m) are present. In B (36 days p. c., 3600 X) a testicular cord is shown. Note the clearly visible basal lamina (bl) which now has been formed by the

sustentacular cells (S)


junction complexes. Degenerative prespermatogonia were very common (Fig. 7 B, arrows). Many interstitial cells had differentiated into Leydig cells with abundant highly eosinophilic cytoplasm and rounded centrally or excentrically located nuclei. Striking ultrastructural features were smooth as well as rough endoplasmic reticulum, polysomes and rounded mitochondria with tubular cristae.

Discussion The results of the present study indicate that the early development of the porcine

gonad follows the general mammalian pattern and confirm previous observations in the same species (PELLINIEMI, 1975). Gonadal ridges began to form at 18 days p.c. on the ventromedial surfaces of the mesonephri. They were composed of a tall, irregular surface epithelium, which intermingled with the underlying mesenchyme, where scattered migrating germ cells were present. These gonadal ridges gradually developed in size, mainly due to growth of the gonadal blastema, where the presence of irregular cellular cords dominated, and by day 25 p. c. indifferent gonads had formed. Germ cells were not yet included in the cords. By day 27 morphologically distinguishable testes were shown. A primitive tunica albuginea was present, and the former gonadal blastema had developed into distinct testicular cords and interstitial tissue. Germ cells were present solely within the cords. The sustentacular cells had at this time a varying morphology with a rather dense cytoplasm and conspicuous desmosomes, and an incipient basal lamina had developed. At 36 days, at the border line between the embryonic and fetal stages, these cells had polarized, with their nuclei located basally. A clearly visible basal lamina was then present and cell contacts were well established, both between these young Sertoli cells and between them and the prespermatogonia.

As described, the cell types involved in the development of the porcine gonad were the surface gonad epithelium (derived from the coelomic epithelium) and the underlying mesenchyme. From these cell types the gonadal blastema appeared to be formed.

The neighbouring mesonephros, with some of its glomeruli adjacent to the gonadal ridge, has been regarded, by several authors, as contributing to gonadal development with several cell types, such as the future interstitial cells of the testis (c. f. BYSKOV, 1986). In man, the upper segment of the mesonephros contributes to the gonadal blastema by releasing cells (podocytes, mesangial cells, capsular cells) from Bowman's capsule and the vascular pole of the Malphigian corpuscle, dedifferentiating or transdifferentiating into phenotypically undifferentiated, mesenchyme-like cells (WARTENBERG, 1981). An impor- tant species difference to be considered in this respect is that while in humans the mesonephros has a rudimentary development and is quite afunctional, the mesonephros in the pig is well developed and functional for a long period of the early fetal life (TIEDEMANN, 1976). A mesonephric contribution of cells for gonadal development would, in the pig, imply dedifferentiation of the yet highly functional mesonephric cells, their migration to a developing new area in the vicinity and their redifferentiation into cells with a totally new morphology and function.

Our group has recently tested the hypothesis that the activity of the enzyme carbonic anhydrase (CA), which is present in many cells of the porcine mesonephros, could be used as a marker for Sertoli cells or their precursor cell type during testicular morphogenesis. No clear CA activity was present in the gonads before they morphologically differentiated into testes, despite the clear CA activity continuously present in the mesonephric nephron, so this study did not cast any light over the problem concerning the origin of the Sertoli cells (RODRIGUEZ-MARTINEZ et al., 1990).

In the mammalian ovary, meiosis is initiated during prenatal development, while in the testis a mechanism, which is not understood in detail, prevents the male germ cell from entering meiotic prophase until puberty. In humans, a dual Sertoli cell system, with dark (i. e. more dense, or with an increased cytoplasmic protein content) and light cells, at the light microscopical level, has been repeatedly proposed (WARTENBERG et al., 1978-89). It

606 HURST, DALIN and RODRIGULZ-MARTINEZ

has been suggested (WARTENBERG et al., 1989) that a balanced proportion of dark and light cells regulates the cell proliferation pattern between meiosis-inducing and meiosis-prevent- ing cells.

In the present study, despite the fact that we used the same fixatives and tissue processing procedures as WARTENBERG’S group, we were unable to distinguish between dark and light Sertoli cell types. The sustentacular, o r young Sertoli, cells were conspicu- ously dense in their cytoplasm, due to their content of rough endoplasmatic reticulum and ribosomes. This cell type, whatever its origin, contributes actively to the most crucial event in gonadal formation represented by the enclosure of germ cells and somatic cells in specific germ-cell compartments. The early Sertoli cells showed no clear cut cell junction complexes of the type found in the mature cells, but desmosomes were present between them from a very early stage, determining the appearance of well-organized testicular cords. Desmosomes were also detected between the Sertoli cells and the developing germ cells (prespermatogonia), which accounts for the general concept that the formation and differentiation of a gonad (i. e. testis) depend upon finely controlled interactions between germ cells and various types of somatic cells. These interactions occur during the period of germ cell migration to the gonad and then, within the gonad, during the formation of definite compartments for further germ cell development.

Another crucial event in testicular development is the differentiation of steroid- secreting cells (Leydig cells) in the extracordal compartment (BYSKOV, 1986). In the pig, they appear soon after gonadal sex differentiation (MOON and HARDY, 1973), and, in our study, they seemed to appear from the same general cell mass (blastema) as the Sertoli cells. Whether or not they originate in the mesonephros or in the resident mesenchyme at the site of the gonadal ridge is not yet clear.

Factors such as growth factors (CLARK et al., 1990), which may be involved in governing the proliferation and differentiation of the sustentacular (Sertoli) cells, are still largely unknown. Studies in this context are being carried out in our laboratory.

Acknowledgements The authors wish to thank Ms. P\SA JANSON for excellent technical assistance and Mr. BRIAN

OGLE from Funbo-Lovsta, the experimental farm of the Swedish University of Agricultural Sciences, for providing us with the animals. This work received financial support from the Swedish Council for Forestry and Agricultural Research.

References BYSKOV, A. G., 1986: Differentiation of Mammalian Embryonic Gonad. Physiol. Reviews 66,

CHIQUOINE, A. D., 1954: The identification, origin and migration of the primordial germ cells in the mouse embryo. Anat. Rec. 118, 135-145.

CLARK, A.M., D. R. HAGEN, and J. M. HAMMOND, 1990: Expression of insulin-like growth factor-I1 mRNA in the porcine testis. J. Reprod. Fert. Abstract series 5, 46 (abstr. 81).

KARNOWSKI, M. J., 1965: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol. 27, 137A.

MERCHANT-LARIOS, H., 1976: The role of germ cells n the morphogenesis and cytodifferentiation in the rat ovary. In: MULLER-BBRAT, N.: Progress in Differentiation Research. North Holland Publ. Amsterdam, 453-462.

MERCHANT-LARIOS, H., 1979: Origin of the somatic cells in the rat gonad: An autoradiographic approach. Ann. Biol. Anim. Biochim. Biophys. 19, 1219- 1229.

MERCHANT-LARIOS, H., 1984: Germ and somatic cell interactions during gonadal morphogenesis. In: VAN BLERKOM, J., and P. M. MOTA (eds.): Ultrastructure of Reproduction. Martinus Nijhoff Publ., Boston, 19-30.

MOON, Y. S., and M. H. HARDY, 1973: The Early Differentiation of the Testis and Interstitial Cells in the Fetal Pig, and Its Duplication in Organ Culture. Amer. J. Anat. 138, 253-268.

71-1 17.


NIEMI, M., M. IKONEN, and A. HERVONEN, 1967: Histochemistry and fine structure of the interstitial tissue in the human foetal testis. In: WOLSTENHOLME, G. E. N., and M. O'CONNOR (eds.): Endocrinology of the Testis. Ciba Fdn. Colloq. Endocrinol., London, Churchill Ltd. 16,

PELLINIEMI, L. J., 1975: Development of embryonic gonad. Ultrastructural and cytogenetic study on

RIDDERSTRALE, Y., E. EKSTEDT, and L. PLOEN, 1985: Localization of carbonic anhydrase in testis.

RODRIGUEZ-MARTINEZ, H., M. HURST, and E. EKSTEDT, 1990: Localization of carbonic anhydrase

TIEDEMANN, K., 1979: Architecture of the mesonephric nephron in pig and rabbit. Anat. Embryo].

UPADHYAY, S., J. M. LUCIANI, and L. ZAMBONI, 1981: The role of the mesonephros in the development of the mouse testis and its excurrent pathways. In: BYSKOV, A. G., and H. PETERS (eds.): Development and function of reproductive organs. Excerpta Med. Amsterdam, 18-27.

WARTENBERG, H., 1978: Human testicular development and the role of the mesonephros in the origin of a dual Sertoli cell system. Andrologia 10, 1-21.

WARTENBERG, H., 1981: The influence of the mesonephric blastema on gonadal development and sexual differentiation. In: BYSKOV, A. G., and H. PETERS (eds.): V Workshop in Development and function of reproductive organs, Copenhagen, July 1981. Excerpta Medica, 3- 12.

WARTENBERG, H., 1983: Structural aspects of gonadal differentiation in mammals and birds. In: MULLER, U., and W. W. FRANKE (eds.): Differentiation. Springer-Verlag, Berlin, 64-71.

WARTENBERG, H., E. RODEMER-LENZ, and C. H. VIEBAHN, 1989: The dual Sertoli cell system and its role in testicular development and in early germ cell differentiation (Pres ermatogenesis). In: HOSTEIN, A. F., K. D. VOIGT, and D. GRXSSLIN (eds.): Reproductive Bi3ogy and Medicine, Diesbach Verlag, 44-57.

WITSCHI, E., 1948: Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal fold. Contrib. Embryo]. 32, 67-80.

ZAMBONI, L., S. UPADHYAY, J. BBZARD, and P. MAUL~ON, 1981: The role of the mesonephros in the development of the sheep testis and its excurrent pathways. In: BYSKOV, A. G., and H. PETERS (eds.): Development and function of reproductive organs. Excerpta Med. Amsterdam, 31 -40.

31-55, J 6A.

the pig. PhD thesis, Turku.

Acta Physiol. Scand. (Suppl. 542), 408.

activity in the developing boar testis. Acta Anat. (Basel), 139, 173-177.

157, 105-112.

embryonic development of the porcine indifferent gonad and testis

Documents