J. Anat. (1977), 123, 3, pp. 589-599 With 14 figures Printed in Great Britain

589

Testis differentiation in the fetal and postnatal ferret RUTH DEANESLY

A.R.C. Institute of Animal Physiology, Babraham, Cambridge, CB2 4AT

(Accepted 1 May 1976) INTRODUCTION

Early embryological work and theories on the development and differentiation of fetal gonads have been recently summarized by Jost, Vigier, Pr6pin & Perchellet (1973) and others, and need not be recapitulated here. Testis differentiation has been examined in the mouse (Russo & de Rosas, 1971; Jean, 1972), the rat (Roosen-Runge & Anderson, 1959; Roosen-Runge, 1961; Mittwoch, Delhanty & Beck, 1969; Jost, 1972), the guinea-pig (Black & Christensen, 1969), the rabbit (Chr6tien, 1966), the dog (Gier & Marion, 1969), the pig (Moon & Hardy, 1973), and the macaque and man (Van Wagenen & Simpson, 1965), but not in the ferret. Pregnancy in the ferret lasts 40-42 days and there may be 5-12 young (Hammond, personal communication). The fetal and postnatal development of the ovaries of the ferret has already been described (Deanesly, 1970), and the present paper is based on the males from the same litters, with some additional younger embryos. As in other mammals, morphological differentiation of the gonad as a testis precedes that of the ovary. MATERIAL AND METHOD

The material examined comprised 32 serially sectioned embryos from day 22 to day 27 of pregnancy and the dissected testes of 27 later fetuses together with 9 young 1-12 days after birth. Fixation of embryos and of some of the individual testes was in Bouin's fluid diluted by 50 % distilled water. Serial sections were cut at 7 ,m and stained with haematoxylin and eosin or PAS. Other dissected testes were fixed by Ciaccio's bichromate-formol-acetic method and sections were stained with haematoxylin and oil red 0 for lipids. Some embryos were weighed after fixation. Embryos processed to show alkaline phosphatase proved useless for studying gonocyte migration, as Chr6tien (1966) observed in the rabbit. Witschi (1948) found that germ cells in the human embryo were large and readily recognizable with routine histological stains, and this appeared to be also true for the ferret. An attempt was made to assess the growth of the gonads just before and after visible sex determination, in view of the suggestion of Mittwoch (1970) that male determination might be effected by increased growth of the undifferentiated gonad. The size of each gonad in 23-27 day embryos was estimated individually by counting serial sections and making tracings from projections of the largest. This method gave a good approximation of gonad volume, but because of variations in embryo size within litters, and differences in the planes of sections of the embryos, it was not possible without a complete series of embryo weights and paper reconstructions, to

590 RUTH DEANESLY establish firm conclusions about the growth of undifferentiated gonads, even from the extensive material available. Certainly such gonads did not fall clearly into large and small categories; only after their differentiation were the testes definitely larger than the ovaries. RESULTS

Observations on serially sectioned embryos Day 22 ofpregnancy: 6 embryos In these embryos the genital ridges, attached to the mesonephros and vascularized from it, contained a few large gonocytes mostly in or near the coelomic epithelium; otherwise they consisted of undifferentiated, mainly stromal cells, some with fibrils. The deeper lying cells merged imperceptibly with the mesonephric stroma. Neither at this stage, nor later, did the cells appear to be arranged in 'cords' as in many descriptions of gonad embryology. Day 23 ofpregnancy: 6 embryos. Day 24: 6 embryos In the present series, the post-fixation weights of 23 and 24 day embryos (136-151 mg) overlapped, and the gonads, still indifferent, but larger and more clearly defined than at day 22, remained broadly attached to the mesonephros and were similar in the two age groups. As before there was no differentiation into cortical and medullary areas, but serial sections gave an impression of cell movements and cell divisions throughout the gonads. (Figs. 2, 3, 4.) Immigration was still taking place and gonocytes had become more numerous; their nuclei varied in diameter from 6 to 9 ,um, probably because of cell divisions, and were found throughout the gonads, not only in the outer regions. Individual gonocytes could be seen encircled by mesenchymal cells, as described by Jost (1972) in the rat, but apparently this did not relate to sexual differentiation or tubule formation, since it was a feature of all embryos (Fig. 4). By 23 days the primordial rete had begun to form within the gonad near the mesonephros. Rete cells, identifiable by their darkly stained, regularly oriented nuclei, formed strands or channels growing out towards the middle of the gonads and coming into contact with deeper lying gonocytes (Figs. 1, 4). In the 24 day embryo these developing rete tubules might be mistaken for the beginnings of testicular ABBREVIATIONS r Rete int Interstitial cells rbc Red blood corpuscles ca Capsule t Tubule

Undifferentiated gonads Fig. 1. 160A. 24 days. Gonad showing attachment to the mesonephros, and deeper staining rete. x 145. Fig. 2. 162cy. 23 days. Gonocytes (arrowed) can be seen in the coelomic epithelium and among deeper mesenchymal elements. Bottom right lies part of the developing rete. x 305. Fig. 3. 160A. 24 days. Part of Figure 1 showing immigrating gonocyte (arrowed), other gonocytes and nucleated red blood corpuscles. x 500. Fig. 4. 160A. 24 days. Part of a section illustrating appearance of cell activity and movement in the gonad and the tendency of gonocytes to become encircled. x 500.

wQv^bxSw~ ~ ~ Ferret testis differentiation 591

'5V ~~'4E,W \o W-*- *

4

*

~

~

~

0e.4 I& fe.

~

~

~

~

~

~

~

~

~

~

~

~

~

SN

sS AX~~~P

., ff

>

#,w

ls;

*-ro

>*^

'svths

~~~ f}

z.*^Z

4;;O s ,.!I

i

"A

592

5

RUTH DEANESLY

Ferret testis differentiation 593 differentiation, but comparison with the early seminiferous tubules at 26 days showed differences in the appearance and orientation of their nuclei and the accompanying cytoplasm. Torrey (1947) found rete development similar in male and female rat embryos at 16 days of pregnancy and later. In ferret embryos at 24 days the developing rete was more distinct and extensive in some than in others, but the differences were not such as would distinguish the sexes. Characteristic of embryos at 24 days, and sometimes at 23 days, was the presence in the gonad stroma, usually, but not always, near the mesonephric border, of conspicuous masses of small, darkly staining nuclei (Fig. 6), each surrounded by a minimum of clear cytoplasm. Some appeared to be lymphocyte nuclei, as did larger, similar ones seen then and later. Other small, dark nuclei, probably pycnotic, occurring in groups, seemed to be those of regressing blood corpuscles, and these were also found in the mesonephros. Day 26 ofpregnancy: 3 males, 6 females In these embryos, 2 days older than the preceding ones and weighing up to 323 mg, the gonads were becoming partly detached from the mesonephros and morphological differentiation had begun. Male gonads (Figs. 5, 6, 7, 9) showed incipient tubule formation, fairly frequent mitoses and eosinophil interstitial cells, while the only change in the other embryonic gonads was the beginning of oogonial aggregation in the cortex. An obvious distinction between the sexes at this time was the general cell enlargement in the testes, associated both with the increased cytoplasm of the future sustentacular or Sertoli cells in the seminiferous tubules and with the enlarged epithelioid, interstitial cells developing from the stroma (Figs. 5, 7). These had round nuclei 6-7 aum in diameter and cells varying up to 12 um in diameter. Another feature of the testes was the formation below the coelomic epithelium of layers of fibroblasts of varying thickness - the future capsule or tunica albuginea. Not infrequently, gonocytes were included within these fibrous layers (Fig. 6) and immigration was still found. Groups of small dark nuclei, similar to those seen earlier, occurred in both testes and ovaries. Epithelioid interstitial cells (Figs. 7, 9) deriving from stroma cells, as in other mammals, occupied a large proportion of the testis but showed no topographic relation to the tubules; groups of them could be seen in the middle of the testis, where no seminiferous tubules had formed - perhaps owing to a local absence of gonocytes. Similar epithelioid interstitial cells do not develop in the ovary until two weeks after birth (Deanesly, 1970). At this stage the rete is in transition: parts of it appear Early testis differentiation Fig. 5. 180E. 26 days. Tubule formation and interstitial cell differentiation. x 280. Fig. 6. 180c. 26 days. Development of fibroblastic capsule and tubules: also shown (top right) is a typical group of lymphocytes. x 500. Fig. 7. 180E. 26 days. Strands of large interstitial cells with round nuclei. PAS stained. x 500. Fig. 8. 170. 27 days. In a testis one day older than that shown in Fig. 5, the tubules are larger and better defined. x 280.

594

9

RUTH DEANESLY

595 Ferret testis differentiation contracted in the hilar area, but its strands extend into the interior of the testis and small rete ducts, better seen on day 27, are beginning to be formed as outgrowths. Although in close contact with gonocytes, interstitial cells, and developing seminiferous tubules, the rete did not appear to be directly involved in their determination or growth. Seminiferous tubules developed below the coelomic epithelium and also in other areas lacking rete. Day 27 ofpregnancy: 2 males, 3 females. Day 28: 2 males By now the testes were considerably larger than litter mate ovaries, and the first stages of differentiation were completed. Seminiferous tubules and interstitial Leydig cells were well defined (Figs. 8, 10). The tubules, surrounded by fibroblasts, had diameters of 35-50 ,um. They contained a layer of Sertoli cells, with clear cytoplasm, nearly filling the lumen, and one or more gonocytes, some in mitosis. A distinct fibrous tunica up to 5 cells deep had now formed below the coelomic epithelium and this still contained gonocytes - as many as 9 in a single section. Interstitial cells were well defined (Figs. 8, 10). They varied in size and shape and were seldom in mitosis. Groups occurred near the middle of the testis. In Ciaccio preparations they stained pink with oil red 0, but not yet the deep orange red of lipids, present by day 32. There appeared to be slight traces of lipid at 28 days, but in general the testes were similar to those at 27 days except for further development of small rete ducts, the future tubuli recti (Figs. 11, 12). Day 30-40 ofpregnancy: 25 males Examination of fetal testes from days 30 to 35 showed the further development of lipid cell inclusions in the Leydig cells; by 32 days, these stained the characteristic orange red with oil red 0 after Ciaccio fixation (Fig. 13). Cells near the exterior continued to be smaller than those in the medulla, size differences being probably associated with the stage of glandular development reached. Up to the end of pregnancy, interstitial cells formed a large proportion of the total volume of the testis. The seminiferous tubules did not show much change during this period; they were few in number and their diameters varied along their length from 45 to 65 ,um (Fig. 11). Gonocytes increased and showed mitoses, and Sertoli cells were further differentiated. By day 35, the rete which had continued to develop consisted of long channels from the hilus connecting with the seminiferous tubules by the small tubuli recti (Fig. 12). No rete cells contained liipid. Birth to 12 days old: 9 males All testes in the first 3 days after birth showed a reduction in the number of Leydig cells; groups of these were replaced by vacuolated fibrous tissue containing Early testis differentiation Fig. 9. 180c. 26 days. General view of differentiating testis, three tubules are arrowed; on the right the rete (r) somewhat compressed. x 270. Fig. 10. 161B. 27 days. Further differentiation of testis tubules, interstitial cells and capsule. x 270.

596

RUTH DEANESLY

13

Ferret testis differentiation

597

congested blood sinuses (Fig. 14). This depletion, which continued for some days, occurred at a time when the testis was growing and the seminiferous tubules were increasing in complexity and appearing more numerous in section. Between the tubules there was increased fibrous tissue, replacing some Leydig cells. A similar reduction of interstitial tissue at around the time of birth was observed by RoosenRunge & Anderson (1959) in the rat, by Russo & de Rosas (1971) in the mouse and by Hayashi & Harrison (1971) and others in man. In mammals, such as the pig (Moon & Hardy, 1973) and the horse (Cole, Hart, Lyons & Catchpole, 1933), interstitial cell regression was observed at different periods of fetal life. DISCUSSION

Certain conclusions have emerged from this study of the developing ferret testis. Between days 22 and 24 of pregnancy, gonad blastemas attached to the mesonephros increased in size but remained morphologically undifferentiated. Beneath coelomic epithelium they consisted of vascularized mesenchymal and stromal tissue into which gonocytes with distinctive large nuclei had immigrated. Some gonocytes were in mitosis, and some tended to become encircled by adjacent mesenchymal cells, a process which did not represent a first stage in testis tubule formation, as suggested by Jost (1972), since it was found in all gonads before sex determination was distinguishable, as well as later. On day 26 the differentiating male gonads showed a striking increase in volume as compared with the ovaries. This was caused both by cell enlargement, especially of developing interstitial cells, and cell division. As Jean (1972) clearly showed in the mouse, the increase in the volume of the fetal gonad between days 13 and 17 of pregnancy, + 925 % in the testes and only 221 % in the ovaries, coincided with its morphological differentiation. Mittwoch, Delhanty & Beck (1969), who assessed gonad size in fetal rats, concluded that the gonads of males, identified by their chromosomes, at 13-5 days, before differentiation, were already larger than those of litter mate females; but their evidence on this point from only 5 male fetuses is hardly conclusive, especially as Jost (1972) finds morphological sex differentiation in the rat at 13-7 days. The fact that fetal testes grow faster than corresponding ovaries after differentiation in rats, mice and ferrets does not support Mittwoch's (1970) hypothesis that male differentiation is actually brought about by more rapid growth of the gonad blastema. Tubule formation, first seen on day 26 of pregnancy, appeared to take place within Testes in older fetuses and newborn young Fig. 11. No. 92. 32 days. Sagittal section through part of testis showing distribution of tubules and interstitial cells and, bottom right, small rete ducts (arrowed). x 156. Fig. 12. No. 58. 35 days. Rete ducts, the tubuli recti (arrowed), in contact with seminiferous tubules. x 315. Fig. 13. No. 45. 37 days. Sagittal section through testis showing distribution of lipid containing interstitial cells. Ciaccio fixed, stained oil red 0. x 59. Fig. 14. No. 50. 2 days post partum. For comparison with similarly stained Fig. 13. The testis is larger, but some of the (arrowed) interstitial cells have lost lipid. x 59.

RUTH DEANESLY 598 about one day, since the testis at 27 days was well organized. The transformation of stromal to epithelioid interstitial cells was equally rapid, although not all stromal cells were affected simultaneously. The 6 females of the 26 day group of embryos had comparatively inactive ovaries, but an oogonial cortex had formed, separated by stromal tissue from the denser medullary areas. In embryos of both sexes the band of tissue joining the gonad to the mesonephros was being reduced, although vascular connexions persisted. The rete, present in all undifferentiated gonads on days 23 and 24, and in testes and ovaries on day 26 and later, developed independently within the gonad blastema. Although in close contact with the other elements of the testis, there were no indications that the rete gave rise to seminiferous tubules. Connexions with them were established later, between days 28 and 35 of pregnancy, by means of the small tubuli recti developing from the rete. SUMMARY

Testis development has been examined in a series of 59 fetal and 9 postnatal ferrets from day 22 of the 40-42 day pregnancy, to 12 days after birth. Developing seminiferous tubules and interstitial cells were first seen on day 26, and were well established one day later. Differentiation was associated with cell enlargement and cell division, and consequently the testes grew distinctly more rapidly at this time than the corresponding ovaries. Up to the end of pregnancy the epithelioid interstitial cells, derived from stromal tissue, formed a large proportion of the testis. Lipid was distinguishable in them, histologically, from about day 30. As in various other mammals, there appeared to be some regression of the interstitial Leydig cells around, the time of birth, when the seminiferous tubules resumed their growth. The intra-gonadal rete was present from day 22 onwards; it established connexions with the seminiferous tubules through the small tubuli recti by day 32. I am much indebted to Mr J. Hammond who supplied and fixed the dated embryos for this work, and to the A.R.C. Institute of Animal Physiology, Babraham, for hospitality and technical assistance. The microphotographs were taken by Mr A. L. Gallup and the embryos processed by Mr L. G. Jarvis. REFERENCES BLACK, V. H. & CHRISTENSEN, A. K. (1969). Differentiation of interstitial cells and Sertoli cells in fetal guinea-pig testes. American Journal of Anatomy 124, 211-238. CHRATIEN, F. C. (1966). ttude de l'origine, de la migration et de la multiplication des cellules germinales chez l'embryon de lapin. Journal of Embryology and Experimental Morphology 16, 591-607. COLE, H. H., HART, C. H., LYONS, W. R. & CATCHPOLE, H. R. (1933). The development and hormonal content of fetal horse gonads. Anatomical Record 56, 275-293. DEANESLY, R. (1970). Oogenesis and the development of the ovarian interstitial tissue in the ferret. Journal of Anatomy 107, 165-178. GIER, H. T. & MARION, G. B. (1969). Development of mammalian testes and genital ducts. Biology of Reproduction 1, 1-23. HAYASHI, H. & HARRISON, R. G. (1971). The development of the interstitial tissue of the human testis. Fertility and Sterility 22, 351. JEAN, C. (1972). D6veloppement volumetrique des gonades du fetus de Souris. Compte rendu des seances de la Societe de biologie 165, 2341-2345.

Ferret testis differentiation

599

JOST, A. (1972). Donnees preliminaires sur les stades initiaux de la differentiation du testicule chez le rat. Archives d'anatomie microscopique et de morphologie experimentale 61, 415-438. JOST, A., VIGIER, B., PREPIN, J. & PERCHELLET, J. P. (1973). Studies on sex differentiation in mammals. Recent Progress in Hormone Research 29, 1-41. MITrwocH, U. (1970). How does the Y chromosome affect gonadal differentiation? Philosophical Transactions of the Royal Society B 259, 113-117. MITrWOCH, U., DELHANTY, J. D. & BECK, F. (1969). Growth of differentiating testis and ovaries. Nature 224, 1323-1325. MOON, Y. S. & HARDY, M. H. (1973). The early differentiation of the testis and intestinal cells in the fetal pig and its duplication in organ culture. American Journal of Anatomy 138, 253-263. ROOSEN-RUNGE, E. C. (1961). The rete testis in the albino rat: its structure, development and morphological significance. Acta anatomica 45, 1-30. ROOSEN-RUNGE, E. C. & ANDERSON, D. (1959). The development of the interstitial cells in the testis of the albino rat. Acta anatomica 37, 125-137. Russo, J. & DE RoSAS, J. C. (1971). Differentiation of the Leydig cell of the mouse testis during the fetal period. An ultra-structural study. American Journal of Anatomy 130, 461-479. TORREY, T. W. (1947). The development of the urinogenital system of the albino rat. III. The urogenital union. American Journal of Anatomy 81, 137-153. VAN WAGENEN, G. & SIMPSON, M. E. (1965). Embryology of the Ovary and Testis. Homo sapiens and Macaca mulatta. New Haven: Yale University Press. WITSCHI, E. (1948). Migration of the germ cells of human embryos from the yolk sac to the primitive gonadal fold. Contributions to Embryology 32, 67-80.

Testis differentiation in the fetal and postnatal ferret.

J. Anat. (1977), 123, 3, pp. 589-599 With 14 figures Printed in Great Britain 589 Testis differentiation in the fetal and postnatal ferret RUTH DEAN...
5MB Sizes 0 Downloads 0 Views