Cytological Studies on Human Spermatogenic and Sustentacular (Sertoli) Cells

Transcription

1 Cytological Studies on Human Spermatogenic and Sustentacular (Sertoli) Cells By Setsuko Ogata Department of Anatomy, Tokyo Women's Medical College, Shinjuku, Tokyo, Japan (Director : Prof. Dr. Kura Kubota) Many cytological studies have been already published on the cytological structure of the human testis. Kubota (1958), from our laboratory, reported that considerable amount of lipoid granules existed in the cytoplasm of sertoli cells. Furtherfore, A r a k a w a (1960) reported on the relationship between age and lipoid and glycogen-like substances observed in the interstitial cells. There are many theories about the functional interpretation of this lipoid and glycogen-like granules, nevertheless, any definite conclusion has not yet been obtained. However, it has been already an undoubted fact that these granules have some relations with endocrin function of testis. I have investigated on the existence and the variation of the distribution according to age, of these lipoid and glycogen-like granules contained in various cells in seminiferous tubules of lacjh human testis, aging from embryo to adult man. Materials and Methods 1) The materials used in this study are as follows ; Normal testes of human embryos of 4, 5, 6, 7, 8 and 10 months old. Normal testes of human young generations of 10, 14 and 17 years old. Normal testes of human adults of 38, 56 and 73 years old. 2) Methods : The following fixation and staining procedures were applid to the materials mentioned above. Firstly, in order to observe morphological structure, paraffin sec- 39

2 40 Setsuko Ogata tions were prepared after fixation in Z e n k e r-formalin solution. Hematoxylin-Eosin and Heide n h a i n's iron hematoxylin staining method were applied to them ; while, after fixation in Z e n k e r- formalin solution and in 100% alcohol, PAS reaction was also employed. Next, paraffin sections were made after fixation in Champ y's solution, and stained by Heidenhai n's iron hematoxylin in order to prove the existence of lipid granules, so that the comparative observations according to age was made at the same time. Lastly, frozen sections were made after fixation in 10% neutral formalin, general lipid stainings, using Sudan III and S m it h- Dietlic h's method and so-called ketosteroid staining method by A s c h b el and S e l i g ma n, were applied to them to examine the nature of the lipid granules. Observation 1) Forms of spermatogenic cells and sertoli cells. In the embryonal specimens, the tubuli seminiferi contorti were filled with spermatogenic cells, and no lumen was recognized in it. Spermatogonium stuck to basement membrane, showing almost round shape. The nucleus appeared spherical, and the chromatin appeared as minute granules. Nucleolus was generally only one in number, and was relatively large. Primary spermatocyte, although sometimes showing a strange form probably by the pressure of its adjacent cells, principally existed in the central part of tubuli seminiferi contorti, and often, a giant cell which may be considered to be the early stage of primary spermatocyte, could be found. This giant cell, considered to be primary spermatocyte, sometimes existed between spermatogonium sticking to the basement membrane. It's nucleus was spherical, and mitochondria, which generally appeared as small granules, surrounds the nucleus. Sertoli cell of embryo existed between spermatogonium and primary spermatocyte, sticking to basement membrane. The form of this cell was not unique and irregular as seen in the adult, even though it was longer. Its whole figure was comparatively clearly seen. The nucleus was oval in shape, with one or two large nucleoli around it, and mitochondria appeared as small granules. The number of sertoli cells found in single seminiferous tubule

3 Cytological Studies on Human Spermatogenic and Sustentacular Cells 41 seemed to be much greater than that in the adult. The tissue of testis, at 10 years old, resembled morphologically to that of embryo that is, no lumen was formed in tubuli seminiferi contorti, and sertoli cell did not present such unique form as that of adult. Tubuli seminiferi contorti were also filled with spermatogenic cells as in the embryonal testis. In case of boys of 14 and 17 years old, the histological feature of the testis tissue was similar to that of adult : that is, lumen was found in tubuli seminiferi contorti, and spermatids were recognized in it. Also, mitosis of primary spermatocyte was actively being done. At 17 years old, mature sperms were recognized : that is, at this age, it was definitely recognized that sperm formation was going on. However, at the same age, each tubuli seminiferi contorti presented various histological aspects, and there are some seminiferous tubules in which no sperm formation was taken place. In case of adults, as to cells in tubuli seminiferi contorti, even though the forms of spermatogonium and primary spermatocyte presented no remarkable differences from those of boys, the cell division of primary spermatocyte was not so frequently seen as in case of boyhood of 14 and 16 years old. However, the form of sertoli cell was characteristic and extremely irregular, and its whole figure was completely obscure. The nucleus was mostly elliptical, and was clearly lacking in chromatin. The nucleoli were clearly recognizable at the peripheral area of the nuclei, which were surrounded by uneven nuclear membrane. The cytoplasm contained abundant components like mitochondria and lipoid granules inside. In case of highly aged testis, the basement membrane of tubuli seminiferi contorti appeared thicker than that of younger one. 2) Cytoplasmic components of spermatogenic cells and sertoli cells. (a) PAS positive granules In case of embryos, the granular structure, considered to be glycogen in nature existed in so-called giant cell, which may be considered as an undifferentiated spermatogenic cell. That is, in the cytoplasm of the giant cell, comparatively equally distributed PAS substances were found, being contrary to the sertoli cells. The amount of PAS substances existing in the giant cell has no relationship to the age of embryo, and also to the stage of cell division. Also, in all the materials examined, the basement membrane of tubuli seminiferi contorti and wall of blood vessel of interstitial tissue

4 42 Setsuko Ogata showed strong PAS reactions. At 10 years old, like in case of embryos, PAS positive substances were seen in the cytoplasm of the giant cell, but not in sertoli cells. In case of 14 and 17 years old, sperm formation was recognized inside of tubuli seminiferi contorti, and the most of the spermatogonium, primary spermatocyte and sertoli cell respectively showed weak PAS reaction. In case of adults, 38, 56, 63 and 73 years old, PAS positive substances in each cell appeared as granules of various sizes. These granules increase their number with age. Regardless of age, very small amount of PAS positive substances in each cell was digested by saliva, but most of them remained undigested. (b) Lipoid granules At 10 years old, as mentioned above, the testis tissue resembled morphologically to that of embryo. The lipoid granule which existed in sertoli cell, spermatogonium, and primary spermatocyte could not be found at that period. At 14 and 17 years old, a great deal of the cells undergoing the mitotic activity, most of them were in metaphase, were recoginzed primary spermatocyte. Lipoid granule found in cells of tubuli seminiferi contorti at this period were of medium size and located in the basal part of cells. They were found numerously in sertoli cell, and only a few of them were recognized in spermatogonium and primary spermtocyte. In case of 38 years old, lipoid granule appeared a little larger and remarkably increased in number, comparing with those in the testis of 14 and 17 years old. Most of them were contained in sertoli cell, like in case of 17 years old. The spermatogonium and primary spermatocyte contained less amount of granules, being distributed evenly in the cytoplasm. - In case of 56, 63 and 73 years old, the same observation as above was obtained. But, with age, the granules increased in number, and showed the variation of the size. Most of lipoid granule found in adult's tubuli seminiferi contorti located in sertoli cells, and only a small amount of them in spermatogenic cells. However, at older ages, many large lipoid granules were seen both in spermatogonium and primary spermatocyte. In order to examine the chemical nature of these lipoid granules, the following staining methods were applied to the frozen sections

5 Cytological Studies on Human Spermatogenic and Sustentacular Cells 43 of the testes from 10, 14, 38, 56, 63 and 73 years old specimens. At 10 years of age, sertoli cells containing large granules which were stained by Sudan III were recognized. But positive result was not obtained clearly by A s c h b e I et S e l i g m a n's reaction. At 14 and 17 years of age, and also in case of adults, following positive results were recognized, that is, red orange by Sudan III staining, black indigo by S m i t h-d i e t 1 i c h's method and indigo purple by A s c h be 1-S e I i g m a n's method. Though, it was impossible to point out which granule had which substance in it, it was supposed that three substances existed most abundantly in sertoli cell. But, at older ages, three substances mentioned above might possibly be encountered in the spermatognium and primary spermatocyte, since there were many granules, which were well fixed with the Champ y's solution, in the spermatogenic cells. Discussion Many cytological studies on testis of human kind and experimental animal have been already published. The author have observed the distribution of lipoid granules and glycogen-like granules in spermatogenic and sertoli cells of normal testis of human embryos and adults. On the close relationship between physiology of human spermatogenic cell and carbohydrate metabolism, Mcleod (1934) and many others have already reported. In author's results, PAS reaction was seen in the giant cell which considered to be primary spermatocyte, and in interstitial cells, in the embryonal stages. In adults, PAS positive substances located almostly in sertoli cells, primary spermatocyte and in spermatogonium. Like in case of embryos, they were also found in interstitial cells of interstitial tissue. Most of these PAS positive substances were resistant to the saliva digestion, accordingly, it is not considered to be glycogen but some complicated substances like mucoprotein or glucoprotein. Referring to some researches on this field, Le blond (1950) stated that mucosaccharides was seen in spermatogenic cells, but no glycogen was found in it ; while, Wolf & Le at hem (1955) reported on the existence of a small amount of glycogen in testis of white rat. Wislocki (1949) found seasonal differences in the con-

6 44 Setsuko Ogata tent of polysaccharides in testis of deer ; that is, at the active stage of reproduction, glycogen appeared in sertoli cells, while, at the resting stage, PAS reaction was found in cells of tubuli seminiferi contorti. A r z a c (1950) reported on the distribution of glycogen in human testis. According to him, small amount of glycogen was clearly seen in the cytoplasm of spermatogonium and sertoli cells of the normal testis, on the contrary, glycogen was abundant in hypoplastic or hypogenital testis. Then, it is considered that, when sperm formation is going on in a high degree, glycogen exists in sertoli cells and spermatogonium, while pathologic or hypofunctional testis amount of glycogen increases depending on the degree of pathologic process. From author's observation, it can not be definitely concluded that, at 17, 38 and 56 years old, when sperm formation or nuclear division is going on actively, PAS reaction is weak, comparing with PAS reaction shown at other ages. But, it might be supposed, as many others have stated, that the glycogen contained in testis, especially in sertoli cells, has an functional meaning as a source of the energy for respiration or movement of mature sperm. The fact, that lipid-like granules exist most numerously in sertoli cells among various cells in seminiferous tubules of human testis, was already reported in detail cytologically by Ito & H i o k i (1940), and electrone microscopically by Kubota & Kushida (1958). Also, K i j i m a (1955) pointed out that fact. Regarding the period when lipoid granule appear in cells of human testis, that is, their relation with age, Lynch & S c o t t (1950) stated, according to their study on 168 specimens of human testis, that in interstitial cells they appeared at the age of 12 years old, and in sertoli cells at the age of 15 years old, where no granules existed in the interstitial cells and no sperm formation was recognized. Ash b el & Seligman (1951), studying on 21 specimens of human testes from 12 week old embryo to 85 years old man, reported that neither active Sudan granules nor active ketosteroid granules were found in sertoli cells and interstitial cells up to 10 years old, and that, at 17 years of age, granules were found only in interstitial cells but not in sertoli cells, though sperm formation was going on at this stage. However, according to K i j i m a, the cells containing a very small amount of lipoid granule were recognized in sertoli cells and in seminiferous epithelial cell, even in the period of embryo and boyhood.

7 Cytological Studies on Human Spermatogenic and Sustentacular Cells 45 In author's observation, the osminophilic granule in sertoli cells of embryos more than 5 months old could not be found in embryos of 4 monts old, and also the size and amount of granules did not show any remarkable change with the development. In case of adult, lipid granules existed in spermatogonium, primary spermatocyte and most abundantly in sertoli cell. These granules were small in size and less in amount at boyhood until 10 years old, comparing with those in adults. But at and after 14 years old they increased and grew into various sizes with age. As regard to the life of these granules, As h b e 1 & Seligman and Lynch & Scott stated that they decreased at and after 40 years old. However, in author's specimens this decreasing tendency at older age could not be observed. Lipid granules contained in spermatogenic cells have been con.- sidered as stored lipid for energy necessary to division and multi- plication at the process of sperm formation. On the other hand, about the functional meanings of lipid granule contained in sertoli cells, there are many theories discussing their relation with hormone. In 1949, B e r t h r o n g, Goodwin & Scott supposed that these granules might be the second hormone secreted from testis, and probably estrogen. A s h b el & Seligman (1951) demonstrated the existence of ketosteroid substances in interstitial cells and in sertoli cells of adult, and reported that the substances existed in interstitial cells was androgen and the substances existed in sertoli cells was the same as estrogen. From author's observation, it was impossible to classify definitely like above ; however, since lipid granule found in sertoli cells showed positive reaction to A s h b e 1 & Seligman method and it can be considered that they are associated with hormone secretion. However, there is another undeniable fact that these granules in sertoli cells existed more abundantly at older age than at younger age when hormone secretion is actively being done. Accordingly, for the research of lipid granule existing in sertoli cells, biochemical study must be tried, to say nothing of morphological study. Above is author's observation added by author's comment on lipoid granule and polysaccharides contained in sertoli and spermatogenic cells. It might be said that in any case of the materials obtained, the individual difference was very great, and quite different aspects were observed in each person at the same age.

8 46 Setsuko Ogata Summary The following conclusion was drown through cytological study on sertoli cells and spermatogenic cells of human testes of embryos from 4 to 10 months old, of boys and adults from 10 to 73 years old. 1) At the period of embryo, sertoli cell does not show irregular form yet, but its whole figure is comparatively clearly seen. Nucleus is elliptical, with uneven nuclear membrane, and there is one or two nucleoli in the nucleus. Tubuli seminiferi contori are filled with primary spermatocytes, with no lumen remained. Occasional giant cells which are considered to be primary spermatocytes are recognized in the lumen. 2) Testis tissue of 10 years old is similar in the histological aspect to that of embryo, as regard to the feature of the lumen and to the appearance of giant primary spermatocytes. 3) Osminophilic granules can be found in sertoli cells as minute granules in embryos more than 5 months old. Though, they disappear temporarily at the age of 10 years, again they come out at the age of 14 years, and hereafter, they increase in number and grow into various sizes with age. Also, as to lipoid granules in sertoli cells of adults, they show positive reaction to the Aschbel et Se I i g m a n's method. Accordingly they are considered to be closely related to hormone secretion of testis. 4) In embryos, minute granules which are considered to be glycogen exist in the giant cell which is supposed to be a primary spermatocyte. And, in the specimens of 14 and 17 years old, the increase of the granules of various sizes were observed in the spermatogenic and sertoil cells, which showed the PAS reaction all over the cytoplasm in diffuse pattern and increased in number with age. As these granules resisted against the saliva digestion, it is considered that they are not mere glycogen, but some complicated compound combined with albumin or other substances, and that they exist and are preserved as source of energy necessary to division of spermatogenic cells at the process of sperm formation. References 1. A r z a c, J. P.: Glycogen in human testicular biopsy material. J. Clin. Endocrinol., 10: 11 (1950).

9 Cytological Studies on Human Spermatogenic and Sustentacular Cells A s h b e 1, R., R. B. Cohen & A.M. Se 1 igman: Histochemical demonstration of Ketosteroids in normal and neoplastic testis. Endocrinol., 49 : 2 (1951). 3. A r a k a w a, K. : Cytological studies on human testis (Interstitial cell). J. of the Tokyo Women's Medical College. 30 : 10 (1960). 4. B e r t hr on g, M., W. E. Good win & W. W. Scott: Estrogen production by the testis. J. Clin. Endocrinol., 9 : 579 (1949). 5. I t o, T. & K. H i o k i : Zur Zytologie der Sertoli Zellen im menschen Hoden. Okajimas Fol. anat. jap., 19 : 3 (1940). 6. K u b o t a, K. & T. Kushida: Electron microscopic studies on human testis. I. The sustentacular cells. J. of the Tokyo Women's Medical College. 28 : 4 (1958). 7. Kijim a, K. : Morphorogical studies on Lipoid of testis and epididymis. Folia Endocrinologia Japonica. 30 : 12 (1955). 8. L y n c h, K. M. & W. W. S cot t : The lipoid concent of the Leydig cell and Sertoll cell in the human testis as related to age, benign prostatic hyperplasia and prostatic cancer. J. Urol., 67: 6 (1950). 9. : The sertoli cell as related to age of man and experimental alteration of the pituitaly gonad axis in the animal with consideration of its role in spermatogenesis. Fertil. and Steril., 2: 35 (1952). 10. : Lipid distribution in the Sertoli cell and Leydig cell of the rat testis as related to experimental alteration of the pituitary-gonad system. Endocrynol., 49 : 8 (1954). 11. L e b 1 o n d, C. P.: Distribution of periodic acid-reactive carbohydrates in the adult rat. Am. J. anat., 86: 1 (1950). 12. Mc 1 e o d, J.: The metabolism of human spermatosa. Proc. Soc. exper. Biol. & Med., 42 : 153 (1939). 13. Mont a g n a, W. & J. B. Hamilton: Histological studies of human testis. I The distoribution of lipid. Anat. Rec., 109 : 4 (1951). 14. : Histological studies of human testis. II The distortion of glycogen and other H104-Schiff reactive substance. Anat. Rec., 112: 237 (1952). 15. W i s 1 o c k i, G. B.: Seasonal change in the testis, epididymides and seminal vesicles of deer investigated by histochemical methods. Endocrinol., 44 : 167 (1949). 16. W o 1 f, R. C. & J. H. Lea them: Hormonal and nutritional influences on the biochemical composition of the rat testis. Endocrinol., 57: 3 (1955). Explanation of Figures 1. Testis of 6 month old emryo, CHAMPY-PAS reaction. x 900 Tubuli seminiferi contorti was filled with spermatogenic cells. Mitochondria appeared as small granules arround the nucleus of spermatogonium. 2. Testis of 8 month old embryo, ZENKER-formalin-PAS reaction. x900 Giant cells with PAS positive reaction. 3. Testis of 10 years old, ZENKER-formalin-PAS reaction. x 900 Diffuse PAS positive reaction were seen in the cytoplasm of primary spermatocyts. 4. Testis of 14 years old, CHAMPY-Fe-Haematoxylin stain. x 900 Lipid granules in the cytoplasm of the sertoli cells. 5. Testis of 38 years old, CHAMPY-PAS reaction. x 900 The granules of PAS positive reaction were seen in the cytoplasm of sertoli cells.

10 48 Setsuko Ogata 6. Testis of 38 years old, CHAMPY-Fe-Haematoxylin stain. x900 Lipoid granules found in the sertoli cells. 7. Testis 56 years old, PAS positive reaction in sertoli cells. x Testis of 73 years old, CHAMPY-Fe-Haematoxylin stain. x900 The large lipoid granules found in the sertoli cells. 9. Testis of 14 years old, formalin fixation, SMITH-DIETLICH reaction. x 900 The lipid granules found in the sertoli cells. 10. Testis of 38 years old, Formalin, SMITH-DIETLICH reaction. x900 The lipid granules found in the spermatocyts. 11. Testis of 56 years old, formalin, ASHBEL and SELIGMAN reaction. x400 The Ashbel and Seligman positive reaction were seen in spermatocytes and sertoli cells.

11 49 Plate I Fig. 1 Fig. 2 Fig. 3 Fig. -1 Fig. 5 Fig. 6 S. Ogata

12 50 Plate II 1,i Fig. 9 Fig. 10 gig. 11 S. Ogata