0040-S 166/90/0022-0113/$10~00
TISSUE & CELL, 1990 22 (2) 11%121 0 1990 Longman Group UK Ltd
VIRANJANIE
K. GUNAWARDANA”
ULTRASTRUCTURAL LOCALISATION OF ALKALINE PHOSPHATASE IN THE INTERTUBULAR TISSUE OF THE TESTIS THE DOMESTIC FOWL Keywords:
Testis, interstitial
tissue, alkaline phosphatase,
ultracytochemistry,
IN
domestic fowl
ABSTRACT. Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue. the Leydig cells, transitional cells and the libroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of tibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.
Introduction
mouse, guinea pig and the dog (McComb, Bowers and Posen, 1979). In view of these inconsistencies it was decided to study the activity of this enzyme in an avian species. Thus in a previous investigation on the distribution of alkaline phosphatase in the testis of the domestic fowl attention was focused primarily on the germinal epithelium (Gunawardana, 1985). It became evident during this study that the interstitial and peritubular tissues displayed prominent enzyme activity. A survey of the available literature revealed a dearth of information on the ultrastructural distribution of this enzyme in the interstitial tissue, but ultracytochemical studies directed towards localizing enzymes in the testis mention the presence of alkaline phosphatase in the peritubular tissue in the rat (Tice and Barrnett, 1963) and in the hamster (Chang, Yokoyama, Brinkley and Mayahara, 1974) even though the cells of the seminiferous tubules are lacking in activity in the latter species. The present study is there-
Alkaline phosphatase is an enzyme which is widely distributed in tissues, and it is characterised by a low substrate specificity and a pH optimum ranging from 8.5-10. Histochemica1 techniques have enabled the detection of this enzyme in a variety of tissues such as bone, kidney, liver and intestine. In the gonad, alkaline phosphatase has found usage as a marker for primodial germ cells (McKay, Hertig, Adams and Danziger, 1953; Blackshaw, 1970). However, studies undertaken to demonstrate alkaline phosphatase activity in the mature mammalian testis have given conflicting results ranging from absence of activity in the germinal cells of the hamster and the rabbit to positive activity in the rat, *Department of Preclinical Studies, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka. Received 9 January 1989. Revised 15 December 1989. 113
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
TISSUE
fore aimed at localising the sites of alkaline phosphatase activity within the intertubular elements of the testis in the domestic fowl.
OF TESTIS
115
tome, and stained with uranyl acetate examination with an electron microscope.
for
Results Materials and Methods Testicular tissue was obtained from sexually mature White Leghorn cocks which were killed by an overdose of pentobarbitone sodium (Nembutal, Abbott Laboratories). For light microscopy, tissues were rapidly frozen in liquid nitrogen and sections 10 p.m thick were cut in a Cryo-Cut II (ReichertJung) maintained at -25°C. Sections were picked up on microscope slides and fixed in 80% ethanol for 1 hr. The sections were brought to water through graded alcohols and were then incubated for 3@45 min in a medium prepared according to Gomori’s method (Culling 1963) using P-glycerophosphate as the substrate. Control studies were carried out using a substrate-free medium. Subsequent procedures were similar to Gomori’s technique except that polyvinylpyrrolidone was used as the mountant. For ultracytochemical studies, material was fixed by immersion in 2% glutaraldehyde in O-1 M cacodylate buffer with 3% sucrose. After fixation for 30min the tissue was sectioned on a vibrotome. Sections 501_~m in thickness were incubated in a medium devised for demonstrating sites of alkaline phosphatase activity (Mayahara, Hirano, Saito and Ogawa, 1967) for periods of time ranging from 1&30min. For control studies sections were incubated in a substrate-free medium. Following incubation the sections were postfixed in 1% osmium tetroxide in cacodylate buffer for 60 min, dehydrated through ethyl alcohol and propylene oxide and embedded in Spurr’s medium (Spurr 1969). Thin sections were cut on an ultra-
Fig. 1. A light microscope bular tissue. x60.
Sections incubated for a period of 30 minutes when examined under the light microscope showed intense staining for alkaline phosphatase in the intertubular tissue (Fig. 1). Ultrastructurally three cell types were recognised in the interstitium and all three types exhibited alkaline phosphatase activity on their cell membranes. The reaction products of enzyme activity were deposited as electron dense beads on the outer surface of the cell membrane. The three cell types are designated as types.1, II and III. The type I cell was irregular (Fig. 2) or elongated (Fig. 3) in shape with a large irregular nucleus almost completely filling the cell. These cells corresponded to fibroblasts. Small or large pinocytotic vesicles containing reaction products were seen in the scanty cytoplasm. The cell classified as type II was irregularly rounded and a few vacuoles showing enzyme activity on their lining membranes were located in the cytoplasm (Fig. 4). This figure also illustrates the formation of a vacuole by the union of short cell processes. In the type III or Leydig cell the cytoplasm was more abundant and vacuolation was a salient feature. The vacuoles had a smooth outline and did not show any enzyme activity (Fig. 5). Mitochondria with tubular cristae were observed between some of the vacuoles. Pinocytotic vesicles were sometimes present, and these contained reaction product (Fig. 2). The blood vessels in the interstitium showed scattered activity in the basement membrane and in some pinocytotic vesicles in the endothelium. More pronounced
section showing intense alkaline phosphatase
activity in the intertu-
Fig. 2. Fibroblast from the interstitium. Note the enzyme activity on the cell membrane. A fragment of Leydig cell cytoplasm with lipid droplets (L) is also shown. Arrows indicate pinocytotic vesicles. X23.000. Fig. 3. Fibroblast from the interstitium. Note the enzyme activity on the cell membrane. Arrow indicates the formation of a pinocytotic vesicle. L-lipid droplet in Leydig cell cytoplasm x34.000.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
activity was seen in the cell processes surrounding the blood vessels (Fig. 6). Moderate enzyme activity was observed on the surface of the red cell. The peritubular tissue is composed of two cell types, the fibroblasts located as an inner cellular layer and the outer layer of myoid cells (Rothwell and Tingari, 1973). Both cell types showed elongated cell processes, but were distinguished from one another on the basis of their cytoplasmic components. The fibroblast cell processes possessed organelles such as endoplasmic reticulum, ribosomes and mitochondria whereas the myoid cell processes had scant organelles and the cytoplasm was filamentous or fibrillar. The myoid cell processes were devoid of any alkaline phosphatase activity (Fig. 7). However, the fibroblast cell processes showed beads of enzyme activity on their cell membranes (Fig. 8). A third category of cell process was similar to the fibroblast cell process but showed prominent activity in pinocytotic vesicles with little or no activity in the cell membrane (Fig. 9). In the cell body of the myoid cells the cell membranes were free of enzyme activity but moderate or faint activity could occasionally be detected in the pinocytotic vesicles (Fig. 10). In sections incubated in a substrate-free medium enzyme activity was absent. Discussion Several investigations have been carried out on the histochemistry of testicular enzymes (Blackshaw 1970). Studies dealing with enzymes in the interstitial tissue have concentrated mainly on those enzymes concerned with steroid synthesis (Woods and Domm, 1966; Blackshaw, 1970). The available reports on the localisation of alkaline phos-
TISSUE OF TESTIS
117
phatase in the interstitial tissue include the work of Lake (1962) on the domestic fowl and Roussel and Stallcup (1966) on the developing bovine testis. Lake, utilising the diazo coupling technique demonstrated that the intertubular tissue had pronounced alkaline phosphatase activity. The present light microscope observations based on the lead capture method of Gomori paralleled the results obtained by Lake (1962). However ultracytochemical studies are required for precise localisation of enzymes and as far as can be ascertained the present work is the only investigation pertaining to the ultrastructural localisation of alkaline phosphatase in the interstitial tissue of the testis in the domestic fowl. The ultrastructure of the interstitial tissue has received the attention of several workers. and a variety of species have been investigated (Burgos, Vitale-Calpe and Aoki. 1970). Connel(1972) referred to the presence of Leydig cells, transitional cells and connective tissue cells in the interstitial tissue of the chick testis. Similar results were recorded by Rothwell (1973) for the developing and adult testis of the domestic fowl. The presence of three cell types is also confirmed by the current findings. Furthermore the positive alkaline phosphatase activity on the cell membranes of all three cell types supports the contention that Leydig cells originate from a fibroblast-like cell through a transitional form to a mature Leydig cell as has been suggested in mammals (Fawcett and Burgos. 1960; Black and Christensen, 1969). On the basis of enzyme activity two types of vacuoles could be identified. It was apparent that the vacuoles lined by the reaction products of alkaline phosphatase activity were derived from the cell membrane. It may be postulated that these vacuoles formed
Fig. 4. Transitional cell showing enzyme activity on the cell membrane. Note the vacuoles (V) lined by reaction product. Arrows indicate the formation of vacuoles. x29.500. Fig. 5. Leydig cell showing enzyme activity on the cell membrane. activity in the lipid droplets L. M-mitochondria. X 14,000.
Note the absence of
Fig. 6. Blood vessel from the interstitium. Note the pronounced activity in the cell process (P) and moderate activity on the red cell (arrow heads). Arrows indicate pmocytotic vesicles. ~23,500.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
TISSUE
either by invagination of the cell membrane or by the union of cell processes as indicated by Fig. 4. This type of vacuole was predominant in the transitional cell. On the other hand. those vacuoles which were free of enzyme activity and distributed abundantly in the Leydig cells were evidently lipid droplets. Both Connel(1972) and Rothwell (1973) considered lipid droplets in the Leydig cells to represent a pool of steroid precursor rather than storage of newly synthesised steroid. In a review dealing with avian male reproduction Lake (1981) has stated that it is probable that the lipid in the Leydig cells represents precursor material involved in steroid production, since they are cholesterol positive and are strongly birefringent. In the light of these observations it is conceivable that the enzyme positive vacuoles in the transitional cells could perhaps reflect the early stages in the intake of steroid precursor into the cell. In this context it is relevant to note here that alkaline phosphatase on membranes is thought to be concerned with active transport across membranes (Kaplan, 1972). and that it is also one of the epithelial enzymes associated with fat absorption by the intestinal epithelium (Yoshizawa, Mizumoto, Nakase and Honjo, 1976). Alkaline phosphatase in transitional cells subserving a similar function cannot be completely overruled. Localisation of alkaline phosphatase in the peritubular tissue of the testis has been reported in the rat (Tice and Barrnett, 1963) and the hamster (Chang et al., 1974). In a previous report on the domestic fowl Gunawardana (1985) noted that the peritubular cell processes demonstrated enzyme activity and it was presumed that the cell processes were those of myoid cells. However the present study indicates that the myoid cells are relati-
Fig. 7. Myold cell processes. x 16,000. Fig. 8. Fibroblast x24.500. Fig. Y. Fibroblast
processes
Note the ftbrillar cytoplasm
vely free of enzyme activity. Those cell proactivity are cesses showing enzyme considered to be fibroblast processes. The distribution of the enzyme in pinocytotic vesicles is comparable to the situation described in the testis of the rat and the hamster. The presence of enzymes in these processes is particularly noteworthy since as mentioned earlier it is generally agreed that alkaline phosphatase is involved in the transmembrane transfer of organic molecules (Kaplan, 1972; Adams, 1983). The kidney, intestine and placenta are among tissues which are well established as containing pronounced alkaline phosphatase activity (Mayahara et al.. 1967; Sheilds, Bates, Yedlin and Best, 1984; Soares, 1987). These are all sites where transmembrane transport is considerable. The interstitium of the testis exemplifies another situation where an efficient transmembrane transfer facility is an essential requirement. In this connection it is pertinent to recall that the seminiferous tubules are not penetrated by blood vessels, and yet, a high concentration of testosterone bathes the seminiferous tubules. This implies the transmembrane transfer of testosterone from the interstitium to the tubules. Furthermore, it appears very likely that material passes from the tubules to the interstitium, since there is now ample evidence to indicate that substances secreted by the Sertoli cells regulate Leydig cell function (Sharpe, 1984; de Kretser, 1986). Thus it seems very probable that alkaline phosphatase in the membranes of the Leydig cells and in the cell processes aid in this two-way transfer of material between Leydig cells and the tubules, possibly the Sertoli cells. This is further substantiated by the earlier finding of alkaline phosphatase in the Sertoli cell membranes in the domestic fowl (Gunawar-
and the absence of enzyme activity.
showing enzyme acttvity on the cell membranes.
processes showing enzyme activity in pinocytotic
Fig. IO. Myoid cells. Cell membranes vesicles. x19.0(x).
119
OF TESTIS
R-ribosomes.
vesicles (arrows).
~28,000.
are free of enzyme activity. Arrows indicate pinocytotic
120
dana, 1985) and in the rat (Kornblatt, Klugerman and Nagy, 1983). The present work also revealed enzyme activity in the blood vessels. This is to be expected since transfer of materials such as hormones takes place between the interstitial cells and the blood vessels as well. Moreover steroid precursors also reach the interstitial cells by way of these blood vessels. Three isozymes of alkaline phosphatase viz. intestinal, liver-bone-kidney and placental have been distinguished in mammals, on the basis of heat sensitivity and inhibition by certain amino acids (Kornblatt et al., 1983). It is likely that isozymes corresponding to intestinal and liver-bone-kidney forms occur also in the domestic fowl. Characterisation of the testicular alkaline phosphatase in the rat has revealed it to be similar to the liver-
GUNAWARDANA
bone-kidney isozyme (Kornblatt etal., 1988). Related studies in the domestic fowl would be of interest, particularly since the studies in the rat appear to refer to an isozyme of alkaline phosphatase which is located within the seminiferous tubules and mainly in the Sertoli cells, thereby implying that similar studies on interstitial cells are lacking. Acknowledgements
I wish to thank Mr M. A. Navaratne for technical assistance. The electron micrographs were taken in the laboratories of the Department of Clinical Veterinary Medicine, University of Cambridge, England. I am grateful to Dr W. F. Blakemore for providing the facilities and to Mr Robert Patterson for his valuable assistance.
Adams, C. S. 1983. Localisation of alkaline phosphatase and NADH diaphorase in the principal cells of the guinea pig epididymis. Acta. Anaf., 116, 146151. Black, V. H.’ and Christensen, A. K. 1969. Differentiation of interstitial cells and Sertoli cells in foetal guinea pig testes. Amer. J. Amt., 124,211-238. Blackshaw, A. W. 1970. Histochemical localization of testicular enzymes. In The Testis (eds A. D. Johnson, W. R. Gomes and N. L. Vandemark), Vol. II, pp.73-123, Academic Press, New York and London. Burgos, M. H., Vitale-Calpe, R. and Aoki, A. 1970. Fine structure of the testis and its functional significance. In The Testis (eds A. D. Johnson, W. R. Games and N. L. Vandemark), Vol. 1, pp. 551-649, Academic Press, New York and London. Chang, J. P., Yokoyama, M., Brinkley, B. R. and Mayahara. H. 1974. Electron microscopic cytochemical study of phosphatases during spermiogencsis in Chinese hamster. Bio[. Reprod., 11,6O-610. Connell, C. J. 1972. The effect of luteinizing hormone on the ultrastructure of the Lcydig cell of the chick. Z. ZeNforsch., 128,13%151. Culling, C. F. A. 1963. Enzymes. In Handbook of Histopathological Techniques, 2nd ed., pp. 269-288, Butterworths. London. Fawcett, D. W. and Burgos, M. H. 1960. Studies on the tine structure of the mammalian testis. 11. The human interstitial tissue. Amer. J. Amt., 107, 245-269. Gunawardana, V. K. 1985. Ultrastructural localisation of alkaline phosphatase in the testis of the domestic fowl. J. Am., 140,711-718. Kaplan, M. M. 1972. Alkaline phosphatase. New-Engl. J. Med., 286,2m202. de Kretser, D. M. 1986. The testis. In Reproduction in Mammals: 3. Hormonal Control of Reproducrion. (eds C. R. Austin and R. V. Short), pp. 76-90. Cambridge University Press, Cambridge. Kornblatt, M. J., Klugerman, A. and Nagy, F. 1983. Characterization and localization of alkaline phosphatase activity in rat testes. Biol. Reprod., 29, 157-164. Lake, P. E. 1962. Histochemical demonstration of phosphomonoesterasc secretion in tht genital tract of the domestic cock. J. Reprod. Fert., 3,356362. Lake, P. E. 1981. Male genital organs. In Form and Function in Bin&. Vol. II. (eds. A. S King and J. McLellans). pp. 1-61. Academic Press, New York, London. Mayahara, H., Hirano, H., Saito, T. and Ogawa, K. 1967. The new lead citrate method for the ultracytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase). Histochemie, 11, 88-96. McComb, R. B., Bowers, G. N. Jr. and Posen, S. 1979. Cited by Kornblatt eral., 1983. McKay, D. G.. Hertig, A. T., Adams, E. C. and Danziger, S. 1953. Histochemical observations on the germ cells of human embryos. Amt. Rec., 117,201-219. Rothwell, B. 1973. The ultrastructure of Leydig cells in the testis of the domestic fowl. J. Amt., 116,245-253.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
TISSUE
OF TESTIS
Rothwell, B. and Tingari, M. D. 1973. The ultrastructure of the boundary testis of the domestic fowl (Gallus domesticus). J. Amt., 114,921-328.
121 tissue of the seminiferous
tubule
in the
Roussel, J. D. and Stallcup, 0. T. 1966. A histochemical study of the development of alkaline phosphatase in the testicle and epididymis of young bovine males. ht. J. ht., 11,215-225. Sharpe, R. M. 1984. Intratesticular factors controlling testicular function. Biol. Reprod., 30,29-49. Sheilds, H. M., Bates, M. L.. Yedlin, S. T. and Best, C. J. 1984. Distribution of immune reactive alkaline phosphatase in the adult rat ileum by immunoperoxidase staining at the light microscope level. Gastroenterology. 87,827-835. Soares, M. J. 1987. Developmental changes in the intraplacental distribution of placental lactogen and alkaline phosphatase in the rat. J. Reprod. Fert., 79,93-98. Spur, A. R. 1969. A low-viscosity epoxy resin embedding medmm for electron mxroscopy. J. I./ho. Res.. 26,31-43. Tice, L. W. and Barrnett, R. J. 1963. The fine structural localization of some testicular phosphatases. Arm. Rec.. 141.4s63. Woods. J. E. and Domm. L. V. 1966. Histochemical identification of the androgen producing cells in the gonads of the domestic fowl and albino rat. Gen. Camp. Endocrinol., 7,559-570. Yoshizawa. Y., Mizumoto, R.. Nakase, A. and Honjo, I. 1976. Histochemical studies on fat absorption in the jejunal mucosa after total pancreatectomy in dog. Amer. J. Surg.. 131,689-696.
TISSUE & CELL, 1990 22 (2) 11%121 0 1990 Longman Group UK Ltd
VIRANJANIE
K. GUNAWARDANA”
ULTRASTRUCTURAL LOCALISATION OF ALKALINE PHOSPHATASE IN THE INTERTUBULAR TISSUE OF THE TESTIS THE DOMESTIC FOWL Keywords:
Testis, interstitial
tissue, alkaline phosphatase,
ultracytochemistry,
IN
domestic fowl
ABSTRACT. Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue. the Leydig cells, transitional cells and the libroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of tibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.
Introduction
mouse, guinea pig and the dog (McComb, Bowers and Posen, 1979). In view of these inconsistencies it was decided to study the activity of this enzyme in an avian species. Thus in a previous investigation on the distribution of alkaline phosphatase in the testis of the domestic fowl attention was focused primarily on the germinal epithelium (Gunawardana, 1985). It became evident during this study that the interstitial and peritubular tissues displayed prominent enzyme activity. A survey of the available literature revealed a dearth of information on the ultrastructural distribution of this enzyme in the interstitial tissue, but ultracytochemical studies directed towards localizing enzymes in the testis mention the presence of alkaline phosphatase in the peritubular tissue in the rat (Tice and Barrnett, 1963) and in the hamster (Chang, Yokoyama, Brinkley and Mayahara, 1974) even though the cells of the seminiferous tubules are lacking in activity in the latter species. The present study is there-
Alkaline phosphatase is an enzyme which is widely distributed in tissues, and it is characterised by a low substrate specificity and a pH optimum ranging from 8.5-10. Histochemica1 techniques have enabled the detection of this enzyme in a variety of tissues such as bone, kidney, liver and intestine. In the gonad, alkaline phosphatase has found usage as a marker for primodial germ cells (McKay, Hertig, Adams and Danziger, 1953; Blackshaw, 1970). However, studies undertaken to demonstrate alkaline phosphatase activity in the mature mammalian testis have given conflicting results ranging from absence of activity in the germinal cells of the hamster and the rabbit to positive activity in the rat, *Department of Preclinical Studies, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka. Received 9 January 1989. Revised 15 December 1989. 113
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
TISSUE
fore aimed at localising the sites of alkaline phosphatase activity within the intertubular elements of the testis in the domestic fowl.
OF TESTIS
115
tome, and stained with uranyl acetate examination with an electron microscope.
for
Results Materials and Methods Testicular tissue was obtained from sexually mature White Leghorn cocks which were killed by an overdose of pentobarbitone sodium (Nembutal, Abbott Laboratories). For light microscopy, tissues were rapidly frozen in liquid nitrogen and sections 10 p.m thick were cut in a Cryo-Cut II (ReichertJung) maintained at -25°C. Sections were picked up on microscope slides and fixed in 80% ethanol for 1 hr. The sections were brought to water through graded alcohols and were then incubated for 3@45 min in a medium prepared according to Gomori’s method (Culling 1963) using P-glycerophosphate as the substrate. Control studies were carried out using a substrate-free medium. Subsequent procedures were similar to Gomori’s technique except that polyvinylpyrrolidone was used as the mountant. For ultracytochemical studies, material was fixed by immersion in 2% glutaraldehyde in O-1 M cacodylate buffer with 3% sucrose. After fixation for 30min the tissue was sectioned on a vibrotome. Sections 501_~m in thickness were incubated in a medium devised for demonstrating sites of alkaline phosphatase activity (Mayahara, Hirano, Saito and Ogawa, 1967) for periods of time ranging from 1&30min. For control studies sections were incubated in a substrate-free medium. Following incubation the sections were postfixed in 1% osmium tetroxide in cacodylate buffer for 60 min, dehydrated through ethyl alcohol and propylene oxide and embedded in Spurr’s medium (Spurr 1969). Thin sections were cut on an ultra-
Fig. 1. A light microscope bular tissue. x60.
Sections incubated for a period of 30 minutes when examined under the light microscope showed intense staining for alkaline phosphatase in the intertubular tissue (Fig. 1). Ultrastructurally three cell types were recognised in the interstitium and all three types exhibited alkaline phosphatase activity on their cell membranes. The reaction products of enzyme activity were deposited as electron dense beads on the outer surface of the cell membrane. The three cell types are designated as types.1, II and III. The type I cell was irregular (Fig. 2) or elongated (Fig. 3) in shape with a large irregular nucleus almost completely filling the cell. These cells corresponded to fibroblasts. Small or large pinocytotic vesicles containing reaction products were seen in the scanty cytoplasm. The cell classified as type II was irregularly rounded and a few vacuoles showing enzyme activity on their lining membranes were located in the cytoplasm (Fig. 4). This figure also illustrates the formation of a vacuole by the union of short cell processes. In the type III or Leydig cell the cytoplasm was more abundant and vacuolation was a salient feature. The vacuoles had a smooth outline and did not show any enzyme activity (Fig. 5). Mitochondria with tubular cristae were observed between some of the vacuoles. Pinocytotic vesicles were sometimes present, and these contained reaction product (Fig. 2). The blood vessels in the interstitium showed scattered activity in the basement membrane and in some pinocytotic vesicles in the endothelium. More pronounced
section showing intense alkaline phosphatase
activity in the intertu-
Fig. 2. Fibroblast from the interstitium. Note the enzyme activity on the cell membrane. A fragment of Leydig cell cytoplasm with lipid droplets (L) is also shown. Arrows indicate pinocytotic vesicles. X23.000. Fig. 3. Fibroblast from the interstitium. Note the enzyme activity on the cell membrane. Arrow indicates the formation of a pinocytotic vesicle. L-lipid droplet in Leydig cell cytoplasm x34.000.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
activity was seen in the cell processes surrounding the blood vessels (Fig. 6). Moderate enzyme activity was observed on the surface of the red cell. The peritubular tissue is composed of two cell types, the fibroblasts located as an inner cellular layer and the outer layer of myoid cells (Rothwell and Tingari, 1973). Both cell types showed elongated cell processes, but were distinguished from one another on the basis of their cytoplasmic components. The fibroblast cell processes possessed organelles such as endoplasmic reticulum, ribosomes and mitochondria whereas the myoid cell processes had scant organelles and the cytoplasm was filamentous or fibrillar. The myoid cell processes were devoid of any alkaline phosphatase activity (Fig. 7). However, the fibroblast cell processes showed beads of enzyme activity on their cell membranes (Fig. 8). A third category of cell process was similar to the fibroblast cell process but showed prominent activity in pinocytotic vesicles with little or no activity in the cell membrane (Fig. 9). In the cell body of the myoid cells the cell membranes were free of enzyme activity but moderate or faint activity could occasionally be detected in the pinocytotic vesicles (Fig. 10). In sections incubated in a substrate-free medium enzyme activity was absent. Discussion Several investigations have been carried out on the histochemistry of testicular enzymes (Blackshaw 1970). Studies dealing with enzymes in the interstitial tissue have concentrated mainly on those enzymes concerned with steroid synthesis (Woods and Domm, 1966; Blackshaw, 1970). The available reports on the localisation of alkaline phos-
TISSUE OF TESTIS
117
phatase in the interstitial tissue include the work of Lake (1962) on the domestic fowl and Roussel and Stallcup (1966) on the developing bovine testis. Lake, utilising the diazo coupling technique demonstrated that the intertubular tissue had pronounced alkaline phosphatase activity. The present light microscope observations based on the lead capture method of Gomori paralleled the results obtained by Lake (1962). However ultracytochemical studies are required for precise localisation of enzymes and as far as can be ascertained the present work is the only investigation pertaining to the ultrastructural localisation of alkaline phosphatase in the interstitial tissue of the testis in the domestic fowl. The ultrastructure of the interstitial tissue has received the attention of several workers. and a variety of species have been investigated (Burgos, Vitale-Calpe and Aoki. 1970). Connel(1972) referred to the presence of Leydig cells, transitional cells and connective tissue cells in the interstitial tissue of the chick testis. Similar results were recorded by Rothwell (1973) for the developing and adult testis of the domestic fowl. The presence of three cell types is also confirmed by the current findings. Furthermore the positive alkaline phosphatase activity on the cell membranes of all three cell types supports the contention that Leydig cells originate from a fibroblast-like cell through a transitional form to a mature Leydig cell as has been suggested in mammals (Fawcett and Burgos. 1960; Black and Christensen, 1969). On the basis of enzyme activity two types of vacuoles could be identified. It was apparent that the vacuoles lined by the reaction products of alkaline phosphatase activity were derived from the cell membrane. It may be postulated that these vacuoles formed
Fig. 4. Transitional cell showing enzyme activity on the cell membrane. Note the vacuoles (V) lined by reaction product. Arrows indicate the formation of vacuoles. x29.500. Fig. 5. Leydig cell showing enzyme activity on the cell membrane. activity in the lipid droplets L. M-mitochondria. X 14,000.
Note the absence of
Fig. 6. Blood vessel from the interstitium. Note the pronounced activity in the cell process (P) and moderate activity on the red cell (arrow heads). Arrows indicate pmocytotic vesicles. ~23,500.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
TISSUE
either by invagination of the cell membrane or by the union of cell processes as indicated by Fig. 4. This type of vacuole was predominant in the transitional cell. On the other hand. those vacuoles which were free of enzyme activity and distributed abundantly in the Leydig cells were evidently lipid droplets. Both Connel(1972) and Rothwell (1973) considered lipid droplets in the Leydig cells to represent a pool of steroid precursor rather than storage of newly synthesised steroid. In a review dealing with avian male reproduction Lake (1981) has stated that it is probable that the lipid in the Leydig cells represents precursor material involved in steroid production, since they are cholesterol positive and are strongly birefringent. In the light of these observations it is conceivable that the enzyme positive vacuoles in the transitional cells could perhaps reflect the early stages in the intake of steroid precursor into the cell. In this context it is relevant to note here that alkaline phosphatase on membranes is thought to be concerned with active transport across membranes (Kaplan, 1972). and that it is also one of the epithelial enzymes associated with fat absorption by the intestinal epithelium (Yoshizawa, Mizumoto, Nakase and Honjo, 1976). Alkaline phosphatase in transitional cells subserving a similar function cannot be completely overruled. Localisation of alkaline phosphatase in the peritubular tissue of the testis has been reported in the rat (Tice and Barrnett, 1963) and the hamster (Chang et al., 1974). In a previous report on the domestic fowl Gunawardana (1985) noted that the peritubular cell processes demonstrated enzyme activity and it was presumed that the cell processes were those of myoid cells. However the present study indicates that the myoid cells are relati-
Fig. 7. Myold cell processes. x 16,000. Fig. 8. Fibroblast x24.500. Fig. Y. Fibroblast
processes
Note the ftbrillar cytoplasm
vely free of enzyme activity. Those cell proactivity are cesses showing enzyme considered to be fibroblast processes. The distribution of the enzyme in pinocytotic vesicles is comparable to the situation described in the testis of the rat and the hamster. The presence of enzymes in these processes is particularly noteworthy since as mentioned earlier it is generally agreed that alkaline phosphatase is involved in the transmembrane transfer of organic molecules (Kaplan, 1972; Adams, 1983). The kidney, intestine and placenta are among tissues which are well established as containing pronounced alkaline phosphatase activity (Mayahara et al.. 1967; Sheilds, Bates, Yedlin and Best, 1984; Soares, 1987). These are all sites where transmembrane transport is considerable. The interstitium of the testis exemplifies another situation where an efficient transmembrane transfer facility is an essential requirement. In this connection it is pertinent to recall that the seminiferous tubules are not penetrated by blood vessels, and yet, a high concentration of testosterone bathes the seminiferous tubules. This implies the transmembrane transfer of testosterone from the interstitium to the tubules. Furthermore, it appears very likely that material passes from the tubules to the interstitium, since there is now ample evidence to indicate that substances secreted by the Sertoli cells regulate Leydig cell function (Sharpe, 1984; de Kretser, 1986). Thus it seems very probable that alkaline phosphatase in the membranes of the Leydig cells and in the cell processes aid in this two-way transfer of material between Leydig cells and the tubules, possibly the Sertoli cells. This is further substantiated by the earlier finding of alkaline phosphatase in the Sertoli cell membranes in the domestic fowl (Gunawar-
and the absence of enzyme activity.
showing enzyme acttvity on the cell membranes.
processes showing enzyme activity in pinocytotic
Fig. IO. Myoid cells. Cell membranes vesicles. x19.0(x).
119
OF TESTIS
R-ribosomes.
vesicles (arrows).
~28,000.
are free of enzyme activity. Arrows indicate pinocytotic
120
dana, 1985) and in the rat (Kornblatt, Klugerman and Nagy, 1983). The present work also revealed enzyme activity in the blood vessels. This is to be expected since transfer of materials such as hormones takes place between the interstitial cells and the blood vessels as well. Moreover steroid precursors also reach the interstitial cells by way of these blood vessels. Three isozymes of alkaline phosphatase viz. intestinal, liver-bone-kidney and placental have been distinguished in mammals, on the basis of heat sensitivity and inhibition by certain amino acids (Kornblatt et al., 1983). It is likely that isozymes corresponding to intestinal and liver-bone-kidney forms occur also in the domestic fowl. Characterisation of the testicular alkaline phosphatase in the rat has revealed it to be similar to the liver-
GUNAWARDANA
bone-kidney isozyme (Kornblatt etal., 1988). Related studies in the domestic fowl would be of interest, particularly since the studies in the rat appear to refer to an isozyme of alkaline phosphatase which is located within the seminiferous tubules and mainly in the Sertoli cells, thereby implying that similar studies on interstitial cells are lacking. Acknowledgements
I wish to thank Mr M. A. Navaratne for technical assistance. The electron micrographs were taken in the laboratories of the Department of Clinical Veterinary Medicine, University of Cambridge, England. I am grateful to Dr W. F. Blakemore for providing the facilities and to Mr Robert Patterson for his valuable assistance.
Adams, C. S. 1983. Localisation of alkaline phosphatase and NADH diaphorase in the principal cells of the guinea pig epididymis. Acta. Anaf., 116, 146151. Black, V. H.’ and Christensen, A. K. 1969. Differentiation of interstitial cells and Sertoli cells in foetal guinea pig testes. Amer. J. Amt., 124,211-238. Blackshaw, A. W. 1970. Histochemical localization of testicular enzymes. In The Testis (eds A. D. Johnson, W. R. Gomes and N. L. Vandemark), Vol. II, pp.73-123, Academic Press, New York and London. Burgos, M. H., Vitale-Calpe, R. and Aoki, A. 1970. Fine structure of the testis and its functional significance. In The Testis (eds A. D. Johnson, W. R. Games and N. L. Vandemark), Vol. 1, pp. 551-649, Academic Press, New York and London. Chang, J. P., Yokoyama, M., Brinkley, B. R. and Mayahara. H. 1974. Electron microscopic cytochemical study of phosphatases during spermiogencsis in Chinese hamster. Bio[. Reprod., 11,6O-610. Connell, C. J. 1972. The effect of luteinizing hormone on the ultrastructure of the Lcydig cell of the chick. Z. ZeNforsch., 128,13%151. Culling, C. F. A. 1963. Enzymes. In Handbook of Histopathological Techniques, 2nd ed., pp. 269-288, Butterworths. London. Fawcett, D. W. and Burgos, M. H. 1960. Studies on the tine structure of the mammalian testis. 11. The human interstitial tissue. Amer. J. Amt., 107, 245-269. Gunawardana, V. K. 1985. Ultrastructural localisation of alkaline phosphatase in the testis of the domestic fowl. J. Am., 140,711-718. Kaplan, M. M. 1972. Alkaline phosphatase. New-Engl. J. Med., 286,2m202. de Kretser, D. M. 1986. The testis. In Reproduction in Mammals: 3. Hormonal Control of Reproducrion. (eds C. R. Austin and R. V. Short), pp. 76-90. Cambridge University Press, Cambridge. Kornblatt, M. J., Klugerman, A. and Nagy, F. 1983. Characterization and localization of alkaline phosphatase activity in rat testes. Biol. Reprod., 29, 157-164. Lake, P. E. 1962. Histochemical demonstration of phosphomonoesterasc secretion in tht genital tract of the domestic cock. J. Reprod. Fert., 3,356362. Lake, P. E. 1981. Male genital organs. In Form and Function in Bin&. Vol. II. (eds. A. S King and J. McLellans). pp. 1-61. Academic Press, New York, London. Mayahara, H., Hirano, H., Saito, T. and Ogawa, K. 1967. The new lead citrate method for the ultracytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase). Histochemie, 11, 88-96. McComb, R. B., Bowers, G. N. Jr. and Posen, S. 1979. Cited by Kornblatt eral., 1983. McKay, D. G.. Hertig, A. T., Adams, E. C. and Danziger, S. 1953. Histochemical observations on the germ cells of human embryos. Amt. Rec., 117,201-219. Rothwell, B. 1973. The ultrastructure of Leydig cells in the testis of the domestic fowl. J. Amt., 116,245-253.
ALKALINE
PHOSPHATASE
IN INTERTUBULAR
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OF TESTIS
Rothwell, B. and Tingari, M. D. 1973. The ultrastructure of the boundary testis of the domestic fowl (Gallus domesticus). J. Amt., 114,921-328.
121 tissue of the seminiferous
tubule
in the
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