Temporal and Spatial Distribution of Alkaline Phosphatase Activity in Male Hypogonadic Rat (hgn/hgn) Testis during Postnatal Development

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1 Temporal and Spatial Distribution of Alkaline Phosphatase Activity in Male Hypogonadic Rat (hgn/hgn) Testis during Postnatal Development Hiroetsu SUZUKI, Masahiro INABA and Katsushi SUZUKI Department of Veterinary Physiology, Nippon Veterinary and Animal Science University, Kyonan-cho, Musashino-shi, Tokyo , Japan (Received 30 October 1997/Accepted 27 January 1998) ABSTRACT. In male hypogonadic mutant rat (hgn/hgn), gonocytes degenerate and peritubular cells form multiple layers around seminiferous tubules during early postnatal testicular development. Alkaline phosphatase (AP) activity has been used as not only a tracer for the primordial germ cells (PGCs) but also a histochemical marker for the peritubular myoid cells. In the present study, we examined the localization of AP activity during the postnatal testicular development in the hgn/hgn and phenotypically normal (+/+ or +/hgn) rat. In the normal testis, high AP activity was located in the surface of the PGCs on 3 days of age. As the PGCs differentiated into spermatogonia, the AP activity drastically decreased in intensity on 7 days and was completely lost by 12 days. In the hgn/hgn, the PGCs showing high AP activity occupied the inside of dilated seminiferous tubules on 3 and 7 days of age. The luminal AP activity declined gradually by 18 days and disappeared on 21 days, when the germ cells completely degenerated in the hgn/hgn testis. In the normal, high AP activity emerged in a layer of the peritubular cells surrounding the tubules on 7 days and afterwards, indicating that the peritubular cells were differentiating into myoid cells. In the hgn/hgn, the peritubular cells formed multiple layers around the tubules and showed weak AP activity on 3 to 18 days of age. On 21 days of age, high AP activity emerged in a single layer of the peritubular cells directly attached to the basement membrane in the hgn/hgn. These results indicate that the PGCs showing AP activity kept remained at later stage in the hgn/ hgn and that the single layer of mature myoid cells showing high AP activity appeared much later in the hgn/hgn testis than in normal. KEY WORDS: alkaline phosphatase, hypogonadism, peritubular myoid cell, primordial germ cell, testicular development. J. Vet. Med. Sci. 60(6): , 1998 Hypogonadic rat (hgn/hgn) shows male sterility [31], female reduced fertility [28], growth retardation [28, 32], and bilateral hypoplastic kidney [29, 33] as a single autosomal recessive trait. The primary cause of the male sterility in the mutant rat appears to exist in the testis in itself [8, 32]. The testicular pathogenesis has prenatally started and the gonocytes are lost during the postnatal stage [32]. Low testosterone and high gonadotropin levels are detected in the adult hgn/hgn serum [8]. Leydig cells make an islet-like conformation around the seminiferous tubules in the adult hgn/hgn testis [31] In the normal rat testis, the primitive sex cords develop into the mature seminiferous tubules during the early postnatal stage. Morphological changes in the tubules include the mitotic quiescence of the primordial germ cells (PGCs), the proliferation of the supporting cells, the formation of well-organized single cell layer of the Sertoli cells along the basement membrane, the reinitiation of the gonocyte mitosis, the movement of the gonocyte to the basement membrane, and the differentiation of the PGCs into the spermatogonia [1, 3, 17, 18, 20, 27]. In the interstitial tissue, the differentiation of the peritubular myoid cells, the maturation of the basement membrane [14, 23, 26], and the appearance of adult type Leydig cells [13] occurred in the developing testis. These events would be regulated by extratesticular factors and various type of cellcell and cell-matrix interactions in developing testis. The postnatal pathogenesis in the hgn/hgn testis includes the failure in the proliferation and distribution of the Sertoli cells, the abnormal cell division of the gonocytes, the degeneration of the gonocytes, and the formation of multiple layers of the peritubular cells around the seminiferous tubules [32]. Alkaline phosphatases (APs) are membranelinked enzymes that hydrolyze orthophosphate monoesters at alkaline ph [4]. In normal murine testis, AP activity is predominantly localized in the gonocytes and the myoid cells [2, 11, 16, 22]. In this study, we examine the temporal and spatial distribution of AP activity in the postnatal hgn/ hgn and normal (+/+ or hgn/hgn) testis to investigate the distribution and development of the hgn/hgn gonocytes and peritubular myoid cells. MATERIALS AND METHODS Animals: Male normal and hgn/hgn rats used in this study were obtained from matings between proven +/hgn males and females derived from the 21st generation of hypogonadic rat (HGN) line maintained in our department [29]. The rats were kept in a conventional animal room, and were fed a mixed grain diet and water ad libitum [31]. Preparation of cryosectioned testis samples: The rats were sacrificed on 3, 7, 12, 18, 21, and 40 days after birth by overdose ether. The testis were removed, weighed to distinguish affected rat from normal one [32, 33], and embedded in a Tissue Tek [Miles Lob.], and frozen in dry ice acetone. Five 10 µm sections were cut in a cryostat. After mounting on poly-l-lysine-coated slides, the sections were dried up by cool air from a drier. The sections were

2 672 H. SUZUKI, M. INABA AND K. SUZUKI fixed in cold acetone/ethanol fixative (1:1 v/v) for 10 min at 20 C. Histochemical localization of alkaline phosphatase: AP activity was detected by a co-coupling method [22]. The sections were soaked in 0.1 M PBS (phosphate buffered saline; ph 7.4), and then were incubated in 0.1 M Tris-HCl (ph 8.5) containing Fast Blue RR (0.6 mg/ml) and Naphthol AS-BI phosphate (1 mg/ml) for 30 min under a dark condition. Then the samples were rinsed in water, counterstained in 1% Nuclear Fast Red, rinsed in water, and mounted in glycerin/gelatin. The substrate for AP was omitted in the negative controls. The samples were examined under a light microscope. RESULTS In both genotypes, AP activity was located in gonocytes, peritubular myoid cells, and vascular endothelium. On 3 days of age, in the normal testis, high AP activity was detected in the PGCs located in the central region of the lumen of the seminiferous tubules (Fig. 1a). The Sertoli cells aligned with a well organized single cell layer along the basement membrane (Fig. 1c). In the hgn/hgn, the APpositive germ cells occupied the inside of the dilated tubules (Fig. 1b, d). The population of the germ cells within a section of the tubules was larger in the hgn/hgn than in the normal. On 7 days of age, in the normal testis, luminal AP activity disappeared in most of the tubules. Some of the peritubular cells expressed AP activity (Fig. 2a, c). In the hgn/hgn, the germ cells within the lumen of the tubules still expressed high AP activity. Most of the cells forming the multiple layers in the peritubular region showed weak AP activity (Fig. 2b, d). On 12 days of age, in the normal, AP activity was not detected at all within the seminiferous tubules. AP activity in the peritubular cells became higher on 12 days than on 7 days (Fig. 3a, c). In the hgn/hgn, weak AP activity still remained within the tubules. Although some of the multi-layered cells around the tubules expressed weak AP activity, normal myoid cells showing high AP activity could not be found (Fig. 3b, d). On 18 and 21 days of age, the location of AP activity was similar as that on the 12 days of age in the normal (Fig. 4a, c; Fig. 5a, c). In the hgn/hgn, as the age advanced, AP activity within the tubules became weaker (Fig. 4b, d). On 21 days of age, the AP activity within the tubular lumen was completely lost and a single layer of the cells showing high AP activity appeared around the basement membrane of the seminiferous tubules in the hgn/hgn (Fig. 5b, d). On 40 days of age, in the normal, high AP activity was recognized in the myoid cells and only a few spermatogonia showed weak AP activity (Fig. 6a, c). In the hgn/hgn, implausible cells remaining within the tubules were negative for AP. Some of the single layered peritubular cells around the tubules expressed high AP activity (Fig. 6b, d). The cells in the outer region of the AP-positive single layered cells showed esterase activity in the hgn/hgn (data not shown), suggesting that they would be adult type Leydig cells [8, 10]. DISCUSSION AP activity has been often used as a marker for PGCs [6, 16, 19]. Some reports have indicated that AP activity is associated with some of the spermatogonia in adult testis [2, 25]. It occurred but very infrequent in the present study, as shown in Fig. 6c. Our results indicated that in the normal rat testis AP activity in most of the germ cells disappeared during the early postnatal stage, when the PGCs move into the basal compartment, colonized there, and differentiated into the spermatogonia. In the hgn/hgn testis, these processes are severely affected [32]. Thus, it is reasonable that the gonocytes remained AP activity at later stage of the postnatal testicular development in the hgn/hgn. Our previous report revealed that abnormal and degenerating mitotic metaphase was often observed in the gonocytes and that most of the gonocytes degenerated until 18 days of age in the hgn/hgn testis [32]. In this study, AP activity remained until 18 days of age in the hgn/hgn testis, suggesting that the remaining germ cells in the hgn/hgn testis would be PGCs and could not differentiate into the normal spermatogonia. The W/+ (white spotting) and Sl/+ (steel) mutant mice have been known to have defects in the migration and proliferation of the PGCs [15, 19]. The grc/ grc (growth and reproduction complex) mutant rat has been reported to have an abnormal arrest in spermatogenesis at the early pachytene stage [5]. The hgn/hgn is very unique to the point of that the PGCs arrived into the gonads but degenerated before entering meiosis. Details of the mechanisms regulating the differentiation from PGCs into spermatogonia have not known yet, since the culture system that proceeds this developmental process has not been established completely [9, 21, 24]. Unknown factors required in this developmental process of the PGCs would be primarily defected or secondarily affected by the dysfunction of testicular somatic cells in the hgn/hgn. In general, AP activity has also been used as a cytochemical marker for the myoid cells [2, 11, 22, 23]. In the normal rat testis, weak AP activity was localized in the single layered cells surrounding the seminiferous tubules at 3 days of age and high AP activity in these cells was emerged at 7 days and afterwards. These results are almost comparable with those previously reported by others [23]. Our previous report indicated that the peritubular cells formed multi-layers around the tubules in the hgn/hgn testis [32]. Therefore, it was expected that the developmental process of the myoid cells would be defective in the hgn/ hgn testis. In this reports, although these multi-layered cells showed weak AP activity, all of them could not develop into the mature myoid cells showing high AP activity. On 21 days of age, however, high AP activity was detected in only the single-layered cells attached to the basement membrane of the tubules in the hgn/hgn testis. Moreover, our recent study indicated that the deposition of desmin, marker for peritubular cells, could not be detected in the

3 AP ACTIVITY IN HYPOGONADIC RAT TESTIS 673 Fig. 1. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 3 days of age (a, b: 200, c, d: 700). In the normal testis, AP activity is recognized in the gonocytes located in the central region of the tubules but not in the Sertoli cells forming palisade-like distribution along the basement membrane. In the interstitial spaces, the vascular endothelium and some of peritubular cells are AP positive (c). In the hgn/hgn, the lumina of the dilated tubules are full of the AP positive germ cells. The Sertoli cells could not form palisade-like distribution. A few layered peritubular cells have weak AP activity (d). T: seminiferous tubules; I: interstitial tissues; G: gonocytes; S: Sertoli cells; P: peritubular cells; B: basement membrane and V: blood vessels. multi-layered peritubular cells in the hgn/hgn testis on 7 and 12 days by the immuno-staining method [30]. These results suggest that most of the multi-layered cells would degenerate or turn into the interstitial fibrous cells and that

4 674 H. SUZUKI, M. INABA AND K. SUZUKI Fig. 2. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 7 days of age. In the normal, in some seminiferous tubules, a single layered peritubular cells show high AP activity, indicating the sign of the differentiation into the mature myoid cells. Weak AP activity remains in a few germ cells on the way that they would be moving from the central region of the tubules to the basal compartment (a, c). In the hgn/hgn, the germ cells showing high AP activity occupied the whole area of the tubular lumen. The cells forming multiple layers in the peritubular region also shows weak AP activity (b, d). Abbreviations except M for peritubular myoid cells and magnifications are the same as Fig. 1. only the peritubular cells attached to the basement membrane could differentiated into the mature myoid cells. At present, we can not explain why the peritubular cells form multiple layers in the hgn/hgn testis. However, it may be possible that the proliferation of the peritubular cells or the contribution of the interstitial cells into the peritubular

5 AP ACTIVITY IN HYPOGONADIC RAT TESTIS 675 Fig. 3. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 12 days of age. In the normal, AP activity completely disappears in the lumen and the single layered myoid cells show the high AP activity in all the cross section of the tubules (a, c). In the hgn/hgn, AP activity is recognized in the germ cells in the tubular lumen and some of the multi-layered cells around the tubules (b, d). Abbreviations and magnifications are the same as Fig. 2. cell population would be in excess since the tubular elongation is severely affected in the hgn/hgn. Recently, we observed that the basement membrane-like structure [7, 12, 14] could not be formed outer side of the peritubular cells during this period in the hgn/hgn testis [30]. This defect would be associated with the failure in the polarization and differentiation of the peritubular cells. The reduced testosterone contents in the hgn/hgn testis on 12

6 676 H. SUZUKI, M. INABA AND K. SUZUKI Fig. 4. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 18 days of age. In the normal, the distribution of AP activity is almost similar as that on 12 days of age and high AP activity is recognized in the myoid cells (a, c). In the hgn/hgn, the intensity of AP activities in the tubular lumen and peritubular tissue is less than that of 12 days of age. Abbreviations and magnifications are the same as Fig. 3. days and afterwards [8, 32] may be associated with the failure of the differentiation of the peritubular cells into the mature myoid cells [11, 12]. REFERENCES 1. Beaumont, H. M. and Mandl, A.M A quantitative study of primordial germ cells in the male rat. J. Embyol. Exp.

7 AP ACTIVITY IN HYPOGONADIC RAT TESTIS 677 Fig. 5. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 21 days of age. In the hgn/hgn, no AP activity remains in the tubular lumen. High AP activity emerges in the peritubular cells forming a single layer attached to the basement membrane of the tubules (d). Abbreviations except R for rete testis and magnifications are the same as Fig. 3. Morphol. 11: Chapin, R. E., Phelps, J. L., Miller, B. E. and Gray, T. B Alkaline hosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture. J. Androl. 8: Clermont, Y. and Perey, B Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 100: Fröhlander, N. and Millan, J Alkaline phosphatases as reporters of developmental and cancerous events. In Vivo 5: Gill, T. J. III and Kunz, H. W Gene complex control-

8 678 H. SUZUKI, M. INABA AND K. SUZUKI Fig. 6. Distribution of AP activity in the normal (a, c) and hgn/hgn (b, d) testis on 40 days of age. Distribution of AP activity is almost similar as that of 21 days of age in both genotypes (a, b). A single layered cells showing high AP activity are found also in the hgn/hgn testis (d). Abbreviations except L for Leydig cells and magnifications are the same as Fig. 3. An arrow shows the spermatogonia with high AP activity. ling growth and fertility linked to the major histocompatibility complex in the rat. Am. J. Pathol. 96: Ginsburg, M., Snow, M. H. and Mclaren, A Primordial germ cells in the mouse embryo during gastrulation. Development 110: Hadley, M. A. and Dym, M Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization. Biol. Reprod. 37: Hakamata,Y., Kikukawa, K., Kamei, T., Suzuki, K., Taya, K.

9 AP ACTIVITY IN HYPOGONADIC RAT TESTIS 679 and Sasamoto, S A new male hypogonadism mutant rat (hgn/hgn): Concentrations of testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the serum and the responsiveness of accessory sex organs to exogenous T, FSH, human chorionic gonadotropin, and luteinizing hormone-releasing hormone. Biol. Reprod. 38: Hofman, M-C., Hess, R.A., Goldberg, E. and Milan, J.L Immortalized germ cells undergo meiosis in vitro. Proc. Natl. Acad. Sci. U.S.A. 91: Hooker, C. W The interstitial tissue of the testis. pp In: The Testis, vol. 1 (Johnson, A. D., Gomes, W. R. and Vandemark, N. L. eds.), Academic Press, New York and London. 11. Hovatta, O Effect of androgens and antiandrogens on the development of the myoid cells of the rat seminiferous tubules (organ culture). Z. Zellforsch. Mikrosk. Anat. 131: Kretser, D.M., Kerr, J.B. and Paulsen, C.A The peritubular tissue in the normal and pathological human testis. An ultrastructural study. Biol. Reprod. 12: Kuopio, T., Tapanainen, J., Pelliniemi, L. J. and Huhtaniemi, I Developmental stage of fetal-type Leydig cells in prepubertal rats. Development 107: Lesson, C. R. and Lesson, T. S The postnatal development and differentiation of the boundary tissue of the seminiferous tubule of the rat. Anat. Rec. 147: Motro, B., Kooy, DVD., Rossant, J., Reith, A. and Berstein, A Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci. Development 113: Macgregor, G. R., Zambrwicz, B.P. and Soriano, P Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 121: Mcguinness, M. P. and Orth, J. M Gonocytes of male rats resume migratory activity postnatally. Eur. J. Cell Biol. 59: Mcguinness, M. P. and Orth, J. M Reinitation of gonocyte mitosis and movement of gonocytes to the basement membrane in testes of newborn rats in vivo and in vitro. Anat. Rec. 233: Mintz, B.and Russel, E. S Gene induced embryological modifications of primordial germ cells in the mouse. J. Exp. Zool. 134: Orth, J. M Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat. Rec. 203: Orth, J. M. and Mcguinness, M. P Neonatal gonocytes co-cultured with Sertoli cells on a laminin-containing matrix resume mitosis and elongate. Endocrinology 129: Palombi, F. and Carlo, C. D Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue. Biol. Reprod. 39: Palombi, F., Faini, D., Salanova, M., Grossi, S.D. and Stefanini, M Development and cytodifferentiation of peritubular myoid cells in the rat testis. Anat. Rec. 233: Rassoulzadegan, M., Paquis-Fluckinger, V., Bertino, B., Sage, J., Jasin, M., Miyagawa, K., Van Heyningern, V., Besmer, P. and Cuzin, F Transmeiotic differentiation of male germ cells in culture. Cell 75: Redi, C.A., Hilscer, B. and Winking, H Stage-dependent enzymatic activities in spermatogenesis of mice with the standard karyotype and of chromosomal variants with impaired fertility. Andrologia 15: Ross, M. H The fine structure and development of the peritubular contractile cell component in the seminiferous tubules of the mouse. Am. J. Anat. 121: Spasford, C. S Changes in the cells of the sex cords and seminiferous tubules during the development of the testis of the rat and mouse. Aust. J. Zool. 10: Suzuki, H., Hakamata, Y., Kamei, T., Kikukawa, K. and Suzuki, K Reproduced fertility in female homozygotes for hgn (male hypogonadism) selected by hgn-associated hypoplastic kidney. Cong. Anom. 32: Suzuki, H. and Suzuki, K Pathophysiology and postnatal pathogenesis of hypoplastic kidney (hpk/hpk) in the male hypogonadic mutant rat (hgn/hgn). J. Vet. Med. Sci. 57: Suzuki, H., Inaba, M. and Suzuki, K The primordial germ cells of male hypogonadic (hgn/hgn) rat fail to differentiate into spermatogonia and multi-layered peritubular cells lack outer basement membrane. Teratology 54: 43A (Abst.) 31. Suzuki, K., Hakamata, Y., Hamada, A., Kikukawa, K., Wada, M. Y. and Imamicni, T Male hypogonadism as a candidate of deficiency of postnatal testicular growth or differentiating foctors: A new autosomal recessive mutation in the rat. J. Hered. 79: Suzuki, K., Hakamata, Y., Suzuki, H., Taya, K. and Sasamoto, S Postnatal alterations of testicular morphology, testicular testosterone contents and plasma hormone levels in male hypogonadic rat (hgn/hgn). J. Reprod. Dev. 39: Suzuki, K., Suzuki, H., Hakamata, Y., Kamei, K. and Kikukawa, K Genetic analysis and histology of hypoplastic kidneys in the male hypogonadic mutant (hgn/hgn) rat. Cong. Anom. 31:

Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos

/. Embryol. exp. Morph. Vol. 64, pp. 133-147, 1981 133 Printed in Great Britain Company of Biologists Limited 1981 Proliferation and migration of primordial germ cells during compensatory growth in mouse