J. Anat. (1982), 14, 2, pp. 351-363 351With 7 figuresPrinted in Great Britain

Morphological and histometric study on the human Sertoli cellfrom birth to the onset of puberty*

MANUtL NISTAL, MARIA ASUNCI6N ABAURREAAND RICARDO PANIAGUA

Department ofMorphology, School of Medicine andDepartment of Cytology, Autonomous University of Madrid, Spain

(Accepted 26 March 1981)

INTRODUCTION

The importance of the Sertoli cell in the process of spermatogenesis has beenemphasized by numerous authors (see Steinberger & Steinberger, 1977), althoughits precise function relative to the developing germ cells is still not well understood.Its resistance to morphological change during changes in the testis has been describedafter irradiation, hypophysectomy, oestrogen and FSH treatment (Clermont &Perey., 1957; Heller, O'Keefe & Heller, 1968). In addition, the Sertoli cell does notundergo mitotic division following sexual maturity although it has been reportedthat Sertoli cells during infancy pass through a period of cellular proliferation(Vilar, 1970). Based on this stability, Sertoli cells have been proposed as a referencecell or constant compensating factor in the quantitation of the germinal epitheliumunder different physiological or pathological conditions (Clermont & Harvey, 1967;Rowley & Heller, 1971).

It has been claimed that the average number of Sertoli cells per transverse tubularsection is 10+ 0-3 in normal adult human testes (Rowley & Heller, 1971). Withregard to the Sertoli cell number during infancy and childhood, Hadziselimovic &Seguchi (1974) have referred to a continuous decrease from approximately 28Sertoli cells per transverse tubular section in the newborn to 10 Sertoli cells in theadult. However, the total Sertoli cell number per testis remains unknown, since arelation between the length and diameter of the seminiferous tubules during infancyand childhood has not been established.The present report concerns the morphology and number of Sertoli cells from

birth to the onset of puberty. In this study, changes in different testicular parametersare investigated, using quantitative histometric techniques successfully used inmammalian testes (Weissbach & Ibach, 1976; Kaler & Neaves, 1978), to elucidatevariations in the absolute Sertoli cell number from birth to the onset of puberty.

MATERIAL AND METHODS

Testes from 60 autopsies were selected for this study. The autopsies were onpatients without testicular pathology who had died from causes other than chronicor hormonal diseases. The total number of autopsies included ten males at each ofthe following ages: newborn, 2, 3, 7, 8 and 11 years.For light microscopy, testes were fixed in 10 % buffered formalin solution (24*Reprint requests to Dr Manuel Nistal, Departmento de Morfologla, Facultad de Medicina,

C/Arzobispo Morcillo, 4. Madrid - 34, Spain.

352 M. NISTAL, MARIA ABAURREA AND R. PANIAGUA

hours). Half of the testes were sectioned through the longitudinal axis (Fig. 1 A);the other half were cut through the transverse equatorial plane (Fig. 1 B). Thesesections were used to measure both testicular axes (a and b) and thus estimate thetesticular parenchymal volume. Slices (2 mm in width) of these testes, parallel tothese planes of section, were later cut, fixed in formalin (48 hours) and embedded inparaffin. Sections were cut at 6,m and stained with haematoxylin and eosin, PASand Masson trichrome.For the histometric study, 50 sections of each testis were selected at random. In

30 randomly selected areas of each section, the following parameters were measured:(1) Number of seminiferous tubules per unit area of section (nt), observed with thex 20 objective over an area of 0 64 mm2. (2) Mean tubular diameter (di) (according toMack, Scott, Ferguson-Smith & Lennox, 1961), using a calibrated vernier ocularmicrometer with the x 40 objective. (3) Sertoli nuclear number per transverse tubularsection (ne), counting only nuclei perfectly focused. (4) Mean Sertoli nuclear axes(c and d) were determined in a similar way, using the same ocular micrometer, withthe x 100 objective. The irregular shape of Sertoli cell cytoplasm led us to considernuclear axes rather than the whole cell axes.The volume of testicular parenchyma after fixation (vt) was estimated considering

it as an ellipsoid. Length (a) and breadth (b) of the testicular parenchyma weremeasured and transformed into volume (nab2/6). This calculation was carried out inall the testes obtained in this study, together with those from 70 other autopsies ofnormal children and infants, sectioned in a similar way.The proportion of the volume, occupied by seminiferous tubules (Pt) was calcu-

lated by multiplying the mean area of the tubules in section (rda?/4) and the meannumber of tubules per unit area (nt), and by dividing this result by the unit area(640000 #sm2). The total seminiferous tubule volume per testis was then estimatedby multiplying this proportion (Pt) and the average testicular parenchymal volume(Vt).

In a similar fashion, estimation of mean Sertoli nuclear volume (v8) was calculatedby application of the same mathematical formula used in the measurement of testisvolume (icd2/6).The proportion occupied by Sertoli cell nuclei (p,) was calculated by multiplying

the area of the nuclei, considering them as ellipses, (icd/4), the mean Sertoli nuclearnumber per tubular section (n3) and the mean number of tubules per unit area (nt),and by dividing this result by the unit area (640000 ,um2). Total Sertoli cell nucleusvolume per testis was then obtained by multiplying the proportion occupied bySertoli cell nuclei (p,) and the average testicular parenchymal volume (vt).

Total Sertoli cell nucleus number per testis was determined by dividing totalSertoli nuclear volume by the average single cell nuclear volume (p,. vt/va).

Sertoli nuclear number per unit testicular parenchyma volume was determined bydividing total Sertoli nuclear number per testis by testicular volume (vt).

Fig. 1. Testes from an infant 7 months old, sectioned showing the long (A) and wide(B) testicular axes (long arrows). The tunica albuginea and the rete testis are not taken intoaccount. H & E. (A) x 4-5; (B) x 12-5.Fig. 2. Development of the seminiferous tubules from birth to 11 years of age. (A) 1 year,(B) 3 years, (C) 8 years, (D) 11 years. The Sertoli cell number per transverse tubular sectiondecreases progressively with age. The mean tubular diameter reaches a minimum at the end of3 years (B), and increases again until puberty (C) and (D). In (D), spermatocytes and somemature Sertoli cells are seen. H & E. (A, B and C) x 340; (D) x 250.

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Fig. 3. Immature Sertoli cells from an infant 3 years old. The nuclei are round, regular inoutline, with a small nucleolus. The cytoplasm shows some strands of rough endoplasmicreticulum, polysomes, a developed Golgi complex and a supranuclear centriole (c). Desmosomes(arrows) are present. x 13 000.

Comparisons of the different parameters directly measured within each age groupwere carried out by analysis of the variance.For electron microscopy, small blocks (1 mm3) of testicular tissue were fixed in

Karnovsky fixative (4 hours) and post-fixed in 1% phosphate-buffered osmiumtetroxide (2 hours), dehydrated in ethanol and embedded in Epon 812. Sections1 ,um thick were stained with toluidine blue. Ultrathin sections were double stainedwith uranyl acetate and lead citrate and subsequently studied in a Philips 300electron microscope.

RESULTSUltrastructure of Sertoli cellsSeminiferous tubules of testes from children between 0 and 11 years of age showed

immature Sertoli cells or pre-Sertoli cells that differed in morphological appearance

354

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Fig. 4. Immature Sertoli cells from a child 9 years old. The nuclei and cytoplasm are elongated.In the supranuclear region the rough endoplasmic reticulum is abundant. L, lumen of semi-niferous tubule; Sp, spermatogonium; arrows, desmosomes. x 9000.

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356 M. NISTAL, MARIA ABAURREA AND R. PANIAGUA

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5$.Fig. 5. Basal region of a mature Sertoli cell from a child 11 years old. The nucleus shows anirregular outline. The smooth endoplasmic reticulum and lipid inclusions are more developedthan in immature cells. Sertoli-Sertoli tight junctions (arrow) are shown. The pair of centrioles(c) is located in the basal cytoplasm. Sp, spermatogoni'im. x 13000.

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Human Sertoli cellfrom birth to pubertyfrom mature Sertoli cells (Figs. 2-4). These latter were only occasionally observedin the testes from the 11 years old males (Fig. 5).Immature Sertoli cells showed a pseudostratified disposition (Fig. 2). They possessed

a round to elongated nucleus, regular in outline and with one small nucleolus,close to the nuclear envelope (Figs. 3, 4). The chromatin was dispersed except for anarrow, peripherally placed dense chromatin band. The cytoplasmic organellesshowed a certain polarity. The supranuclear cytoplasm, facing the lumen, containedabundant rough endoplasmic reticulum cisternae, a well developed Golgi complex,and vesicles (Fig. 4). The basal cytoplasm was characterized by the presence ofmicrofilament bundles and pinocytotic vesicles. Mitochondria and some tubules orvesicles of smooth endoplasmic reticulum were homogeneously distributed through-out the cytoplasm. Crystals of Charcot-Bottcher, special configurations of theendoplasmic reticulum and lipofuchsin pigment granules were not seen. Lysosomesand lipid droplets, although present, were scarce. Sertoli-Sertoli junctional com-plexes, as described in mature Sertoli cells (Fawcett, 1975), were not seen in theseimmature cells, but desmosomes were frequently found (Figs. 3, 4).

In specimens from 8 to 11 years of age, the nuclei of Sertoli cells showed a moreirregular outline, and the nucleoli were more prominent. These showed the centralspherical body characteristic of mature Sertoli cells (Bustos-Obregon & Esponda,1974), surrounded by a dense trabecular network including granular and fibrillarcomponents, although it was less developed than in mature Sertoli cells. In thecytoplasm, lipid inclusions were more numerous and the smooth endoplasmicreticulum more developed. Junctional complexes began to appear. Centrioles,located usually in the supranuclear region in immature Sertoli cells (Fig. 3), seemedto migrate towards the basal nuclear pole. Occasional mature Sertoli cells showedthe centrioles in this basal position and the characteristic Sertoli-Sertoli tightjunctions (Fig. 5). A distinction between clear and dark Sertoli cells was not observed.

Although until 4 years of age the seminiferous tubules did not show an apparentlumen by light microscopy, the lumen was seen by electron microscopy in all testesfrom birth. Mitotic figures of Sertoli cells were found only in newborn testes and,even there, was an occasional finding.

Histometric studyResults of histometric study are shown in Tables 1, 2 and 3 and in Figures 6 and 7.The mean of the long testicular axis increased with age (Table 1). According to

the analysis of the variance performed (Table 3) differences for this parameterbetween correlated ages were all significant, except between 2 and 3 years. Meantesticular breadth increased with age, although differences between correlated ageswere significant only from 3 years onwards. Thus, increase in testicular volume withage was to be noted from 3 years onwards (Table 1).The proportion of testis occupied by seminiferous tubules decreased slightly

from 0 to 3 years, remaining afterwards about 62-5 %. However, total seminiferoustubule volume per testis increased progressively from 3 years (Table 1).The mean length of Sertoli cell nuclei increased with age, mainly from 7 years

(Table 2). Differences between averages of correlated groups were significant, exceptfor 2-3 and 3-7 years (Table 3). Differences between mean widths were significantexcept between 7 and 8 years. In spite of the evidence that differences between somegroups were not significant, the values have been admitted because they progressivelyincreased, and transformed into volumes, showed considerable differences.

357

358 M. NISTAL, MARIA ABAURREA AND R. PANIAGUA

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Age (years)

Fig. 6. Graphic representation of Sertoli cell nuclear number per testis and per unit volume-of testicular parenchyma.

Table 1. Testicular volume and seminiferous tubule volume(Testicular parenchyma axes (a and b) were estimated on 130 testes. Mean tubular diameter

(dt) and number of tubules per unit area of testis (nt) were estimated on 60 testes (50 sectionsof each testis). Testicular volume (vt), proportion of testis occupied by seminiferous tubules(Pt) and total seminiferous tubules volume per testis were estimated as indicated in the Table.Standard deviations are expressed only for direct estimations. Confidence limits 95 %.) C

Proportion TotalV'Number of of testis tubule

Testicular parenchyma tubules per occupied by volumeaxes (mm) Testicular Mean tubular unit area s. tubule per tes-

Age volume (mm3) diameter (Am) (640000 utm') p_ d? nt tis (mnlo(years) longa wide b vt = ircb dt nt t 4x640000 Pt.vt

0 13-8+0-15 4*7+0-18 160 84+1-01 80+1 01 0-691 1102 14-1+0i16 4-7+0*20 163 72+0-92 104+1-21 0-662 108*-3 14-0+0*11 4-7+0-14 162 68+0 87 110+1-30 0-620 1007 15-7±0d16 5-9±0d17 286 81±1-02 78+0-98 0-627 179 A8 16X0+0X20 6-1±0X14 312 82±1V08 75+0-87 0-618 192

11 16-8+0-11 7-2+0-15 462 90+1-13 64±0-78 0-637 294 1

The proportion of testis occupied by Sertoli cell nuclei decreased with age. This 41decrease was more marked from 3 to 7 years (Table 2).

Total Sertoli cell nucleus volume per testis also increased with age, parallel to the sincrease of Sertoli cell nucleus volume (Table 2).

Sertoli cell nucleus number was calculated for unit volume of testis as well as thetotal number of nuclei per testis (Table 2 and Fig. 6). Whereas the number of nuclei o-per unit volume decreased markedly with age (from 810 to 278 millions), the totalnumber remained about 128 millions from birth to the onset of puberty.

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The Sertoli cell nuclear number per transverse tubular section decreased with age(Table 2 and Fig. 7), from 24 to 12, while both mean tubular diameter and semini-ferous tubule number per unit area showed a complementary increase (Table I andFig. 7).

DISCUSSION

For this study, normal testes have been selected corresponding to those ageswhich preceding authors (Mancini, 1968; Vilar, 1970) have considered to show thechanges that testes undergo during infancy and childhood. It has been reported thatduring the first 3 years of postnatal life the human testis undergoes a period ofmaturation which becomes more marked at puberty (Mancini, 1968; Vilar, 1970).

Ultrastructural findings concerning the form of the immature Sertoli cell aresimilar to those reported by Hadziselimovic & Seguchi (1974). These authorsdistinguished two types of immature Sertoli cells in relation to nuclear shape:round and long. Both types of nuclei have been seen in the present study, but it isnot clear that two types of immature Sertoli cells exist. Because ultrastructuraldifferences between cells with round nuclei and cells with long nuclei were notmarked, and in many sections both cell types could not be distinguished, it ispossible that one cell type was sectioned in varying planes. Thus, it is consideredthat only one immature Sertoli cell type exists.The principal cytological characteristics of immature Sertoli cells (abundant

rough endoplasmic reticulum, a well developed Golgi complex, and vesicles) arelacking in mature Sertoli cells (Fawcett, 1975), and some authors have suggestedthat immature Sertoli cells might produce hormones of a non-steroid nature (Gondos,1977).

360

Human Sertoli cellfrom birth to puberty 361The distinction between clear and dark Sertoli cells reported by some authors

(Vilar, 1970) was not observed in this study. It is possible that dark cells were theconsequence of imperfect fixation of testicular tissue (Chemes et al. 1977). Leydigcells were also classified into two types, clear and dark; however, Christensen (1970)claimed that in perfusion-fixed testes dark cells did not appear. In specimens around11 years of age, immature Sertoli cells showed cytological features more commonlyseen in mature Sertoli cells. However, truly mature Sertoli cells were only occasionallyobserved in testes, from specimens at 11 years of age. This is in agreement with theresults of Hadziselimovic & Seguchi (1974) who did not find mature Sertoli cellsin specimens under 13 years of age.

It has been reported that the mitotic activity of Sertoli cells is pronounced duringfetal and early postnatal life but ceases a few days after birth (Steinberger & Stein-berger, 1977). In the present study, mitotic figures were seen only in the testes of thenewborn specimens.The results of the present histometric studies corroborate the previous observa-

tions of Hadziselimovic & Seguchi on the progressive diminution of the number ofSertoli cells per transverse tubular section from birth (28 cells) to the onset ofpuberty (14 cells). The differences between the results of Hadziselimovic & Seguchiand those reported in the present paper may be attributed to the different thicknessesof sections used for counting.The constant Sertoli cell number per testis is in keeping with the other histo-

metric data. From birth to 3 years of age, the testicular volume undergoes slightchanges. The diminution of the mean tubular diameter (dt), which corroborates theresults of Scorer & Farrington (1971), is balanced by the increase in the number oftubules per testicular unit area (nt). The decrease in the Sertoli cell number pertransverse tubular section (from 24 to 12) is balanced by the increase in the numberof tubules per unit area (nt) (from 80 to 110).From 3 to 11 years of age, the increase in the testicular volume (Vt) is considerable

(from 162 to 462 mm3). The seminiferous tubules also increase their diameter (dt)(Scorer & Farrington, 1971) and this increase corresponds to the decrease in thenumber of seminiferous tubules per unit area (nt). Therefore, although tubulesbecome longer and more convoluted (Mancini, 1968; Bergada, 1970), the ratioseminiferous tubules/interstitium is maintained. Because the total Sertoli cell numberper testis does not increase, these cells must be more widely spaced and, in conse-quence, the Sertoli cell number per transverse tubular section (ne) must diminish.

In conclusion, there is a progressive decrease in the Sertoli cell number pertransverse tubular section (nj) as well as per unit volume of testicular parenchyma,mainly from 3 years onwards. However, this is the result of the progressive increasein the testicular volume as well as in the length and width of the seminiferous tubules,without change in the Sertoli cell number per testis. This number may be consideredas a constant, not only for adult testes but also for postnatal developing testes.Whether or not the small amount of circulating gonadotropins detected during

infancy and childhood could be responsible for the increases in both testicularvolume and seminiferous tubule volume remains to be clarified. Several authorshave measured these gonadotropins in the plasma and urine of children by radio-immunoassay (Bergadag 1970; Kulin & Santner, 1977) and have pointed out thatduring childhood mean FSH levels rise from 15 to 77 m-i.u./h, and mean LH levelsrise from 18 to 38 m-I.u./h. In addition, it has been shown that the Sertoli cell isthe primary target of FSH (Means, 1977) and that the FSH-LH balance regulates

362 M. NISTAL, MARIA ABAURREA AND R. PANIAGUA

Sertoli cell differentiation (Chemes, Dym & Raj, 1979). It may be that the increasein the gonadotropin levels during childhood is also responsible for the increase inthe Sertoli cell nucleus volume observed in the postnatal testis.The results of the present study corroborate previous reports emphasizing the

stability of postnatal Sertoli cells (Rowley & Heller, 1971; Steinberger & Steinberger,1977). It is considered that the estimation of a Sertoli cell number for each age duringinfancy and childhood may be useful in the evaluation of the seminiferous epithelium.

SUMMARY

In order to evaluate the changes in the number and form of the Sertoli cell frombirth to the onset of puberty, a histometric and ultrastructural study was carriedout in normal children. Ultrastructural findings revealed that immature Sertolicells, present from birth to puberty, show round to elliptical nuclei, with regularoutlines and small nucleoli. The cytoplasm exhibits well developed rough endo-plasmic reticulum, prominent supranuclear Golgi complexes and vesicles at theluminal face; smooth endoplasmic reticulum was scarce. Histometric study revealeda progressive decrease in the Sertoli cell number per transverse tubular section aswell as per unit area of testicular parenchyma, mainly from 3 years onwards.However, this decrease seemed to be the result of the progressive increase in thetesticular volume as well as in the length and width of the seminiferous tubules,without change in the total Sertoli cell number per testis. This number may beconsidered constant, not only for adult testes but also for postnatal developingtestes.

REFERENCES

BERGADA, C. (1970). The effect of gonadotropins on the prepubertal testis. In Advances in ExperimentalMedicine and Biology, Vol. 10. The Human Testis (ed. E. Rosemberg & C. A. Paulsen), pp. 393-403.New York: Plenum Press.

BUSTOS-OBREG6N, E. & ESPONDA, P. (1974). Ultrastructure of the nucleus of human Sertoli cells innormal and pathological testes. Cell and Tissue Research 152, 467-475.

CHEMES, H. E., DYM, M., FAWCETr, D. W., JAVADPOUR, N. & SHERINS, R. (1977). Patho-physiologicalobservations of Sertoli cells in patients with germinal aplasia or severe germ cell depletion. Ultra-structural findings and hormone levels. Andrologia 17, 108-123.

CHEMES, H. E., DYM, M. & RAJ, H. G. M. (1979). Hormonal regulation of Sertoli cell differentiation.Biology of Reproduction 21, 251-262.

CHRISrENSEN, A. K. (1970). Fine structure of testicular interstitial cells in humans. In Advances in Experi-mental Medicine and Biology. Vol. 10. The Human Testis (ed. E. Rosemberg & C. A. Paulsen), pp.75-93. New York: Plenum Press.

CLERMONT, Y. & PEREY, B. (1957). Quantitative study of the cell population of the seminiferous tubulesin immature rats. American Journal ofAnatomy 100, 241-268.

CLERMONT, Y. & HARVEY, S. C. (1967). Effects of hormones on spermatogenesis in the rat. Ciba Collo-quium on Endocrinology 16, 173-196.

FAWCETT, D. W. (1975). Ultrastructure and function of the Sertoli cell. In Handbook ofPhysiology. MaleReproductive System. Vol. 5, Section 7. Endocrinology (ed. D. W. Hamilton & R. 0. Greep). Baltimore:Waverley Press.

Gom)os, B. (1977). Testicular development. In The Testis, vol. iv (ed. A. D. Johnson & W. R. Gomes),pp. 1-37. New York: Academic Press.

HADZISELIMOVIC, F. & SEGUCHI, H. (1974). Ultramikroskopische Untersuchungen an Tubulus Semini-ferous bei Kindern von der Geburt bis zur Pubertat. II. Entwicklung und Morphologie der Sertolizellen.Verhandlungen der anatomischen Gesselschaft, Anatomischer Anzeiger 68, 140-161.

HELLER, G. V., O'KEEFE, K. B. & HELLER, C. G. (1968). Effects of follicle stimulating hormone (FSH)on Sertoli cells in the hypophysectomized rat. Clinical Research 16, 113-123.

KALER, L. W. & NEAVES, W. B. (1978). Attrition of the human Leydig cell population with advancing age.Anatomical Record 192, 512-518.

KULIN, H. E. & SANTNER, S. J. (1977). Timed urinary gonadotropin measurements in normal infants,children and adults, and in patients with disorders of sexual maturation. Jouirnal of Pediatrics 90,760-765.

Human Sertoli cell from birth to puberty 363MACK, W. S., Scorr, L. S., FERGUSON-SMITH, M. A. & LENNOX, B. (1961). Ectopic testis and truedescended testis. A histological comparison. Journal of Pathology and Bacteriology 82, 439-443.

MANCINI, R. E. (1968). Bases histofisiol6gicas de la funci6n testicular. In Testiculo Humano (ed. Fun-daci6n para el Fomento de los estudios endocrinol6gicos y metab6licos de Rosario), pp. 11-45.Buenos Aires: Panamericana M6dica.

MEANS, A. R. (1977). Mechanism of action of follicle stimulating hormone (FSH). In The Testis, vol. IV(ed. A. D. Johnson & W. R. Gomes), pp. 163-168. New York: Academic Press.

RowLEY, M. J. & HELLER, C. G. (1971). Quantitation of the cells of the seminiferous epithelium of thehuman testis employing the Sertoli cells as a constant. Zeitschrift fur Zellforschung und mikroskopischeAnatomie 115, 461-472.

SCORER, C. G. & FARRINGTON, G. H. (1971). Histological studies of the undescended testes. In Con-genital Deformities ofthe Testis and Epididymis. London: Butterworth.

STEINBERGER, A. & STEINBERGER, E. (1977). The Sertoli cells. In The Testis, vol. IV (ed. A. D. Johnson &W. R. Gomes), pp. 371-399. New York: Academic Press.

VILAR, 0. (1970). Histology of the human testis from neonatal period to adolescence. In Advances inExperimental Medicine and Biology. vol. 10. The Human Testis (ed. E. Rosemberg & C. A. Paulsen),pp. 95-111. New York: Plenum Press.

WEISSBACH, L. & IBACH, B. (1976). Quantitative parameters for light microscopy assessment of thetubuli seminiferi. Fertility and Sterility 27, 836-847.