INTERNATIONALJOURNAL OFANDROLOGY

8 (1985) 295-312

Deparhnent of Cytology and Histology1, Faculty ofBiology,

University of Sahmunca, Salumunca,
D e p a m e n t of Electron Mi~roscOpr~, Uniuersitary Clinical Hospital,
University of Salumunca,
and Deparhnat of Morphology3, School of Medicine,
Autonomous University ofMadrid, Madrid, Spain

Ultrastructural changes in Sertoli cells

in ageing humans
BY

R. Paniagual, P. Amat2, M. NistaP

and A. Martin2

Ultractructural study of seminiferous tubules in ageing men revealed a varying

degree of spermatogenetic arrest associated with changes in the Sertoli cells.
Approximately half of the Sertoli cells showed a normal mature nuclear
appearance although the cytoplasm was altered morphologically. These cells
were classified as containing abundant lipid droplets (30 %), containing large
cytoplasmic vacuoles fdled with an amorphous material similar to that in the
tubule lumen and surrounded by junctional specializations (8%), multinuclea-
ted Sertoli cells (4%), or Sertoli cells with numerous mitochondria displaying
tubular cristae (2%).The remaining 7 % of Sertoli cells had an immature
nuclear appearance and sparse development of the cytoplasmic organelles;
these cells probably represent dedifferentiated Sertoli cells. Although indivi-
dual differences were marked, a correlation between the increase in gonado-
trophin levels and changes in both germ cell development and Sertoli cell
structure was observed.

Kq words: ageing human testes - Sertoli cell - seminiferous tubules.

Advancing age is associated with ageing of the somatic tissues and is also characteri-
zed by declining sexual function. The decrease in testosterone levels and the
associated increase in gonadotrophin levels found by sever& authors in elderly men

Received on February 1Ith, 1985.

295
(Stearns et al. 1974; Baker et al. 1976; Mannelli et al. 1979; Mastrogiacomo et al.
1982) have suggested that the decline in testicular function is a type of primary
testicular failure (Nieschlag et al. 1983). Morphological changes in the ageing testis
have also been reported. Mean testis length and volume (Stearns et al. 1974)as well
as sperm quality (Schwartz et al. 1983)seem to decrease with advancing age and the
peritubular and interstitial connective tissue undergo fibrosis (Kothari & Gupta
1974). Alterations in the seminiferous epithelium include maturation arrest at
spermatid, spermatocyte or spermatogonium level (Lynch & Scot 1950), reduced
numbers of dark Type A spermatogonia, localized multilayered arrangements of
spermatogonia, and an increased number of multinucleated germ cells (Holstein &
Hubmann 1980). The Sertoli cells of these testes show an increase in glycogen and
lipid content (Lynch & Scott 1950), and a decrease in number (Harbitz 1973a).
Studies on Leydig cells have reported an increase (Kothari & Gupta 1974; Honer6
1978) or a decrease (Harbitz 1973b; Kaler & Neaves 1978) in cell number and a
diminished response to stimulation with human chorionic gonadotrophin (Nankin
et al. 1981; Davidson et al. 1983).
Ultrastructural studies on ageing testes are rare and most of them have examined
principally germ cells (Holstein & Hubmann 1980). Only Schulze & Schulze (1981)
have reported the presence of multinucleated Sertoli cells in ageing testes. The
present paper reports on several morphological Sertoli cell types found in the testes
of elderly men.

Materials and Methods

Testicular biopsies were obtained approximately 2 h after death from 42 elderly

men (65 to 89 years of age) and 10 young adults (27 to 42 years of age) who had not
suffered from testicular, endocrine or related pathology including varicocele. In
addition, orchidectomy specimens were obtained from 26 patients (66 to 82 years of
age) suffering from prostatic carcinoma and who had received no previous
hormonal or drug treatments and were not suffering from testicular pathology.
Testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH)
levels in serum were measured before orchidectomy using double-antibody radio-
immunoassay techniques. Two separate blood samples were obtained from each

Figs. I and 2.
Fig. 1. Normal mature Sertoli cells in a seminiferous tubule with complete spermatogenesis.
T h e Sertoli cell nuclei are irregular in outline and contain well-developed nucleoli (nu). The
cytoplasm possesses a moderate amount of lipid droplets (li). Spermatogonia (star) and
spermatids (arrow) are seen. x 2350.
Fig. 2. Mature Sertoli cells containing abundant weakly osmiophilic lipid droplets (li) in a
seminiferous tubule with maturation arrest at the level of spermatocytes. A Charcot-Bottcher
crystal (arrow) is seen. x 3250.

296
 patient at least 15 min apart. Normal values were 6.3 0.3 ng/ml (testosterone);
_+

5.5 k 0.5 mIU/ml (LH); and 4.9 f 0.3 mIU/ml (FSH). All the subjects studied were
of proven fertility. Workers dealing with toxic agents were excluded.
Testicular specimens were fEed in 4 % cacodylate-buffered glutaraldehyde,
postfEed in 1% veronal-buffered osmium tetroxide, and embedded in araldite.
Ultrathin section were double stained with uranyl acetate and lead citrate and
examined with a Philips-201 electron microscope.
The 9% distribution of Sertoli cell types was calculated on 20 low-power electron
microscopic fields (648 pm2) from each testis. Electronmicrographs were taken at
2000 X, and augmented to 10000 X. Sinse individual differences were very
marked, only the total mean for each group of testes was calculated.

Results
Cyto logzcalfeatures
The appearance of seminiferous tubules in elderly men varied considerably among
testes. Whereas some testes showed complete spermatogenesis, others exhibited
maturation arrest at the level of spermatids, spermatocytes or spermatogonia.
Sertoli-cell-only tubules were also seen. The tunica propria appeared either normal
or thickened. Areas of sclerosed tubules were observed occasionally. Ultrastructural
examination revealed the presence of 6 morphologically distinct Sertoli cell types.
1. Sertoli cells with a normal, mature ultrastructural appearance. These cells
showed irregularly outlined nuclei with a well-developed tripartite nucleolus. The
cytoplasm contained abundant smooth endoplasmic reticulum, Charot-Bottcher
crystals, concentric annulate lamellae, elongated electron-dense mitochondria, lipid
droplets, residual bodies, and a group of rough endoplasmic reticulum cisternae
arranged in parallel at the basal portion of the cytoplasm. Inter-Sertoli junctional
specializationswere also present (Fig. 1).
2 . Sertoli cells with abundant lipid droplets Some of these showed a weakly
osmiophilic content (Figs. 2 and 3) and were surrounded by mitochondria or
endoplasmic reticulum (Fig. 3). Other droplets were larger, electron-lucent and
formed more conspicuous accumulations (Figs. 4 and 5 ) . In these accumulations
~~ ~~

Figs. 3 -5.
Fig. 3. Cytoplasmic portion of a Sertoli cell with abundant lipid droplets (li) showing
ultrastructural features of mature Sertoli cells, such as an irregularly outlined nucleus,
annulate lamellae (AL) and inter-Sertoli junctional specializations (large arrow). Mitochon-
dria (small arrow) and endoplasmic reticulum (open arrow) can be seen associated with lipid
droplets. x 6500.
Fig. 4. Sertoli cells with large electron-lucent lipid droplets (li) in a seminiferous tubule with
hypospermatogenesis. x 3250.
Fig. 5. Accumulation of electron-lucent lipid droplets (li) in the Sertoli cells cytoplasm.
Among the lipid droplets some mitochondria (arrow) can be seen. x 6500.

298
299
mitochondria were associated with the lipid droplets (Fig. 5). The lipid-free
cytoplasm exhibited the usual organelles of mature Sertoli cells including Charcot-
Bottcher crystals (Fig. 2), annulate lamellae and inter-Sertoli junctional speciali-
zations (Fig. 3). The nucleus was like that of mature Sertoli cells (Figs. 2-4).
3 . Sertoli cells with abundant large cytoplasmic vacuoles containing an amor-
phous material, similar to that found in the tubule lume (Figs. 6 and 7 ) .These cells
also exhibited slender cytoplasmic projections into the tubule lumen and abundant
lipid droplets. Both vacuoles and cytoplasmic projections showed the characteristic
subsurface cisternae and microfiaments of Sertoli cell junctional specializations
(Fig. 7 ) . The remaining cytoplasmic characteristics, as well as the nuclear pattern,
were similar to those of mature Sertoli cells.
4. Multinucleate Sertoli cells showing a mature appearance according to the
nuclear morphology. The cytoplasm exhibited swollen smooth endoplasmic reti-
culum and mitochondria, and lipid droplets (Figs. 8 and 9).
5. Sertoli cells with abundant mitochondria. These were elongated and displayed
tubular cristae (Figs. 10-12). Lipid droplets were also abundant. Lysozomes and
Charcot-Bottcher crystals were present (Fig. 12). The endoplasmic reticulum was
present in normal or decreased amounts.
6. Immature-like Sertoli cells. These cells were more differentiated than pre-
pubertal Sertoli cells but differed from mature Sertoli cells in the following
features: The nuclei were less irregularly outlined and did not contain a well-
developed nucleolus (Figs. 13 and 14). The cytoplasm contained less abundant
smooth endoplasmic reticulum than did mature Sertoli cells and showed no
annulate lamellae. Charcot-Bottcher crystals and inter-Sertoli junctional speciali-
zations were present. The latter extended, from the basal cytoplasm up to near the
tubule lumen in tubules with severe germinal hypoplasia. Prominent interdigi-
tations were also observed in these tubules (Figs. 14 and 15).
The relative distribution of these Sertoli cell types in the different types of
seminiferous tubules is shown in Table 1.
Most Sertoli cells in control testes from young men showed a normal ultrastruc-
tural appearance, although some of the other cell types, mainly with abundant lipid
droplets, were found in a small proportion (Table 1).

Figs. 6 and 7.
Fig. 6. Multivacuolated mature Sertoli cells in a testis with maturation arrest at spermatocyte
(star) level. The vacuolar content (asterisk) is similar to that in the tubule lumen (TL). Sertoli
cell cytoplasmic projections (arrows)into the tubule lumen are seen. x 3250.
Fig. 7. Higher magnification of vacuoles showing an amorphous material surrounded by
membrane. The vacuoles are surrounded by junctional specializations of Sertoli cells
(arrows).TL: tubule lumen. x 6500.

300
30 1
Hormonal analyses
Hormone assays performed in orchidectomized patients revealed normal (4.5- 5.3
mIU/ml, 12 patients), slightly increased (6.6-7.3 mIU/ml, 8 patients) or elevated
(9.4- 12.6 mIU/ml, 6 patients) FSH levels. A correlation was observed between the
increase in FSH levels and the degree of damage to seminiferous epithelium
including Sertoli cell alterations. LH levels were increased (6.1-8.3 mIU/ml) in 12
patients who also exhibited increased FSH levels. Testosterone levels were normal
(5.6-6.7 ng/ml, 17 patients) or slightly decreased (4.8-5.3 nglml, 9 patients who also
showed elevated gonadotrophin levels).

Discussion

The results of this study indicate that testicular alterations with advancing age affect
not only germ cells but also Sertoli cells, and there was a positive correlation
between the degree of germ cell damage and the proportion of altered Sertoli cells.
This agrees with the literature on the primordial role of Sertoli cells in spermato-
genesis (Nistal & Paniagua 1984).
The most conspicuous morphological feature of ageing Sertoli cells was the
abundance of lipid droplets, affecting nearly half of the ageing Sertoli cell
population, since all of the altered Sertoli cell types presented abundant lipid
droplets, except for immature-like Sertoli cells. Sertoli cells with abundant lipids
were also present in younger males but in a much lower proportion. The
accumulation of lipid seems to be a gradual process, starting at 15 years of age and
increasing with age (Lynch & Scott 1950).The increased lipid content in Sertoli cells
has been considered to be derived from phagocytized germ cells, since lipid is
abundant in seminiferous tubules with many degenerating germ cells (Gravis &
Weaker 1978; Schulze 1984) but not in tubules with germinal aplasia, such as those
of men with hydrogonadotrophic hypogonadism (De Kretser 1968), or several
types of hypergonadotrophic hypogonadism including Klinefelter’s syndrome
(Nistal et al. 1982),the Sertoli-cell-onlysyndrome (Chemes et al. 1977; De Kretser et
al. 1981) and cryptorchidism (Schulze et al. 19’76).Many of the lipid droplets in the
ageing Sertoli cells correspond to the type of electron-lucent lipids which have been
considered to represent residual bodies (Schulze 1984). Since many of the tubules
with these lipids in ageing testes showed maturation arrest of spermatogenesis and

Figs. 8 and 9.
Fig. 8. Part of a multinucleated Sertoli cell in a Sertoli-cell-onlytubule showing a cytoplasmic
portion that contains 3 nuclei with a normal mature nucleolus (nu), lipid droplets (li), and
swollen smooth endoplasmic reticulum (asterisk). x 3250.
Fig. 9. Higher magnification of Fig. 8 showing the absence of cell membranes between the
three nuclei. X 7450.

302
303
many degenerating germ cells, it is possible that the origin of lipid accumulation in
these tubules may be the degenerating germ cells. However, numerous lipid
droplets of the weakly osmiophilic type were also observed even in some Sertoli-cell-
only tubules from ageing testes, and an increase in lipids has also been reported in
some testicular disorders with germ cell aplasia (Nistal & Paniagua 1984). Alterna-
tively, if Sertoli cells synthesize steroid hormones, as has been postulated (Fawcett
1975; Steinberger & Steinberger 1977), then it is possible that the Sertoli cells have
stopped steroid hormone synthesis and the accumulated lipid represents unused
precursors (Gravis & Weaker 1978).
In testes from ageing men, vacuolated Sertoli cells were usually found in
seminiferous tubules with germinal hypoplasia. These vacuoles were surrounded by
a membrane and do not seem to correspond to dilated smooth endoplasmic
reticulum since this organelle exhibited its characteristic tubular pattern in the
Sertoli cell cytoplasm. The vacuoles contained no lipids but did display an
amorphous content similar to that found in the tubule lumen. In addition, these
vacuoles were surrounded by junctional specializations of Sertoli cells. A similar
finding has been reported by Kerr et al. (1979) in rat testes after surgical induction
of cryptorchidism, and by Flickinger (1981) in vasectomized hamsters. Kerr et al.
(1979) interpreted these vacuoles as dilations of the basal intercellular space
between opposing inter-Sertoli cell junctions. The vacuoles of ageing Sertoli cells
probably do not have this origin, since they appear at different levels of the Sertoli
cell cytoplasm including locations near the lumen in tubules with germinal
hypoplasia. In addition, the menbranous complexes originating from the vacuoles
in the study of Kerr et al. (1979) were not seen in the present study. The
explanation of Flickinger (1981) seems to be more appropiate for the vacuoles in
ageing sertoli cells. This author assumed that similar vacuoles are dilations of the
extracellular space developed from premature exfoliation of germ cells. This agrees
with the presence of Sertoli cell junctional specializations which are commonly
found at the Sertoli cell-spermatocyte or Sertoli cell-spermatid junctions (Russell
1980).
Multinucleated Sertoli cells have only been reported in ageing testes and have
been said to result from either mitosis without karyokinesis or cell fusion (Schulze &
Schulze 1981). An increased number of Sertoli cells has been reported in adult
animals after X-irradiation (Nebel & Murphy 1960), after artificial cryptorchidism
(Clegg 1963), in cultured rat Sertoli cells after FSH stimulation (Griswold et al.

Figs.10-12.
Fig. 10. Sertoli cell with abundant mitochondria displayingtubular cristae (star). x 6500.
Fig. 11. Cross-sectioned Sertoli cell with abundant mitochondria and lipid droplets (star),
surrounded by normal Sertoli cells. x 3250.
Fig. 12. Higher magnification of Fig. 11 showing mitochondria with tubular cristae,
abundant lipid droplets, and a cross-sectioned Charcot-Bottchercrystal (arrow). x 16 000.

304
 305
Andrology 8,4 20
 1976) and after FSH administration following hypophysectomy (Murphy 1965);
however, definitive morphometric studies have not been carried out and such
increases might be only the result of the decrease in length of the seminiferous
tubules, as has been reported in several studies (Clermont & Morgentaler 1955;
Steinberger & Steinberger 1977; Nistal et al. 1982). According to Harbitz (1973a)
the number of Sertoli cells declines with ageing. It is therefore possible that
multinucleated Sertoli cells could originate from cell fusion. Certainly, those in the
present study presented several characteristics of cell degeneration such as dilations
of the smooth endoplasmic reticulum and membranous organelles. As has been
reported in some cryptorchid human testes (Amat et al. 1985), it is possible that
degenerating Sertoli cells tend to fuse, thus forming a syncytium. Since multi-
nucleated germ cells (Holstein & Hubmann 1980) and multinucleated Leydig cells
(personal observation) are frequently observed in ageing testes, it may thus be
concluded that ageing induces the formation of multinucleated cells in the human
testis, though whether this results from karyokinesis or cell fusions remains to be
proven.
Sertoli cells with numerous mitochondria have not been described in the testis.
Conspicuous accumulations of mitochondria are frequent in the basal cytoplasm of
Sertoli cells. However, in the present study, Sertoli cells with abundant mitochon-
dria at different levels of the cytoplasm were observed in small numbers in testes
from ageing men but not in testes from young men. Cells with abundant
mitochondria are a normal component of parathyroid and gastric glands, renal
tubules and ductus deferens (Hoffer 1976; Paniagua et al. 1981), where they are
probably involved in electrolyte and water transport. It is unlikely that Sertoli cells
with abundant mitochondria have a similar function. The presence of mitochondria
with tubular cristae in conjunction with abundant lipid droplets might imply
increased steroid hormone production, although the amount of smooth endo-
plasmic reticulum does not seem to be increased. Since further data are not
available, these cells may only be considered as another form of Sertoli cell
alteration caused be ageing. This may represent the morphological expression of
DNA alterations with age, or a metaplastic transformation similar to that observed
in other glands such as the thyroid and parathyroid.
All the above-mentioned forms of altered Sertoli cells commonly displayed a

Fig$. 13-15.
Fig. 13. Immature appearance of Sertoli cells in a testis with maturation arrest at the level of
spermatogonia. The nuclei (N) are less irregularly shaped and the nucleolus less developed
than in mature Sertoli cells. x 3250.
Fig. 14. Higher magnification of immature-like Sertoli cells showing regularly outlined
nuclei with sparse cytoplasmic organelles, interdigitations (star) and extensive inter-Sertoli
junctional specializations (arrow). X 6500.
Fig. 15. Higher magnification of inter-Sertoli junctional specializations (arrow) seen in
Fig. 14. X 16 500.

306
307
 Table 1.
Relative distribution of Sertoli cell types in the testes of normal young men (= controls) and in ageing men.

Total % % distribution in ageing testes

I
Sertoli cell type Sertoli-
Controls Ageing testes complete cell-only
03
0 I I I I I I I
co
Normal mature 93 49 15.7 12.5 9.3 6.9 4.6
With abundant lipids 6 30 3.2 6.8 8.9 7.7 3.4
Vacuolated 0.5 8 - 0.2 1.8 2.6 3.4
Multinucleate - 4 - - 1.1 1.1 1.8
With abundant mitochondria - 2 - - - 0.3 1.7
Immature-like 0.5 7 - - 0.8 1.9 4.3

Sertoli cell percentages are based on calculations from 20 low-power electron microscopic fields (648 pmZ)from each testis.
nuclear development characteristic of adult Sertoli cells. However, immature-like
Sertoli cells which had a less irregularly shaped nucleus and a less developed
nucleolus than did mature Sertoli cells have also been observed in this study. Three
types of immature Sertoli cells can be found in the human testis: 1) cells that have
never undergone maturation, as in hypogonadotrophic hypogonadism (de Kretser
1968), in male pseudohermaphroditism (Smith et al. 1967; Okon et al. 1980),and in
the so-called ‘hypoplastic zones’ of cryptorchid testes (Hedinger et al. 1967),which
exhibited a prepubertal appearance according to electron microscopy (Schulze
1984); 2) cells which have undergone incomplete maturation and which appear as
intermediate between completely undifferentiated and mature Sertoli cells, such as
those reported in several hypergonadotrophic hypogonadial states (Nistal et al.
1982), and in cryptorchidism (Schulz et al. 1976; Hadziselimovic 1977; Schulze
1984); 3) presumably mature Sertoli cells that have undergone transformation into
immature cells, such as those reported after long-term oestrogen administration
(Rodriguez-Rigau et al. 1977; Schulze 1984). Since the immature-like Sertoli cells
were rare in young adult testes but not in ageing testes, these cells probably
correspond to the latter type of immature cells, and may be considered as
dedifferentiated Sertoli cells which still exhibit several signs of previous maturation
such as the presence of Charcot-Bottcher crystals, inter -Sertoli junctional speciali-
zations and a developed multilayered basal lamina (Hadziselimovic 1977). It is
probable that nuclear dedifferentiation may represent a more advanced step in
Sertoli cell damage caused by ageing, which is observed first in the cytoplasm.
It is concluded that the decline of testicular function with advancing age affects
the Sertoli cells. These cells begin to accumulate lipids, derived either from
phagocytized degenerated germ cells or from unused steroid hormone precursors.
The premature exfoliation of germ cells gives rise to cytoplasmic projections and
invaginations (vacuoles). Altered Sertoli cells tend to form syncytia either by cell
fusion or by karyokinesis. Finally, the nuclei, which have been well preserved
morphologically from degeneration changes, may then undergo dedifferentiation.
The presence of prominent interdigitations and extensivejunctional specializations
in the adjacent Sertoli cells in tubules with severe germinal hypoplasia is probably
related to the loss of germ cells, as occurs with seminiferous tubules in Sertoli-cell-
only syndrome (Chemes et al. 1977; de Kretser et al. 1981).
The causes of these alterations are still unknown although both hormonal and
vascular alterations may be involved. With advancing age a progressive decrease in
testosterone levels has been reported (Baker et al. 1976; Mannelli et al. 1979;
Davidson et al. 1983). This decrease has been attributed to a drop in free
testosterone levels (Royer et al. 1984),which represents the biologically active form
of testosterone (Vermeulen et al. 1972). It is possible that this deficiency is partially
involved in the involution of germ cells and Sertoli cells, and may play some role in
the increase in gonadotrophin levels and the partial regression of secondary sex
characteristics in aged men. Vascular alterations such as arteriosclerosis, which is

309
f r e q u e n t in aged men, may reduce the metabolic activily of cells involved i n
spermatogenesis, including Sertoli cells, through a n ischaemic mechanism (Lea-
them 1977). Ischaemic lesions affecting Sertoli cells would lead to m o r e pro-
nounced alterations i n the seminiferous epthelium.

Acknowledgement
This work was supported by a grant from the ‘Comisih Asesora de Investigaci6n Cientifica y
TCcnica’, Madrid, Spain.

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Author’s address:
Ricardo Paniagua, Departemento de Citologa e Histolosa, Facultad de Biolosa,
Universidad de Salamanca, E.-37008-Salamanca,Spain.

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