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Original Studies |
Department of Cell Biology and Genetics (M.P.M., R.P.), University of Alcalá, E-28871 Madrid; Department of Morphology, School of Medicine (J.R., F.M.G.), Autonomous University, E-28029 Madrid, Spain
Address all correspondence and requests for reprints to: Maria P. De Miguel, Department of Cell Biology and Genetics, University of Alcala, E-28871 Alcala de Henares, Madrid, Spain.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OSM was initially characterized by its ability to inhibit growth of several tumor cell lines (5). Later, it was shown that OSM could also modify the functions of normal tissues (6). In the testis, the role OSM has been demonstrated in Sertoli cells of neonatal rats (7). In mouse testicular cell cultures, it has been reported that OSM stimulates both the proliferation of primordial germ cells (PGCs) in fetal testes (8) and, later, the start of spermatogenesis, in postnatal testes (7). In addition this cytokine has also been found in rat Leydig cells at all ages studied (7).
As it has been hypothesized that both PGCs and gonocytes are the precursor cells of several germ cell tumors (seminoma and carcinoma in situ, respectively), we investigated the presence of OSM in the Sertoli cells of germ cell tumors. In addition, the observation of OSM expression in rat Leydig cells, even when gonadotropins were low, prompted us to investigate this expression in human testicular disorders showing different types of Leydig cell alterations. Because no previous studies on OSM in human testes had been reported, our study included normal human testes at different ages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because rat testes show positive immunostaining to the same antibody used here in the Sertoli cells (fetal and newborn testes) and Leydig cells (all ages) (7), sections of fetal (18 days postcoitum), newborn, and adult rat testes were immunostained as the human sections and used as positive controls.
Because a large number of Leydig cells displayed positive immunostaining in some of the testes studied, a semiquantitative evaluation was carried out. For each testis, the percentage of immunostained Leydig cells was calculated by counting at least 50 Leydig cells per section. From the average values for each testis, the mean and standard deviation (SD) for the testis group was obtained. Although most Leydig cells were easily distinguished from other interstitial cells by their round nucleus and ovoid cytoplasm, in order to get a more accurate identification of these cells, two additional sections from each testis were immunostained with testosterone, according to the method described by Nistal et al. (9, 10, 11).
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Results |
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. With the exception of the androgen
insensitivity syndrome, within each testis group the immunostaining
pattern was very similar in all testes. Normal testes
Fetal testes showed seminiferous tubules that were lacking lumen
and comprised of Sertoli cells, gonocytes, and fetal spermatogonia. The
interstitium was abundant and contained large Leydig cell clusters and
numerous blood vessels. An intense immunoreaction to OSM was detected
in most Sertoli cells and Leydig cells (Fig. 1, A and B).
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Child testes presented a histologic pattern similar to that of newborn
testes, although fetal Leydig cells had disappeared. No OSM
immunoreaction was detected (Fig. 1D).
All pubertal testes showed spermatogenetic development to spermatocytes
in most seminiferous tubules and even spermatids in some tubules.
Complete spermatogenesis was never observed. Leydig cells were present
in varying numbers. Immunoreaction to OSM was only occasionally
observed (in less than 2% of Leydig cells) with independence of the
spermatogenetic development of tubules (Fig. 1E).
Adult testes presented complete spermatogenesis in most tubules, and
Leydig cell clusters were scattered among the tubules. Most of the
Leydig cells exhibited OSM immunoreaction in their cytoplasm (Fig. 1, F
and G). Comparison of the normal adult testes obtained during surgery
with the autopsy specimens showed neither histological nor
histochemical changes.
Aging testes showed smaller tubules than those of adult testes, and a
variable degree of spermatogenetic development: from tubules with only
Sertoli cells and spermatogonia to tubules with complete
spermatogenesis. The predominant patterns were complete spermatogenesis
in one subject testis, germ cell development to spermatocytes and round
spermatids in 6 testes, and tubules with Sertoli cells and
spermatogonia in one testis. OSM immunoreaction was observed in the
Leydig cells, even in testes with marked germ cell depletion, although
immunolabeling was weaker than in adult testes in all cases (Fig. 1H).
Nontumoral disorders
Cryptorchid testes showed a variable testicular pattern: three
testes showed Sertoli-cell-only tubules; in six testes the seminiferous
tubules contained Sertoli cells and spermatogonia; and the remaining
two testes presented germ cell development up to primary spermatocytes
and scanty round spermatids. In all cases, the Leydig cells formed
clusters as well as large nodules. Most Leydig cells appeared
immunostained to OSM, even in the hyperplastic nodules (Fig. 2A).
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The testes of men with androgen insensitivity syndrome presented a
diffuse lesion consisting of small seminiferous tubules, which were
devoid of apparent lumen and which contained Sertoli cells and isolated
spermatogonia (2 testes) or Sertoli cells only (3 testes). Groups of
tubules were separated by wide spaces filled by a fibrous, storiform
connective tissue that contained large Leydig cell clusters. In two
specimens with Sertoli-cell-only tubules, the testes contained nodules
(hamartomas) that stood out from the surrounding testicular parenchyma
and consisted of very small seminiferous tubules with immature Sertoli
cells and hyalinized lamina propria. Numerous Leydig cells were found
among the tubules in all cases. OSM labeling in Leydig cells was
observed in three subjects (two with Sertoli-cell-only tubules and one
with spermatogonia). However, the distribution of labeling varied
widely, from intense to absent, within the same testis and with
independence of the lesion zone (diffuse lesion or hamartomatous
nodule) and the tubular pattern (Sertoli-cell-only tubules or tubules
with spermatogonia) (Fig. 2, C and D).
Tumors
Leydig cells in testes with CIS cells showed a normal (as in
adults) OSM immunoreaction (Fig. 2E). In the testicular germ cell
tumors studied here, the Leydig cells in the testicular parenchyma that
surrounded the neoplastic cells immunostained to OSM in a manner
similar to that of normal testes, with independence of the degree of
spermatogenesis alteration. Within the testicular tumor, no Leydig
cells were usually identifiable. However, in testes with intratubular
seminoma, the recognizable Leydig cells in the testicular interstitium
that was not infiltrated by neoplastic cells, showed no OSM
immunoreaction (Fig. 2F).
In the Leydig cell tumors, neoplastic cells showed a strong reaction to
OSM (Fig. 2G).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the rat testis, OSM was immunodetected in the Sertoli cells from the fetal period to the development of B spermatogonia—that is, at the onset of spermatogenetic development (7). We have detected positive OSM immunostaining in human Sertoli cells exclusively in the fetal testis. If the results obtained in rats were transposable to the human testis, the Sertoli cells of newborns and those of infants younger than 4 yr of age should show OSM immunoreaction, because B spermatogonia does not appear before 4 yr of age (12). This suggests that, in the human testis, another member of the IL-6 family could be involved in the initiation of the spermatogenetic process. In fact, IL-6 has been shown to be produced by human Sertoli cells (13).
We have studied OSM immunoreaction in germ cell tumors in order to ascertain whether the formation of these neoplastic cells was associated with a putative re-expression of OSM by the Sertoli cells. However, we failed to find immunostained Sertoli cells in the well-preserved areas of the testicular parenchyma.
In the newborn testis, like in the rat testis (7), both fetal and adult Leydig cells showed immunoreaction to OSM. In the present study, no OSM immunoreaction was observed in infantile testes. The so-called infantile Leydig cells, which populate the testicular interstitium during childhood, display an intermediate ultrastructural pattern between mesenchymal and true Leydig cells and are weakly immunoreactive to testosterone (9). It has been proposed that these cells could be an involutive stage from fetal Leydig cells, which would undergo dedifferentiation (9, 10). Because proliferation and maintenance of Leydig cell differentiation is modulated by LH and growth factors (14), it is possible that OSM could be one of those growth factors and that its absence is associated with the loss of Leydig cell differentiation. In addition, it is also possible that in infancy the OSM signal in Leydig cells is substituted by another family member, such as LIF, as demonstrated in rodent testes (15), or IL-6. IL-6 production by Leydig cells has been reported in rats (16) and humans (13).
In pubertal boys whose testes have not yet reached complete spermatogenesis, a number of well-differentiated, testosterone-positive Leydig cells have been demonstrated (9). In the present study, none of the pubertal testes showing these characteristics were positively immunostained to OSM. These results suggest that histochemically detectable Leydig cell production of OSM is only performed by well-differentiated and functionally active Leydig cells. This hypothesis is also sustained by the decrease in OSM reactiveness (from 64% to 37%) in the senile testis, in which Leydig cells undergo a certain dedifferentiation with diminution of the steroidogenic function (17).
The occurrence of morphological and functional alterations of Leydig cells in cryptorchid testes is controversial. Ultrastructural alterations have been reported (18), and it has also been documented that human cryptorchidism is sometimes associated with anomalies in the hypothalamic-pituitary-gonadal axis, which would lead to alterations in Leydig cells (19). In contrast, Mancini et al. (20) failed to find morphological differences between the Leydig cells of human cryptorchid testes and those of normal testes. Present results suggest a normal Leydig cell differentiation in cryptorchid testes because OSM expression in these Leydig cells—even in areas where Leydig cell hyperplasia was observed—was similar to that of normal adult testes.
In Klinefelter’s syndrome, in addition to morphologically normal Leydig cells, altered Leydig cells have been reported (17) and the number of testosterone-positive Leydig cells has found to be decreased (11). The absence of OSM suggests a nonfunctional state of the Leydig cells in these patients. Endocrinological studies revealed that androgens levels are under half of the normal values (21).
The histologic testicular pattern in the cases of androgen insensitivity syndrome studied here fits with previous studies (22). OSM immunostaining revealed intensely stained Leydig cells together with unstained Leydig cells in some patients and complete absence of immunostaining in other patients. This agrees with previous descriptions of varying numbers of morphologically altered Leydig cells in this syndrome (23) and important alterations in the regulation of the hypothalamic-hypophyseal-testicular axis (24). In this syndrome, estrogen production has been found to be increased 2-fold, and this has been attributed to an increased stimulation of Leydig cells by LH (25), whereas testosterone values remain normal or slightly elevated (24).
Functioning testicular Leydig cell tumors secreted active steroids, resulting in increased estradiol and normal-to-low testosterone values in peripheral blood, although LH levels were low (26). Ultrastructural studies on Leydig cell tumors revealed a decreased amount of smooth endoplasmic reticulum, although all other cytologic features were typical of normal Leydig cells (27). The presence of OSM in these cells, as in the adult normal testes, corroborated the idea that neoplastic Leydig cells did not undergo dedifferentiation and are functionally active.
It has been proposed that CIS cells give rise to seminoma (28) and, thereafter, to other types of germ cell tumors (48). The presence of androgen receptors in CIS and seminoma cells suggests an involvement of Leydig cells in the development of testicular germ cell tumors (29). Because the Leydig cells among tubules with CIS cells showed normal OSM immnoexpression, whereas those in intratubular seminoma did not immunostain, it was tempting to speculate that OSM might be indirectly (through Leydig cells) involved in such tumor development and that OSM expression in Leydig cells was lost during this progression. In other tumor progressions, breast cancer for instance, OSM, IL-6, and LIF have stimulated the activity of 17ß-HSD (30).
A gradient effect has been described in the impairment of spermatogenesis near malignant neoplasms, suggesting a direct factor-mediated influence of tumor on spermatogenesis (31). Interestingly, OSM has been shown to inhibit the growth of a variety of solid tumors (6) and, together with LIF and IL-6, to induce the differentiation of myeloid leukemia cells (32). The presence of specific receptors for these factors in testicular tumors would give more information about the possible implication of this family of growth factors in testicular tumorigenesis.
In conclusion, OSM expression in the human testis does not match that of rodents, as it is not present in the human Sertoli cells at the onset of spermatogenesis and because a down-regulation does take place in the human infantile Leydig cells. The presence of this protein in the Leydig cells of newborn and adult normal testes suggests a role in the maintenance of Leydig cell differentiation. In general, the appearance of OSM in these cells coincides with those Leydig cells that are functionally active and well differentiated.
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Footnotes |
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Received July 24, 1998.
Revised October 7, 1998.
Accepted November 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase deficiency, testicular feminization, and
related disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds.
The metabolic basis of inherited disease. McGraw-Hill, New York;
1919–1944.
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