Expression of cytokeratins 8 and 18 in human Sertoli cells of immature and atrophic seminiferous tubules*

Differentiation (1990) 43: 6 6 7 0

Differentiation Ontogeny and Neoplasia 0 Springer-Verlag 1990

Expression of cytokeratins 8 and I8 in human Sertoli cells of immature and atrophic seminiferous tubules * Peter Stosiek' **, Michael Kasper', and Uwe Karsten' Department of Pathology, District Hospital, DDR-8900 Gorlitz, German Democratic Republic Central Institute of Molecular Biology, Academy of Sciences of the GDR, DDR-1115. Berlin-Buch, German Democratic Republic Accepted in revised form January 6 , 1990

Abstract. Tissues of human testes, either normal (23 specimens of various developmental stages), or affected by pathological conditions (19 specimens of dystopia, atrophia and/or oligospermia) were immunohistochemically examined for the expression of different cytokeratins, using mainly frozen material. Cytokeratins 8 and 18 were found in varying amounts in Sertoli cells of fetal, prepubertal and senile testes and in all cases of pathological alteration. Cytokeratins were completely absent only in normal, mature seminiferous tubules. Therefore, the immunohistochemical detection of cytokeratins in Sertoli cells seems to provide a sensitive marker for immature or damaged testes.

Introduction The characteristic intermediate filament (IF) protein of normal human seminiferous tubuli cells, including Sertoli cells, is vimentin [3, 121. This corresponds to the mesodermal origin of gonadal tissue [30]. On the other hand it is now well established that I F expression is differentiation-regulated. Considering this, cytokeratins (CKs) could have been expected to be present in at least some of these cells, because they show certain typical features of epithelial cells. Although Sertoli cells lack desmosomes [12], they possess specialized tight junctions [lo], a necessary prerequisite for the blood-testis barrier [4,27]. Whereas CK expression has been clearly demonstrated to occur transiently in developing rat Sertoli cells [25], few data from human material are available. Miettinen et al. [24] observed some CK-positive cells lining atrophic and, to a lesser extent, nonatrophic seminiferous tubules. In line with our earlier studies on the coexpression of CK and vimentin in a subset of epithelia [18], we were interested in a more-detailed investigation of the expression of CKs in human gonadal tissues of different developmental stages including nonmalignant pathological conditions. Methods

samples (atrophic, dystopic testes and oligospermia, see Table 1) were derived from surgery. The later had been divided into two parts. One part was routinely formalin-fixed and embedded in paraffin. The other was cryosectioned. In the case of paraffin-embedded material only monoclonal antibodies to formalin-resistant epitopes could be employed (lu5, VZM 3B4), and the sections had to be pretreated with protease [19]. In the case of testicular tumors, noninfiltrated parenchyma in the neighborhood of the tumor mass was studied. Zmmunohistochemistry. The following monoclonal antibodies (Mabs) to IFs were employed: A45-B/B3 (broadrange CK [16]); lu-5 (broad-range CK [31]); C10 (CK 8, unpublished, Dr. Bartek, Brno, CSSR); TROMA 1 (CK 8 [6]); SK2-27 (CKs 14, 16, and 17 [22]); E 3 (CK 17 [14]); CK-2 (CK 18, Boehringer, Mannheim, FRG); A53-BIA2 (CK 19 [17]); BA 16 (CK 19 [2]); C 18 (CK 7, unpublished, Dr. Bartek, Brno, CSSR); VZM3B4 (Vimentin, Progen, Heidelberg, Federal Republic of Germany). A modified immunoperoxidase technique was employed as described 1201. Briefly, the unfixed, air-dried, frozen sections were treated after incubation with 1% normal goat serum and washed in phosphate-buffered saline (PBS) with 0.3% H20, for 30 min, then incubated with the primary monoclonal antibodies at room temperature for 30 min. Then followed the sequence : polyclonal goat antibody to mouse (30 min), monoclonal antiperoxidase (30 min), 0.002% peroxidase solution (30 min). All washes were performed in PBS and the color was developed with diaminobenzidine. Controls were performed by replacing the primary antibody with PBS or irrelevant hybridoma supernatant. Double labelling was performed in immunofluorescence mode using first a polyclonal guinea-pig vimentin antiserum G P 8 (obtained from Dr. R. Moll, Mainz, FRG) made visible with fluorescein isothiocyanate (F1TC)-labelled goat antiserum to guinea-pig Ig (Staatliches Institut fur Immunpraparate und Nahrmedien, Berlin) and, secondly, Mab A45BIB3 with TRITC-labelled goat antibody to mouse Ig (TAG0 Diagnostics, Burlingame, USA). For details of the method see Franke et al. [l I].

Tissues. Normal testes (fetal, prepubertal, mature and senile) were obtained from autopsy, taken 8-12 h postmortem, frozen, and cryosectioned (4 pm). The remaining tissue

Results

* Dedicated to the 60th birthday of Prof. Wolfgang Remmele, Wiesbaden, FRG ** To whom offprint request should be sent

The results are summarized in Table 1. Epithelia lining the seminiferous tubules expressed vimentin at all developmental stages (Fig. 2 a) and pathological conditions examined,

67

Table 1. Results of immunohistology with tissue sections from normal and nonmalignant pathological specimens from human testis N"

Tissue/ condition

Age

Fetal testis, normal

41h month 4 gestation

Inmature testis, normal

Iday2 years

5

Mature testis, normal

2944 years

9

Dystopic testis

5-20 years

3

Atrophic testis in the vicinity of testicular cancer

25-52 years

12

Oligospermia

2627 years

4

Senile testis

70-88 years

5

A45lu-Sb CK18 BIB3 CK CK7 CK )broad) (broad)

CK8

TROMAI SK-2-27 E3 CK8 CK14, CK17 16,17

CK-2 CK18

nd

+++

nd

nd

+++

Frozen section

CIO

A53- BA16 VIM3/ Tissue B/A2 CK19 Bdb fixation CK19 Vim -

+,

+++ ++

nd

nd

++

++

-

++ ++

-

-

++

-

-

+++

Frozen section

-to

nd

nd

-to

-

nd

+++

Frozen section

++

-

-

+++

Frozen section

+to

-

-

+++

Frozen section

-

+ +++

-

nd

nd

nd

-

++ ++

-

+to

-to

-

+

+to

-

+++ ++

nd

+++

~

~

+

+++

++

+

-to

nd

nd

nd

nd

nd

nd

nd

nd

+++

Frozen section

-to

nd

nd

nd

nd

nd

nd

nd

nd

nd

+++

Frozen section

+to

+++

nd, not done; only a few single cells positive; positive; -, negative

-

+ +, 10-90%

of Sertoli cells positive;

+ + +, all epithelia cells lining the tubules

a In two cases with strong CK positivity of Sertoli cells antibodies to CKs4 and 13 (kind gifts of Dr. van Muijen) were tested with negative results Only tested on formalin-fixed and paraffin-embedded material (see Methods)

as did the nonepithelial parts. In contrast, CK expression was restricted to epithelia and, in addition, to distinct developmental stages. CKs 8 and 18 were found in both fetal and prepubertal testes in epithelial cells lining the seminiferous tubules (gonadal germ cells) with lower intensity (Figs. 1d, 2b) than in pathologically altered adult tissue (see below). In mature, normal parenchyma of the testis, CKs disappeared almost entirely. Only in some atrophic tubules could some single CK-positive Sertoli cells be detected (not shown). Interestingly, several types of atrophy of the seminiferous epithelium led to reexpression or, alternatively, continued expression of CKs. The later was the case with pressure atrophy as a result of testicular dystopia (undescended testes), the former being a consequence of pressure exerted by the proximity of a growing and infiltrating malignant tumor (Fig. 1a). In both cases CKs and 18 were identified. The CK-positive cells were Sertoli cells (see Fig. 1b). The cases of oligospermy and senile atrophy showed that other, independent conditions (mechanical pressure and degenerative processes of the testes) can also lead to inappropriate CK expression in Sertoli cells (Fig. 1b, c). Double-immunofluorescence experiments confirmed that CK and vimentin were present in the same cells (not shown). Discussion

Sertoli cells perform a great variety of functions [21]. They provide nutritional and mechanical support to spermatogenetic cells, contribute to the microenvironment of germ cells, and are responsible for the formation of the bloodtestis barrier. The latter function includes precisely controlled resorption and secretion processes [l, 7, 9, 26, 291.

In fetal and infantile testes, Sertoli cells are not yet fully differentiated with respect to their functions and histological appearance. In later prepubertal stages they are histologically distinguishable from spermatogenic cells by their basal location and by their clear cytoplasm containing a considerable amount of glycogen IS]. Their nuclei are elongated with a voluminous nucleolus. Most characteristic are deep invaginations of the nuclear and cell membrane. Fully differentiated Sertoli cells provide a histological indicator for maturity of the testis [15]. Under conditions of damage and age-dependent changes leading to atrophy of spermatogenic cells, Sertoli cells remain in place, still lining the seminiferous tubules. The predominant intermediate filament protein of the seminiferous tubules, vimentin, has been examined in detail during spermatogenesis. Mali et al. [23] reported a cyclic, stage-dependent regulation of vimentin expression during spermatogenesis, and Spruill et al. [28] examined the hormone-dependent phosphorylation of vimentin by the action of a CAMP-dependent protein kinase. We demonstrate in this report: 1. The expression of CKs 8 and 18 together with vimentin in immature Sertoli cells 2. The retained or regained expression of these CKs under pathologic conditions leading to atrophy of spermatogenic cells The expression of CK in cells of epithelial differentiation independent of their histogenetic derivation is a well-established phenomenon. It is much more difficult to explain why CKs are lost in Sertoli cells at maturation, a fact that is also true in the rat [25]. Although stage-specific expression has already been observed in certain tissues [8, 191, the mechanism of down-regulation is not understood.

Fig. 1a-d. Seminiferous tubule in the vicinity of a growing seminoma. All Sertoli cells are positive for cytokerdtin with antibody ,445-B/53. b Seminiferous tubule in a senile testis of a 76-year-old man. Note the fibrillar staining of cytokeratin-18-positive Sertoli cells. Antibody A45-BIB3. c Cytokeratin-I 8-positive Sertoli cells in a senile testis of a 85-year-old man. Antibody CK-2. d Expression of cytokeratins in the testis of a 4-month-old fetus. Antibody A45-BIB3. Immunoperoxidase technique (a, c, d) ham, 50 pm. Imrnunofluorescence method (b); bars, 20 pm

69

Fig. 2a, b. Double-label-immunofluorescence microscopy performed on cryostat sections of human testis from a 10-month-old child using guinea-pig antibodies to vimentin (a) and the monoclonal cytokeratin antibody A45-EIE3 (b), bars 20 pm

It appears that the structures and/or functions exerted by CKs are not compatible with the specific functions of Sertoli cells at maturity. If so, it could well be that the CK expression in mature Sertoli cells is actively suppressed. In atrophic Sertoli cells, this suppressive activity might be missing. A superficially similar situation is the coexpression of CK and vimentin in pathologically altered renal tubular epithelia [13], but in this case primarily CK-positive epithelia additionally express vimentin after tissue injury. Nevertheless, it must remain open whether the coexpression of CK and vimentin in Sertoli cells must be interpreted as a regressive change (for other explanations of coexpression see Kasper et al. [20] or as progressive functional metaplasia. An important aspect worth investigating in more detail is the possible use of immunohistochemistry with CK Mabs as an aid in the early diagnosis of pathologic processes in the testis. The feasibility of this approach remains to be established. however.

Acknowledgements. We thank the following colleagues for their generous supply of monoclonal antibodies: Dr. J. Bartek, Brno, CSSR ( E A 16, C l O ) ; Dr. P. Leoncini, Siena, Italy (SK2-27); Dr. F. Gudat, Basel, Switzerland (lu-5); Dr. R. Moll, Mainz, FRG (VZM3E4); and Dr. G.A. Bannikov, USSR (E3).

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