Localization of human follicle-stimulating hormone in the testis

^a Institute of General Biology of the University of Siena and Center for the Study of Germinal Cells, C.N.R., 53100 Siena, Italy
^b Ares Services S.A., CH-1211 Geneva 20, Switzerland

	ABSTRACT

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Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [¹³¹I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.—Baccetti, B., Collodel, G., Costantino-Ceccarini, E., Eshkol, A., Gambera, L., Moretti, E., Strazza, M., Piomboni, P. Localization of human FSH in the testis. FASEB J. 12, 1045–1054 (1998)

Key Words: FSHR mRNA • FSH antibody • human testis cells • mouse testis cells • ultrastructure

	INTRODUCTION

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FOLLICLE-STIMULATING HORMONE (FSH)² is a glycoprotein produced by the hypophysis, required for initiation and regulation of spermatogenesis in the male and for the ovulation in the female. Its biochemical action on the target tissue is initiated by interaction of the hormone with a cell surface receptor. This binding is followed by the activation of adenylate cyclase via a guanine nucleotide binding protein (G-protein) and subsequent stimulation of protein kinase activity (GRK). An enzyme of this group (GRK4) was localized in human testes and spermatozoa by Sallese et al. (1). These FSH receptors are monomeric glycoproteins members of the G-protein-coupled receptor superfamily (2), which includes the muscarinic receptor (3), a surface protein largely diffused in the mammalian spermatozoa.

During development of the rat testis, the response to FSH was studied by detecting the expression of the FSH receptor (FSHR) mRNA (4). This expression occurs primarily (at approximately 5 days of age) in Sertoli cells, where FSH is an important mitogen (5). Subsequently, the mitotic rate decreases and the primary action of FSH concerns Sertoli cell maturation (6). The localization of FSH in the isolated rat Sertoli cells was demonstrated immunocytochemically by transmission electron microscopy (TEM) on the plasma membrane and in cytoplasmic vesicles (7). The presence of FSH in the germinal cells is still controversial because previous investigators all used light microscopy.

We decided to investigate the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions. This was always carried out at the submicroscopical level by using an antigenicity conservative procedure that is different from the ones used by previous investigators. This research was undertaken in order to assess the presence of FSH in the various cell types of the testis. FSH is, in fact, strongly implicated in the control of male germ cell maturation and apoptosis, and consequently is involved in male fertility.

	MATERIALS AND METHODS

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Animals
Balb/c mice, 7 and 24 days old, were injected with 1 and 10 I.U. of human FSH (Serono, Suisse) and then killed 24 h postinjection. The testes were immediately removed and fixed for the electron microscopy procedure.

Patients
Testicular bioptic tissue was obtained from a fertile patient undergoing surgery for a contralateral testicular seminoma. The patient submitted to semen and hormonal analyses. The serum level of FSH was tested twice before the operation and found to be normal. The specimen was processed for electron microscopy.

Cell culture
Y1 cell line, obtained from a mouse adrenal tumor was stably transfected with human FSH receptor cDNA (gift from Serono, Suisse). Cells were cultured in vitro with Ham's F10 containing 15% horse serum, 2.5% fetal bovine serum, 2 mM L-glutamine, and 80 µg/ml of G418. When cells reached 80% confluence, the plates were washed twice with 8 ml of assay medium (Ham's F10 medium supplemented with 1 mg/ml bovine serum albumin, 2 µM L-glutamine). The cells were then cultured in assay medium alone or with the addition of human FSH (Serono, Suisse), 10 I.U., for 20 min, 40 min, and 4 h. At the end of the incubation, the cells were gently dislodged from the petri dish surface by using a nonenzymatic cell dissociation solution (Sigma, St. Louis, Mo.). The cells were centrifuged and the pellet was fixed for the electron microscopy procedure.

Fluorescence immunocytochemistry
Human ejaculated spermatozoa, washed in phosphate-buffered saline (PBS), were smeared on glass slides, air dried, rinsed in PBS, and treated with cold methanol for 20 min at -20°C and cold acetone at 20°C for 5 min. After air drying, the slides were rinsed in PBS and saturated for 20 min with PBS-bovine serum albumin (BSA) 1% containing 5% normal goat serum (NGS). The specimens were then incubated overnight at 4°C with the anti-human FSH monoclonal antibody (Chemicon, El Segundo, Calif.) diluted 1:100 in PBS/0.1% BSA/1% NGS. After washing (3x10 min in PBS), the specimens were treated for 1 h at room temperature with FITC goat anti-mouse immunoglobulin G (IgG) (Sigma) diluted 1:100. After extensive washing, the slides were mounted in glycerol containing 5% n-propylgallate and observed with a Leitz Aristoplan light microscope equipped with fluorescence apparatus.

Electron microscopy
Ultrastructure
Human testicular biopsy was fixed for 2 h at 4°C in Karnovsky fixative, rinsed overnight in 0.1 M, pH 7.2, cacodylate buffer, postfixed in 1% buffered OsO₄, dehydrated, and embedded in Epon Araldite. Ultrathin sections were cut by Supernova Reichert-Jung ultramicrotome, and stained with uranyl acetate and lead citrate. The observations were made with a Philips CM10 transmission electron microscope.

Immunocytochemistry
Human testicular biopsy, mice testes, and Y1 cells were fixed at 4°C in 3% p-formaldehyde/0.1% glutaraldehyde in PBS, rinsed overnight in PBS with 5% sucrose, and for 30 min in PBS containing 50 mM NH₄Cl. After washing in PBS, the small blocks of material were dehydrated and embedded in Lowicryl K4M at -35°C, according to Carlemalm et al. (8). Ultrathin sections, mounted on formwar-coated gold grids, were saturated for 20 min with Tris-buffered saline (TBS) -1% BSA containing 5% NGS and treated overnight at 4°C with the anti-human FSH monoclonal antibody (Chemicon) diluted 1:140 in TBS-0.1% BSA-1% NGS. After incubation, the specimens were washed three times in TBS with 0.1% BSA-0.05% Tween 20 and then incubated for 1 h at RT with the goat anti-mouse IgG antibody conjugated with colloidal gold. After deep rinsing (washed three times for a total 60 min in TBS-BSA, TBS, and distilled water), the grids were stained with uranyl acetate, then observed and photographed with the CM10 electron microscope (Philips, Mt. Vernon, N.Y.). Control sections were treated in the same way, omitting the primary antibody.

In situ hybridization
The mRNA encoding for the FSH receptor was detected by a 45-mer cDNA antisense oligonucleotide complementary to the 1441–1486 base pair region of the FSH receptor sequence (25) (5'-AAT-CCA-GCC-CAT-CAC-CAT-GAT-ACT-GGC-AGC-ATG-GCG-GAG-CTG-CAC-3'). The 3' end-biotinylated oligonucleotide was purchased from Genenco Medical (Rome, Italy). The biotinylated probe was suspended in hybridization buffer containing 4x SSC (0.6 M sodium chloride, 0.06 M sodium citrate), 50% freshly deionized formamide, 10% just-prepared dextran sulfate, 0.5 mg/ml Escherichia coli tRNA, and 1 mg/ml salmon sperm DNA.

Ultrathin sections of Lowicryl-embedded samples (human testis, mouse testis, and Y1 cells), mounted on formwar-coated gold grids, were rehydrated with sterile 2x distilled water. The probe was denatured for 3 min at 80°C and immediately cooled at 0°C. The grids, previously treated with K proteinase 10 µg/ml in PBS, were incubated in the hybridization solution containing the probe at a concentration of 475 ng/ml. Incubation was performed by floating the grids on 20 µl drops in a moist chamber for 16 h at 37°C. After incubation, the grids were washed in 50% formamide: 4x SSC (2x 5 min, at 37°C); 4x SSC (2x 5 min, at 37°C); 0.2x SSC (2x 5 min, at RT); distilled water (2 min at RT). Control sections were treated in the same way, omitting the DNA probes.

The hybridized specimens were treated with mouse monoclonal anti-biotin antibody (IgG) (Boehringer-Mannheim, Mannheim, Germany) recognized by a colloidal gold-conjugated goat anti-mouse IgG. For the control of the secondary gold-conjugated antibody, incubation with the anti-biotin antibody was omitted. All sections were carefully rinsed (washed three times in TBS-BSA, TBS, and distilled water for a total of 45 min) and observed with electron microscopy.

	RESULTS

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a: FSH in human testes and ejaculated spermatozoa
In human testes, the cytoplasm of the Sertoli cells and germinal cells, from spermatogonia to round spermatids, has the peculiar characteristic of being full of numerous invaginations and vesicles connected to the plasma membrane ( Fig. 1A). Examined by conventional transmission electron microscopy, the vesicles appear circular or tubular, the first being cross sections of the second ones. The diameter of the vesicles varies from 0.1 to 0.2 µm. This picture suggests the presence of an intense phenomenon of endocytosis, occurring mostly in the peripheral cytoplasmic area of the cell ( Fig. 1A).

View larger version (155K): [in this window] [in a new window]   Figure 1. A) TEM micrograph of a cross section of human testicular tissue embedded in Epon-Araldite. A Sertoli cell (SC) is connected with two spermatogonia (SP) and lines the basal lamina (BL). The cytoplasm of the Sertoli cell as well as that of the spermatogonia contains many tubules and vesicles (EV), which are the expression of endocytotic processes. The Sertoli cells are characterized by irregular nucleus (SN), dark and small mitochondria (SM), and lipidic vacuoles; the young germinal cells have round nuclei (GN) and large and pale mitochondria (GM). B) TEM micrograph of a cross section of Lowicryl-embedded human testicular tissue. The sections have been treated with the anti-human FSH monoclonal antibody. Two Sertoli cells (SC) are evident; a cluster of gold granules is evident near the nucleus (SN) of one and appears to be contained in a dense endocytotic vesicle (EV). A) x8,000; B) x44,000.

In sections of Lowicryl-embedded material, treated with anti-FSH monoclonal antibody and labeled with a secondary antibody colloidal gold-conjugated, we found habitual clusters of granules in the peripheral area (from 3 to 12) localized in the vesicles ( Fig. 1B; Fig. 2A–D ). The labeling is absent in controls, where incubation with the primary antibody was omitted.

View larger version (208K): [in this window] [in a new window]   Figure 2. TEM micrographs of Lowicryl-embedded human testicular tissue treated with the anti-human FSH monoclonal antibody. A) A human spermatogonium, with a round nucleus (GN) and large mitochondria (GM), contains clusters of gold granules (arrows) localized near the plasma membrane (PM). B) Enlargement of panel A showing that the gold particles are located in endocytotic vesicles (EV) or in a depression of the plasma membrane (arrow). C) Gold particles decorate an endocytotic vesicle (EV) located near the spermatocyte nucleus (GN), surrounded by mitochondria (GM). D) Gold granules label a circular vesicle (EV) in the cytoplasm of a round spermatid, which shows a nucleus (GN) with almost dense chromatin and a well-shaped acrosome (A). A) x13,000; B) x60,000; C) x42,000; D) x23,000.

The presence of FSH in the cytoplasm of both germinal and Sertoli cells is substantiated by in situ hybridization detecting the mRNA of the FSH receptor, carried out on the same Lowicryl-embedded human testicular material. According to this technique, the colloidal gold granules are grouped in the perinuclear area and at the periphery of the cells, where the FSHR mRNA is expressed, in Sertoli as well as in germinal cells ( Fig. 3). The reaction is also positive inside the nucleus, mainly at the periphery of the nucleoplasm, in the form of a linear arrangement of granules. The reaction is totally negative in both kinds of controls.

View larger version (148K): [in this window] [in a new window]   Figure 3. TEM micrographs of human testicular tissue embedded in Lowicryl and cross sectioned. By in situ hybridization, detection of the mRNA for the FSHR is revealed by gold granules (arrows) localized as follows: A, B) Sertoli cell cytoplasm; C, D) spermatocyte cytoplasm; E, F) young spermatid cytoplasm containing an evident acrosome (A); G) late spermatid cytoplasm. SN, Sertoli cell nucleus; GN, germinal cell nucleus; GM, germinal cell mitochondria. A) x13,500; B) x54,000; C) x23,000; D) x54,000; E) x19,000; F) x38,000; G) x42,000. Panel B is an enlargement of panel A; panel D is an enlargement of panel C; panel F is an enlargement of panel E (dotted regions).

We investigated the presence of human FSH in ejaculated human spermatozoa, which are almost devoid of endocytotic vesicles. Labeling with the anti-hFSH monoclonal antibody, examined by fluorescent immunocytochemistry, was completely negative.

b: Human FSH injected in mouse
This experiment was carried out in order to verify the observations referred to in section a. We used two groups of young mice, 7 and 24 days old, the first devoid of endogenous FSH, the second with advanced spermatogenesis. Localization of the injected human FSH was tested by immunocytochemistry. Clusters of colloidal gold granules were observed in Lowicryl-embedded testicular material after treatment by anti-human FSH monoclonal antibody. In the first group of mice, the particles were localized in the cytoplasm of spermatogonia contained in endocytotic vesicles ( Fig. 4A). In the second group, the particles were present in the same regions of germinal and Sertoli cells described in section a, namely, in the cytoplasm contained in endocytotic vesicles ( Fig. 4B–D).

View larger version (158K): [in this window] [in a new window]   Figure 4. TEM micrographs of cross sectioned mouse testicular tissue embedded in Lowicryl. A) Anti-human FSH monoclonal antibody recognizes the human FSH in vivo injected in a 7-day-old mouse. Colloidal gold particles (arrow) are localized in a cytoplasmic vesicle (EV) of a spermatogonium, whose nucleus (GN) contains granular chromatin; it is surrounded by mitochondria (GM). B) The same antibody is bound to a cytoplasmic vesicle (EV) in a spermatogonium from a 24-day-old mouse that was injected with human FSH. The germinal nucleus (GN) is surrounded by mitochondria (GM). C) Cytoplasmic localization of the human FSH, contained in two endocytotic (EV) vesicles (arrow), is confirmed in a round spermatid where the acrosomal vesicle (A) is evident. D) In a 24-day-old mouse, the FSH molecule is identified by a cluster of gold particles (arrow) contained in a vesicle (EV) that is located in the cytoplasmic extension of a Sertoli cell, inserted between two mature elongate spermatids. The latter show dense nuclei (GN) and almost mature acrosomes (A). A) x30,000; B) x45,000; C) x30,000; D) x60,000.

c: Human FSH in vitro administrated to the Y1 cell line
This experiment was carried out as a control for the localization of human FSH by using the Y1 cell line stably transfected with FSH receptor cDNA ( Fig. 5A) and devoid of endogenous FSH. The cells were treated in vitro with human FSH. Forty minutes after treatment, these cells clearly demonstrated a reaction to the administered FSH, assuming a round shape ( Fig. 5B). On these FSH-treated cells, we applied two techniques: the immunochemical detection of human FSH molecule by the specific monoclonal antibodies and the in situ hybridization for localization of the mRNA for FSHR. The first procedure revealed the presence of human FSH bound to the plasma membrane ( Fig. 5C) and in the dense cytoplasmic vesicles ( Fig. 5D), as described for human and mouse testes, respectively, in sections a and b ( Fig. 5C, D). The second procedure identified the messenger for FSHR in the nucleus and cytoplasm of Y1 cells ( Fig. 5E, F).

View larger version (160K): [in this window] [in a new window]   Figure 5. A, B) Light microscopy micrographs of the Y1 cell line before (A) and after (B) in vitro treatment with human FSH. The cells react to the FSH, assuming a round shape 20–40 min after treatment. C, D) TEM micrographs of Y1 cells, 40 min after the hFSH treatment, embedded in Lowicryl and sectioned. Anti-human FSH monoclonal antibody [revealed by the presence of gold granules (arrows)] recognizes the FSH molecule on the cell surface (PM) or in an endocytotic vesicle (EV). M, mitochondrion. E, F) In situ hybridization with a DNA biotinylated probe detecting the FSHR mRNA in Y1 cells. In panel E, gold particles (arrow) are present in the nucleus (N); in panel F, gold granules (arrow) are localized in the cytoplasm between the nucleus (N) and the plasma membrane (PM). A, B) x300; C) x75,000; D, E) x42,000; F) x60,000.

	DISCUSSION

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The FSH molecule was found primarily in rat and mouse Sertoli cells. The role of FSH on Sertoli and Leydig cell mitosis and maturation was demonstrated in men, mice, and rats by detecting the localization of anti-FSH antibodies (10–15) or of [¹³¹I]-labeled FSH (16, 17) by injecting fluorochrome-tagged FSH (18), or by detecting FSHR mRNA (4, 13, 14, 19, 20) or anti-FSHR antibodies (21). Even in Sertoli cells, internalization and degradation after endocytosis were demonstrated at the TEM level by [¹³¹I]iodo-FSH autoradiography (22, 23).

For many years, localization was believed to be restricted to the plasma membrane, where it was claimed that the receptor was localized. Some investigators subsequently detected the hormone in Sertoli cell cytoplasm by autoradiographic and immunocytochemical procedures, and others were able to reveal the presence of endocytotic vesicles by autoradiography applied to electron microscopy (22), and immunocytochemistry (7). Moreover, by in situ hybridization at the light microscopic level, the FSHR mRNA has been consistently found in the Sertoli cytoplasm, both in rats (20) and men (14). The data presented here largely substantiate this latter view. We used two different models (mice and human Sertoli cells) studied by two different techniques: immunocytochemistry and in situ hybridization. The novelties were the use of electron microscopy on Lowicryl-embedded material and the multiple experimental approach. The hypothesis of FSH internalization by endocytosis has been largely supported by our ultrastructural observations. In normal human testes, we have detected the FSH molecule, by anti-FSH monoclonal antibodies, in the cytoplasmic region of the Sertoli cells contained in endocytotic vesicles. The FSHR mRNA has been detected by in situ hybridization in the Sertoli cytoplasm. Human FSH injected in 24-day-old mice was observed localized in the same areas where endogenous FSH was found in the human normal testis.

The submicroscopical distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has been clarified by us, improving on evidence in the literature.

The presence of FSH in the germinal cells has been controversial. All the previous investigators used light microscopy: Orth and Christensen (17) localized [¹²⁵I]-labeled FSH on spermatogonial surface; Yoon and Golimbu (24) immunocytochemically localized hFSH in the luminal, basal, and interstitial area of human seminiferous tubules affected by Sertoli barrier disorders; Kulkarni et al. (12) immunocytochemically localized FSH in spermatogonia of immature marmoset testis; Boitani et al. (25) found that FSH is essential for the progression of type A spermatogonia up to the stage of pachytene spermatocites in cultures of rat testes. On the other hand, Wahlstrom et al. (13) failed to localize FSH in human and rat germinal cells. The same negative results were achieved by Bockers et al. (14), using immunocytochemistry and in situ hybridization for FSHR mRNA in testicular tissue of infertile men.

The most important data offered by our research have therefore concerned the germinal cells in normal human testis and in the testes of 7-day-old mice, still devoid of endogenous FSH, as well as of 24-day-old ones, both injected with human FSH. All of our observations demonstrate the presence of FSH and of the FSHR mRNA: the first on the plasma membrane and in the endocytotic vesicles; the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia or spermatocytes up to round spermatids. In this respect, the way of penetration was the endocytosis via membrane vesicles in which the FSHR molecule is localized, where its messenger is largely present in the cytoplasm, and it is responsible for the binding and subsequent internalization of the FSH molecule. Using low microscopical resolutions, previous investigators failed to find FSH or its receptor in the human or rat germinal cells (13, 14); others obtained uncertain positive immunocytochemical signals in rat (16, 17) as well as in marmoset testes (12).

As a control of our techniques, we carried out a parallel experiment: human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The human FSH molecule has been localized by monoclonal antibodies on the plasma membrane and in cytoplasmatic vesicles. By using in situ hybridization, FSHR mRNA was found in the nucleus or scattered in the cytoplasm. Moreover, we observed that these cells reacted to the administered FSH by assuming a round shape.

We can conclude that the FSH is present in Sertoli cells and in round germinal cells (both expressing the FSHR), that FSH penetrates in a similar way in both kind of cells by endocytosis, and that it therefore is subsequently localized in both cells in the same membranous organelles.

	FOOTNOTES

 
¹ Correspondence: Institute of General Biology of the University of Siena and Center for the Study of Germinal Cells, C.N.R., Via T. Pendola 62, 53100 Siena, Italy. E-mail:

² Abbreviations: BSA, bovine serum albumin; NGS, normal goat serum; FSH, follicle-stimulating hormone; G-protein, guanine nucleotide binding protein; FSHR, FSH receptor; TEM, transmission electron microscopy; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; IgG, immunoglobulin G.

Received for publication December 1, 1997. Accepted for publication March 5, 1998.

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