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Testis |
Department of Cell Biology, Georgetown University Medical Center, Washington, DC 20057
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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growth factors, male reproductive tract, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no evidence for the expression of the receptors on Sertoli cells. In addition, the male transgenic mice exhibited spermatogenic arrest and were found to be infertile. Thus, we hypothesized that Sertoli cell-secreted VEGF has a role in the process of spermatogenesis. The process of spermatogenesis begins with the type A spermatogonium. Type A spermatogonial cells can either proliferate and renew themselves into other type A spermatogonia or proliferate and differentiate sequentially into intermediate spermatogonia, type B spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and eventually spermatozoa [12]. In the present study, we examined the expression of VEGFR-1 and VEGFR-2 on germ cell types within the seminiferous tubule of normal mouse testis by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult males and mothers with 6-day-old male pups belonging to Balb-C strain of mice were obtained from the Charles River Breeding Laboratories (Wilmington, MA). The animals were maintained in the Research Resource Facility at Georgetown University Medical Center on a 12L:12D cycle and were given access to food (rodent laboratory chow 5001; Ralston-Purina, St. Louis, MO) and water ad libitum. All animal care procedures were carried out according to the National Research Council's Guide for the Care and Use of Laboratory Animals. The experimental protocols used in this study were approved by the Animal Care and Use Committee of Georgetown University.
Tissue Collection and Processing
Testes were excised from immature and adult males killed by carbon dioxide inhalation and fixed in Bouin fixative solution for 24 h at room temperature. They were embedded in paraffin following dehydration in ascending concentrations of alcohol. Sections of 5 µm thickness were cut and mounted on glass slides.
Isolation of Sertoli Cells and Different Germ Cell Types From the Mouse Testis
Sertoli cells were isolated from 6-day-old mouse testis by the method standardized in our laboratory for rats with minor modifications [13]. Briefly, decapsulated testes were minced for a short period and suspended in Dulbecco Modified Eagle Medium (DMEM) containing 500 U/ml collagenase (CLS II) and 5 µg/ml DNAse (type I) for 10 min in a shaking water bath (90 cycles/min) at 34°C. The DMEM contained 1.2 mM calcium and 0.8 mM magnesium. The mixture was allowed to settle. The supernatant was discarded. The sediment, which mostly consisted of long seminiferous cord fragments, was washed twice with DMEM and resuspended in DMEM containing 500 U/ml collagenase, 5 µg/ml DNAse, and 1 mg/ml hyaluronidase (type III) for 20–30 min in a shaker water bath at 34° C. The digestion was terminated when the peritubular myoid layer was observed to have fallen away from the base of the Sertoli cells. The seminiferous cord fragments, which were now much smaller in size, were washed twice with DMEM and subjected to a third digestion for approximately 15–20 min with collagenase (500 U/ml), hyaluronidase (1 mg/ml), and DNAse (5 µg/ml) until small clumps of 10–50 Sertoli cells were obtained. The clumps were pelleted at low-speed centrifugation, washed twice in DMEM, and resuspended in DMEM/F12 medium supplemented with ITS (insulin [5 µg/ml], transferrin [5 µg/ml], and selenium [5 ng/ml]) and 100 ng/ml FSH. The purity of each preparation was established by staining for smooth muscle 
-actin (a marker for contaminating peritubular myoid cells), c-kit (a marker for contaminating type A spermatogonial cells) and 3ß-hydroxysteroid dehydrogenase (a marker for contaminating Leydig cells). The purity of Sertoli cells used in the studies ranged between 96% and 98%. Sertoli cell yield from 40 mice (6 days old) was approximately 100 million. The cells were seeded on laminin-coated coverslips at a density of 
5 x 105 cells per well in 24-well plates and incubated at 34°C for 3 days.
Type A spermatogonia were isolated from 6-day-old mouse testis by the STA-PUT technique standardized in our laboratory and characterized by immunocytochemistry using a c-kit receptor antibody [14]. Briefly, the decapsulated testes were suspended in DMEM/F12 containing collagenase (1.5 mg/ml) and DNAse (1 µg/ml), and incubated at 34°C for 15 min in a shaking water bath operated at 100 cycles/min. After two washes in DMEM/F12 medium, seminiferous cord fragments, mostly devoid of interstitial cells, were incubated in DMEM/F12 medium containing collagenase (1.5 mg/ml), hyaluronidase (1.5 mg/ml), trypsin (0.5 mg/ml), and DNAse (1 µg/ml) for 20–30 min using the conditions described above. The dispersed cells were washed twice with medium and filtered through 80 µm and 40 µm nylon mesh (Tetco Inc., Briarcliff Manor, NY), successively. The cells of the dissociated epithelium were then separated by sedimentation velocity at unit gravity at 4°C, with use of a 2–4% BSA gradient in DMEM/F12 medium. The cells were bottom loaded into an SP-120 chamber in a volume of 30 ml, and a BSA gradient was generated using 275 ml of 2% and 4% BSA. The cells were allowed to sediment for a standard period of 2.5 h, and then 35 fractions of 15-ml volume were collected at 90-sec intervals.
The cells in each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation, and then resuspended in DMEM/F12 medium. Type A spermatogonia were further purified from contaminating Sertoli cells by differential plating in the presence of 5% horse serum for 4 h (95–99% pure). The purity of the cells was established by immunostaining for c-kit (specific for spermatogonia), smooth muscle -actin (a marker for contaminating peritubular myoid cells), and 3ß-hydroxysteroid dehydrogenase (a marker for contaminating Leydig cells). The contaminating cells were either peritubular myoid cells or occasionally, Leydig cells. Sertoli cell contamination as determined by staining with Oil O-Red (Sigma Chemical Company, St. Louis, MO) was very minimal. Approximately 10 million spermatogonia are obtained from 80 mice (6 days old). Using the same technique, both pachytene spermatocytes and round spermatids were isolated from adult mice. Generally, we obtained 90%–95% pure populations of pachytene spermatocytes and round spermatids. Contaminating cells in round spermatid population were either type B spermatogonia or preleptotene spermatocytes as determined by morphology. Spermatozoa were isolated by mechanical expression from the epididymis of adult mice.
Immunofluorescence Staining
The sections of testis mounted on glass slides were deparaffinized and rehydrated before staining. For the visualization of VEGF and its receptors, the sections were initially blocked with normal goat serum for 30 min at room temperature. Subsequently the sections were incubated overnight at 4°C with the primary antibody against VEGF (mouse monoclonal antibody, # sc7269, Santa Cruz Biotechnology Company, Santa Cruz, CA) or VEGFR-2 (rat monoclonal antibody, #MAB1669, Chemicon International, Temecula, CA) or VEGFR-1 (rabbit polyclonal antibody, # sc316, Santa Cruz Biotechnology). At the end of incubation, sections were washed and treated with either Texas Red-conjugated or fluorescein isothiocyanate-conjugated secondary antibodies against the immunoglobulins of species in which primary antibodies were raised (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. The sections were mounted with cover glasses and Permount (Electron Microscopy Sciences, Fort Washington, PA) and visualized in a fluorescence microscope (Carl Zeiss, Inc., Gottingen, Germany). Appropriate controls for immunostaining included lack of primary and secondary antibodies.
Freshly isolated Sertoli cells, type A spermatogonia, and other differentiated germ cell types were centrifuged at 90 x g for 5 min onto glass slides. The cells were fixed and permeabilized with ice-cold methanol for 3 min and then washed with phosphate buffered saline. Immunofluorescence staining was performed as described above for the testicular sections.
RT-PCR
Total RNA was extracted from the whole testis, isolated Sertoli cells, type A spermatogonia, pachytene spermatocytes, and round spermatids using the TRIzol Reagent isolation kit (Invitrogen Life Technologies, Carlsbad, CA). The first strand of cDNA was synthesized following the protocol of the Superscript first-strand synthesis system for RT-PCR (Invitrogen Life Technologies). To ascertain the quality of the first strand of cDNA synthesized, a control PCR with a set of primers for ubiquitously expressed glyceraldehydes phosphate dehyrdrogenase (GAPDH) was performed. The amplified product corresponded to the expected size (452 bp). The following set of three primers was used to amplify the cDNA for the products of VEGF and its receptors: mouse VEGF (826 bp) 5' GGA CCC TGG CTT TAC TGC 3' and 5' CGG GCT TGG CGA TTT AG 3'; mouse VEGFR-2 (1792 bp) 5' CCT GGC TGA CCC GAT TCC 3' and 5' TCC CGC TTT GTT GAT GGC 3'; mouse VEGFR-1 (283 bp) 5' GGT GCC CGC TCT TTG 3' and 5' TGT CTC AGT GGG GAT TGC 3'. Prior to PCR cyclic amplification, the reaction mixtures were incubated at 94° C for 4 min. PCR amplification of the cDNA was performed in a Peltier thermal cycler (MJ Research Inc., Waltham, MA) for a total of 35 cycles: 94°C for 45 sec, 58°C for 30 sec, and 72°C for 1
min. This was followed by eventual extension at 70°C for 10 min. The PCR products were separated on 1.5% agarose gel using 1x Tris-acetate-EDTA buffer. The smaller size products, less than 500 bp, were separated on 2% agarose gel. For sequencing purposes, the products of PCR were separated by polyacrylamide gel electrophoresis, and the individual bands were excised. They were purified and subjected to sequence analysis with an automated sequencer (Applied Biosystems, Foster City, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Paraffin-embedded sections of normal adult mouse testis were immunostained with a mouse monoclonal antibody against VEGF. Immunofluorescence was observed both in the interstitial space and within the seminiferous tubules (Fig. 1A). Sertoli cells exhibited characteristic streaks (arrowheads) of immunofluorescence from the base to the lumen of the seminiferous tubule. None of the germ cell types in the seminiferous tubule showed immunofluorescent staining. The cells in the interstitial space appeared to express VEGF strongly (arrow). Sections stained with nonimmune mouse serum, in the absence of primary or secondary antibody, showed no immunofluorescence (Fig. 1B).
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VEGF Is Synthesized by Sertoli Cells Within the Seminiferous Tubules
To determine whether Sertoli cells synthesize VEGF within the seminiferous tubule, Sertoli cells were isolated from immature mice testes (6 days old). Figure 2 demonstrates the expression of VEGF in sections of 6-day-old mouse testis. In similarity with adult mouse testis, VEGF immunostaining was observed both in the interstitial space and seminiferous tubules (Fig. 2A). In the absence of a lumen in the seminiferous cords of 6-day-old mouse testis, Sertoli cells present at the periphery and in the center of the cords showed a strong expression (arrowheads) of VEGF. Spermatogonia interspersed between Sertoli cells at the base of the tubule showed no immunofluorescence (arrows). Interstitial cells showed very high levels of immunofluorescence for VEGF (asterisk). Nonspecific fluorescence was not observed when stained with nonimmune mouse serum (Fig. 2B). Following confirmation of expression of VEGF by Sertoli cells in situ at 6 days of age, we isolated Sertoli cells and examined for the specific localization of VEGF in cultured cells. Immediately after isolation, Sertoli cell purity ranged between 96% and 98%. The contaminants were peritubular myoid cells and type A spermatogonia. Sertoli cells were cultured for 3 days in the presence of cytosine arabinoside and then treated with 20 mM HEPES for 3 min to remove any attached residual germ cells. Following this procedure, the purity of the Sertoli cell preparation was closer to 100% with occasional contamination with peritubular myoid cells. Cultured Sertoli cells were stained for VEGF with Texas Red-labeled secondary antibody and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Fig. 2C). Although the cytoplasm of Sertoli cells exhibited a red immunofluorescence (arrows), the nuclei were stained blue.
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Total RNA isolated from these cells was reverse transcribed, and the resultant cDNA was used to perform PCR with specific primers for mouse VEGF. The results of the study are shown in Figure 2D. With specific primers designed to amplify an 826-bp fragment of VEGF, two bands migrating around molecular size of 800 bp were observed on agarose gel in the whole testis and isolated Sertoli cells, suggesting that Sertoli cells synthesize VEGF within the seminiferous tubule of testis. Because the bands corresponding to VEGF were in close molecular size range, the bands were separated far apart on a 3.5% polyacrylamide gel, purified, and sequenced using an automated sequencer. The upper band corresponded to expected 826 bp. The lower band was identical in sequence to the upper band until 370 bp from the start of the forward primer. Similarly, 240 bp from the start of the reverse primer were identical to the upper band. There appeared to be a deletion as well as base substitutions in the intervening sequence. Although immunohistochemistry for VEGF did not reveal the expression of VEGF in germ cell types, RT-PCR for VEGF was performed with the total RNA isolated from type A spermatogonia, pachytene spermatocytes, and round spermatids. The specific primer set used for amplifying an 826-bp fragment of VEGF was unable to generate the expected PCR product from all the three germ cell types (data not shown).
Type A Spermatogonia Synthesize and Express VEGFR-2 but Not VEGFR-1
To determine the potential target cells of VEGF secreted by Sertoli cells, we investigated the expression of receptors for VEGF, i.e., VEGFR-1 and VEGFR-2, on germ cell types within the seminiferous tubules. Because 6-day-old mouse testis consists of only type A spermatogonia and Sertoli cells in the seminiferous tubule, we examined the cross-sections by immunofluorescent staining for VEGFR-1 (Fig. 3) and VEGFR-2 (Fig. 4). There was no evidence for the presence of VEGFR-1 either on Sertoli cells or on type A spermatogonia (Fig. 3A). However, VEGFR-1 immunostaining was strong in the interstitial cell population. The specificity of immunostaining was established by deleting the primary antibody and substituting with nonimmune rabbit serum (Fig. 3B). Interestingly, VEGFR-2 was specifically localized to type A spermatogonia (Fig. 4A). Nonimmune rat serum showed lack of immunofluorescence in tissue sections stained in parallel (Fig. 4B). Using a double immunostaining for VEGF and VEGFR-2, simultaneous localization of VEGF in Sertoli cells and VEGFR-2 in type A spermatogonia was observed (Fig. 4C). In addition, colocalization of VEGF and VEGFR-2 in the interstitial cell population was also observed. We confirmed these results in both isolated populations of Sertoli cells and type A spermatogonia.
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Type A spermatogonia exhibited localization of VEGFR-2 specifically in the cytoplasm (Fig. 4D). In contrast, type A spermatogonia did not express VEGFR-1 (Fig. 3C). Furthermore, we investigated the synthesis of these receptors by type A spermatogonia using RT-PCR with specific primer sets. From Fig. 4E, it is apparent that type A spermatogonia synthesized mRNA for VEGFR-2. A 1792-bp band corresponding to the primer set for VEGFR-2 is visible both in the whole testis and isolated type A spermatogonia. Since PAGE is more efficient, the RT-PCR product for VEGFR-2 was separated on a 3.5% polyacrylamide gel. The expected size band of 1792 was excised, purified, and sequenced with specific primers to amplify approximately 500-bp fragments overlapping with each other. The sequence data matched exactly with the DNA sequence for VEGFR-2. In contrast, Figure 3D shows the expression of mRNA for VEGFR-1 in only the whole testis but not in the isolated type A spermatogonia. A 283-bp band corresponding to the primer set for VEGFR-1 is apparent in the whole testis. Appearance of the band in whole testis confirms the presence of VEGFR-1 in the cells of interstitial space outside the seminiferous tubule (Fig. 3A). The RT-PCR band for VEGFR-1 (283-bp size) was sequenced, and the sequence information matched with the corresponding DNA sequence for VEGFR-1.
VEGFR-2 Expression Is Absent in the Seminiferous Tubule of the Adult Mouse Testis
In the previous experiment with immature mouse testis, it was apparent that VEGFR-2 is synthesized and expressed by type A spermatogonia. To determine whether VEGFR-2 expression is maintained during the process of differentiation of spermatogonia into spermatocytes and spermatids, we examined cross-sections of adult mouse testis for VEGFR-2 expression. A strong immunofluorescent staining of interstitial cells for VEGFR-2 was observed (Fig. 5A). Surprisingly, no immunofluorescence was observed within the seminiferous tubule. Neither more differentiated spermatogonia nor differentiating germ cell types, i.e., pachytene spermatocytes, round spermatids, elongating spermatids, and spermatozoa, exhibited any fluorescence. When the primary antibody was substituted with nonimmune rat serum, immunofluorescent staining of interstitial cells was completely lost (Fig. 5B).
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Gain of VEGFR-1 Expression by the Differentiating Germ Cells in Adult Mouse Testis
Since VEGF is expressed in the seminiferous tubule of adult mouse testis (Fig. 1), we hypothesized that VEGF may be acting on the germ cell types using an alternate receptor for VEGF. Thus, we examined cross-sections of testis with a specific antibody against VEGFR-1. Interestingly, VEGFR-1 was not present in spermatogonia and pachytene spermatocytes but localized exclusively to the acrosomal region of round spermatids (Fig. 6A) and spermatozoa (Fig. 6B). A testis section stained in parallel with nonimmune rabbit serum instead of primary antibody was negative (Fig. 6C), suggesting the specific nature of immunostaining observed in round spermatids and spermatozoa.
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To further confirm this observation, we isolated populations of pachytene spermatocytes, round spermatids, and epididymal spermatozoa from adult mice and stained them for VEGFR-1. VEGFR-1 was absent in pachytene spermatocytes (Fig. 7A) but specifically localized to round spermatids (Fig. 7B) and spermatozoa (Fig. 7C).
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To determine exactly when VEGFR-2 synthesis stops and VEGFR-1 synthesis begins, we performed RT-PCR with total RNA isolated from type A spermatogonia, pachytene spermatocytes, and round spermatids. Interestingly, VEGFR-2 mRNA expression was observed only in type A spermatogonia but not in pachytene spermatocytes or round spermatids (Fig. 8A). In contrast, VEGFR-1 mRNA expression was observed in pachytene spermatocytes and round spermatids but not in type A spermatogonia (Fig. 8B).
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Expression of VEGFR-1 and VEGFR-2 Is Inversely Correlated During the Postnatal Development of Mouse Testis
A representative photograph of the products of RT-PCR performed with the total RNA obtained from testes of 6-, 21-, 42-, and 65-day-old mice is illustrated in Figure 9. In Fig. 9A, 6-day-old mice exhibit a very low level of expression of VEGFR-1 mRNA (lane 1). As the age of the mice increases, there is an increase in the relative intensity of the band corresponding to VEGFR-1 mRNA (Fig. 9B). The intensity is highest in the adult mouse testes (lane 7). In contrast, VEGFR-2 mRNA is highly expressed only on Day 6 of postnatal development (Fig. 9C, lane 1). By 21 days of age, the intensity of the bands decreased significantly (lane 3). Subsequent to Day 21 until adulthood, the levels remained low (lanes 3 and 4). The age-dependent decrease in the relative intensity of VEGFR-2 mRNA is shown in the bar diagram (Fig. 9D). Thus, there is an inverse correlation between the expressions of two isoforms of VEGFR during postnatal development of the mouse testes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In accordance with two previous reports [9, 11], the presence of VEGF protein in the testis was confirmed in the present study. We also specifically localized VEGF to Sertoli cells and the Leydig cell population in the adult testis. Furthermore, using specific primers for mouse VEGF, it was observed that VEGF is being actively synthesized by Sertoli cells under normal culture conditions. The purity of the Sertoli cell preparation used in these studies was very close to 100%. Immediately following isolation, the purity of the cells ranged between 96% and 98% as determined by staining for contaminating peritubular myoid cells (smooth muscle -actin), type A spermatogonia (c-kit), and Leydig cells (3ß-hydroxysteroid dehydrogenase). The principal contaminants were peritubular myoid cells and type A spermatogonia. The expansion of peritubular myoid cells in culture were inhibited in the presence of cytosine arabinoside and the attached type A spermatogonia at the end of 3 days culture were removed by treatment with Hepes buffer. Thus, the products of RT-PCR for VEGF were specific to Sertoli cells and not due to the presence in contaminating cell types.
To exert an effect on the process of spermatogenesis, VEGF must act on the germ cell types. The action of VEGF is mediated through three isoforms of VEGF receptors, i.e., VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and VEGFR-3 (Flt-4) [2, 10]. Seminiferous tubules in 6-day-old mouse testis consist of only type A spermatogonia and Sertoli cells. There are several subtypes of spermatogonia in more mature and adult testis of rodents. These include single, paired, aligned, and types A1–A4 subtypes of type A spermatogonia [16–21]. Type A spermatogonia found in 6-day-old mice are mostly at the single subtype stage and could be considered as stem cells. It is possible to identify these cells in sections based on their morphological features as well as to isolate them as a highly enriched population by the procedure standardized in our laboratory [14]. In cross-sections of immature testis, we observed a lack of immunostaining for VEGFR-1 in type A spermatogonial stem cells. This result was surprising in the light of recent reports that hematopoietic stem cells express VEGFR-1 but not VEGFR-2, and inhibition of VEGFR-1 prevented the cycling of hematopoietic stem cells [22–24]. However, VEGFR-1 mRNA expression in pachytene spermatocytes and round spermatids has been reported in adult wild-type mice and transgenic mice overexpressing VEGF [11].
On the basis of this study, we immunostained cross-sections of adult mouse testis with a specific antibody against VEGFR-1, and interestingly, immunofluorescence was specifically localized to round spermatids, elongating spermatids, and the heads of spermatozoa but to none of the spermatogonial subtypes. Furthermore, we confirmed the expression of VEGFR-1 mRNA in isolated pachytene spermatocytes and round spermatids by RT-PCR and the translated protein in round spermatids and epididymal spermatozoa by immunohistochemistry. Although Ergun et al [9] detected VEGFR-1 mRNA expression in the adult human testis in accordance with our results in the adult mouse testis, their immunohistochemistry data in testis sections suggest the presence of VEGFR-1 protein only in Sertoli cells, Leydig cells, and perivascular cells. We also observed VEGFR-1 expression in Leydig cells and perivascular cells by immunohistochemistry. However, Sertoli cells either in tissue sections or isolated from immature mouse testis did not exhibit any immunostaining for VEGFR-1. In addition, RT-PCR of total RNA isolated from immature Sertoli cells with specific primers for VEGFR-1 did not show the presence of VEGFR-1 mRNA.
In agreement with our results, in situ studies for VEGFR-1 in adult wild-type mouse testis performed by Korpelainen et al. [11] did not reveal localization of VEGFR-1 in Sertoli cells. It appears that Sertoli cells derived from either immature or adult Sertoli cells do not express VEGFR-1. In the study by Ergun et al. [9], localization of VEGFR-1 in Sertoli cells was solely by immunohistochemistry of tissue sections. There was no demonstration of VEGFR-1 in isolated Sertoli cells. In addition, a lack of expression in germ cell types unlike in the present study and one by Korpelainen et al. [11] is surprising. Whether this difference is due to the nature and specificity of antibodies used or the immunohistochemistry technique employed is not known. Our data with mRNA expression for VEGFR-1 in isolated pachytene spermatocytes and round spermatids are in agreement with the data by Korpelainen et al. [11] in wild-type and transgenic mice. The presence of VEGFR-1 mRNA and lack of immunostaining for VEGFR-1 in pachytene spermatocytes suggest that transcription of the gene begins during the process of meiosis, but the protein appears to be expressed only in haploid germ cells. Similar findings on the initiation of transcription of several genes during differentiation through meiosis and subsequent translation into protein products in haploid germ cells have been reported previously [25].
The above results suggested that postmeiotic germ cells probably use VEGFR-1 for interacting with VEGF secreted by Sertoli cells. However, it was not clear whether VEGF secreted by Sertoli cells during the early stage of postnatal testicular development has any target cell types expressing an alternate VEGF receptor subtype. As mentioned before, during early stages of development, only Sertoli cells and type A spermatogonia are present. Both these cell types are proliferating, although the rate of proliferation varies, with spermatogonia proliferating faster [21].
VEGFR-2 receptor is the most well-studied receptor and has been demonstrated to have a role in survival and proliferation of endothelial cells [26]. Thus, we hypothesized that VEGFR-2 is probably expressed either on Sertoli cells or on type A spermatogonia. Immunofluorescent staining indicated the presence of VEGFR-2 only on type A spermatogonia but not on Sertoli cells in cross sections of 6-day-old mouse testis. This is the first report of VEGFR-2 expression in type A spermatogonia during early postnatal development. Expression of VEGFR-2 on type A spermatogonia was also confirmed using isolated type A spermatogonia from 6-day-old mice. Furthermore, using specific primers for VEGFR-2, we demonstrated the expression of mRNA for VEGFR-2 in spermatogonia but not in Sertoli cells. However, VEGFR-2 was also expressed by interstitial cells. Contrary to our findings, the expression of VEGFR-2 in Sertoli cells but not in spermatogonia of adult human testes has been reported [9]. In the present study, Sertoli cells either in tissue sections or isolated from immature mouse testis did not exhibit any immunostaining for VEGFR-2. In addition, RT-PCR of total RNA isolated from immature Sertoli cells with specific primers for VEGFR-2 did not show the presence of VEGFR-1 mRNA. In agreement with our results, in situ studies for VEGFR-2 in adult wild type mouse testis performed by Korpelainen et al. [11] did not reveal localization of mRNA for VEGFR-2 in both Sertoli cells and germ cell types.
Interestingly, we also observed lack of immunostaining for VEGFR-2 in type A spermatogonia in adult mouse testes, suggesting that only the population of type A spermatogonial stem cells possess VEGFR-2 and not more differentiated types of spermatogonia observed in the adult testis. Although type A spermatogonial stem cells present in the adult testis should have stained for VEGFR-2, the observed lack of expression could be attributed to a relatively low number of these cells. In addition to a lack of expression in spermatogonia, none of the advanced germ cell types and Sertoli cells showed any immunofluorescence for VEGFR-2. However, VEGFR-2 expression was evident in Leydig cells in agreement with previously published results [9, 11]. Based on immunohistochemistry and mRNA localization data, it appears that Sertoli cells of the immature or adult mouse testis do not express VEGFR-2. The apparent discrepancy observed in the expression in the adult human testis [9] could be due to the nature and specificity of antibodies and the immunohistochemistry technique employed.
The foregoing discussion suggests that VEGFR-2 is expressed in type A spermatogonial stem cells and VEGFR-1 is expressed in more advanced germ cell types. In contrast, both VEGFR-1 and VEGFR-2 are expressed in the interstitial cell population. During postnatal development of the testis, a relatively constant number of somatic cells exists in comparison with the exponential numbers of germ cell types. Thus, we investigated the change in the pattern of synthesis of VEGFR-1 and VEGFR-2 during postnatal development of testis with RT-PCR using specific primers for each subtype of receptor. Around Day 6, when type A spermatogonial stem cells predominate, only VEGFR-2 mRNA expression was strong. By Day 21, when pachytene spermatocytes and a few round spermatids are present, VEGFR-2 levels decline substantially, whereas VEGFR-1 mRNA expression begins. By Day 42, when there is an increase in the advanced germ cell population (i.e., pachytene spermatocytes, round spermatids, elongating spermatids, and spermatozoa), there is a further increase in VEGFR-1 mRNA levels. In comparison, VEGFR-2 mRNA levels decrease to negligible levels. In the fully mature adult testis (Day 65) containing predominantly more differentiated spermatogonia and advanced germ cell types, VEGFR-1 mRNA levels are relatively high and VEGFR-2 mRNA levels are very low. There appears to be an inverse correlation between the expression of the two receptor subtypes. Although spermatogonial stem cells express VEGFR-2, the advanced germ cell types express VEGFR-1. This suggests that VEGF may elicit different signals for proliferation and differentiation of undifferentiated spermatogonia and spermatocytes through a switch in the receptor subtypes during male germ cell differentiation.
In summary, our results indicate that VEGF is expressed by Sertoli cells, and the receptors, VEGFR-2 and VEGFR-1, are expressed in type A spermatogonial stem cells and advanced germ cell types, respectively. VEGF probably exerts a differential effect of proliferation or differentiation based on the receptor subtype during the process of spermatogenesis. Functional studies are underway to establish the specific role of VEGF during the process of spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Neelakanta Ravindranath, Department of Cell Biology, Georgetown University Medical Center, Medical Dental Building, SE 216, 3900 Reservoir Road NW, Washington, DC 20057. FAX: 202 687 1823; ravindrn{at}georgetown.edu
Received: 6 January 2003.
First decision: 17 February 2003.
Accepted: 8 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. Samson, F. V. Peale Jr., G. Frantz, N. Rioux-Leclercq, E. Rajpert-De Meyts, and N. Ferrara Human Endocrine Gland-Derived Vascular Endothelial Growth Factor: Expression Early in Development and in Leydig Cell Tumors Suggests Roles in Normal and Pathological Testis Angiogenesis J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4078 - 4088. [Abstract] [Full Text] [PDF]
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