Mol. Hum. Reprod. Advance Access originally published online on October 11, 2007
Molecular Human Reproduction 2007 13(11):815-820; doi:10.1093/molehr/gam064
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Krüppel-like factor 4 expression in normal and pathological human testes
1 Stem Cell Research Group, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany 2 Institute of Anatomy, Developmental Biology, University of Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany 3 Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany 4 School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia 5Department of Urology and Pediatric Urology, University Hospital, Rudolf-Buchheim-Strasse 7, 35385 Giessen, Germany 6Present address: Departments of Animal Science, McGill Nutrition and Food Science Centre, McGill University, MacDonald Campus, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, QC, Canada H9X 3V9
7 Correspondence address. Tel: +49-551-3851-132; Fax: +49-551-3851-288; E-mail: rbehr{at}dpz.eu
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Abstract |
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Krüppel-like factor 4 (KLF4) is a transcription factor involved in many cellular and developmental processes such as terminal differentiation of cells and carcinogenesis. Mice lacking KLF4 die post-natally due to skin barrier deficiencies and exhibit several additional cellular defects. The adult rodent testis expresses high levels of Klf4 mRNA. Using in situ hybridization, we previously localized most of the Klf4 mRNA to round spermatids in mice. Moreover, in rodent Sertoli cells, Klf4 is strongly inducible by FSH. Here, we show by northern blot analysis that the human testis also strongly expresses KLF4. Applying immunohistochemistry, we localized KLF4 protein to the nuclei of round spermatids during normal spermatogenesis stages II–IV. Analysing round spermatid maturation arrests, strong cytoplasmic staining could be seen in two samples. We failed to detect KLF4 in human Sertoli cells. Most human Leydig cells expressed KLF4 at high levels in the nucleus. However, some individual Leydig cells lacked KLF4, suggesting different functional states of the Leydig cells. The strong expression of KLF4 in the human testis and the importance of KLF4 in several mouse tissues suggest a significant role for KLF4 in the human testis. A first hint at a role for KLF4 during spermiogenesis could be the altered subcellular localization of the protein during arrested spermiogenesis.
Key words: KLF4/Leydig cell/spermatid/spermatogenesis/testis
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Introduction |
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Krüppel-like factor 4 (KLF4, formerly GKLF, gut-enriched Krüppel-like factor) is a zinc finger transcription factor which is essential for post-natal survival in the mouse (Segre et al., 1999). KLF4 is essential for proper differentiation of keratinocytes, epithelial cells of the tongue (Segre et al., 1999), goblet cells in the colon (Katz et al., 2002) and the gastric epithelium (Katz et al., 2005). Moreover, Klf4 expression has been localized to several other cell types (Garrett-Sinha et al., 1996; Shields et al., 1996; Panigada et al., 1999; Foster et al., 2000; Ehlermann et al., 2003). KLF4 has also been reported to be aberrantly expressed in some types of tumors such as breast, gastric and colon cancer (Foster et al., 2000; Chen et al., 2002; Dang et al., 2003; Pandya et al., 2004; Katz et al., 2005; Wei et al., 2005). Also, KLF4 has been characterized as a tumor suppressor or oncogene, with its function dependent on the molecular environment in which KLF4 was acting (Rowland et al., 2005; Rowland and Peeper, 2006). Subcellular localization of KLF4 is mostly nuclear. However, cytoplasmic localization of KLF4 has also been described in some cell types (Chiambaretta et al., 2004; Pandya et al., 2004). Recently, KLF4 has also been shown to be an important regulator of the developmental potency of pluripotent cells (Li et al., 2005; Nakatake et al., 2006; Takahashi and Yamanaka, 2006; Okita et al., 2007; Wernig et al., 2007). In summary, KLF4 appears to have important roles in several different cellular and developmental contexts.
Klf4 is closely related to Klf5 (formerly Iklf, intestinal Krüppel-like factor) with respect to its gene structure and nucleotide sequence of the coding region (Kaczynski et al., 2003; Ghaleb et al., 2005). However, interestingly, both proteins appear to have contrasting effects on the cell cycle (Ghaleb et al., 2005). Although KLF5 is considered to promote proliferation, KLF4 is rather involved in cell cycle arrest and the fully differentiated status of a cell.
We have shown by non-radioactive in situ hybridization and northern blotting that Klf4 mRNA is strongly expressed in post-meiotic germ cells of the mouse testis (Behr and Kaestner, 2002). However, northern blot analysis revealed that Klf4 mRNA is also present in the prepubertal mouse testis (Godmann et al., 2005), i.e. before meiotic and post-meiotic germ cells appear, suggesting that other cell types besides spermatids also express Klf4. Supporting this data, a strong induction of Klf4 mRNA upon stimulation with FSH has been shown in rat primary Sertoli cell cultures (Hamil and Hall, 1994; McLean et al., 2002) and in the hypogonadal hpg mouse, which lacks post-meiotic germ cells (Sadate-Ngatchou et al., 2004). In the present study, we report for the first time the expression of KLF4 in the human testis at the mRNA level by northern blot analysis, as well as at the protein level by immunohistochemistry. We describe the KLF4 protein distribution in the normal human testis, in testicular biopsy samples exhibiting round spermatid maturation arrest as well as in samples of patients with increased FSH levels.
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Materials and Methods |
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Northern blot analysis
The northern blot used to analyze KLF4 mRNA expression in several human tissues was obtained from BD Biosciences Clontech (Heidelberg, Germany; Cat. No. 636805). Approximately, 2 µg poly A+ RNA per lane was electrophoresed on a denaturing formaldehyde 1.0% agarose gel, blotted onto a positively charged nylon membrane and fixed by UV irradiation. The membrane was prehybridized at 68°C for 60 min in ExpressHyb buffer (BD Biosciences Clontech, Heidelberg, Germany). P32-dCTP was incorporated into the human KLF4-probe using the High Prime solution from Roche Molecular Biochemicals (Mannheim, Germany). Hybridizations were performed at 68°C for 16 h. Membranes were washed three times for 20 min each time in 2xSSC (standard saline citrate)–0.1% sodium dodecyl sulphate (SDS) at room temperature (RT) and twice for 30 min each in 0.1xSSC–0.1% SDS at 50°C. The membrane was exposed to an X-ray film (Kodak) for 4 h at –80°C in an exposure cassette with intensifier screens. After removing the KLF4 probe by two washes in 0.1% SDS at 90°C, the blot was hybridized to a ‘β-actin’ probe for normalization and quantification purposes. Relative expression levels for each organ were determined using the Scion Image analysis software (Scion Image 4.0.3.2 [EC] alpha, Scion Corporation, MD, USA).
Testicular tissue, histological analysis and hormone levels
Ethical approval was granted by the ethics committee of the Justus-Liebig-University, Giessen (decision 75/00). All patients had given written informed consent for such investigations to be performed. In total, 19 testicular biopsies from 19 different testes from 15 different patients were analyzed. In five patients with obstructive azoospermia after vasectomy, biopsies were carried out for diagnostic purposes during vasectomy reversal. The remaining 10 patients (22–44 years) were azoospermic but wished to father a child. Hence, testicular biopsies were carried out for diagnostic reasons to decide whether the patients exhibited obstructive or non-obstructive azoospermia (NOA). All patients were diagnosed NOA. Testicular tissue was fixed by immersion in Bouin’s fixative and embedded in paraffin using standard techniques. For histological evaluation, 5 µm paraffin sections were stained with hematoxylin. Histological evaluation revealed normal spermatogenesis (Bergmann and Kliesch, 1998) in the five control cases. These samples served as normal controls for immunohistochemistry. Among the patients showing NOA, histological evaluation (Bergmann and Kliesch, 1998) exhibited impaired spermatogenesis with complete lack of sperm due to a total arrest of spermatogenesis at the level of round spermatids in four patients (six biopsies). Histological analysis of the remaining tissue samples revealed hypospermatogenesis, spermatogenic arrest at the level of spermatocytes and Sertoli cell-only (SCO) syndrome, respectively.
Immunohistochemistry
Bouin’s-fixed and paraffin-embedded specimens were sectioned at 5 µm. Sections were rehydrated and an antigen retrieval step was performed by microwaving the sections in 0.05 M citrate buffer for 10 min. Endogenous peroxidase was inhibited by incubation with peroxidase blocking reagent (DakoCytomation Carpinteria, CA, USA, LSAB+ system-HRP, K0679). Non-specific binding of the first antibody was blocked by a 30 min incubation step in 5% (w/v) BSA in 0.05 mol/l Tris–HCl, 0.15 mol/l NaCl, pH 7.6 (TBS). A rabbit polyclonal antibody raised against the 180 N-terminal amino acids of KLF4 of human origin (SC-20691; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:600 dilution in 5% BSA in TBS. All incubation steps were done in a humid chamber and incubations with the primary antibodies were performed overnight at 4°C. DakoCytomation Universal LSAB Plus-kit (K0679) including biotinylated second antibody polymer and horse-radish peroxidase (HRP) conjugated streptavidin was employed for detection of bound primary antibody. 3,3prime;-diaminobenzidine (DAB) chromogen was used as substrate for the HRP and Mayer’s hematoxylin as counterstain. Control stains were carried out omitting the primary antibody. Indistinguishable KLF4 staining was also obtained with the anti-KLF4 antibody (AF3640) from R&D systems (Minneapolis, MN, USA) at a 1:1000 dilution using the same protocol. However, all results shown in here were obtained with the above-mentioned antibody from Santa Cruz. Androgen receptor was detected similarly using the anti-androgen receptor antibody (N-20, sc-816; Santa Cruz Biotechnology Inc., CA, USA).
Immunohistochemical double staining
Tissue sections were processed as described earlier including the application of the KLF4 antibody. This incubation step was followed by two washes for 5 min each in TBS. Alkaline phosphatase (AP)-labeled polymer from the EnVision Doublestain System from DakoCytomation (Carpinteria, CA, USA, order number K1395) was applied for 30 min at RT followed again by two wash steps in TBS. Bound AP was visualized using the AP color substrate. After another wash step, double-stain block was applied for 3 min at RT followed by two washes in TBS. Anti-INSL3 (RA15) antibody (Ivell et al., 1997) was applied in a 1:2000 dilution in TBS plus 5% BSA overnight at 4°C followed by two washes in TBS at RT. Biotinylated polyclonal rabbit anti-rat antibody (E0467, Dakocytomation, Hamburg, Germany) was used at a 1:200 dilution for 90 min at RT to detect INSL3. After washing again in TBS a streptavidin-horse-radish conjugate (SA-5004, Vector laboratories, Burlingame, CA, USA) was applied for 20 min at a 1:200 dilution at RT. After another two washes in TBS, bound INSL3 antibody was visualized using DAB. Mayer’s hematoxylin was used to counterstain for 10 sec. Tissue sections were mounted in Faramount aqueous mounting medium (DakoCytomation). Control stains were carried out omitting the primary antibody.
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KLF4 mRNA is highly expressed in the human testis
As Fig. 1 shows, KLF4 mRNA exhibited significant expression in the human spleen, testis, ovary, colon and peripheral blood leukocytes (PBL). Small intestine, prostate and thymus showed only weak or almost no signals (<10% of colon). Normalization of KLF4 signals to ‘β-actin’ signals revealed strongest expression in the colon followed by testis, PBL, ovary and spleen (Fig. 1b). The size of the major transcript was
3.6 kb. Interestingly, the human testis exhibits, similar to mouse (Shields et al., 1996; Behr and Kaestner, 2002; Godmann et al., 2005), an additional smaller band, which is not present in the samples from the other tissues analyzed. Moreover, we show here for the first time a significant expression of KLF4 mRNA in the ovary.
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KLF4 protein is expressed in round spermatids
We localized KLF4 protein to round spermatids (Fig. 2a). During normal spermatogenesis, KLF4 was restricted to the nucleus of spermatids (Fig. 2a). The protein became detectable during stage II, whereas stage I was immunonegative. Stage III spermatids displayed highest staining intensity. Stage IV spermatids were still KLF4-positive but already exhibited reduced labeling. When spermatids continued to elongate during stage V, KLF4 protein was no longer detectable. A schematic overview over the KLF4-expressing germ cell stages is given in Fig. 3. No KLF4 signals could be observed in Sertoli cells. However, the nuclei of a few peritubular myoid cells also showed KLF4 antigenicity.
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KLF4 in patients with round spermatid maturation arrest
We analyzed six biopsies with round spermatid maturation arrest for the expression of KLF4. All samples tested expressed KLF4 in arrested spermatids (Fig. 2c and d). However, although in four samples KLF4 was restricted to the nucleus (Fig. 2c), two samples also showed clear cytoplasmic KLF4 staining in addition to nuclear staining (Fig. 2d).
KLF4 is expressed in most, but not all Leydig cell nuclei
In most Leydig cells, we obtained moderate or strong signals for KLF4 in the nuclei (Fig. 4a). Interestingly, some of the Leydig cells within a cluster of cells were devoid of KLF4. Most Leydig cells also showed weak cytoplasmic staining (Fig. 4a). To demonstrate that the cells lacking KLF4 protein in the nucleus were indeed Leydig cells, we performed double staining for both INSL3 (Fig. 4b), a specific Leydig cell marker (Ivell et al., 1997), and KLF4 on the same tissue section (Fig. 4c). This analysis definitely demonstrated that there were indeed different subpopulations of Leydig cells as shown by their KLF4 protein expression with the majority of the Leydig cells strongly expressing KLF4. Another population showed only moderate or weak KLF4 signals and several other Leydig cells were devoid of KLF4. Figure 4d demonstrates that there are also androgen receptor-positive and -negative Leydig cells in the human testis.
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Discussion |
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This is the first report on KLF4 expression in the human testis. We show that KLF4 mRNA is highly abundant in the human testis. The additional smaller band occurring in the northern blot is most likely due to alternative usage of polyadenylation signals, which we have previously described in the mouse (Godmann et al., 2005). Post-meiotic germ and Leydig cells express KLF4 protein at significant levels. In the mouse, KLF4 is essential for post-natal survival since skin lacking KLF4 fails to establish its barrier function, and the pups die within hours due to dehydration (Segre et al., 1999). Also, the integrity of the epithelium of the tongue (Segre et al., 1999) and the gastric epithelium depends on KLF4 (Katz et al., 2005). Moreover, KLF4 is necessary for the specification of colonic goblet cells (Katz et al., 2002) and proper function of the eye (Swamynathan et al., 2007). Altogether, the data from mice indicate that KLF4 has an important role for normal functions of different cell types. Therefore, the significant expression of KLF4 in the human testis suggests that KLF4 might also have an important role for normal testicular function.
Spermiogenesis comprises the post-meiotic morphological and biochemical differentiation process which results in the terminally differentiated male germ cell, the spermatozoon. With regard to its cell biological complexity and its transcriptome spermiogenesis is unique (Shima et al., 2004; Wu et al., 2004). One of the main regulators of spermiogenesis is the transcription factor cAMP response element modulator (CREM). Mice lacking the CREM gene fail to produce sperm due to an arrest at the early stage of spermiogenesis (Blendy et al., 1996; Nantel et al., 1996). Many genes, which are necessary for or highly expressed during spermiogenesis, are not transcribed in the absence of CREM (Beissbarth et al., 2003). We have also shown that round spermatid maturation arrest can be associated with absence of CREM in round spermatids (Steger et al., 1999; Weinbauer et al., 1998). Since KLF4 is necessary for post-proliferative terminal differentiation of different types of cells and since it is highly expressed in spermatids, it is tempting to speculate that KLF4 might also represent an important regulator of spermiogenesis. However, none of the human tissue samples exhibiting round spermatid maturation arrest analyzed in this study lacked KLF4 expression. But, interestingly, two of the round spermatid maturation arrest samples analyzed here showed clear cytoplasmic staining (Fig. 2d) in addition to nuclear signals. This might be a finding of major relevance. It has previously been shown that subcellular localization of KLF4 (nuclear versus cytoplasmic) has a strong influence on the prognosis of the clinical outcome of breast cancer (Pandya et al., 2004). Localization of KLF4 in the nucleus of breast cancer cells was strongly correlated with an aggressive phenotype in early stage infiltrating ductual carcinoma indicating that localization of KLF4 can serve as a marker for fundamentally different biological states of a cell. This supports the view that KLF4 localization in spermatids may help to discriminate between different causes for arrested spermatogenesis. The causes for arrested spermiogenesis can be very different and complex and, since we analyzed only a few samples with blocked spermatogenesis, our results do not exclude that a subset of round spermatid maturation arrests, which was not included in our samples, may also be due to a lack of KLF4 in spermatids.
In rodent Sertoli cells, Klf4 expression can be induced by FSH (Hamil and Hall, 1994; McLean et al., 2002; Sadate-Ngatchou et al., 2004). However, in the normal human testis as well as in the samples showing spermatogenic arrests, we were not able to detect KLF4 signals in Sertoli cells. We additionally tested some biopsy samples from patients exhibiting a SCO and increased FSH levels (10–32 IU, normal range 1–7 IU/l) for KLF4-immunogenicity in Sertoli cells. But also in these testis tissue samples, we could not detect KLF4. However, it must be noticed that FSH-dependent gene induction in a primary culture of healthy Sertoli cells or in the hpg mouse is a fundamentally different situation compared with patients with increased FSH levels. In the experimental setting, a high dose of FSH acts on functionally normal Sertoli cells. In contrast, in patients with high FSH serum levels, these levels are usually high because of a defective Sertoli cell (Bergmann et al., 1994). Thus, the data obtained before in rodents or with rodent Sertoli cells cannot be transferred to patients with high FSH levels. Anyway, to date, it remains unclear under which physiological or pathological conditions or whether KLF4 protein is induced at all in human Sertoli cells. As the importance of FSH for spermatogenesis in primates including man is different from that in rodents (McLachlan et al., 2002), it is conceivable that also the downstream effects of FSH signaling, including induction of Klf4, differ between primates and rodents.
Leydig cells are the major source of testosterone in the male and also express by themselves the androgen receptor. However, not all Leydig cells express the AR at the same time indicating different physiological states of the cells (Vornberger et al., 1994 and Fig. 4d for illustration purposes of the human testis). A similar finding was obtained in the present study when human testes were stained for KLF4. We detected strong KLF4 signals in many, but not all Leydig cells presumably indicating different physiological states. So far, the functional significance of the heterogeneous KLF4 expression in Leydig cells remains unclear. However, our data support the view that morphologically similar adjacent Leydig cells within a cluster of cells can be very different at the molecular level. This is also corroborated by previous reports by Davidoff et al. (1993) and Schulze et al. (1991), who also found several proteins differentially expressed between individual Leydig cells of the adult human testis. Since KLF4 expression is associated with the fully differentiated state of cells, it is tempting to speculate that the KLF4-negative Leydig cells might not be fully differentiated and constitute an immature progenitor population of adult Leydig cells. Future work including investigations on the proliferative state of the KLF4-negative Leydig cells is necessary to unravel and define functional differences between morphologically similar Leydig cells within a single cluster of these cells.
Interestingly, the KLF4 expression in human Leydig cells represents a substantial difference in KLF4 expression compared with the mouse testis. Although in the mouse testis, the mRNA was detected by in situ hybridization only in spermatids and by northern blot and microarray analyses also in cultured Sertoli cells, there is so far no indication for KLF4 expression in mouse Leydig cells. Thus, our finding of strong KLF4 protein expression in human Leydig cells may document a fundamental difference in the molecular regulation of Leydig cell maturation and/or function in rodents and primates.
Although a putative role for KLF4 in human Sertoli cells still remains unclear, our data clearly demonstrate that KLF4 is strongly expressed in the human testis suggesting that KLF4 might have important roles in post-meiotic germ cells as well as in human Leydig cells.
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Deutsche Forschungsgemeinschaft (Be2296/4) to R.B.
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Acknowledgement |
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We thank Isabell Kromberg and Marion Niebeling for technical assistance.
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Submitted on July 19, 2007; resubmitted on August 23, 2007; accepted on September 4, 2007.
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