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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1858-1865
Copyright © 2003 by The Endocrine Society

Transcription Factor GATA-6, Cell Proliferation, Apoptosis, and Apoptosis-Related Proteins Bcl-2 and Bax in Human Fetal Testis

Ilkka Ketola, Jorma Toppari, Tommi Vaskivuo, Riitta Herva, Juha S. Tapanainen and Markku Heikinheimo

Children’s Hospital and Program for Developmental and Reproductive Biology, Biomedicum Helsinki, University of Helsinki (I.K., M.H.), 00014 Helsinki, Finland; Departments of Physiology and Pediatrics, University of Turku (J.T.), 20520 Turku, Finland; Departments of Obstetrics and Gynecology (T.V., J.S.T.) and Pathology (R.H.), University of Oulu, 90220 Oulu, Finland; and Department of Pediatrics, Washington University (M.H.), St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Markku Heikinheimo, M.D., Ph.D., Biomedicum Helsinki, 5th floor, Room B525b, P.O. Box 63, University of Helsinki, 00014 Helsinki, Finland. E-mail: markku.heikinheimo{at}helsinki.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The GATA family of transcription factors have been implicated in regulating the development and function of many organs. Furthermore, they have been linked to signaling cascades regulating cell fate through apoptosis. GATA-6 has been shown to be expressed in the gonads, but its cell-specific expression in the testis has remained unclear. We have studied GATA-6 expression in human fetal testis using in situ hybridization and immunohistochemistry and compared these results with the expression of the apoptosis-related proteins Bcl-2 and Bax. Furthermore, apoptosis was studied by thymidine deoxyribose-mediated deoxy-UTP nick end labeling assay, and cell proliferation by Ki-67 immunohistochemistry. GATA-6 mRNA and protein were expressed in Sertoli and Leydig cells early in gestation. Apoptotic cells were scanty between wk 16 and 40, and proliferation significantly ceased during the third trimester, supporting the view that only a little tissue remodeling occurs in the late fetal testis. Bax was present throughout the fetal period, whereas Bcl-2 expression decreased toward term. Neither of these factors correlated to the extent of apoptosis, and thus their role in the regulation of apoptosis in the fetal testis remains open. Despite strong expression, GATA-6 did not correlate with apoptosis or cell proliferation and is therefore unlikely to be directly involved in these processes in the human fetal testis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PROPER TISSUE DEVELOPMENT requires carefully coordinated cellular differentiation, proliferation, growth, and apoptosis. Apoptotic germ cell death is an important mechanism directing testicular development and function in vivo. Bcl-2 family members appear to be key regulators of this process (1, 2, 3). However, relatively little is known about the role of apoptosis and Bcl-2 family proteins in the development of human fetal testis (4, 5, 6). GATA transcription factors are involved in the regulation of differentiation, proliferation, and apoptosis in multiple tissues (7, 8). In particular, the results of previous work have indicated that GATA-1 (9), GATA-4 (10), and GATA-6 (11) are expressed during critical periods of murine testicular development (12, 13, 14). Hence, together with other factors, they may serve a role in early testicular development and function.

GATA-1 is expressed in postnatal Sertoli cells, but not in other testicular cells (12), whereas GATA-4 is already expressed at the urogenital ridge stage, and it becomes localized later in the Sertoli and Leydig cells as well as in peritubular myoid cells (13, 14). Studies in mice have suggested that GATA-6 is mainly expressed in the testicular seminiferous tubules from late fetal to adult testis (14), but the precise cell types expressing GATA-6 have remained unclear. Even less is known about the expression and role of GATA-6 in the human testis. For example, there are no reports on the spatiotemporal expression of GATA-6 in fetal or adult human testis (15). On the other hand, GATA-6 is known to be abundantly expressed in human fetal and adult ovary, and it may function in the regulation of somatic cell fate in the female gonad (16, 17, 18).

The results of in vitro trans-activation studies suggest that GATA-1 and GATA-4 are important regulators of testicular gene expression (13, 14, 19, 20, 21, 22, 23, 24), whereas the testicular genes regulated by GATA-6 are not known. Several lines of evidence have linked GATA proteins to apoptosis. Disruption of Gata1 leads to apoptosis in erythroid cell lines (25, 26, 27, 28). Furthermore, GATA-1 has also been shown to regulate the apoptosis-related proteins Bcl-xL and Bcl-2 (29, 30). Studies of Gata4 and Gata6 knockout cell lines and mice have revealed that the lack of these proteins leads to apoptotic cell death in cardiomyocytes and embryonic endoderm (31, 32).

Our earlier work suggests a link between murine ovarian follicular apoptosis and down-regulation of GATA-4 (33). In the human ovary, GATA-4 may also be involved in the process protecting fetal granulosa cells from apoptosis together with Bcl-2 family proteins (18). Likewise, the close family member of GATA-4, GATA-6, which is abundant in the mouse ovary and testis, might also have a role in determining cell fate in the gonads. This hypothesis has not previously been tested in the murine or human testis. Of the many apoptosis-regulating proteins, Bcl-2 and Bax are expressed in adult human testis, and they have been proposed to have a role in regulating cellular differentiation and maturation of somatic and germs cells (34, 35). In the fetal testis, Bcl-2 and Bax expression have been detected during the first half of embryonal development, although their exact roles during fetal testicular development have remained unresolved (5, 6). The testicular expression pattern of Bcl-2 and Bax after midgestation has not been studied before.

Although GATA-4 has been studied in detail during human testicular gonadogenesis and disease (36), virtually nothing is known about the role of GATA-6 in the human testis. Given that GATA-6 has been proposed to have a role in the regulation of apoptosis (32, 37) and the cell cycle machinery (38, 39), we have now explored in detail the possible spatial and temporal relationships among GATA-6 expression, cell proliferation, apoptosis, and the apoptosis-related proteins Bcl-2 and Bax during human fetal testicular development.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Fetal testicular samples

Fetal testicular tissue samples at wk 16–22 (n = 9) were obtained after spontaneous or therapeutic abortions because of maternal disease and at autopsy (wk 27–40; n = 5) at the Department of Obstetrics and Gynecology, University of Oulu (Oulu, Finland). The five autopsy samples were from neonates who died because of perinatal asphyxia or infection in 48 h after birth. The karyotype and testicular morphology were normal in every sample, and samples with detectable autolysis were excluded from the study. The studies were accepted by the ethics committees of the Children’s Hospital of University of Helsinki and University of Oulu and were conducted according to the recommendations of the Declaration of Helsinki.

Cloning of human GATA-6 and GATA-4 cDNAs

A 712-bp human GATA-6 cDNA was synthesized by PCR from human granulosa cell total cDNA using oligonucleotides 5'-ATG ACT CCA ACT TCC ACC TCT and 5'-CAG CCT CCA GAG ATG TGT AC, designed according to a human GATA-6 cDNA (GenBank no. NM-005257) as described previously (40). In brief, the cycling conditions for PCR were as follows: 94 C for 15 sec, 56 C for 30 sec, and 72 C for 1 min, for 30 cycles, followed by final extension for 20 min at 72 C. Similarly, a 575-bp human GATA-4 cDNA was synthesized from the same source using oligonucleotides 5'-CTC CTT CAG GCA GTG AGA GC and 5'-GAG ATG CAG TGT GCT CGT GC, designed according to a human GATA-4 cDNA (GenBank no. NM-002052) (40). The cycling conditions for PCR were the same as those described above, except that the annealing temperature was 58 C. The human GATA-6 and GATA-4 PCR products were ligated into pCR2.1-TOPO vectors and subcloned into the EcoRI site of pGEM-7Zf+/- vectors (Promega Corp., Madison, WI). The sequences of the cloned PCR products were verified using an ABI PRISM 377 DNA sequencer (Perkin-Elmer, PE Applied Biosystems, Foster City, CA).

In situ hybridization

Testicular samples were fixed in formalin and subjected to in situ hybridization as previously described (41). Tissue sections (8 µm) were incubated with 1 x 106 cpm 33P-labeled antisense riboprobe (1000–3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) in a total volume of 80 µl. The riboprobes for GATA-6 and GATA-4 were prepared as described above. The expression patterns for GATA-6 and GATA-4 were studied using adjacent tissue sections, whenever possible, in in situ hybridization and immunohistochemistry. In situ hybridization and immunohistochemical studies were repeated at least twice. Samples were analyzed by light-/darkfield and phase contrast microscopy (DMRXA microscope, Leica Corp., Heerbrugg, Switzerland).

Immunohistochemistry

Testicular samples were fixed in formalin and embedded in paraffin. Sections (8 µm) of formalin-fixed testicular samples were deparaffinized in xylene, hydrated gradually through graded alcohols, and subjected to immunohistochemistry using 1:200 dilution rabbit polyclonal antihuman GATA-6 IgG (sc-9055, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), goat polyclonal antimouse GATA-4 IgG (sc-1237, Santa Cruz Biotechnology, Inc.), 1:100 dilution mouse monoclonal antihuman Ki-67 IgG (DAKO Corp., Glostrup, Denmark), or nonimmune IgG as the primary antibody. Samples with primary antibody were incubated at 37 C for 1 h. An avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA), 3,3-diaminobenzedine (Sigma-Aldrich, St. Louis, MO) was used as the chromogen and the development reaction occurred in the presence of 0.03% hydrogen peroxide. Bcl-2 was detected by using 1:25 dilution monoclonal mouse antihuman Bcl-2 (DAKO Corp., Glostrup, Denmark) and Bax by using 1:500 dilution polyclonal rabbit antihuman Bax (BD PharMingen, San Diego, CA). Biotinylated rabbit antimouse and goat antirabbit immunoglobulins were used as secondary antibodies for Bcl-2 and Bax, respectively. For Bcl-2 and Bax, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol, and sections with primary antibodies were incubated overnight at 4 C. The samples from different gestational weeks for each antibody were stained in one experiment, and each experiment was repeated two to four times.

In situ DNA 3'-end labeling

Apoptosis was qualitatively identified in the testes using an in situ thymidine deoxyribose-mediated deoxy-UTP nick end labeling (TUNEL) DNA 3'-end labeling kit (Oncor, Gaithersburg, MD). Paraffin sections of testes were rehydrated through an alcohol series. The permeability of the cell membranes was increased by incubating the sections in 400 µg proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) in 200 ml PBS for 15 min. Endogenous peroxidase activity was inhibited by quenching the samples for 5 min in 5% hydrogen peroxide. DNA fragmentation was identified by applying terminal transferase enzyme with digoxigenin-labeled nucleotides to the samples and incubating for 1 h under coverslips. Antidigoxigenin antibody was used to recognize the digoxigenin-labeled nucleotide chains attached to the 3'-ends of sample DNA. A color reaction was produced with diaminobenzidine in the presence of 0.03% hydrogen peroxide. The tissue sections used in in situ hybridization, immunohistochemistry, and TUNEL assay were counterstained with hematoxylin.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
GATA-6 mRNA is expressed in fetal human testis

We used mRNA in situ hybridization to explore GATA-6 expression in the fetal testis. From gestational wk 16–40, GATA-6 mRNA was detected in both testicular cords and interstitium (Fig. 1Go, A, B, and E–H). The signal intensity was highest during the early second trimester, and it diminished with advancing weeks. GATA-6 mRNA expression was most intense in the testicular cords at midgestation (wk 18 in Fig. 1Go), and thereafter it appeared to be uniform and unchanged throughout the testis until wk 40 (Fig. 1Go, G and H, and data not shown). GATA-4 mRNA was also expressed in both testicular cords and interstitium (Fig. 1Go, C and D). Signal intensity was highest in testicular cords in the earliest specimens (wk 16), and it remained slightly stronger in cords compared with interstitium (data not shown). When the mRNA expression patterns of GATA-6 and GATA-4 were compared at 16 wk, GATA-6 was abundantly expressed in both interstitium and testicular cords, whereas GATA-4 expression was mainly restricted to the developing testicular cords (Fig. 1Go, B and D).



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  		Figure 1. mRNA in situ hybridization of GATA-6 (A, B, and E–H) and GATA-4 (C and D) in the fetal testis. Gestational wk 16 (A–D), 18 (E and F), and 23 (G and H) are shown. GATA-6 mRNA expression was highest at wk 16 (B), and it diminished with advancing gestational weeks (F and H). At wk 18, GATA-6 was expressed predominantly in the testis cords (F), and thereafter it appeared to be uniform throughout the testis (H). Arrowheads point out the demarcation area where seminiferous cords can be seen. Magnification, x50 (A–D) and x100 (E–H). Bar, 100 µm.

 
GATA-6 protein is localized to the Sertoli and Leydig cells in the fetal testis

In immunohistochemistry, GATA-6 protein was present in Sertoli and Leydig cells from wk 16–40 (Fig. 2Go, A, C, and D). However, a subset of the somatic cells did not express GATA-6, and the relative number of GATA-6-positive Sertoli and Leydig cells showed a decreasing trend with advancing fetal development. Peritubular myoid cells were occasionally GATA-6 positive, although the overwhelming majority remained negative. The fetal germ cells, i.e. gonocytes, remained GATA-6 negative. In Sertoli and Leydig cells, the staining intensity for GATA-6 was at its strongest between wk 16–23.



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  		Figure 2. Immunohistochemistry of GATA-6 (A, C, and D) and GATA-4 (B) proteins in the fetal testis. Gestational wk 16 (A, B, and inset in C), 23 (C), and 40 (D) are shown. Between wk 16–23 (A and C), GATA-6 protein expression in Sertoli and Leydig cells was at its strongest, and the number of GATA-6-positive cells decreased with advancing fetal development (D). The inset in C shows a preimmune control for GATA-6. Magnification, x400 (A–D) and x200 (inset in C). Bar, 50 µm.

 
For comparison, we also analyzed GATA-4 expression in the same fetal testicular samples (Fig. 2BGo and data not shown). GATA-4 protein was detected in Sertoli and Leydig cells as well as in myoid cells. In testicular cords, some of the cells, judged to be gonocytes by their morphology, remained GATA-4 negative. In the testicular cords and interstitium, GATA-4-positive cells outnumbered GATA-6-positive cells at every fetal age studied.

Apoptosis and cell proliferation in the fetal testis

Apototic cells, as indicated by TUNEL positivity, were present in the testicular cords and interstitium from the second trimester to term (Fig. 3Go, A, C, and E). It was not possible to verify specific cell types undergoing apoptosis at this stage of development. At wk 16, when only a few apoptotic cells were observed, Ki-67-positive proliferative cells were scarce in the testicular cords as well (Fig. 3BGo). However, Ki-67 positivity was more abundant in the interstitium (Fig. 3BGo). The number of apoptotic cells was higher at midgestation than earlier, whereas the number of proliferating cells remained stable (Fig. 3Go, C and D). At term, no Ki-67-positive cells were observed, and apoptosis was also minimal at this point (Fig. 3Go, E and F).



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  		Figure 3. TUNEL assay (A, C, and E) and immunohistochemistry of Ki-67 protein (B, D, and F) in the fetal testis. TUNEL- and Ki-67-positive cells indicate apoptotic and proliferative cells, respectively. Gestational wk 16 (A and B), 27 (C and D), 40 (E and F), and 18 (inset in F) are shown. Arrows point out representative apoptotic cells, and arrowheads show proliferating cells; nonspecific staining is seen as a dark brown color (D). Note that there are no Ki-67-positive cells in wk 40 fetal testis (F). The inset in F shows a preimmune control for Ki-67. Magnification, x200 (A, B, and inset in F) and x400 (C–F). Bar, 50 µm.

 
Bcl-2 expression ceases with advancing fetal age, whereas Bax expression remains constant

Bcl-2 was expressed in the interstitial cells, presumably Leydig cells, between wk 16 and 23 (Fig. 4Go, A and C). Weak Bcl-2 expression was seen in the peritubular myoid cells as well. After wk 27, Bcl-2 protein was no longer detectable in any testicular cell compartment (Fig. 4EGo and data not shown). Abundant cytoplasmic Bax expression was detected at 16 wk in both testicular cords and interstitium, and it remained intensive throughout fetal development (Fig. 4Go, B, D, and F). Peritubular myoid cells did not express Bax protein.



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  		Figure 4. Immunohistochemistry of Bcl-2 and Bax in the fetal testis. Gestational wk 17 (A and B), 23 (C and D), and 40 (E and F) are shown. Bcl-2 is expressed in Leydig and myoid cells until wk 23 (A and C), but not thereafter (E). Bax protein was abundantly expressed in the interstitium and testicular cords between wk 16–40 (B, D, and F). Preimmune controls for Bcl-2 and Bax are shown in G (16 wk) and H (21 wk), respectively. Magnification, x200. Bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study GATA-6 expression was detected in a subset of human fetal testicular cord Sertoli cells and interstitial Leydig cells. The expression pattern of GATA-6 partially overlapped that of GATA-4 (36). The differences in the expression of these related factors may reflect distinct functions for them in the fetal human testis. In the heart and gut, GATA-4 and GATA-6 are also expressed in a partially overlapping manner, and they have been considered to fulfill distinct functions in regulating gene expression in these organs (42, 43). The role of GATA-4 in gene regulation and gonadal development is fairly well established (44). However, studies have not revealed any testicular target genes for GATA-6, although many results suggest that it has an essential role in the proper development of several other organs in which it is expressed. GATA-6 regulates tissue differentiation at the earliest stages of embryonal development, and disruption of the Gata6 gene leads to early embryonal lethality (32, 45). Moreover, GATA-6 has been linked to apoptosis and regulation of cell cycle machinery (32, 37, 38, 39).

The overall rate of apoptosis and proliferation in the fetal testis was low throughout the gestational weeks studied (wk 16–40). Sertoli cell apoptosis was at its highest at the end of the second trimester, coinciding with the declining serum FSH levels (46). The link between diminishing FSH and abundant apoptosis suggests a role for FSH in the demise of fetal Sertoli cells and provides a possible mechanism for the declining Sertoli cell number from midgestation to term (4). The possible role of FSH in inhibiting testicular apoptosis is supported by the observation that FSH effectively blocks hypophysectomy-induced apoptosis in the rat testis (47). FSH has been shown to be important for Sertoli cell proliferation (48). There are no data, however, on FSH receptor expression in human fetal testis. Therefore, studies on FSH receptor expression in fetal testis would be required to make final conclusions on the importance of FSH-mediated signals in the apoptosis of human fetal testis. To shed more light on the function of GATA-6 in the fetal testis, we investigated whether GATA-6 expression can be linked to apoptosis and/or cell proliferation during fetal testicular development. This was not, however, the case, as the expression pattern of GATA-6 did not match the patterns of apoptosis or cell proliferation. These findings suggest that GATA-6 is unlikely to be involved in pro- or antiapoptotic processes in the fetal testis.

Our results on cell proliferation in the fetal testis are in agreement with those of earlier studies implying that testicular cell proliferation occurs mainly during the first trimester. Germ cell proliferation has been shown to cease between wk 12–18 (49), and the number of fetal Leydig cells remains constant between wk 13–24, decreasing thereafter (50). Information on Sertoli cell proliferation in humans is scanty. The very few proliferating cells in fetal testicular cords between wk 16–40 indicate that fetal Sertoli cell proliferation is most intense before the second trimester. The number of proliferative cells in the interstitium was, however, more abundant than that in the testicular cords. Studies in rodents have shown that the development of other testicular cell types is dependent on Sertoli cell differentiation (51). Hence, it is plausible to propose that human Sertoli cell differentiation and proliferation precede those of other cell types. Significant Sertoli cell proliferation occurs during infancy and puberty, resulting in an almost 15-fold increase in the number of Sertoli cells from fetal to adult testis (52).

Previous investigators have demonstrated the expression of Bcl-2 and Bax in human fetal testis up to midgestation (5, 6). In the present study we extended these analyses to term. Taking the previous and current results together, we can conclude that Bcl-2 expression changes in a cell-specific manner along with fetal testicular development. During the first trimester it was expressed in Sertoli and Leydig cells. In the second trimester Bcl-2 was expressed in Leydig cells and peritubular myoid cells. Finally, in the third trimester it was no longer detectable in any testicular cell type. Bcl-2 was not expressed in male germ cells in the present study. In contrast, in adult human testis, Bcl-2 expression has been reported in germ cells (35). In human fetal testis, Bcl-2 is expressed when major testicular morphogenetic and developmental changes take place, and it may protect somatic cell types from apoptosis during their proliferation and maturation. During the second trimester testicular development reaches a quiescent steady state level, and Bcl-2 is no longer needed or, alternatively, other factors fulfill its function.

The current results on Bax expression are in accordance with those from a previous report in which Bax expression was studied up to midgestation (6). The present report, however, is the first on Bax expression in human fetal testis during the second half of gestation. The activity of Bax protein is dependent on its intracellular localization (53, 54, 55), and Bax expression in the fetal testis does not necessarily reflect the amount of biologically active Bax protein. This may be the case in fetal testis, as the number of apoptotic cells was low even in the presence of abundant Bax protein. In the human adult testis, Bax is expressed in germ cells, and it has been proposed to have a role in germ cell differentiation and maturation (36).

Several investigators have emphasized the role of GATA-4 in gene regulation and differentiation in male gonads from mice to humans (44, 56). In the mouse, GATA-6 is expressed in the somatic cell compartment (14, 57). This study shows for the first time that GATA-6 is also expressed in human fetal testis. Recent in vitro data suggest that this transcription factor may regulate several gonadal genes (57), but the in vivo relevance of these findings remains to be explored. The present data suggest that GATA-6 is unlikely to be involved in the cell proliferation or Bcl-2/Bax-regulated apoptosis in fetal testes. However, it is possible that other molecules are involved in testicular somatic and germ cell apoptosis during the fetal period. Therefore, the relationship of GATA-6 to other anti- and proapoptotic mechanisms remains to be explored in human fetal testis.


   Footnotes
 
This work was supported by the Finnish Pediatric Research Foundation, the Finnish Medical Foundation, and the Finnish Cultural Foundation (to I.K.); the Sigrid Jusélius Foundation (to J.S.T., and M.H.); Oulu University Central Hospital (to J.S.T. and T.V.); the Academy of Finland (to J.S.T. and J.T.); the Finnish Research Program on Environmental Health, of the Academy of Finland and Turku University Central Hospital (to J.T.); University Central Hospital in Helsinki (to I.K. and M.H.); and Helsinki University Research Foundation (to I.K.) and Funds (to M.H.).

Abbreviations: TUNEL, Thymidine deoxyribose-mediated deoxy-UTP nick end labeling.

Received October 23, 2002.

Accepted January 10, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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