320 K. NISHINO JOURNAL ET OF AL. EXPERIMENTAL ZOOLOGY 286:320–327 (2000) Establishment of Fetal Gonad/Mesonephros Coculture System Using EGFP Transgenic Mice KOICHIRO NISHINO, MINORU KATO, KOU YOKOUCHI, KEITARO YAMANOUCHI, KUNIHIKO NAITO, AND HIDEAKI TOJO* Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan ABSTRACT In developing mouse embryos, the Sertoli cells, Leydig cells, and seminiferous cords are differentiated in the XY gonads. The migration of mesonephric cells into the gonads is required during the developmental stage for seminiferous cord formation in the male gonads. In previous experiments, an organ coculture system has been used to examine morphologically developing gonads. However, by the process used in this system for fixing and staining the gonad/ mesonephros complexes for examination, the kinetics of cell migration and the character of migrating cells cannot be observed. In the present study, we established an improved organ coculture system, using transgenic mice ubiquitously expressing Enhanced Green Fluorescent Protein (EGFP). In this system, time-dependent morphological changes in male-specific migration were observable in the gonad/mesonephros complex. The cell migration occurred at around 20 hr of coculture and began to spread at 25 hr with increases in the number of migrating cells occurring at 45 hr of coculture. No degenerative changes were detected at the end of coculture. Our results indicate that the present coculture system is very useful for investigating the mechanism of cell migration, as well as the characteristics of the migrating cells, in developing gonads. J. Exp. Zool. 286:320–327, 2000. © 2000 Wiley-Liss, Inc. In mice, the gonads of male embryos at 12.5 days post coitus (dpc) are dramatically differentiated and form cord-like structures containing pre-Sertoli cells and primordial germ cells. In contrast, the histological structures of female gonads are little changed at this time. Several previous studies using the primary culture or organ culture of male gonads have shown that Sertoli cells, Leydig cells, and seminiferous cords are differentiated in vitro (Magre and Jost, ’84; Patsavoudi et al., ’85; Jost and Magre, ’88; Karl and Capel, ’98), and that the seminiferous cord formation requires cell migration from the adjacent mesonephros. It has been reported that some of the stromal cells in male gonads are of mesonephric origin (Buehr et al., ’93). Merchant-Larios et al. (’93) demonstrated, in a study using an organ coculture system at the male gonad and the 3H-thymidine labeled mesonephros, that Sertoli and Leydig cells are differentiated without the mesonephros, while endothelial and peritubular myoid-like cells migrate into the male gonad from the mesonephros. It was also shown that both male and female mesonephric cells or even limb bud cells migrate into the male gonad when they are cocultured with male gonads (Buehr et al., ’93; 2000 WILEY-LISS, INC. Merchant-Larios et al., ’93; Moreno-Mendoza et al., ’95). Martineau et al. (’97) and Brennan et al. (’98) verified, by using their own coculture system of a transgenic mouse ubiquitously expressing β-galactosidase, that cell migration from the mesonephros to the gonad is male-specific and that the migration is dependent on induction signals from the male gonad. These organ coculture systems of a gonad grafted on another individual mesonephros have contributed to the morphological analysis of gonad development. In these previous experiments, however, the gonad/mesonephros complexes were fixed and stained for examination, which meant that the kinetics of cell migration and the character of migrating cells, such as the differential ability of cells or gene expression in the cells, could not be examined. In the present study, we established an improved organ coculture system using Grant sponsor: Ministry of Education, Science, Sports and Culture of Japan Grant-in-Aid for Scientific Research (A); Grant number: 07556063. *Correspondence to: Hideaki Tojo, Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: [email protected] Received 16 February 1999; Accepted 2 June 1999 COCULTURE OF FETAL GONAD AND MESONEPHROS transgenic mice ubiquitously expressing the enhanced green fluorescent protein (EGFP) under the control of a CMV enhancer and β-actin promoter. The green signal of EGFP can be readily detected in a live organ under a fluorescent microscope without fixing and staining. The present coculture system enabled us to observe the time-dependent morphological changes that occur in the male-specific migration of cells in the gonad/mesonephros complex. The present coculture system showed a two-phase migration of the mesonephros cells. MATERIALS AND METHODS EGFP-transgenic mice Mice of BDF1 (C57BL/DBA F1) and ICR strains were purchased from a dealer (Japan SLC, Shizuoka, Japan), and were kept under the regulated temperature (22–25°C), humidity (40–60%), and illumination cycles (14 hr light, and 10 hr dark) throughout the experiments. The pCX-EGFP plasmid containing CMV enhancer, chicken β-actin promoter, and 733 bp cDNA encoding EGFP was a generous gift from Dr. M. Okabe, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan (Okabe et al., ’97). Briefly, the 3.2kbp insert fragment of the EGFP gene was prepared by digesting pCX-EGFP plasmid with Sal I and BamHI (Takara, Otsu, Japan). The DNA fragments were purified by the cesium chloride ultracentrifugation method, and approximately 4.0 µg/ ml of the linear fragments were used for microinjection into the pronuclei of fertilized eggs. The 7to 8-week-old F1 females (C57BL/DBA) were superovulated by an injection of 7.5 IU of pregnant mare’s serum gonadotropin (PMSG; Teikokuzoki, Tokyo, Japan), followed 48 hr later by the injection of 7.5 IU of hCG. Fertilized eggs were collected from the oviducts of the females that had been mated with males of the same strain the day after mating. Microinjection was performed through the use of an inverted microscope equipped with a micromanipulator (Narishige, Tokyo, Japan). Approximately 2 pl of DNA solution were injected into the pronuclei of collected eggs and the microinjected embryos were cultured for 4 days in M16 medium (Sigma, St. Louis, MO) until they reached the morula or blastocyst stage. The EGFP-positive embryos were then transferred to the uteri of the pseudo-pregnant female ICR strain female mice (>10 weeks old). The transgenic mice were identified by both Southern blotting and PCR analyses using DNA extracted from the tail tips of 4-weekold pups. The expression of EGFP was determined 321 by northern blotting and by observing the fluorescence on the entire body of transgenic mice using an excitation light. The several transgenic lines ubiquitously expressing EGFP were used for the present experiments. Organ cultures The 12.5-dpc mouse embryos were collected from the pregnant females (C57BL/DBA F1) that had been mated with the transgenic males. To determine whether the collected embryos expressed the transgene, the embryos were examined for the expression of EGFP under a fluorescent stereomicroscope (Leica MZFLIII/CLS150, Leica AG, Heerbrugg, Switzerland) using an excitation light. The whole gonad and mesonephros were dissected from all embryos, and the gonad and mesonephros were then separated using a 27-gauge needle in ice-cold PBS under a stereomicroscope (Martineau et al., ’97). Gonads separated from non-transgenic littermates and mesonephroi from EGFP transgenic pups were placed onto ice-cold F12/DME medium (Gibco BRL, Grand Island, NY) containing 1.2 g/liter of NaHCO3, 10% fetal bovine serum, 105 units/liter of penicillin, 100 mg/liter of streptomycin and 50 µl of gentamicin. Gonad and mesonephros were placed in a groove on a 1.5% agar plate (Bacto Agar: Difco, Detroit, MI) in 35 mm culture dishes containing the culture medium without serum. Excess medium was removed from the groove to promote adhesion of the gonad to the mesonephros. One hour after the start of the coculture, gonad/mesonephros complexes were added to 2 ml of the culture medium and incubated for 45 hr under a humidified atmosphere of 5% CO2 in air at 37°C. The culture media were exchanged at 2– 4 hr, 20 hr, and 40 hr of coculture. Cell migrations from the mesonephros expressing EGFP into the gonads of non-transgenic embryos were directly observed every 5 hr without fixation under the fluorescent stereomicroscope. Photographs were taken under UV light with a Leica GFP2 filter. Hematoxylin-eosin (HE) staining Gonad/mesonephros complexes cocultured for 45 hr were fixed with 4% paraformaldehyde in PBS for 2 hr. After dehydration through an ethanol series, the samples were embedded in paraffin, sectioned at 5.0 µm, and stained with Mayer’s hematoxylin and eosin solution. The preparation was examined under a light-inverted microscope. 322 K. NISHINO ET AL. RESULTS Transgenic mice ubiquitously expressing EGFP In the transgenic mice ubiquitously expressing EGFP, green signals of EGFP were observed in the heart, liver, kidney, testis, and muscle upon irradiation by the excitation light (Fig. 1). Deep green fluorescence was also observed under a fluorescent stereomicroscope in the whole-mount specimens of gonads and mesonephroi collected from the 12.5-dpc transgenic fetuses (Fig. 2). In the present experiment, the sex of the 12.5dpc embryos was identified by the presence or absence of testis cords, and male or female gonads that were separated from the nontransgenic littermates were attached to the mesonephroi of the nonsexed EGFP transgenic pups. Only the mesonephros of transgenic embryos expressed green fluorescence under a fluorescent stereomicroscope, but the gonads of non-transgenic embryos did not express fluorescence (Fig. 3). Observation of male-specific cell migration into gonads To determine whether the mesonephros cells migrated into the gonads, organ complexes cultured for 35 hr were directly observed under a fluorescent stereomicroscope using an excitation light. We found that mesonephric cells migrated into the male gonad (Fig. 4A), whereas no cell migration of such cells into the female gonad was observed (Fig. 4B). This finding of male-specific migration corresponds with that in previous studies, and confirms that the present system using EGFP transgenic mice enabled observation of the cell migration in the gonad/mesonephros complex. To survey temporal morphological changes of the male-specific migration in detail, the kinetics of the cell migration were examined every 5 hr starting at 15 hr of coculture (Fig. 5). Figure 5A–F shows a visual field representing typical migration kinetics. No sign of the cell migration from the mesonephros into the gonad was observed at 15 hr of coculture (Fig. 5A). Cell migration was first detected as a penetration of green signals into the gonads at around 20 hr of coculture (Fig. 5B), and at 25 hr of coculture (Fig. 5C), the green signals elongated and became clear. Although the elongated green signal had not spread at 30 hr, a deep green circle appeared in the gonad along with the green signal (Fig. 5D), and the number of deep green circles on the line had increased at 35 hr (Fig. 5E). At 40–45 hr of coculture, the green circles began to accumulate at the top of the green signal (Fig. 5F). Histological analysis The section of 45-hr cocultured male gonad/mesonephros complex revealed cord-like structures comparable to the 12.5-dpc normal male gonads. The histological examination of cocultured gonads did not indicate degenerative changes during the in vitro culture (Fig. 6). DISCUSSION The purpose of the present study was to establish an experimental coculture system that did not require fixing and staining, by which the cell migration from mesonephroi to gonads could be observed. For this purpose, we generated transgenic mice in which ubiquitous expression of EGFP, visible under excitation fluorescence, was used as a signal. The transgenic mice produced in the present study expressed EGFP strongly in the adult heart, testis, and muscle, though diffusely in the liver and kidney. This difference in the expression patterns of EGFP might be characteristic of the chicken β-actin promoter activity. Our results are in agreement with those reported previously (Okabe et al., ’97); the strong and uniform expression of EGFP in early-stage embryos they reported were also shown in the mesonephroi and gonads of 11.5–12.5-dpc embryos in the present study. Cell migrations from the mesonephroi into the gonads of 11.5-dpc mouse embryos have been reported by Buehr et al. (’93), Merchant-Larios et al. (’93), and Martineau et al. (’97). Martineau et al (’97) showed that this cell migration was malespecific. The present study, in which a clear malespecific cell migration was also observed, confirms their results. These previous reports did not clarify the point at which cell migration occurred in their respective coculture systems. Our results show that migration is initiated at around 20 hr of coculture. The green signal of EGFP was elongated between 20 and 25 hr of culture, and the signal did not spread until the end of culture (45 hr after the start of coculture). The present findings suggest that the migration process consists of two phases, the first of which, evidenced by the elongation of the very thin green line, indicates migration from the mesonephros into gonad. The second phase, during which deep green circles appear on the green line, show an increase in number of migratory cells throughout the culture period and a peak accumulation at the top of the COCULTURE OF FETAL GONAD AND MESONEPHROS Fig. 1. Adult tissues of EGFP transgenic (Tg) mice. Left panels indicate the light field and right panels indicate the dark field under fluorescence. Green signals were observed 323 in all of the adult tissues. A and B, heart; C and D, liver; E and F, kidney; G and H, testis; I and J, muscle. 324 K. NISHINO ET AL. Fig. 2. The 12.5-dpc fetuses collected from pregnant nonTg females mated with EGFP Tg males (A). Strong green fluorescence is observed in the whole mount of Tg fetuses (B, right), but not expressed in the non-Tg littermate (B, left). The fetal gonad and mesonephros (C) also strongly express green fluorescence (D). Fig. 3. The grafted male gonad of a 12.5-dpc non-Tg littermate on mesonephros from the Tg embryo (A). Under an excitation light, only the mesonephros, not the gonad, expresses green fluorescence (B). COCULTURE OF FETAL GONAD AND MESONEPHROS 325 Fig. 4. The male or female gonad/mesonephros at 35 hr of coculture. The mesonephros cells migrate into the male gonad (A), whereas no cell migration is observed into the female gonad (B). initial green signal between 25 hr and 45 hr of culture. Although the cells corresponding to the initial green signal and to the green circle were not identified in the present study, it is thought that several types might migrate during the coculture. The relationship between cell migration and components of the extracellular matrix (ECM) has been well investigated in several tissues. The construction and disintegration of ECM by proteolytic enzymes and their inhibitors are important for the formation, maintenance, and remodeling of the tissues. Testicular cord formation, Sertoli cell differentiation and migration, and germ cell development are also regulated by ECM (Hadley et Fig. 5. The time-dependent morphological changes in male-specific migration in a gonad/mesonephros complex. A– F are a higher magnification of the same area. No sign of cell migration into the gonad was detected at 15 hr of coculture (A). Cell migration was first detected as a penetration of green signals into the gonads at around 20 hr of coculture (B), and the green signals elongated and became clear at 25 hr of coculture (C). At 30 hr, although the range of the elongated green signals had not spread, a deep green circle appeared in the gonad along with each green signal (D), and at 35 hr, the number of green circles increased in a line on each green signal (E). The green circles accumulated at the top of each green signal at 45 hr of coculture (F). The arrows indicate an elongated green. Arrowheads indicate deep green circles. 326 K. NISHINO ET AL. Fig. 6. The paraffin section of a 45 hr-cocultured male gonad/mesonephros complex stained with hematoxylin and eosin. Cord-like structures found in the section did not show any degenerative changes during in vitro culture. al., ’85; Tung and Fritz, ’86). Therefore, one possible explanation is that cells initially migrated, then stromal cells probably determine the migration site and produce specific ECM proteins. Several proteinases such as matrix metalloproteinases (MMPs) and plasminogen activators are expected to play a key role in testicular development (Lacroix et al., ’77; Marzowski et al., ’85; Vihko et al., ’87; Sang et al., ’90a,b; Ailenberg et al., ’91). Similarly, tissue inhibitors of metalloproteinases (TIMPs) have also been reported to be an important factor in the testicular developmental processes (Ailenberg et al., ’91; Ulisse et al., ’94; Grima et al., ’96). Therefore, MMPs and TIMPs may be involved in the initial invasion of mesonephros cells. Other types of stromal cells might migrate along with the initial ECM guide. Further studies, however, are necessary to elucidate the mechanism of cell migration from mesonephroi to the gonads. No degenerative changes were observed in the gonad/mesonephros complexes cocultured up to 45 hr in the present system. In addition, cord-like structures comparable to the 12.5-dpc normal male gonads were observed in an organ complex, showing that EGFP does not have deteriorative effects on tissue development and indicating that the present coculture system was subphysiological. It has been reported that the migrated cells from the mesonephros are located outside the testicular cord and differentiate to myoid cells surrounding seminiferous tubules or endothelial cells in the vasculature, but not to Leydig cells, since they do not express steroidogenic enzymes. The differentiation capabilities of the migrated cells, however, were not fully examined in our investigation. In our preliminary experiment, we were able to find migrated cells expressing EGFP that were conclusively derived from the mesonephros in the primary culture; only gonads were collected from the gonad/mesonephros complexes cocultured for 45 hr and digested with trypsin for the subsequent primary cell culture. This culture system may be very useful for investigating the differentiation and gene-expression abilities of migrated cells expressing EGFP. In conclusion, we have successfully established a coculture system, without fixing and staining, that uses transgenic mice ubiquitously expressing EGFP in which the cell migration from the mesonephroi to the gonads can be observed. This system is subphysiological and can be used for subsequent primary culture containing migrated cells. We are currently investigating the mechanisms of cell migration and the characteristics of the migrating cells using this coculture system. ACKNOWLEDGMENT We thank Dr. M. Okabe, Research Institute for Microbial Diseases, Osaka University, Japan, for providing the EGFP-expression vector (pCXEGFP) used in this study. LITERATURE CITED Ailenberg M, Stetler-Stevenson WG, Fritz IB. 1991. Secretion of latent type IV procollagenase and active type collagenase by testicular cells in culture. J Biochem 279:75–80. Brennan J, Karl J, Martineau J, Nordqvist K, Schmahl J, Tilmann C, Ung K, Capel B. 1998. Sry and the testis: molecular pathways of organogenesis. J Exp Zool 281:494–500. Buehr M, Gu S, McLaren A. 1993. Mesonephric contribution to testis differentiation in the fetal mouse. Development 117:273–281. Grima J, Calcagno K, Yan Cheng C. 1996. cDNA cloning, and developmental changes in the steady-state mRNA level of rat testicular tissue inhibitor of metalloproteinase-2 (TIMP2). J Androl 17:263–275. Hadley HA, Byers SW, Suarez-Quian CA, Kleinman HK, Dym M. 1985. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J Cell Biol 101:1511–1522. Jost A, Magre S. 1988. Control mechanism of testicular differentiation. Philos Trans R Soc Lond B 322:55–61. Karl J, Capel B. 1998. Sertoli cells of the mouse testis originate from the coelomic epithelium. Dev Biol 203:323–333. Lacroix M, Smith FE, Firtz IB. 1977. Secretion of plasminogen activator by Sertoli cell-enriched culture. Mol Cell Endocrinol 9:227–236. Magre S, Jost A. 1984. Dissociation between testicular organogenesis and endocrine cyto-differentiation of Sertoli cells. Proc Natl Acad Sci USA 81:7831–7834. Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R, Capel B. 1997. Male-specific cell migration into the developing gonad. Curr Biol 7:958–968. Marzowski J, Sylvester SR, Gilmont RR, Griswold MD. 1985. COCULTURE OF FETAL GONAD AND MESONEPHROS Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity. Biol Reprod 32:1237–1245. Merchant-Larios H, Moreno-Mendoza N, Buehr M. 1993. The role of the mesonephros in cell differentiation and morphogenesis of the mouse fetal testis. Int J Dev Biol 37:407–415. Moreno-Mendoza N, Herrera-Munoz J, Merchant-Larios H. 1995. Limb bud mesenchyme permits seminiferous cord formation in the mouse fetal testis but subsequent testosterone output is markedly affected by the sex of the donor stromal tissue. Dev Biol 169:51–56. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimura Y. 1997. Green mice as a source of ubiquitous green cells. FEBS Lett 407:313–319. Patsavoudi E, Magre S, Castanier M, Scholler R, Jost A. 1985. Dissociation between testicular morphogenesis and functional differentiation of Leydig cells. J Endocrinol 105:235–238. Sang Q-X, Dym M, Byers SW. 1990a. Secreted metallo- 327 proteinases in testicular cell culture. Biol Reprod 43: 946–955. Sang Q-X, Stetler-Stevenson WG, Liotta LA, Byers SW. 1990b. Identification of type IV collagenase in rat testicular cell culture: influence of peritubular-Sertoli cell interactions. Biol reprod 43:956–964. Tung PS, Fritz IB. 1986. Extracellular matrix components and testicular peritubular cells influence the rate and pattern of Sertoli cell migration in vitro. Dev Biol 113:119–134. Ulisse S, Farina AR, Piersanti D, Tiberio A, Cappabianca L, D’Orazi G, Jannini EA, Malykh O, Stetler-Stevenson WG, D’Armiento M, Alberto Gulino A, Mackay AR. 1994. Follicle-stimulating hormone increases the expession of tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 and induces TIMP-1 AP-1 site binding complex(es) in prepubertal rat Sertoli cells. Endocrinology 135:2479–2487. Vihko KK, Toppari J, Parvinen M. 1987. Stage-specific regulation plasminogen avtivator secretion in the rat seminiferous epithelium. Endocrinology 120:142–145.