Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal... Research article 117 Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis 1 2 3 3 3 Shinichiro Chuma , Mito Kanatsu-Shinohara , Kimiko Inoue , Narumi Ogonuki , Hiromi Miki , 4 1 1 3 2, Shinya Toyokuni , Mihoko Hosokawa , Norio Nakatsuji , Atsuo Ogura and Takashi Shinohara * Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki 305-0074, Japan Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan *Author for correspondence (e-mail: takashi@mfour.med.kyoto-u.ac.jp) Accepted 27 October 2004 Development 132, 117-122 Published by The Company of Biologists 2005 doi:10.1242/dev.01555 Summary Primordial germ cells (PGCs) are derived from a Thus, fetal male germ cell development is remarkably population of pluripotent epiblast cells in mice. However, flexible, and the maturation process, from epiblast cells little is known about when and how PGCs acquire the through PGCs to postnatal spermatogonia, can occur in the capacity to differentiate into functional germ cells, while postnatal testicular environment. Primordial germ cell keeping the potential to derive pluripotent embryonic germ transplantation techniques will also provide a novel tool to cells and teratocarcinomas. In this investigation, we show assess the developmental potential of PGCs, such as those that epiblast cells and PGCs can establish colonies of manipulated in vitro or recovered from embryos harboring spermatogenesis after transfer into postnatal seminiferous lethal mutations. tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination Key words: Germ cell, Epiblast, Primordial Germ Cell (PGC), Pro- using spermatozoa or spermatids derived from PGCs spermatogonia, Spermatogonia, Testis, Spermatogenesis, harvested from fetuses as early as 8.5 days post coitum. Transplantation, Microinsemination Introduction 2001; Hajkova et al., 2002). Therefore, the characteristics of PGCs change during development before they mature into Mammalian germ cells undergo unique genetic and cellular postnatal germ cells. changes as they develop and differentiate to form functional Spermatogenesis is initiated shortly after birth (Russell et al., gametes. A population of pluripotent epiblast cells at around 1990; Meistrich and van Beek, 1993). Pro-spermatogonia 6.5 days post coitum (dpc) gives rise to primordial germ cells resume mitosis as spermatogonia, at around postnatal day 5, (PGCs), which become identifiable as a cluster of cells at the then enter into meiosis as spermatocytes and produce base of the allantois at 7.25 dpc (Ginsburg et al., 1990; Tam spermatids, which develop into spermatozoa. Spermatogonial and Zhou, 1996; Tsang et al., 2001). During development, the stem cells are a subpopulation of spermatogonia and have the number of PGCs increases from 40 cells at 7.5 dpc to 25,000 unique ability to self-renew as well as to differentiate to cells at 13.5 dpc, and they migrate through the developing produce spermatozoa (Meistrich and van Beek, 1993; de Rooij hindgut and mesentery to reach the urogenital ridge (UGR) at and Russell, 2000). These cells continue to divide throughout around 10.5 dpc. By 13.5 dpc, PGCs in the male genital ridge the life of the animal, and can be identified by their ability to enter into mitotic arrest and become pro-spermatogonia, while generate and maintain colonies of spermatogenesis following germ cells in the female arrest at meiotic prophase I (reviewed transplantation into the seminiferous tubules of infertile by McLaren, 2003). Primordial germ cells show different recipient testes (Brinster and Zimmermann, 1994). Using this features at different developmental stages. For example, assay, several groups have shown that pro-spermatogonia in migratory-stage PGCs exhibit a higher frequency of conversion developing fetal testes can differentiate into spermatogonial into embryonic germ cells, pluripotent cells that resemble stem cells when transferred into the adult testis (Ohta et al., blastocyst-derived embryonic stem cells, than do PGCs in the 2004; Jiang and Short, 1998). However, it is unknown if gonads (Matsui et al., 1992; Resnick et al., 1992; Labosky et germline cells at earlier stages of development can produce al., 1994). In addition, epigenetic changes characteristic to germline cells also occur in PGCs. Erasure of parental genomic spermatogonial stem cells or spermatogenic colonies after transplantation. imprints on both paternal and maternal alleles in PGCs commences near the time of their settlement in the UGR at 10.5 In this investigation, we sought to determine the potential of dpc, and new imprints are imposed in pro-spermatogonia germline cells from earlier embryos to develop into before birth (Szabo and Mann, 1995; Ueda et al., 2000; Surani, spermatogonial stem cells, using immature recipient animals. Development 118 Development 132 (1) Research article Epiblast cells or PGCs were transplanted into infertile mouse testes fixed in 4% paraformaldehyde in PBS were stained with Rhodamine-conjugated Peanut agglutinin (PNA) (Vector, testes and examined for their ability to re-populate the Burlingame, CA) for acrosomes, and with Hoechst 33258 (Sigma, St seminiferous tubules. Louis, MO) for nuclei. Microinsemination Materials and methods Microinsemination was undertaken by intracytoplasmic injection into Collection of donor cells C57BL/6 × DBA/2 F1 oocytes (Kimura and Yanagimachi, 1995). Donor cells were collected from pregnant C57BL/6 mice that were Embryos that were constructed using spermatozoa or elongated maintained in a controlled environment with 12:12 light:dark cycles spermatids derived from 8.5 dpc or 12.5 dpc PGCs were transferred from 08.00 h to 20.00 h (SLC, Shizuoka, Japan). The day when a into the oviducts of pseudopregnant ICR females after 24 or 48 hours copulation plug was found was designated as 0.5 dpc. In some in culture, respectively. Live fetuses retrieved on day 19.5 were raised experiments, we used a transgenic mouse line C57BL/6 Tg14 (act- by lactating ICR foster mothers. EGFP) OsbY01 (designated Green) provided by Dr M. Okabe (Osaka Genotyping of offspring and bisulfite sequencing of University, Osaka, Japan) (Okabe et al., 1997). The spermatogonia, imprinted genes spermatocytes and round spermatids of these mice express the enhanced green fluorescent protein (EGFP) gene, which gradually PCR fragments of the Kit gene encompassing the W point mutation or decreases after meiosis. The sex of embryos was determined using the W mutation (Nocka et al., 1990; Hayashi et al., 1991) were genotyping based on the Ube1 PCR method (Chuma and Nakatsuji, amplified using genomic DNA from mice derived from PGC trans- 2001), and only male embryos were used in this study. Due to the plantation, or from a W/W mouse as a control heterozygote for both limitations of cell recovery and the number of animals that could be mutations. PCR primers were 5′-CATTTATCTCCTCGACAAC- injected per day, the sex check was not performed in experiments CTTCC-3′ and 5′-GCTGCTGGCTCACAATCATGGTTC-3′ for W using 6.5 dpc embryos. Cells for transplantation were obtained from genotyping, and 5′-AGATGGCAACTCGAGACTCACCTC-3′ and 5′- whole embryonic ectoderm with primitive endoderm at 6.5 dpc, or TGCCCCCACGCTTTGTTTTGCTAA-3′ for W genotyping. germ-cell-containing tissues by dissection of the posterior thirds of Amplified products were gel extracted and directly sequenced. 8.5 dpc embryos, the mesenteries and guts of 10.5 dpc embryos, the Bisulfite genomic sequencing of differentially methylated regions UGRs of 10.5 dpc embryos, and the genital ridges of 11.5, 12.5, 14.5 (DMRs) of the Igf2r and H19 imprinted genes was carried out as and 16.5 dpc embryos. Tissues from each developmental stage were described (Ueda et al., 2000; Lee et al., 2002; Lucifero et al., 2002). dissociated by enzymatic digestion using 0.25% trypsin with 1 mmol/l Briefly, genomic DNAs were isolated from the offspring derived from EDTA (Invitrogen, Carlsbad, CA) for 10 minutes. Cells were PGC transplantation, and treated with sodium bisulfite, which suspended in Dulbecco’s modified Eagle’s medium, supplemented as deaminates unmethylated cytosines to uracils, but does not affect 5- previously described (Ogawa et al., 1997). methylated cytosines. Polymerase chain reaction amplification of each DMR from bisulfite-treated genomic DNAs was carried out using Transplantation into recipient testes primer sets as described (Ueda et al., 2000; Lucifero et al., 2002), and v v v Donor cells were transplanted in histocompatible W/W or W /W mice DNA sequences were determined. (W mice, obtained from SLC, Shizuoka, Japan). Only 5- to 10-day- old male mice were used as recipients. W mutants lack endogenous spermatogenesis (Silvers, 1979), because of mutations in the Kit gene Results (Nocka et al., 1990; Hayashi et al., 1991). Recipient animals were Epiblasts with primitive endoderms (6.5 dpc) or tissues placed on ice to induce hypothermic anesthesia, and returned to their containing fetal germ cells were collected from different stages dams after surgery (Shinohara et al., 2001). Approximately 2 µl of cell of embryos (posterior third of 8.5 dpc embryos, mesenteries suspension were introduced into each testis by injection via the and guts of 10.5 dpc embryos, or gonads of 10.5 to 16.5 dpc efferent duct (Ogawa et al., 1997). embryos) (Fig. 1A). The cells were dissociated enzymatically Histological analysis and single cell suspensions were transplanted into the Three to four months after transplantation, the recipient testes were seminiferous tubules of recipient immature W mice. Although fixed in 10% neutral-buffered formalin (Wako Pure Chemical W mice have a very small number of spermatogonia (Ohta et Industries, Osaka, Japan) and processed for paraffin sectioning. al., 2003), spermatogenesis is arrested at the point of Sections were stained with hematoxylin and eosin. Two histological undifferentiated type A spermatogonia and no differentiating sections were prepared from the testes of each animal and viewed at germ cells are found due to defects in the Kit gene (Nocka et 400× magnification to determine the extent of spermatogenesis. The al., 1990; Hayashi et al., 1991) (Fig. 1B). Therefore, any numbers of tubule cross-sections with or without spermatogenesis spermatogenesis detected in the recipient testis must be derived (defined as the presence of multiple layers of germ cells in the from the donor cells. Although the concentration of cells seminiferous tubule) were recorded for one histological section from each testis. Meiosis was detected by immunofluorescence staining injected varied due to the more limited recovery of cells from using anti-synaptonemal complex protein 3 (SCP3) antibody (Chuma early-stage fetuses, the cell viability, or percentage of tubules and Nakatsuji, 2001) and Alexa 488-conjugated anti-rabbit filled with donor cells, was similar in all experiments. The immunoglobulin G antibody (Molecular Probes, Eugene, USA). recipient mice were sacrificed 3 to 4 months after Periodic acid Schiff (PAS) staining (Muto Pure Chemicals, Tokyo, transplantation, and the testes were examined histologically for Japan) was carried out to examine acrosome formation in spermatids. the presence of spermatogenesis. This time period represents In experiments using Green mice, recipient testes were recovered 10 three to four spermatogenic cycles in mice (Meistrich and van to 11 weeks after donor cell transplantation, and analyzed by Beek, 1993; de Rooij and Russell, 2000), which would allow observing EGFP signals under fluorescence microscopy. Donor cells sufficient time for the development of sperm from were identified specifically because host testis cells had no spermatogonial stem cells. endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied the entire circumference of the tubule and At least three experiments were performed using cells was at least 0.1 mm long (Nagano et al., 1999). Cryosections of the harvested from each stage of embryonic development, and the Development Differentiation potency of PGC 119 Fig. 1. Spermatogenesis and teratogenesis from fetal germ cells and epiblast cells. (A) Embryos at 6.5 and 8.5 dpc, a mid-part of 10.5 dpc embryo, and a male gonad and mesonephros at 12.5 dpc. Dotted lines demarcate regions used for transplantation. At 10.5 dpc, the urogenital ridges (asterisk) and mesentery with gut (arrow) were dissected separately. (B) A section of a W male testis (control recipient) stained with HE. Spermatogenesis is absent. (C) W mouse testis after transplantation of 8.5 dpc PGCs. Spermatozoa (arrow) are present in the center of the seminiferous tubule. (D) Anti-SCP3 immunostaining (green) of W testis after transplantation of 8.5 dpc PGCs, counterstained with Hoechst 33258 dye (blue). Inset, higher magnification view of the same sample. (E) Transplantation of epiblast cells at 6.5 dpc. Spermatocyte (arrow) and round spermatid (arrow head) were found. Inset shows acrosomes stained with PAS (red) in round spermatids. (F) W mouse testis transplanted with 8.5 dpc PGCs from Green mice embryos. Colonization of the recipient seminiferous tubule by EGFP (+) donor cells (green) was observed (arrow). (G) A section of the same testis as in (F), stained with Rhodamine-PNA (red) for acrosomes and with Hoechst 33258 dye (blue) for nuclei. Spermatogenenic colonies derived from EGFP (+) donor cells are present (arrow). (H) Higher magnification view of (G), showing spermatogonia (arrowhead) residing at the base of the seminiferous tubule, and elongated spermatids (arrow) with acrosomes (red), shedding the EGFP (+) cytoplasm. (I) Teratoma from epiblast cells at 6.5 dpc. Muscle, dermoid cyst and neuronal tissue are observed. (J) Spermatozoa (arrow) clustered around a Sertoli cell, released from a recipient testis of 8.5 dpc PGCs. (K) An offspring developed from an oocyte injected with a sperm derived from 8.5 dpc PGCs. (L) DNA sequences of the Kit gene around the W (left panels) and W (right panels) point mutations of an offspring derived from transplantation of 8.5 dpc PGCs (upper panels) and a W/W mouse as a control heterozygote (lower panels). Scale bars: 1 mm in F; 10 µm in H; 20 µm in J; 50 µm in others. Table 1. Donor cell colonization in W recipient mice Number of Number of % tubule cross- Number of Donor Number of transplanted testes with section with testes with age (dpc) recipient testes cells (×10 /testis) spermatogenesis (%) spermatogenesis* teratoma (%) 6.5 27 0.4±0.2 2 (7.4) 0.1±0.1 4 (14.8) 8.5 13 7.2±0.6 9 (69.2) 3.8±0.1 4 (30.8) 10.5 (mes+gut) 12 12.8±1.4 4 (33.3) 0.9±0.5 0 10.5 (UGR) 15 11.4±2.4 11 (73.3) 7.2±0.1 0 11.5 13 2.4±1.0 8 (61.5) 2.4±0.7 0 12.5 13 9.0±1.6 11 (84.6) 15.7±4.5 0 14.5 12 20 12 (100) 56.5±10.8 0 16.5 12 20 12 (100) 59.3±7.5 0 Values are mean±s.e.m. Results from at least three separate experiments. Testes were analyzed 3-4 months after transplantation. Mes+gut, mesentery and gut; UGR, urogenital ridge. *Percentage of tubule cross-sections containing spermatogenesis/total tubule cross-sections examined in each testis. results are summarized in Table 1. Overall, 69 of 117 (59%) injection of donor cells (Fig. 1B). Spermatogenesis observed recipient testes showed spermatogenesis, but no differentiating in the recipient testes originated from spermatogonial stem germ cells were found in control testes that did not receive an cells, because other spermatogenic cells do not have the Development 120 Development 132 (1) Research article Table 2. Development of oocytes injected with spermatogenic cells derived from PGCs In vitro development (%) Donor age Number of Number of Implantation Number of (dpc) reconstituted eggs One cell Two cell Four cell eggs transferred sites (%) offspring (%) 8.5* 186 78 (41.9) 105 (56.5) NA 105 47 (25.2) 20 (10.8) 12.5 182 8 (4.4) 25 (13.7) 130 (71.4) 130 88 (48.3) 29 (15.9) Combined results using elongated spermatids and spermatozoa. *Combined results from two different recipient testes. NA, not applicable because cells were transferred at the two-cell stage. capacity for self-renewal and disappear by 35 days after allowed specific identification of donor cells, because transplantation (Russell et al., 1990; Brinster and endogenous host testis cells had no detectable fluorescence. Zimmermann, 1994; Shinohara et al., 2001). Spermatogenesis Four experiments were performed, and a total of 16 testes were in the recipient testes that received fetal germ cells (8.5-16.5 microinjected with donor cells. Approximately 6 to 12 × 10 dpc) was morphologically normal (Fig. 1C), and the kinetics cells were transplanted into each testis. When the recipients of spermatogenic colonization were generally comparable to were analyzed 10 to 11 weeks after transplantation, 4 of 16 results obtained after transplantation of postnatal (25%) testes had spermatogenic colonies that showed EGFP spermatogonial stem cells (Shinohara et al., 2001). All stages signals (Fig. 1F). The average number of colonies per testis of spermatogenic cells, including mature spermatozoa, were was 0.3 ± 0.1 (mean ± s.e.m.). Histological analysis of the found in recipient testes. Synchrony of meiosis in seminiferous EGFP (+) colonies showed the presence of apparently normal tubules was confirmed by immunostaining for SCP3, a spermatogenesis (Fig. 1G,H). component of the synaptonemal complex (Fig. 1D). While these results indicate that all types of donor cells The age of the donors had a significant effect on the number differentiated into spermatogonial stem cells, differentiation of of recipient seminiferous tubules with observable donor cells was not restricted to the germline lineage. spermatogenesis. Whereas only 0 to 11% of the tubules showed Consistent with findings of previous studies (Illmensee and spermatogenesis in testes that received 8.5 dpc PGCs, Stevens, 1979), testes receiving epiblast cells or 8.5 dpc PGCs spermatogenesis derived from donor cells at later stages of formed teratomas (Fig. 1I). Epiblast cells produced larger gonadal development was generally more extensive, and tumors than did 8.5 dpc PGCs, and some of the seminiferous recipient testes grew larger due to the increased production of germ cells. In one case, transplantation of 16.5 dpc pro-spermatogonia resulted in 96% of the tubules with evident spermatogenesis, and spermatozoa were transported to the epididymis (data not shown). Spermatogenesis was also found in the recipients that were injected with cells from epiblasts with primitive endoderms. Although we could not identify mature spermatozoa histologically, spermatogenesis was found in two different testes that received injections of 6.5 dpc epiblast and primitive endoderm cells. The spermatogonia, spermatocytes and round spermatids that developed in the recipient seminiferous tubules appeared morphologically normal (Fig. 1E), and acrosome formation in round spermatids was confirmed by PAS staining (Fig. 1E, inset). To confirm the donor origin of spermatogenesis, we used Green mice that ubiquitously express the EGFP transgene. Donor cells were collected from the posterior thirds of 8.5 dpc embryos that showed EGFP signals. This Fig. 2. DMR methylation of the Igf2r and H19 genes in offspring derived from transplantation of 8.5 and 12.5 dpc PGCs. DNA methylation was analyzed by bisulfite genomic sequencing. Both male and female offspring of each stage of donor PGCs were analyzed. Individual lines represent sequenced clones. Black ovals indicate methylated cytosine-guanine sites (CpGs) and white ovals indicate unmethylated CpGs. Development Differentiation potency of PGC 121 tubules were dilated and broken. Cells from three germ layers, tubules, while spermatogenesis did not occur from PGCs from including ciliated epithelium, muscle, neuron and bone, were 12.5 dpc embryos. Another group reported that germ cells from found in these testes. Interestingly, spermatogenesis was day 0-3 mouse pups did not show spermatogenic colonies after occasionally observed in tubules not affected by tumorigenesis, transplantation into adult testes (McLean et al., 2003). In our indicating that the same population of donor cells could cause study, however, epiblast cells at 6.5 dpc and PGCs at 8.5 to 16.5 spermatogenesis and teratogenesis. No teratomas were found dpc produced spermatogenesis after transplantation into in the recipients of gonadal PGCs or pro-spermatogonia. immature seminiferous tubules at postnatal day 5 to 10. This To examine whether germ cells generated from PGCs were difference might simply be ascribed to structural differences; fully functional, we performed microinsemination, a technique immature Sertoli cells lack tight junctions and may allow commonly used to produce offspring from infertile animals and migrating transplanted cells easier access to the stem cell humans (Kimura and Yanagimachi, 1995; Palermo et al., niches, which are distributed nonrandomly in the seminiferous 1992). Donor PGCs were collected from 8.5 or 12.5 dpc tubules (Chiarini-Garcia et al., 2001). Alternatively, immature embryos, and transplanted into the testes of W mice. Four testis may express factors that support survival and months after transplantation, spermatozoa or elongated differentiation of epiblast cells and PGCs, while mature testis spermatids were collected from tubule fragments by may not. Because PGCs have chemotaxic activity (Godin et al., mechanical dissociation (Fig. 1J), and microinjected into 1990), we speculate that some of the transplanted cells oocytes. The results of the microinsemination experiments are migrated into the niches of immature seminiferous tubules, summarized in Table 2. Spermatogenic cells derived from 8.5 where they could survive. PGCs and epiblast cells may have dpc PGCs appeared to be less competent for egg activation, then switched their cell cycle fate to function as because a significant number of eggs receiving elongated spermatogonial stem cells. Both male and female PGCs enter spermatid/spermatozoa from 8.5 dpc PGCs stayed at into meiosis in the absence of the male gonadal environment, metaphase II or the metaphase II-anaphase transition and did but do not if they lodge there (McLaren and Southee, 1997; not develop into the 2-cell stage after 24 hours. However, Chuma and Nakatsuji, 2001). Somatic cells in the fetal testis offspring were obtained from oocytes inseminated with are assumed to produce a substance that inhibits the meiotic spermatid/spermatozoa derived from both stages of PGCs (Fig. transition of PGCs. Given our results, it appears that 1K). Genotyping of offspring showed that they did not carry seminiferous tubules of newborn mice may also have similar W or W mutations in the Kit gene (Nocka et al., 1990; Hayashi meiosis-inhibiting activity. et al., 1991), demonstrating the offspring were derived from Erasure of parental genomic imprints commences in PGCs wild-type donor PGCs that had been transplanted into the testis at around the time of their arrival in the UGR (Szabo and Mann, (Fig. 1L). No apparent abnormality was seen in any of the 1995; Surani, 2001; Hajkova et al., 2002). However, it has not offspring, and they were fertile. Bisulfite sequencing analysis been clear whether this epigenetic event depends on induction of the offspring showed no obvious fluctuations in the from the UGR or is programmed autonomously in PGCs. As methylation status of the DMRs of paternally methylated H19 the offspring from PGC transplantations were viable and and maternally methylated Igf2r genes (Fig. 2). apparently healthy, epigenetic modifications, including erasure of parental genomic imprints, should have occurred appropriately in transplanted PGCs. This was corroborated by Discussion the normal methylation patterns exhibited in the DMRs of the This study demonstrates that not only migrating PGCs but also Igf2r and H19 genes. Because the establishment of paternal epiblast cells can differentiate into spermatogonial stem cells methylation proceeds after birth in the normal testis (Ueda et and produce spermatogenesis after transfer into postnatal testis. al., 2000), it seems unlikely that the postnatal testis has the The ability to initiate and maintain spermatogenesis after ability to erase parental methylation. Therefore, our results transfer into the infertile testis fulfills the criteria for the suggest that PGCs may have the autonomous program for the identification of spermatogonial stem cells (Brinster and erasure of parental methylation before reaching the UGR. Zimmermann, 1994). Developmental processes that occur Primordial germ cell transplantation will provide a new during differentiation of spermatogonial stem cells from experimental approach for the study of PGC development. epiblast cells include induction of PGCs among epiblast cells, Primordial germ cells harvested from embryonic lethal mutants migration and proliferation of PGCs, erasure of parental as early as gastrulation can be traced for their differentiation genomic imprints, and G1 (G0) arrest in the developing male capacities by transplantation into recipient testis. Similarly, gonad. Nonetheless, our results demonstrate that these PGCs manipulated in vitro, such as those cultured with growth developmental events do not necessarily require embryonic factors or transfected with vectors (De Miguel et al., 2002; somatic environments, and most processes of male germline Watanabe et al., 1997), can now be assessed for their effects differentiation can take place in postnatal testis. This flexibility on subsequent differentiation in vivo. Such functional studies of fetal germ cell differentiation may be related to, and partly would help elucidate factors that regulate male germline account for, the recent success in the derivation of sperm from development in mammals. embryonic stem cells in vitro (Toyooka et al., 2003; Geijsen et al., 2004). We thank Dr Masaru Okabe for kindly providing C57BL/6 Tg14 The important factor that contributed to the results of our (act-EGFP) OsbY01 mice, Dr Fumitoshi Ishino for helpful advice on experiments is the use of immature postnatal testes as bisulfite sequencing, and Drs Yasuhisa Matsui and Yoshito Kaziro for recipients. Recently, Ohta et al. (Ohta et al., 2004) showed that critical reading of the manuscript. This research was supported by a pro-spermatogonia from 14.5 dpc embryos completed grant from the Japanese Ministry of Education, Science, Sports and spermatogenesis when transplanted in mature seminiferous Culture. Development 122 Development 132 (1) Research article References differentiation of spermatogonial stem cells in the W/W mutant mouse testis. Biol. Reprod. 69, 1815-1821. Brinster, R. L. and Zimmermann, J. W. (1994). Spermatogenesis following Ohta, H., Wakayama, T. and Nishimune, Y. (2004). Commitment of fetal male germ-cell transplantation. Proc. Natl. Acad. Sci. USA 91, 11298- male germ cells to spermatogonial stem cells during mouse embryonic development. Biol. Reprod. 70, 1286-1291. Chiarini-Garcia, H., Hornick, J. R., Griswold, M. D. and Russell, L. D. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y. (2001). Distribution of type A spermatogonia in the mouse is not random. (1997). ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, Biol. Reprod. 65, 1179-1185. 313-319. Chuma, S. and Nakatsuji, N. (2001). Autonomous transition into meiosis of Palermo, G., Joris, H., Devroey, P. and van Steirteghem, A. C. (1992). mouse fetal germ cells in vitro and its inhibition by gp130-mediated Pregnancies after intracytoplasmic injection of single spermatozoon into an signaling. Dev. Biol. 229, 468-479. oocyte. Lancet 340, 17-18. De Miguel, M. T., Cheng, L., Holland, E. C., Federspiel, M. J. and Resnick, J. L., Bixler, L. S., Cheng, L. and Donovan, P. J. (1992). Long- Donovan, P. J. (2002). Dissection of the c-kit signaling patheway in mouse term proliferation of mouse primordial germ cells in culture. Nature 359, primordial germ cells by retroviral-mediated gene transfer. Proc. Natl. Acad. 550-551. Sci. USA 99, 10458-10463. Russell, L. D., Ettlin, R. A., Sinha Hikim, A. P. and Clegg, E. D. (1990). de Rooij, D. G. and Russell, L. D. (2000). All you wanted to know about Histological and Histopathological Evaluation of the Testis (ed. L. D. spermatogonia but were afraid to ask. J. Androl. 21, 776-798. Russell, R. A. Ettlin, A. P. Sinha Hikim and E. D. Clegg), pp. 1-40. Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K. and Daley, Clearwater, FL: Cache River Press. G. Q. (2004). Derivation of embryonic germ cells and male gametes from Shinohara, T., Orwig, K. E., Avarbock, M. R. and Brinster, R. L. (2001). embryonic stem cells. Nature 427, 148-154. Remodeling of the postnatal mouse testis is accompanied by dramatic Ginsburg, M., Snow, M. H. and McLaren, A. (1990). Primordial germ cells changes in stem cell number and niche accessibility. Proc. Natl. Acad. Sci. in the mouse embryo during gastrulation. Development 110, 521-528. USA 98, 6186-6191. Godin, I., Wylie, C. C. and Heasman, J. (1990). Genital ridges exert long- Silvers, W. K. (ed.) (1979). The Coat Colors of Mice, pp. 206-223. New York, range effects on mouse primordial germ cell numbers and direction of NY: Springer. migration in culture. Development 108, 357-363. Surani, M. A. (2001). Reprogramming of genome function through epigenetic Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., inheritance. Nature 414, 122-128. Walter, J. and Surani, M. A. (2002). Epigenetic reprogramming in mouse Szabo, P. E. and Mann, J. R. (1995). Biallelic expression of imprinted genes primordial germ cells. Mech. Dev. 117, 15-23. in the mouse germ line: implications for erasure, establishment, and Hayashi, S., Kunisada, T., Ogawa, M., Yamaguchi, K. and Nishikawa, S. mechanism of genomic imprinting. Genes Dev. 9, 1857-1868. (1991). Exon skipping by mutation of an authentic splice site of c-kit gene Tam, P. P. and Zhou, S. X. (1996). The allocation of epiblast cells to in W/W mouse. Nucleic Acids Res. 19, 1267-1271. ectodermal and germ-line lineages is influenced by the position of the cells Illmensee, K. and Stevens, L. C. (1979). Teratomas and chimeras. Sci. Am. in the gastrulating mouse embryo. Dev. Biol. 178, 124-132. 240, 120-133. Tsang, T. E., Khoo, P. L., Jamieson, R. V., Zhou, S. X., Ang, S. L., Jiang, F.-X. and Short, R. V. (1998). Different fate of primordial germ cells Behringer, R. and Tam, P. P. (2001). The allocation and differentiation of and gonocytes following transplantation. APMIS 106, 58-63. mouse primordial germ cells. Int. J. Dev. Biol. 45, 549-555. Kimura, Y. and Yanagimachi, R. (1995). Mouse oocytes injected with Toyooka, Y., Tsunekawa, N., Akasu, R. and Noce, T. (2003). Embryonic testicular spermatozoa or round spermatids can develop into normal stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci. USA 100, 11457-11462. offspring. Development 121, 2397-2405. Ueda, T., Abe, K., Miura, A., Yuzuriha, M., Zubair, M., Noguchi, M., Labosky, P. A., Barlow, D. P. and Hogan, B. L. M. (1994). Mouse embryonic Niwa, K., Kawase, Y., Kono, T., Matsuda, Y. et al. (2000). The paternal germ (EG) cell lines: transmission through the germ line and differences in methylation imprint of the mouse H19 locus is acquired in the gonocyte the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene stage during foetal testis development. Genes Cells 5, 649-659. compared with embryonic stem (ES) cell lines. Development 120, 3197- Watanabe, M., Shirayoshi, Y., Koshimizu, U., Hashimoto, S., Yonehara, S., Eguchi, Y., Tsujimoto, Y. and Nakatsuji, N. (1997). Gene transfection Lee, J., Inoue, K., Ono, R., Ogonuki, N., Kohda, T., Kaneko-Ishino, T., of mouse primordial germ cells in vitro and analysis of their survival and Ogura, A. and Ishino, F. (2002). Erasing genomic imprinting memory in growth control. Exp. Cell Res. 230, 76-83. mouse clone embryos produced from day 11.5 primordial germ cells. Development 129, 1807-1817. Lucifero, D., Mertineit, C., Clarke, H. J., Bestor, T. H. and Tasler, J. M. (2002). Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79, 530-538. Matsui, Y., Zsebo, K. and Hogan, B. L. M. (1992). Derivation of pluripotent embryonic cells from murine primordial germ cells in culture. Cell 70, 841- McLaren, A. (2003). Primordial germ cells in the mouse. Dev. Biol. 262, 1- McLaren, A. and Southee, D. (1997). Entry of mouse embryonic germ cells into meiosis. Dev. Biol. 187, 107-113. McLean, D. J., Friel, P. J., Johnston, D. S. and Griswold, M. D. (2003). Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol. Reprod. 69, 2085-2091. Meistrich, M. L. and van Beek, M. E. A. B. (1993). Spermatogonial stem cells. In Cell and Molecular Biology of the Testis (ed. C. Desjardins and L. L. Ewing), pp. 266-295. New York, NY: Oxford University Press. Nagano, M., Avarbock, M. R. and Brinster, R. L. (1999). Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol. Reprod. 60, 1429-1436. Nocka, K., Tan, J. C., Chiu, E., Chu, T. Y., Ray, P., Traktman, P. and Besmer, P. (1990). Molecular bases of dominant negative and loss of 37 v 41 function mutations at the murine c-kit/white spotting locus: W , W , W and W. EMBO J. 9, 1805-1813. Ogawa, T., Aréchaga, J. M., Avarbock, M. R. and Brinster, R. L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111-122. Ohta, H., Tohda, A. and Nishimune, Y. (2003). Proliferation and Development http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Loading next page...
 
/lp/the-company-of-biologists/spermatogenesis-from-epiblast-and-primordial-germ-cells-following-0gDqSrjO2t

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
The Company of Biologists
Copyright
© 2021 The Company of Biologists. All rights reserved.
ISSN
0950-1991
eISSN
0950-1991
DOI
10.1242/dev.01555
Publisher site
See Article on Publisher Site

Abstract

Research article 117 Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis 1 2 3 3 3 Shinichiro Chuma , Mito Kanatsu-Shinohara , Kimiko Inoue , Narumi Ogonuki , Hiromi Miki , 4 1 1 3 2, Shinya Toyokuni , Mihoko Hosokawa , Norio Nakatsuji , Atsuo Ogura and Takashi Shinohara * Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki 305-0074, Japan Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan *Author for correspondence (e-mail: takashi@mfour.med.kyoto-u.ac.jp) Accepted 27 October 2004 Development 132, 117-122 Published by The Company of Biologists 2005 doi:10.1242/dev.01555 Summary Primordial germ cells (PGCs) are derived from a Thus, fetal male germ cell development is remarkably population of pluripotent epiblast cells in mice. However, flexible, and the maturation process, from epiblast cells little is known about when and how PGCs acquire the through PGCs to postnatal spermatogonia, can occur in the capacity to differentiate into functional germ cells, while postnatal testicular environment. Primordial germ cell keeping the potential to derive pluripotent embryonic germ transplantation techniques will also provide a novel tool to cells and teratocarcinomas. In this investigation, we show assess the developmental potential of PGCs, such as those that epiblast cells and PGCs can establish colonies of manipulated in vitro or recovered from embryos harboring spermatogenesis after transfer into postnatal seminiferous lethal mutations. tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination Key words: Germ cell, Epiblast, Primordial Germ Cell (PGC), Pro- using spermatozoa or spermatids derived from PGCs spermatogonia, Spermatogonia, Testis, Spermatogenesis, harvested from fetuses as early as 8.5 days post coitum. Transplantation, Microinsemination Introduction 2001; Hajkova et al., 2002). Therefore, the characteristics of PGCs change during development before they mature into Mammalian germ cells undergo unique genetic and cellular postnatal germ cells. changes as they develop and differentiate to form functional Spermatogenesis is initiated shortly after birth (Russell et al., gametes. A population of pluripotent epiblast cells at around 1990; Meistrich and van Beek, 1993). Pro-spermatogonia 6.5 days post coitum (dpc) gives rise to primordial germ cells resume mitosis as spermatogonia, at around postnatal day 5, (PGCs), which become identifiable as a cluster of cells at the then enter into meiosis as spermatocytes and produce base of the allantois at 7.25 dpc (Ginsburg et al., 1990; Tam spermatids, which develop into spermatozoa. Spermatogonial and Zhou, 1996; Tsang et al., 2001). During development, the stem cells are a subpopulation of spermatogonia and have the number of PGCs increases from 40 cells at 7.5 dpc to 25,000 unique ability to self-renew as well as to differentiate to cells at 13.5 dpc, and they migrate through the developing produce spermatozoa (Meistrich and van Beek, 1993; de Rooij hindgut and mesentery to reach the urogenital ridge (UGR) at and Russell, 2000). These cells continue to divide throughout around 10.5 dpc. By 13.5 dpc, PGCs in the male genital ridge the life of the animal, and can be identified by their ability to enter into mitotic arrest and become pro-spermatogonia, while generate and maintain colonies of spermatogenesis following germ cells in the female arrest at meiotic prophase I (reviewed transplantation into the seminiferous tubules of infertile by McLaren, 2003). Primordial germ cells show different recipient testes (Brinster and Zimmermann, 1994). Using this features at different developmental stages. For example, assay, several groups have shown that pro-spermatogonia in migratory-stage PGCs exhibit a higher frequency of conversion developing fetal testes can differentiate into spermatogonial into embryonic germ cells, pluripotent cells that resemble stem cells when transferred into the adult testis (Ohta et al., blastocyst-derived embryonic stem cells, than do PGCs in the 2004; Jiang and Short, 1998). However, it is unknown if gonads (Matsui et al., 1992; Resnick et al., 1992; Labosky et germline cells at earlier stages of development can produce al., 1994). In addition, epigenetic changes characteristic to germline cells also occur in PGCs. Erasure of parental genomic spermatogonial stem cells or spermatogenic colonies after transplantation. imprints on both paternal and maternal alleles in PGCs commences near the time of their settlement in the UGR at 10.5 In this investigation, we sought to determine the potential of dpc, and new imprints are imposed in pro-spermatogonia germline cells from earlier embryos to develop into before birth (Szabo and Mann, 1995; Ueda et al., 2000; Surani, spermatogonial stem cells, using immature recipient animals. Development 118 Development 132 (1) Research article Epiblast cells or PGCs were transplanted into infertile mouse testes fixed in 4% paraformaldehyde in PBS were stained with Rhodamine-conjugated Peanut agglutinin (PNA) (Vector, testes and examined for their ability to re-populate the Burlingame, CA) for acrosomes, and with Hoechst 33258 (Sigma, St seminiferous tubules. Louis, MO) for nuclei. Microinsemination Materials and methods Microinsemination was undertaken by intracytoplasmic injection into Collection of donor cells C57BL/6 × DBA/2 F1 oocytes (Kimura and Yanagimachi, 1995). Donor cells were collected from pregnant C57BL/6 mice that were Embryos that were constructed using spermatozoa or elongated maintained in a controlled environment with 12:12 light:dark cycles spermatids derived from 8.5 dpc or 12.5 dpc PGCs were transferred from 08.00 h to 20.00 h (SLC, Shizuoka, Japan). The day when a into the oviducts of pseudopregnant ICR females after 24 or 48 hours copulation plug was found was designated as 0.5 dpc. In some in culture, respectively. Live fetuses retrieved on day 19.5 were raised experiments, we used a transgenic mouse line C57BL/6 Tg14 (act- by lactating ICR foster mothers. EGFP) OsbY01 (designated Green) provided by Dr M. Okabe (Osaka Genotyping of offspring and bisulfite sequencing of University, Osaka, Japan) (Okabe et al., 1997). The spermatogonia, imprinted genes spermatocytes and round spermatids of these mice express the enhanced green fluorescent protein (EGFP) gene, which gradually PCR fragments of the Kit gene encompassing the W point mutation or decreases after meiosis. The sex of embryos was determined using the W mutation (Nocka et al., 1990; Hayashi et al., 1991) were genotyping based on the Ube1 PCR method (Chuma and Nakatsuji, amplified using genomic DNA from mice derived from PGC trans- 2001), and only male embryos were used in this study. Due to the plantation, or from a W/W mouse as a control heterozygote for both limitations of cell recovery and the number of animals that could be mutations. PCR primers were 5′-CATTTATCTCCTCGACAAC- injected per day, the sex check was not performed in experiments CTTCC-3′ and 5′-GCTGCTGGCTCACAATCATGGTTC-3′ for W using 6.5 dpc embryos. Cells for transplantation were obtained from genotyping, and 5′-AGATGGCAACTCGAGACTCACCTC-3′ and 5′- whole embryonic ectoderm with primitive endoderm at 6.5 dpc, or TGCCCCCACGCTTTGTTTTGCTAA-3′ for W genotyping. germ-cell-containing tissues by dissection of the posterior thirds of Amplified products were gel extracted and directly sequenced. 8.5 dpc embryos, the mesenteries and guts of 10.5 dpc embryos, the Bisulfite genomic sequencing of differentially methylated regions UGRs of 10.5 dpc embryos, and the genital ridges of 11.5, 12.5, 14.5 (DMRs) of the Igf2r and H19 imprinted genes was carried out as and 16.5 dpc embryos. Tissues from each developmental stage were described (Ueda et al., 2000; Lee et al., 2002; Lucifero et al., 2002). dissociated by enzymatic digestion using 0.25% trypsin with 1 mmol/l Briefly, genomic DNAs were isolated from the offspring derived from EDTA (Invitrogen, Carlsbad, CA) for 10 minutes. Cells were PGC transplantation, and treated with sodium bisulfite, which suspended in Dulbecco’s modified Eagle’s medium, supplemented as deaminates unmethylated cytosines to uracils, but does not affect 5- previously described (Ogawa et al., 1997). methylated cytosines. Polymerase chain reaction amplification of each DMR from bisulfite-treated genomic DNAs was carried out using Transplantation into recipient testes primer sets as described (Ueda et al., 2000; Lucifero et al., 2002), and v v v Donor cells were transplanted in histocompatible W/W or W /W mice DNA sequences were determined. (W mice, obtained from SLC, Shizuoka, Japan). Only 5- to 10-day- old male mice were used as recipients. W mutants lack endogenous spermatogenesis (Silvers, 1979), because of mutations in the Kit gene Results (Nocka et al., 1990; Hayashi et al., 1991). Recipient animals were Epiblasts with primitive endoderms (6.5 dpc) or tissues placed on ice to induce hypothermic anesthesia, and returned to their containing fetal germ cells were collected from different stages dams after surgery (Shinohara et al., 2001). Approximately 2 µl of cell of embryos (posterior third of 8.5 dpc embryos, mesenteries suspension were introduced into each testis by injection via the and guts of 10.5 dpc embryos, or gonads of 10.5 to 16.5 dpc efferent duct (Ogawa et al., 1997). embryos) (Fig. 1A). The cells were dissociated enzymatically Histological analysis and single cell suspensions were transplanted into the Three to four months after transplantation, the recipient testes were seminiferous tubules of recipient immature W mice. Although fixed in 10% neutral-buffered formalin (Wako Pure Chemical W mice have a very small number of spermatogonia (Ohta et Industries, Osaka, Japan) and processed for paraffin sectioning. al., 2003), spermatogenesis is arrested at the point of Sections were stained with hematoxylin and eosin. Two histological undifferentiated type A spermatogonia and no differentiating sections were prepared from the testes of each animal and viewed at germ cells are found due to defects in the Kit gene (Nocka et 400× magnification to determine the extent of spermatogenesis. The al., 1990; Hayashi et al., 1991) (Fig. 1B). Therefore, any numbers of tubule cross-sections with or without spermatogenesis spermatogenesis detected in the recipient testis must be derived (defined as the presence of multiple layers of germ cells in the from the donor cells. Although the concentration of cells seminiferous tubule) were recorded for one histological section from each testis. Meiosis was detected by immunofluorescence staining injected varied due to the more limited recovery of cells from using anti-synaptonemal complex protein 3 (SCP3) antibody (Chuma early-stage fetuses, the cell viability, or percentage of tubules and Nakatsuji, 2001) and Alexa 488-conjugated anti-rabbit filled with donor cells, was similar in all experiments. The immunoglobulin G antibody (Molecular Probes, Eugene, USA). recipient mice were sacrificed 3 to 4 months after Periodic acid Schiff (PAS) staining (Muto Pure Chemicals, Tokyo, transplantation, and the testes were examined histologically for Japan) was carried out to examine acrosome formation in spermatids. the presence of spermatogenesis. This time period represents In experiments using Green mice, recipient testes were recovered 10 three to four spermatogenic cycles in mice (Meistrich and van to 11 weeks after donor cell transplantation, and analyzed by Beek, 1993; de Rooij and Russell, 2000), which would allow observing EGFP signals under fluorescence microscopy. Donor cells sufficient time for the development of sperm from were identified specifically because host testis cells had no spermatogonial stem cells. endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied the entire circumference of the tubule and At least three experiments were performed using cells was at least 0.1 mm long (Nagano et al., 1999). Cryosections of the harvested from each stage of embryonic development, and the Development Differentiation potency of PGC 119 Fig. 1. Spermatogenesis and teratogenesis from fetal germ cells and epiblast cells. (A) Embryos at 6.5 and 8.5 dpc, a mid-part of 10.5 dpc embryo, and a male gonad and mesonephros at 12.5 dpc. Dotted lines demarcate regions used for transplantation. At 10.5 dpc, the urogenital ridges (asterisk) and mesentery with gut (arrow) were dissected separately. (B) A section of a W male testis (control recipient) stained with HE. Spermatogenesis is absent. (C) W mouse testis after transplantation of 8.5 dpc PGCs. Spermatozoa (arrow) are present in the center of the seminiferous tubule. (D) Anti-SCP3 immunostaining (green) of W testis after transplantation of 8.5 dpc PGCs, counterstained with Hoechst 33258 dye (blue). Inset, higher magnification view of the same sample. (E) Transplantation of epiblast cells at 6.5 dpc. Spermatocyte (arrow) and round spermatid (arrow head) were found. Inset shows acrosomes stained with PAS (red) in round spermatids. (F) W mouse testis transplanted with 8.5 dpc PGCs from Green mice embryos. Colonization of the recipient seminiferous tubule by EGFP (+) donor cells (green) was observed (arrow). (G) A section of the same testis as in (F), stained with Rhodamine-PNA (red) for acrosomes and with Hoechst 33258 dye (blue) for nuclei. Spermatogenenic colonies derived from EGFP (+) donor cells are present (arrow). (H) Higher magnification view of (G), showing spermatogonia (arrowhead) residing at the base of the seminiferous tubule, and elongated spermatids (arrow) with acrosomes (red), shedding the EGFP (+) cytoplasm. (I) Teratoma from epiblast cells at 6.5 dpc. Muscle, dermoid cyst and neuronal tissue are observed. (J) Spermatozoa (arrow) clustered around a Sertoli cell, released from a recipient testis of 8.5 dpc PGCs. (K) An offspring developed from an oocyte injected with a sperm derived from 8.5 dpc PGCs. (L) DNA sequences of the Kit gene around the W (left panels) and W (right panels) point mutations of an offspring derived from transplantation of 8.5 dpc PGCs (upper panels) and a W/W mouse as a control heterozygote (lower panels). Scale bars: 1 mm in F; 10 µm in H; 20 µm in J; 50 µm in others. Table 1. Donor cell colonization in W recipient mice Number of Number of % tubule cross- Number of Donor Number of transplanted testes with section with testes with age (dpc) recipient testes cells (×10 /testis) spermatogenesis (%) spermatogenesis* teratoma (%) 6.5 27 0.4±0.2 2 (7.4) 0.1±0.1 4 (14.8) 8.5 13 7.2±0.6 9 (69.2) 3.8±0.1 4 (30.8) 10.5 (mes+gut) 12 12.8±1.4 4 (33.3) 0.9±0.5 0 10.5 (UGR) 15 11.4±2.4 11 (73.3) 7.2±0.1 0 11.5 13 2.4±1.0 8 (61.5) 2.4±0.7 0 12.5 13 9.0±1.6 11 (84.6) 15.7±4.5 0 14.5 12 20 12 (100) 56.5±10.8 0 16.5 12 20 12 (100) 59.3±7.5 0 Values are mean±s.e.m. Results from at least three separate experiments. Testes were analyzed 3-4 months after transplantation. Mes+gut, mesentery and gut; UGR, urogenital ridge. *Percentage of tubule cross-sections containing spermatogenesis/total tubule cross-sections examined in each testis. results are summarized in Table 1. Overall, 69 of 117 (59%) injection of donor cells (Fig. 1B). Spermatogenesis observed recipient testes showed spermatogenesis, but no differentiating in the recipient testes originated from spermatogonial stem germ cells were found in control testes that did not receive an cells, because other spermatogenic cells do not have the Development 120 Development 132 (1) Research article Table 2. Development of oocytes injected with spermatogenic cells derived from PGCs In vitro development (%) Donor age Number of Number of Implantation Number of (dpc) reconstituted eggs One cell Two cell Four cell eggs transferred sites (%) offspring (%) 8.5* 186 78 (41.9) 105 (56.5) NA 105 47 (25.2) 20 (10.8) 12.5 182 8 (4.4) 25 (13.7) 130 (71.4) 130 88 (48.3) 29 (15.9) Combined results using elongated spermatids and spermatozoa. *Combined results from two different recipient testes. NA, not applicable because cells were transferred at the two-cell stage. capacity for self-renewal and disappear by 35 days after allowed specific identification of donor cells, because transplantation (Russell et al., 1990; Brinster and endogenous host testis cells had no detectable fluorescence. Zimmermann, 1994; Shinohara et al., 2001). Spermatogenesis Four experiments were performed, and a total of 16 testes were in the recipient testes that received fetal germ cells (8.5-16.5 microinjected with donor cells. Approximately 6 to 12 × 10 dpc) was morphologically normal (Fig. 1C), and the kinetics cells were transplanted into each testis. When the recipients of spermatogenic colonization were generally comparable to were analyzed 10 to 11 weeks after transplantation, 4 of 16 results obtained after transplantation of postnatal (25%) testes had spermatogenic colonies that showed EGFP spermatogonial stem cells (Shinohara et al., 2001). All stages signals (Fig. 1F). The average number of colonies per testis of spermatogenic cells, including mature spermatozoa, were was 0.3 ± 0.1 (mean ± s.e.m.). Histological analysis of the found in recipient testes. Synchrony of meiosis in seminiferous EGFP (+) colonies showed the presence of apparently normal tubules was confirmed by immunostaining for SCP3, a spermatogenesis (Fig. 1G,H). component of the synaptonemal complex (Fig. 1D). While these results indicate that all types of donor cells The age of the donors had a significant effect on the number differentiated into spermatogonial stem cells, differentiation of of recipient seminiferous tubules with observable donor cells was not restricted to the germline lineage. spermatogenesis. Whereas only 0 to 11% of the tubules showed Consistent with findings of previous studies (Illmensee and spermatogenesis in testes that received 8.5 dpc PGCs, Stevens, 1979), testes receiving epiblast cells or 8.5 dpc PGCs spermatogenesis derived from donor cells at later stages of formed teratomas (Fig. 1I). Epiblast cells produced larger gonadal development was generally more extensive, and tumors than did 8.5 dpc PGCs, and some of the seminiferous recipient testes grew larger due to the increased production of germ cells. In one case, transplantation of 16.5 dpc pro-spermatogonia resulted in 96% of the tubules with evident spermatogenesis, and spermatozoa were transported to the epididymis (data not shown). Spermatogenesis was also found in the recipients that were injected with cells from epiblasts with primitive endoderms. Although we could not identify mature spermatozoa histologically, spermatogenesis was found in two different testes that received injections of 6.5 dpc epiblast and primitive endoderm cells. The spermatogonia, spermatocytes and round spermatids that developed in the recipient seminiferous tubules appeared morphologically normal (Fig. 1E), and acrosome formation in round spermatids was confirmed by PAS staining (Fig. 1E, inset). To confirm the donor origin of spermatogenesis, we used Green mice that ubiquitously express the EGFP transgene. Donor cells were collected from the posterior thirds of 8.5 dpc embryos that showed EGFP signals. This Fig. 2. DMR methylation of the Igf2r and H19 genes in offspring derived from transplantation of 8.5 and 12.5 dpc PGCs. DNA methylation was analyzed by bisulfite genomic sequencing. Both male and female offspring of each stage of donor PGCs were analyzed. Individual lines represent sequenced clones. Black ovals indicate methylated cytosine-guanine sites (CpGs) and white ovals indicate unmethylated CpGs. Development Differentiation potency of PGC 121 tubules were dilated and broken. Cells from three germ layers, tubules, while spermatogenesis did not occur from PGCs from including ciliated epithelium, muscle, neuron and bone, were 12.5 dpc embryos. Another group reported that germ cells from found in these testes. Interestingly, spermatogenesis was day 0-3 mouse pups did not show spermatogenic colonies after occasionally observed in tubules not affected by tumorigenesis, transplantation into adult testes (McLean et al., 2003). In our indicating that the same population of donor cells could cause study, however, epiblast cells at 6.5 dpc and PGCs at 8.5 to 16.5 spermatogenesis and teratogenesis. No teratomas were found dpc produced spermatogenesis after transplantation into in the recipients of gonadal PGCs or pro-spermatogonia. immature seminiferous tubules at postnatal day 5 to 10. This To examine whether germ cells generated from PGCs were difference might simply be ascribed to structural differences; fully functional, we performed microinsemination, a technique immature Sertoli cells lack tight junctions and may allow commonly used to produce offspring from infertile animals and migrating transplanted cells easier access to the stem cell humans (Kimura and Yanagimachi, 1995; Palermo et al., niches, which are distributed nonrandomly in the seminiferous 1992). Donor PGCs were collected from 8.5 or 12.5 dpc tubules (Chiarini-Garcia et al., 2001). Alternatively, immature embryos, and transplanted into the testes of W mice. Four testis may express factors that support survival and months after transplantation, spermatozoa or elongated differentiation of epiblast cells and PGCs, while mature testis spermatids were collected from tubule fragments by may not. Because PGCs have chemotaxic activity (Godin et al., mechanical dissociation (Fig. 1J), and microinjected into 1990), we speculate that some of the transplanted cells oocytes. The results of the microinsemination experiments are migrated into the niches of immature seminiferous tubules, summarized in Table 2. Spermatogenic cells derived from 8.5 where they could survive. PGCs and epiblast cells may have dpc PGCs appeared to be less competent for egg activation, then switched their cell cycle fate to function as because a significant number of eggs receiving elongated spermatogonial stem cells. Both male and female PGCs enter spermatid/spermatozoa from 8.5 dpc PGCs stayed at into meiosis in the absence of the male gonadal environment, metaphase II or the metaphase II-anaphase transition and did but do not if they lodge there (McLaren and Southee, 1997; not develop into the 2-cell stage after 24 hours. However, Chuma and Nakatsuji, 2001). Somatic cells in the fetal testis offspring were obtained from oocytes inseminated with are assumed to produce a substance that inhibits the meiotic spermatid/spermatozoa derived from both stages of PGCs (Fig. transition of PGCs. Given our results, it appears that 1K). Genotyping of offspring showed that they did not carry seminiferous tubules of newborn mice may also have similar W or W mutations in the Kit gene (Nocka et al., 1990; Hayashi meiosis-inhibiting activity. et al., 1991), demonstrating the offspring were derived from Erasure of parental genomic imprints commences in PGCs wild-type donor PGCs that had been transplanted into the testis at around the time of their arrival in the UGR (Szabo and Mann, (Fig. 1L). No apparent abnormality was seen in any of the 1995; Surani, 2001; Hajkova et al., 2002). However, it has not offspring, and they were fertile. Bisulfite sequencing analysis been clear whether this epigenetic event depends on induction of the offspring showed no obvious fluctuations in the from the UGR or is programmed autonomously in PGCs. As methylation status of the DMRs of paternally methylated H19 the offspring from PGC transplantations were viable and and maternally methylated Igf2r genes (Fig. 2). apparently healthy, epigenetic modifications, including erasure of parental genomic imprints, should have occurred appropriately in transplanted PGCs. This was corroborated by Discussion the normal methylation patterns exhibited in the DMRs of the This study demonstrates that not only migrating PGCs but also Igf2r and H19 genes. Because the establishment of paternal epiblast cells can differentiate into spermatogonial stem cells methylation proceeds after birth in the normal testis (Ueda et and produce spermatogenesis after transfer into postnatal testis. al., 2000), it seems unlikely that the postnatal testis has the The ability to initiate and maintain spermatogenesis after ability to erase parental methylation. Therefore, our results transfer into the infertile testis fulfills the criteria for the suggest that PGCs may have the autonomous program for the identification of spermatogonial stem cells (Brinster and erasure of parental methylation before reaching the UGR. Zimmermann, 1994). Developmental processes that occur Primordial germ cell transplantation will provide a new during differentiation of spermatogonial stem cells from experimental approach for the study of PGC development. epiblast cells include induction of PGCs among epiblast cells, Primordial germ cells harvested from embryonic lethal mutants migration and proliferation of PGCs, erasure of parental as early as gastrulation can be traced for their differentiation genomic imprints, and G1 (G0) arrest in the developing male capacities by transplantation into recipient testis. Similarly, gonad. Nonetheless, our results demonstrate that these PGCs manipulated in vitro, such as those cultured with growth developmental events do not necessarily require embryonic factors or transfected with vectors (De Miguel et al., 2002; somatic environments, and most processes of male germline Watanabe et al., 1997), can now be assessed for their effects differentiation can take place in postnatal testis. This flexibility on subsequent differentiation in vivo. Such functional studies of fetal germ cell differentiation may be related to, and partly would help elucidate factors that regulate male germline account for, the recent success in the derivation of sperm from development in mammals. embryonic stem cells in vitro (Toyooka et al., 2003; Geijsen et al., 2004). We thank Dr Masaru Okabe for kindly providing C57BL/6 Tg14 The important factor that contributed to the results of our (act-EGFP) OsbY01 mice, Dr Fumitoshi Ishino for helpful advice on experiments is the use of immature postnatal testes as bisulfite sequencing, and Drs Yasuhisa Matsui and Yoshito Kaziro for recipients. Recently, Ohta et al. (Ohta et al., 2004) showed that critical reading of the manuscript. This research was supported by a pro-spermatogonia from 14.5 dpc embryos completed grant from the Japanese Ministry of Education, Science, Sports and spermatogenesis when transplanted in mature seminiferous Culture. Development 122 Development 132 (1) Research article References differentiation of spermatogonial stem cells in the W/W mutant mouse testis. Biol. Reprod. 69, 1815-1821. Brinster, R. L. and Zimmermann, J. W. (1994). Spermatogenesis following Ohta, H., Wakayama, T. and Nishimune, Y. (2004). Commitment of fetal male germ-cell transplantation. Proc. Natl. Acad. Sci. USA 91, 11298- male germ cells to spermatogonial stem cells during mouse embryonic development. Biol. Reprod. 70, 1286-1291. Chiarini-Garcia, H., Hornick, J. R., Griswold, M. D. and Russell, L. D. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y. (2001). Distribution of type A spermatogonia in the mouse is not random. (1997). ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, Biol. Reprod. 65, 1179-1185. 313-319. Chuma, S. and Nakatsuji, N. (2001). Autonomous transition into meiosis of Palermo, G., Joris, H., Devroey, P. and van Steirteghem, A. C. (1992). mouse fetal germ cells in vitro and its inhibition by gp130-mediated Pregnancies after intracytoplasmic injection of single spermatozoon into an signaling. Dev. Biol. 229, 468-479. oocyte. Lancet 340, 17-18. De Miguel, M. T., Cheng, L., Holland, E. C., Federspiel, M. J. and Resnick, J. L., Bixler, L. S., Cheng, L. and Donovan, P. J. (1992). Long- Donovan, P. J. (2002). Dissection of the c-kit signaling patheway in mouse term proliferation of mouse primordial germ cells in culture. Nature 359, primordial germ cells by retroviral-mediated gene transfer. Proc. Natl. Acad. 550-551. Sci. USA 99, 10458-10463. Russell, L. D., Ettlin, R. A., Sinha Hikim, A. P. and Clegg, E. D. (1990). de Rooij, D. G. and Russell, L. D. (2000). All you wanted to know about Histological and Histopathological Evaluation of the Testis (ed. L. D. spermatogonia but were afraid to ask. J. Androl. 21, 776-798. Russell, R. A. Ettlin, A. P. Sinha Hikim and E. D. Clegg), pp. 1-40. Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K. and Daley, Clearwater, FL: Cache River Press. G. Q. (2004). Derivation of embryonic germ cells and male gametes from Shinohara, T., Orwig, K. E., Avarbock, M. R. and Brinster, R. L. (2001). embryonic stem cells. Nature 427, 148-154. Remodeling of the postnatal mouse testis is accompanied by dramatic Ginsburg, M., Snow, M. H. and McLaren, A. (1990). Primordial germ cells changes in stem cell number and niche accessibility. Proc. Natl. Acad. Sci. in the mouse embryo during gastrulation. Development 110, 521-528. USA 98, 6186-6191. Godin, I., Wylie, C. C. and Heasman, J. (1990). Genital ridges exert long- Silvers, W. K. (ed.) (1979). The Coat Colors of Mice, pp. 206-223. New York, range effects on mouse primordial germ cell numbers and direction of NY: Springer. migration in culture. Development 108, 357-363. Surani, M. A. (2001). Reprogramming of genome function through epigenetic Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., inheritance. Nature 414, 122-128. Walter, J. and Surani, M. A. (2002). Epigenetic reprogramming in mouse Szabo, P. E. and Mann, J. R. (1995). Biallelic expression of imprinted genes primordial germ cells. Mech. Dev. 117, 15-23. in the mouse germ line: implications for erasure, establishment, and Hayashi, S., Kunisada, T., Ogawa, M., Yamaguchi, K. and Nishikawa, S. mechanism of genomic imprinting. Genes Dev. 9, 1857-1868. (1991). Exon skipping by mutation of an authentic splice site of c-kit gene Tam, P. P. and Zhou, S. X. (1996). The allocation of epiblast cells to in W/W mouse. Nucleic Acids Res. 19, 1267-1271. ectodermal and germ-line lineages is influenced by the position of the cells Illmensee, K. and Stevens, L. C. (1979). Teratomas and chimeras. Sci. Am. in the gastrulating mouse embryo. Dev. Biol. 178, 124-132. 240, 120-133. Tsang, T. E., Khoo, P. L., Jamieson, R. V., Zhou, S. X., Ang, S. L., Jiang, F.-X. and Short, R. V. (1998). Different fate of primordial germ cells Behringer, R. and Tam, P. P. (2001). The allocation and differentiation of and gonocytes following transplantation. APMIS 106, 58-63. mouse primordial germ cells. Int. J. Dev. Biol. 45, 549-555. Kimura, Y. and Yanagimachi, R. (1995). Mouse oocytes injected with Toyooka, Y., Tsunekawa, N., Akasu, R. and Noce, T. (2003). Embryonic testicular spermatozoa or round spermatids can develop into normal stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci. USA 100, 11457-11462. offspring. Development 121, 2397-2405. Ueda, T., Abe, K., Miura, A., Yuzuriha, M., Zubair, M., Noguchi, M., Labosky, P. A., Barlow, D. P. and Hogan, B. L. M. (1994). Mouse embryonic Niwa, K., Kawase, Y., Kono, T., Matsuda, Y. et al. (2000). The paternal germ (EG) cell lines: transmission through the germ line and differences in methylation imprint of the mouse H19 locus is acquired in the gonocyte the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene stage during foetal testis development. Genes Cells 5, 649-659. compared with embryonic stem (ES) cell lines. Development 120, 3197- Watanabe, M., Shirayoshi, Y., Koshimizu, U., Hashimoto, S., Yonehara, S., Eguchi, Y., Tsujimoto, Y. and Nakatsuji, N. (1997). Gene transfection Lee, J., Inoue, K., Ono, R., Ogonuki, N., Kohda, T., Kaneko-Ishino, T., of mouse primordial germ cells in vitro and analysis of their survival and Ogura, A. and Ishino, F. (2002). Erasing genomic imprinting memory in growth control. Exp. Cell Res. 230, 76-83. mouse clone embryos produced from day 11.5 primordial germ cells. Development 129, 1807-1817. Lucifero, D., Mertineit, C., Clarke, H. J., Bestor, T. H. and Tasler, J. M. (2002). Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79, 530-538. Matsui, Y., Zsebo, K. and Hogan, B. L. M. (1992). Derivation of pluripotent embryonic cells from murine primordial germ cells in culture. Cell 70, 841- McLaren, A. (2003). Primordial germ cells in the mouse. Dev. Biol. 262, 1- McLaren, A. and Southee, D. (1997). Entry of mouse embryonic germ cells into meiosis. Dev. Biol. 187, 107-113. McLean, D. J., Friel, P. J., Johnston, D. S. and Griswold, M. D. (2003). Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol. Reprod. 69, 2085-2091. Meistrich, M. L. and van Beek, M. E. A. B. (1993). Spermatogonial stem cells. In Cell and Molecular Biology of the Testis (ed. C. Desjardins and L. L. Ewing), pp. 266-295. New York, NY: Oxford University Press. Nagano, M., Avarbock, M. R. and Brinster, R. L. (1999). Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol. Reprod. 60, 1429-1436. Nocka, K., Tan, J. C., Chiu, E., Chu, T. Y., Ray, P., Traktman, P. and Besmer, P. (1990). Molecular bases of dominant negative and loss of 37 v 41 function mutations at the murine c-kit/white spotting locus: W , W , W and W. EMBO J. 9, 1805-1813. Ogawa, T., Aréchaga, J. M., Avarbock, M. R. and Brinster, R. L. (1997). Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111-122. Ohta, H., Tohda, A. and Nishimune, Y. (2003). Proliferation and Development

Journal

DevelopmentThe Company of Biologists

Published: Jan 1, 2005

References