Animal Care and Mating

All mice were maintained under specific-pathogen-free conditions at the Bioresource Center (RIKEN, Japan). They were housed under controlled lighting conditions (daily light period, 0700 h to 2100 h). Female mice were mated with males overnight and were checked for vaginal plugs the next morning (0.5 day postcoitum [dpc] = noon of the day when a vaginal plug was detected). All animal experiments were conducted in accordance with the guidelines of the RIKEN Tsukuba Institute.

Transplantation of PGCs Reaggregated with Gonadal Somatic Cells Under the Kidney Capsule

Gonadal cell reaggregation and transplantation were performed according to the protocol described by Qing et al. [13], with minor modifications. Briefly, C57BL/6NCrSlc (B6) females (Japan SLC, Shizuoka, Japan) were mated with green fluorescent protein (GFP)-expressing transgenic C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb males [official symbol, Tg(CAG-EGFP)131Osb] or DBA/2CrSlc males (Japan SLC) to obtain B6-GFP or B6D2F1 (BDF1) embryos, respectively.

Genital ridges (gonads with adjacent mesonephros) were collected from 12.5-dpc embryos, and each gonad was separated from the mesonephros by using a dissecting microscope and sharp needles. The sex of the isolated gonad was determined by its appearance. Four gonads of the same sex were mixed in a 1.5-ml centrifuge tube and dispersed in a 100-μl solution of 0.25% trypsin plus 0.2% collagenase IV at 37°C for 15 min, followed by pipetting 5–10 times. After the addition of 900 μl of culture medium 199 (Sigma, Tokyo, Japan) containing 10% fetal bovine serum (Biowest, France) and 1% antibiotics (penicillin-streptomycin; Invitrogen, Tokyo, Japan), cells were resuspended in 100 μl of culture medium. After the addition of phytohemagglutinin-P (final concentration, 35 μg/ml; Sigma), cell suspensions were incubated at 37°C for 10 min. Subsequently, the suspensions were centrifuged twice at 300 × g for 4 min to make reaggregated pellets as grafts. The reaggregated grafts were gently picked up with a truncated 200-μl micropipette, placed on isopore membrane filters (pore size, 3.0 μm; Millipore, Bedford, MA) in culture medium, and cultured for 12–18 h at 37°C. Finally, grafts were transplanted under the kidney capsules of bilaterally ovariectomized recipient female mice of matched strains. The number of transplants per single kidney was one or two.

Microinjection of Round Spermatids Derived from Transplanted PGCs

Four weeks after transplantation, the grafts that had been reconstructed by using male gonads were removed from the kidney capsules of recipient mice and dissociated mechanically, as described for intact mouse testes [14]. Briefly, the grafts were placed in erythrocyte lysing buffer (155 mM NH₄Cl, 10 mM KHCO₃, 2 mM ethylenediamine tetraacetic acid, pH 7.2) at 4°C, the presumed tunica albuginea was removed, and the seminiferous tubule masses were transferred into cold (4°C) phosphate-buffered saline (PBS) supplemented with 5.6 mM glucose, 5.4 mM sodium lactate, and 0.1 mg/ml polyvinyl alcohol (GL-PBS). The tubules were cut into small pieces and pipetted gently to disperse cells into the GL-PBS. Subsequently, the cell suspension was filtered through a 38-μm nylon mesh, washed twice by centrifugation (300 × g for 4 min), and resuspended in 50 μl of GL-PBS. We found presumptive round spermatids in these suspensions but not elongated spermatids or spermatozoa. Therefore, in the next series of experiments, we performed round spermatid injection (ROSI), as described below.

To collect oocytes for ROSI, 7- to 10-wk-old female BDF1 mice (Japan SLC) were superovulated via injections of equine chorionic gonadotropin (7.5 IU and 2.5 IU), followed by injections of human chorionic gonadotropin (hCG; 7.5 IU and 2.5 IU) 48 h later. Mature metaphase II (MII) oocytes were collected from the oviducts 15–17 h after hCG injection. Cumulus cells were released by treating cultures with 0.1% hyaluronidase in potassium-modified simplex optimization medium (KSOM) [15]. The oocytes were transferred to fresh KSOM and incubated at 37°C in an atmosphere of 5% CO₂ in air for up to 90 min before ROSI.

ROSI was performed with a piezo-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan) [16]. The cover of a plastic dish (Falcon product no. 1006; Becton Dickinson, Franklin Lakes, NJ) was used as a microinjection chamber. Several small drops (∼4 μl) of bovine serum albumin (BSA)-free HEPES-buffered KSOM (Hepes-KSOM), with or without 10% polyvinylpyrrolidone (PVP), were placed on the bottom of the plate and covered with mineral oil. Graft-derived cells were placed in one of the PVP droplets. Before oocytes were injected with the nuclei of round spermatids, they were activated by treatment with Ca²⁺-free KSOM containing 2.5 mM SrCl₂ for 20 min at 37°C. Oocytes that had reached telophase II at 40–90 min after the onset of activation treatment were each injected with a round spermatid. They were kept in Hepes-KSOM at room temperature (24°C) for 10 min before culture in KSOM at 37°C under 5% CO₂ in air.

In Vitro Maturation of GV Oocytes Derived from Transplanted PGCs and ICSI

Grafts reconstructed with female gonads were picked up from recipient kidneys 4 wk after transplantation. Grafts containing several follicles were mechanically processed to isolate GV oocytes and then subjected to in vitro maturation (IVM), as described previously [17]. Briefly, after antral follicles in the grafts were punctured with sharp needles in a Hepes-buffered 1:1 mixture of TYH (119.3 mM NaCl, 4.78 mM KCl, 1.26 mM CaCl₂H₂O, 1.19 mM KH₂PO₄, 25.07 mM NaHCO₃, 5.56 mM glucose, 1 mM pyruvic acid, 4 mg/ml BSA, 5 mg/100 ml streptomycin, 7.5 mg/100 ml penicillin G) and minimal essential medium alpha (TaM) (Invitrogen), the GV oocytes were collected using a filtered mouth pipette and subjected to IVM for 15–18 h in TaM at 37°C under 5% CO₂ in air. Oocytes that reached the MII stage via IVM were subjected to ICSI, as the zona pellucida of graft-derived oocytes was hardened during IVM and fertilization was not possible using conventional in vitro fertilization (data not shown).

ICSI was performed using freeze-thawed sperm from a B6 or Japanese Fancy 1 (JF1/Msf) strain. Frozen sperm (kept for several months in a deep freezer at −80°C) suspended in human tubal fluid medium were thawed by a 3-min incubation in a water bath at 37°C and were stored at 4°C for up to 120 min. The thawed sperm were placed in one of the PVP droplets on the microinjection chamber. The head of a single spermatozoon was separated from its tail by applying a few piezo pulses to the head–tail junction. The isolated sperm head was injected into an oocyte derived from the grafts stored in Hepes-buffered KSOM. Manipulated oocytes were kept at room temperature for 10 min before culturing in KSOM at 37°C under 5% CO₂ in air.

Embryo Transfer and Delivery of Offspring

Micromanipulated embryos that reached the two-cell stage after 24 h of culture in KSOM were transferred into the oviducts of Day-1 pseudopregnant ICR mice (CLEA Japan, Tokyo, Japan). On Day 20, the recipient female mice were examined for the presence of fetuses, and live pups were nursed by lactating ICR foster mothers.

Histological Analyses

Some of the transplanted grafts removed on Day 28 or 56 after transplantation were fixed overnight in 4% paraformaldehyde in PBS or Bouin solution and were routinely embedded in paraffin. Embedded samples were sectioned (4 μm) and subjected to hematoxylin-eosin (HE) staining and immunohistochemistry. For immunohistochemistry, rehydrated sections were pretreated in a microwave for 10 min in the presence of 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were then blocked for 1 h at room temperature in 0.5% blocking reagent (Perkin-Elmer Japan, Yokohama, Japan). Primary antibody incubations were carried out overnight at 4°C in blocking solution, using rabbit anti-mouse Vasa homologue (MVH) protein antibody (1:5000; a gift from Dr. Toshiaki Noce) [18], rabbit anti-heat-shock protein 1-like (HSPA1L)/Hsc70t antibody (1:2000; a gift from Dr. Hirokazu Fujimoto) [19], and rabbit anti-synaptonemal complex protein 3 (SYCP3) antibody (1:1000; Ab code 15092; Abcam, Japan). The immunoreactions of each primary antibody were visualized using biotin-labeled secondary antibodies and a Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA).

Measurement of Serum Hormone Levels

Blood samples were collected from anesthetized host mice at 4 wk after transplantation, using cardiac puncture, and then centrifuged to separate the serum. Serum testosterone levels were determined using an enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer's instructions. For quantification of serum estradiol levels, samples were applied to a liquid chromatography-tandem mass spectrometry apparatus (ASKA Pharma Medical Co., Ltd., Kawasaki, Japan). Age- and sex-matched nontransplanted animals with or without ovariectomy were used as controls. Data were analyzed statistically using a Student t test, and significance was set at a P value of <0.05.

Gonadal Cells Formed Testis- or Ovary-Like Tissue Structures Under the Kidney Capsule

We transplanted 12.5-dpc PGCs, which had just initiated sex-dependent differentiation, ectopically, together with age-matched somatic cells, to examine the extent to which they could undergo normal development. Before transplantation, four 12.5-dpc gonads were completely dissociated to single cells by enzymatic treatment (Fig. 1C). The mean number of cells collected from a single male and female gonad was 2.90 × 10⁵ ± 0.07 × 10⁵ SEM (including 1.00 × 10⁵ ± 0.09 × 10⁵ PGCs) and 1.84 × 10⁵ ± 0.08 × 10⁵ (including 6.24 × 10⁴ ± 0.07 × 10⁴ PGCs), respectively (n = 4 gonads). Cells were reaggregated overnight as a single graft and transplanted under the kidney capsules of bilaterally ovariectomized female mice (Fig. 1A). In the first series of experiments, we used gonads from B6-GFP fetuses to distinguish transplanted reaggregated tissues from recipient host cells. Male and female reaggregated grafts developed testis- and ovary-like tissues, respectively, within 4 wk after transplantation (Fig. 1D and E). Similar tissue regeneration and development activities were observed with BDF1-derived donor cells for both sexes (Fig. 1F). After this time point, however, the male testis-like tissues became swollen and vacuolar, whereas the female ovary-like structures were atrophic in appearance (Fig. 2). Teratoma formation was not observed in host animals at any time point analyzed. These results indicate that embryonic gonadal cells of both sexes reconstructed the gonadal structure ectopically under the kidney capsule and that their maximum development was reached at around 4 wk after transplantation. In some experiments, we reaggregated gonadal cells from different donor strains (e.g., GFP-expressing and non-GFP-expressing animals) and confirmed chimeric distribution in the reconstructed tissues (Fig. 1G).

FIG. 1.

Transplantation of PGCs with gonadal somatic cells under the kidney capsule. A) Schematic shows the transplantation procedure used in this study. Two pairs of sex-matched gonads at 12.5 dpc were dissociated, reaggregated, and transplanted under the kidney capsules of ovariectomized female recipient mice. B) Appearance of male and female gonads (g) with adjacent mesonephros (m) of 12.5-dpc embryos is shown. C) Microscopic appearance of gonadal dissociates of 12.5-dpc female gonads. Arrows and inset indicate PGCs with their characteristic lamellipodia. D–F) Gross appearance of grafts at 4 wk after transplantation under the kidney (kid) capsule is shown. Male and female grafts formed testis-like (white arrowheads) and ovary-like (black arrowheads) tissues, respectively, in both B6-GFP (D and E) and BDF1 (F) strains. G) A testis-like tissue generated by mixing gonadal cells from different strains (GFP and non-GFP mice). The chimeric composition of the entire tissue was confirmed by GFP fluorescence in the gross appearance (upper right panel) and in a histological section (lower panel). Bar = 100 μm.

FIG. 2.

Male (A–F) and female (G–I) grafts collected at different time points (>4 wk after transplantation) are shown. A) Gross appearance of a testis-like tissue (white arrowhead) from BDF1 mice at 8 wk posttransplantation. The size of this structure was much larger than that observed at 4 wk (see Fig. 1F). As shown in an HE-stained section (B), the enlargement was caused by extraordinarily swollen seminiferous tubules that contained fluid (FC). In the periphery, some seminiferous tubules maintained their normal epithelial structure and underwent spermatogenesis, even at 8 wk after transplantation (C), as shown by positive staining for DDX4 (D), SYCP3 (E), and HSPAL1 (F). (G and H) An ovary-like tissue from BDF1 mice shows a hemorrhagic appearance at 5 wk after transplantation. In addition to a few large blood-containing follicles (or corpuses; asterisks), many follicles exhibited a red spot (arrows), indicating previous ovulation. Although a few oocytes were collected from the remaining follicles, they were completely degenerated (I). Bar = 100 μm.

Male PGCs from BDF1 Mice, but Not from B6-GFP Mice, Completed Meiosis Under the Kidney Capsule

Next, we histologically examined the testis-like tissues reconstructed in males. The HE-stained sections largely resembled those of normal adult testes, containing many seminiferous tubules and mesenchymal cells that filled the interstitial spaces (Fig. 3A–H). However, we found that the development of germ cells within the seminiferous tubules was greatly affected by the genotype of the donor cells (Fig. 3A–H). The seminiferous tubules derived from B6 donor cells contained only a few cell layers (Fig. 3A), whereas those from BDF1 donor cells formed thick epithelia with several cell layers (Fig. 3E). For precise identification of germ cells, we stained the sections by using an antibody to MVH/DDX4, a marker of spermatogonia-to-round spermatid stages [18]. As expected, the thick epithelia of BDF1-derived tissues were strongly stained for DDX4; in contrast, those of B6-GFP-derived tissues showed no positively stained cells, except for the occasional presence of presumptive spermatogonia (Fig. 3B). Furthermore, the seminiferous tubules from BDF1 tissues were positive for the SYCP3 protein (a marker of meiotic cells) and for the HSPA1L/Hsc70t protein (a marker of elongated spermatids), suggesting the completion of meiosis in these testis-like tissues. We found presumptive round spermatids with characteristically small nuclei and nucleolus-like structures in HE-stained sections of BDF1 tissues (Fig. 3I). In the seminiferous tubules containing germ cells, the mean frequency of round spermatids was 0.33% ± 0.09% SEM (n = 4) of the germ cell population. At 8 wk after transplantation, the testis-like tissues were occupied mostly by large vacuoles originating from extraordinarily swollen seminiferous tubules. However, there were several seminiferous tubules containing round spermatids in the peripheral areas of these testis-like tissues, similar to those observed at 4 wk, indicating that spermatogenic waves continued in the reconstructed tissues from under the kidney capsule for at least 8 wk (Fig. 2).

FIG. 3.

Histological views of testis-like tissues at 4 wk after transplantation and collection of round spermatids are shown. B6-GFP-derived (A–D) and BDF1-derived (E–H) tissues were very different regarding their spermatogenic ability. As shown by HE staining (A and E) and immunostaining for DDX4 (B and F; wide-range germ cell marker from spermatogonia to spermatids), SYCP3 (C and G; meiotic cell marker), and HSPA1L (D and H; elongated spermatid marker), only a few presumptive spermatogonia survived in B6-GFP-derived tissues (B, arrowheads), whereas BDF1-derived tissues contained thick spermatogenic layers with postmeiotic spermatogenic cells. Presumptive spermatids were observed in an HE-stained section (I, arrowheads and inset) and dissociated suspension (J, arrowheads and inset) of BDF1-derived tissues. K) Newborn offspring produced by ROSI using these presumptive round spermatids from BDF1 grafts. Bars = 100 μm.

Production of Normal Pups from Round Spermatids Derived from Transplanted PGCs

To examine the developmental potential of presumptive spermatids in BDF1 testis-like tissues, we performed ROSI into in vivo-derived BDF1 oocytes. We mechanically dissociated the testis-like tissues at 4 wk after transplantation and observed the isolated cells with an inverted microscope. Using differential interference contrast microscopy, we identified normal appearing round spermatids with characteristically round and small nuclei and nucleolus-like structures but not elongated spermatids in the cell suspension (Fig. 3J). In four replicated ROSI experiments, the majority (84.6%) of oocytes survived injection, and 74% of oocytes developed to the two-cell stage on the following day (Table 1). After the transfer of 135 two-cell embryos into pseudopregnant female mice, eight pups (three males and five females [5.9% per transfer]) were born at term (Fig. 3K and Table 1). One pup died within a day, but the remaining seven pups grew to adulthood and showed a variety of coat colors, as expected from the BDF1 × BDF1 mating (black, brown, diluted black, and diluted brown), and normal fertility when mated with mice of the opposite sex.

TABLE 1.

In vitro maturation of oocytes and development of embryos after microinsemination with PGC-derived male and female gametes.

Female PGCs Developed into GV Oocytes Under the Kidney Capsule and Matured In Vitro

We also undertook histological examination of the ovary-like tissues derived from female 12.5-dpc gonadal cells. At 4 wk after transplantation, an ovarian mass from each of the B6 and the BDF1 strains contained many antral follicles (Fig. 4A and E). Histologically, there seemed to be no distinct boundary between the cortex and the medulla, and large antral follicles were also observed in the central area of the tissue. Interestingly, we found several polyovular follicles (containing more than one oocyte in a single follicle) in both strains (Fig. 4A and E, asterisks). Mechanical dissociation of the ovary-like tissues enabled the collection of a mean of 20 ± 14.2 SEM (n = 8) and of 28.4 ± 9.7 (n = 11) GV oocytes from B6-GFP- and BDF1-derived tissues, respectively (total, 165 and 312, respectively [Fig. 4B and F]). These oocytes were fully grown, with diameters of 65–70 μm. After undergoing IVM for 15–18 h, >50% of oocytes were extruded from the first polar body and reached MII, regardless of genotype (Fig. 4C and G and Table 1). At 5 wk after transplantation, ovary-like tissues showed a hemorrhagic appearance, with many follicles exhibiting a red spot, indicative of previous ovulation (Fig. 2G and H). Although a few oocytes were collected from the remaining follicles, these cells were completely degenerated (Fig. 2I). This finding suggests that only a single wave of oogenesis occurred in the reconstructed ovary-like tissues.

FIG. 4.

Histological observation of ovary-like tissues at 4 wk after transplantation and collection of oocytes. The histological appearance of B6-GFP-derived (A) and BDF1-derived (E) tissues was indistinguishable from each other. HE staining of sections. Asterisks indicate polyovular follicles. B and F) GV oocytes isolated from B6-GFP and BDF1 grafts via mechanical dissociation. C and G) MII oocytes obtained via in vitro maturation of these GV oocytes. MII oocytes in both groups developed into the 2-cell stage at a similar rate after ICSI (D: 2-cell stage from GFP-bearing B6-GFP oocytes). However, embryos from BDF1, but not B6-GFP mice, developed into normal offspring after embryo transfer (H; see Table 1). Bar = 100 μm.

Production of Pups from BDF1 Graft-Derived Oocytes

To examine the developmental potential of the MII oocytes derived from ovary-like grafts, these oocytes were subjected to ICSI. As shown in Table 1, more than half of the sperm-injected oocytes survived in both the B6-GFP and the BDF1 strains (58.7% and 65.0%, respectively), and most of them developed to the two-cell stage (70.3% and 69.0%, respectively) (Fig. 4D). After these two-cell embryos were transferred into recipient female mice, only 4.8% of B6-GFP/ICSI embryos implanted in the uteri of recipients and no live embryos reached term (Table 1). In contrast, 25.0% (26/104) of the BDF1/ICSI embryos transferred underwent implantation, and six pups (two males and four females) were obtained at term (5.8% per transferred embryo [Fig. 4H and Table 1]). One female pup died after 1 wk, but the remaining five pups grew to adulthood, and both sexes showed normal fertility.

Serum Hormone Levels in Host Animals

To determine whether the testis- and ovary-like tissues generated under the kidney capsules were endocrinologically functional, we measured the serum levels of testosterone and estradiol in the host animals. As shown in Figure 5, the serum concentrations of these hormones were significantly higher in host animals than in the nontransplanted, ovariectomized animals, although they were still lower than that of intact controls. This finding clearly indicates that the testis- and ovary-like tissues generated under the kidney capsules were also endocrinologically functional.

FIG. 5.

Serum hormone levels of host animals that received male (A) and female (B) gonadal cells under their kidney capsule. The testosterone and estradiol levels in the host animals (TP) were significantly higher compared with those detected in nontransplanted ovariectomized animals (OVX) in both groups, whereas they did not reach the levels detected in intact males and females, respectively (Cont). Asterisks indicate the presence of significant differences between the groups (P < 0.05).