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Testis; |
Laboratories of Anatomy3 and Experimental Animal Sciences,4 Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Department of Veterinary Radiology,5 School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, 069-8501 Japan
ABSTRACT
Although mammals produce either sperm or eggs depending on their sex, we found oocytes in the testes of newborn MRL/MpJ male mice. In the present study, we report the morphological characteristics of testicular oocytes, the postnatal change of oocyte number per testis, and the expression of a few oocyte-specific genes in the testes of MRL/MpJ mice. The testicular oocytes had a diameter of 50–70 µm and were surrounded by zonae pellucidae, which were observed between oocytes and follicular epithelial cells. Ultrastructurally, the testicular oocytes contained numerous microvilli and cortical granules, receiving cytoplasmic projections from follicular epithelial cells. The testicular oocytes appeared as early as at birth, and the largest number was found on Day 14. The testicular oocytes were detected in only MRL strains and B6MRLF1, but not in C57BL/6, C3H/He, BALB/c, DBA/2, A/J, and MRLB6F1. The expression of the oocyte-specific genes Zp1, Zp2, Zp3, and Omt2a was detected in testes from MRL/MpJ mice. These results suggest that newborn male MRL/MpJ mice with XY chromosomes can produce oocytes in their testes and that one of the genes causing this exists on the Y chromosome.
follicular development, meiosis, mouse, MRL/MpJ, oocyte development, ovary, sex differentiation, testicular oocyte, testis
It has been generally believed that in mammals, males produce only sperm, and oocytes are only produced in females. It is also known that vertebrates born as either males or females never change their sex during their lifetimes. However, an exception to this rule is the substantial number of naturally occurring teleosts [1, 2]. Interestingly, it has recently been demonstrated that sperm-producing germ cells in rainbow trout could be differentiated into oocytes with biotechnological modifications [3]. Although some genetic abnormalities of sexual differentiation in mammals, including experimental chimeras, can cause the appearance of oocytes in the testes or the development of ovotestis, it has never been reported that apparently healthy and fertile male animals can produce oocytes during spermatogenesis.
The sex of a mammal is determined when an ovum containing a haploid genome with an X chromosome is fertilized by a sperm possessing a haploid genome with either an X or Y chromosome. However, the anatomical determination of sex occurs later in development as gonads first arise as bipotential primordia with the plasticity to develop into ovaries or testes. Determination of the bipotential primordia into male gonads requires expression from Sry (sex-determining region on Y), which initiates the differentiation of Sertoli cells, as well as their structural organization into a testis cord [4]. If the primordial gonad proceeds to development along the ovarian cascade despite being in an XY animal, this could result in sexual reversal of the animal, or the development of ovotestes. This has been observed in animals with translocations or deletions of the Sry gene, or animals with delayed Sry expression [4–6]. Another factor contributing to the plasticity of sexual determination is the fact that the differentiation of germ cells into oogonia or prospermatogonia is directed by signals from somatic cells in the gonads, not by the germ cells themselves [7]. These findings led us to hypothesize that genital glands have the potential to produce differentiated germ cells that are usually found in the opposite sex.
The MRL/MpJ inbred mouse strains, MRL/MpJ-+/+ (M+) and MRL/MpJ-Faslpr/Faslpr, hereafter referred to as MRL/MpJ-lpr/lpr (lpr), originate from crosses initiated in the 1960s that used a number of standard inbred strains [8]. The homozygous mutant lpr (lymphoproliferation) has a defect in Fas expression and develops a massive generalized enlargement of lymphatic tissues, as well as several autoimmune diseases. The M+ strain also displays these phenotypes, but in milder forms. The MRL/MpJ strains are also known to have several unique characteristics in regenerative wound healing, such as those observed in ear punch closure and in cardiomyocyte regeneration [9, 10], and in the development of several autoimmune diseases, such as systemic lupus erythematosus, polyarteritis nodosa, rheumatoid arthritis, and systemic sclerosis. These results suggest that these phenotypes are due to the MRL genetic background but not to the lpr allele [11]. In addition, the testis of MRL/MpJ mice is known to have at least the following unique characteristics: metaphase-specific apoptosis of meiotic spermatocytes [12, 13] and heat shock resistance of spermatocytes, as found in experimental cryptorchidism [14]. These two phenotypes are attributed to mutations in exonuclease 1, which has an important role in DNA repair [15, 16]. To investigate other characteristics of spermatogenesis in MRL/MpJ mice, we examined the differentiation process from prospermatogonia to spermatogonia in postnatal developing mouse testes. We found oocytelike cells in the seminiferous tubules of newborn MRL/MpJ male mice and report here the morphological characteristics of these testicular oocytes. We also present observations on the postnatal change of oocyte number per testis and show the expression of oocyte-specific genes in the testes of MRL mice. Finally, we discuss mechanisms by which these oocytes could have developed in the testicular environment of MRL mice.
Several inbred mouse strains and F1, C57BL/6 (B6), C3H/He, BALB/c, DBA/2, A/J, MRL/MpJ-+/+ (M+), MRL/MpJ-lpr/lpr (lpr), B6MRLF1 (F1 between female B6 and male MRL), and MRLB6F1 (F1 between female MRL and male B6) were used in the present study. Eight-week-old male and female mice purchased from Japan SLC (Shizuoka, Japan) were maintained with free access to food and water in our facility. In the handling of experimental animals, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals," Hokkaido University, Graduate School of Veterinary Medicine. In this study, 10 to 124 newly born mice from each strain were obtained by free breeding and were killed at 0–50 days after birth by detruncation or cervical dislocation. For embryo collection, timed mating was established by housing females with males overnight. At noon the following day, females were checked for the presence of vaginal plugs, which denoted pregnancy, and the embryos were recorded as being in Embryonic Day 0.5 (E0.5) of development. The testes and ovaries of MRL mice and the testes of B6 mice at E14.5, E16.5, and E18.5 were used as samples.
To examine testicular oocytes, two types of specimens—fresh whole-mount preparations and fixed serial sections—were prepared. For whole-mount preparations, the testes of 0- to 50-day-old lpr and M+ mice were removed and were immediately mounted on glass slides with coverslips. They were then crushed to make whole-mounted single tubular sheets and were observed under a differential interference microscope (BX50F4; Olympus). For fixed preparations, the testes of 0- to 18-day-old and the ovaries of 0- to 8-day-old lpr and M+ mice were removed and immediately fixed with Bouin solution for 24 h, cut into 5-µm-thick serial paraffin sections, and stained with hematoxylin-eosin (HE) or periodic acid-Schiff (PAS). Some testis sections were obtained from whole-mount preparations after microscopic observation.
To examine the ultrastructure of testicular oocytes, testes of 4-day-old lpr mice were immediately fixed with 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C for 6 h. The testes were then postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer at room temperature for 2 h, dehydrated with graded alcohol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and they were observed with an electron microscope (JEM-1210; JOEL).
To estimate the oocyte score, the average number of oocytes per testis found at each age from Days 0–50 after birth was calculated. Paraffin-embedded serial sections were made from the testes of 0- to 4-day-old mice, and whole-mount preparations were made from the testes of 8- to 50-day-old mice. The number of testicular oocytes was counted in these samples. To determine the existence of testicular oocytes in other strains, whole-mount preparations of testes from B6, C3H/He, BALB/c, DBA/2, and A/J age matched to the testes of lpr mice that had shown the highest oocyte score were used. The B6MRLF1 and MRLB6F1 strains were also examined to determine whether this phenotype is recessive or dominant.
To confirm the existence of the zona pellucida and the initiation of meiosis, and to determine the function of observed follicular epithelial-like cells, we used immunohistochemistry to detect the presence of zona pellucida protein 3 (ZP3), synaptonemal complex protein 3 (SYCP3), and forkhead box L2 (FOXL2). Briefly, the deparaffinized sections were treated with antigen-unmasking solution, Target Retrieval Solution (Dako Cytomation, Carpinteria, CA), or with 10 mM citrate buffer, which was autoclaved for 20 min at 105°C. The samples were then incubated in 3% hydrogen peroxidase/methanol solution for 10 min to block endogenous peroxidase activity. Sections blocked in normal goat serum were incubated with goat anti-ZP3 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and sections blocked in normal rabbit serum were incubated with rabbit anti-SYCP3 antibody (1:750; GeneTex, San Antonio, TX) or rabbit anti-FOXL2 antibody (1:50; Sigma, St. Louis, MO). All primary antibody stainings were done at 4°C overnight. Negative controls were left in the normal serum blocks and were not incubated with antibodies. The sections then were treated with a secondary biotinylated anti-goat or anti-rabbit immunoglobulin G (IgG) antibody (Chemicon, Temecula, CA; Nichirei, Tokyo, Japan) for 30 min, then in streptavidin-peroxidase (Nichirei) for 30 min, and finally in 3,3'-diaminobenzidine-H2O2 solution. Some sections were counterstained with either HE or PAS.
RT-PCR for Oocyte-Specific Genes
To examine the expression of oocyte-specific genes, total RNA was obtained from the testes and ovaries of 4- to 13-day-old B6 and lpr mice using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Complementary DNA was then synthesized with ReverTra Ace (Toyobo, Osaka, Japan), and PCR was carried out with ExTaq (Takara, Tokyo, Japan) under the following PCR conditions: 5 min at 95°C, 35 cycles of 40 sec at 95°C, 30 sec at 62°C, and 1 min at 72°C, followed by 5 min at 72°C. The PCR primers for oocyte-specific genes used in the present study are shown in Table 1. The zona pellucida in mice is composed of the zona pellucida proteins ZP1, ZP2, and ZP3. ZP3 and ZP2 act as primary and secondary sperm receptors, respectively, whereas ZP1 cross-links the filaments composed of ZP2/ZP3 dimers [17]. Oocyte maturation alpha, Omt2a, is a protein that acts after oocyte maturation and the onset of meiosis [18]. Since these genes are produced by growing oocytes, but not by follicular epithelial cells in mice [18–21], they were selected as oocyte-specific markers. The amplification of these oocyte-specific gene transcripts was verified by sequencing with a cycle sequencing kit containing fluorescent terminators employing standard methods (Applied Biosystems, Foster City, CA) and a model 377 automatic sequencer (Applied Biosystems).
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Testicular Oocytes in Whole-Mount Preparations and in Paraffin Sections
Testicular oocytes were found in whole-mount preparations of testes obtained from lpr mice and in testes from M+ mice aged 8 to 30 days after birth (Fig. 1). Testicular oocytes coexisted with gonocytes and spermatogonia in the seminiferous tubules, mostly in the neighborhood of the rete testis. When there were two or more oocytes in a single seminiferous tubule, they were located close to each other. Because of their unique size, 50–70 µm in diameter, they were easily distinguishable from somatic cells and sperm-producing cells. Each oocyte had an abundant cytoplasm and a large nucleus with one or two distinct nucleoli, and each oocyte was surrounded by a zona pellucida-like structure, which was observed between the oocyte and follicular epithelial-like cells. Follicular epithelial-like cells, sometimes consisting of a few layers, were clearly distinguishable at Days 14–30.
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Since no testicular oocytes were detected in whole-mount preparations of testes obtained from Days 0 and 4, we wondered whether paraffin-embedded serial sections of 0- to 4-day-old testes better preserved any testicular oocytes that might exist. To our surprise, testicular oocytes were found as early as at birth; they were well developed, with diameters between 30 and 50 µm, and were larger than the gonocytes and somatic cells of the testes (Fig. 2A). The testicular oocytes contained large oval nuclei with distinct nucleoli and abundant cytoplasm stained lightly by eosin, and they appeared similar to ovarian oocytes in female mice of the same age. However, the testicular oocytes obtained from 4- to 16-day-old mice were partially surrounded by follicular epithelial-like cells (Fig. 2, B and C). Each of these follicular epithelial-like cells had a nucleus with an irregular shape containing distinct nucleoli similar to those in Sertoli cells. The testicular oocytes appeared in the following stages: 1) surrounded by layers of squamous to cuboidal follicular epithelial-like cells and resembling the appearance of the primordial to secondary ovarian follicle (Fig. 2, D and E); 2) containing two nuclei; these were observed in 16-day-old M+ mice (Fig. 2F); and 3) in the process of degeneration with vacuolization of cytoplasm, disappearance of nuclear envelope, and karyolysis; these were observed in 16-day or older mice (Fig. 2, F and G). Although the follicular epithelial-like cells formed a multilayer similar to that observed in early-stage secondary follicles, they never a formed follicular antrum or a polar body-like structure.
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Ultrastructural Characteristics of Testicular Oocytes
Unlike spermatogonia and Sertoli cells, the testicular oocytes did not attach to the basement membrane (Fig. 3A). Under electron microscopy, the zona pellucida appeared discontinuous (Fig. 3B). Where there were gaps in the zona pellucida, follicular epithelial-like cells directly contacted the oocyte (Fig. 3B). Where the zona pellucida was intact, the oocyte extended numerous microvilli through about half the thickness of the zona pellucida, and the follicular epithelial-like cells attached to the cell membrane of the oocyte with slender cytoplasmic processes penetrating the zona pellucida-like structure (Fig. 3, C and D). In the cytoplasm of the testicular oocyte, a round, highly dense matrix bound by a single smooth membrane was found just beneath the cell membrane; this appeared similar to the cortical granules found in ovarian oocytes (Fig. 3, D and E). These cortical granules were divided into two types by their diameters. They were located mainly in two regions. The smaller granules appeared to be synthesized in the juxtanuclear region, and the larger granules appeared to be synthesized near the cell membrane. The testicular oocytes contained Golgi complexes observed as multiple aggregates of small vesicles, and flattened tubules similar to those involved in the synthesis and formation of cortical granules in ovarian oocytes (Fig. 3, F and G).
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Number of Testicular Oocytes in Postnatal Development and in Mouse Strains
The number of testicular oocytes per testis was counted in whole-mount preparations and in complete serial sections. The oocytes were detected in about half of the mice examined (65 of 124), and the maximum number of oocytes detected in one testis was 12. The appearance of oocytes in the testis peaked on Day 14 after birth with an oocyte score of approximately 1.2, as shown in Figure 4. No oocytes were observed on Days 40 and 50 after birth. The testes of other inbred strains and F1 mice age matched to lpr mice with the highest oocyte score (14 days old) were examined (Table 2). We found no oocytes in these other inbred strains of mice and in MRLB6F1 mice. However, F1 progeny from female B6 mice mated to male MRL mice were shown to have testicular oocytes with a lesser oocyte score than that of the MRL strains.
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Zona Pellucida and Follicular Epithelial Cells in Testicular Oocytes
The zona pellucida-like structures showed a positive reaction to the PAS stain. They were observed as discontinuous lines on Days 0 and 4 after birth, and then as continuous lines on Day 8, much like ovarian oocytes of the same age (Fig. 5, A–F). Additionally, zona pellucida-like structures were shown to express ZP3 by immunostaining (Fig. 5G) and at the same level as that in ovarian oocytes (Fig. 5H). However, the follicular epithelial-like cells of testicular ocytes were not immunostained with FOXL2 (Fig. 5, J and K), whereas the follicular epithelium in ovaries was stained (Fig. 5L).
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Initiation of Meiosis in Fetal Testes of MRL Mice
The immunoreactivity of SYCP3 was detected in germ cells within the testis cords of MRL testes on E14.5, E16.5, and E18.5, similar to that detected in female germ cells in the fetal ovary at E16.5 (Fig. 5, M–R). The testis cord containing these germ cells is located mostly in the neighborhood of the rete testis or at the edge of the testis. The meiotic germ cells were close together within the same testis cord, as observed with testicular oocytes after birth. Some germ cells of B6 testes at E14.5 showed weak immunopositivity to SYCP3 (Fig. 5P); however, there were no immunopositive cells at E16.5 (Fig. 5Q).
Expression of Oocyte-Specific Genes in Testicular Oocytes
The expression of oocyte-specific genes in MRL testes was examined by RT-PCR and compared to the expression in B6 testes (Fig. 6). The lpr testes expressed all of the oocyte-specific genes examined in the present study; however, the intensities of expression were weaker than those found in the ovaries. Expression of the oocyte-specific genes became stronger on Day 13 in comparison to Day 4. The testicular expression of Zp1 and Omt2a was detected only in lpr mice, whereas the expression of Zp2 and Zp3 was also observed in testes from B6 mice, and these results were confirmed by sequencing (data not shown). The expression of Zp2 and Zp3 in B6 mice was weaker than in lpr mice.
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Testicular Oocytes in XY Fertile Males
The existence of testicular oocytes has been reported in several cases. Testicular oocytes were first found in the testes of chimeric mice in 1968 [22]. Then, testicular oocytes were observed in the fetal testes after being transplanted under a kidney capsule of adult female hosts [23, 24]. Testicular oocytes also existed in the testes of XXsxr sex-reversed mice [25]. The most recent report of testicular oocytes was found in the testes of XX
XY chimeric mice [26]. However, to date, this is the first report of the appearance of testicular oocytes in XY fertile males.
Although the testicular oocytes were found as early as birth in paraffin sections, they were not detected in the whole-mount preparations of testes before Day 8. The detection of testicular oocytes in whole-mount preparations may have required the maturation of the zona pellucida, and the discontinuity of the zona pellucida on Day 4 may explain why testicular oocytes were not detected in 4-day-old whole-mount testes.
Sertoli cells originate from the same the precursors as granulosa cells [27], and they can transdifferentiate into follicular cells under the influence of ovarian factors [28]. These findings suggest that the follicular epithelial-like cells surrounding testicular oocytes originated from Sertoli cells. However, we cannot rule out the possibility that they are undifferentiated bipotential epithelial cells. The testicular oocytes and follicular epithelial-like cells appear to progress along the maturation process until the early secondary follicle stage in the testicular environment; however, their development was delayed and limited compared with oocytes and follicular cells in the ovaries. The following two reasons were suggested for this: 1) Sertoli cells mature and form tight junctions among adjacent Sertoli cells after the fetal and neonatal proliferation period [29], indicating that they are no longer able to transdifferentiate into follicular epithelial cells, and 2) proliferation of follicular epithelial cells was prevented by the initiation of spermatogenesis because of the competition for space. Therefore, it was suggested that the maturation of Sertoli cells and initiation of spermatogenesis prevented the growth of follicular epithelial cells.
Some irregular characteristics were observed in the testicular oocytes. The testicular oocytes without follicular epithelial cells found on Day 0 already contained zona pellucida, which in the ovary is believed to be formed only during the developmental process from primary to secondary follicles. Additionally, binuclear testicular oocytes were observed; these were thought to be a result of the resumption of meiosis, a nuclear division in an oocyte without subsequent cytokinesis. Normally, ovarian germ cells initiate prophase of meiosis by E13.5 and are arrested in the diplotene stage of meiotic prophase I by the surrounding follicular epithelial cells until puberty [30, 31]. The existence of abnormal binuclear oocytes in the testes and the fact that the follicular epithelial-like cells of testicular oocytes lacked FOXL2, an important factor for ovarian somatic cell differentiation, follicular development, and maintenance [32, 33], indicated that the follicular epithelial-like cells do not have the abilities to regulate oocyte growth the way follicular epithelial cells in the ovary do.
The oocyte score decreased over time, and degenerating testicular oocytes appeared on Day 16 after birth and later. Also, there were no testicular oocytes observed after Day 30. The testicular environment changes to induce the entry of germ cells into meiosis after birth, whereas oocytes are arrested in the diplotene stage from birth until puberty [30]. It is possible that the changing testicular environment led to the degeneration of testicular oocytes and induced the elimination of testicular oocytes by Sertoli cells, which have the ability to phagocytose abnormal germ cells.
Expression of Zp1, Zp2, and Zp3 were detected in lpr testes on Days 4 and 13, and the presence of Zp3 was also confirmed at the protein level by immunohistochemistry. The timing and levels of the Zp genes' expression coincided with the formation and completion of the zona pellucida in growing testicular oocytes, as observed in paraffin sections. The zona pellucida glycoproteins serve as sperm receptors and induce acrosome reactions [17], and Omt2a is known to have a role after the onset of meiosis [18]. The expression of the Zp genes and Omt2a indicated that testicular oocytes might have the ability to fuse with sperm or even be fertilized as an oocyte. However, there are many other oocyte-specific genes, such as Bmp15, Zar1, and Gdf9, that have important roles in germ cell development, differentiation of granulosa cells, oocyte maturation, fertilization, and early embryonic development [34]. Thus, further studies are needed to confirm the expression of oocyte-specific genes in individual testicular oocytes and compare them with those in ovarian oocytes.
Why Do Testicular Oocytes Appear in MRL Mice?
Since both MRL strains, MRL/MpJ-+/+ and MRL/MpJ-lpr/lpr, had testicular oocytes, the development of testicular oocytes appears to be dependent on the MRL genetic background, but not on the Fas gene [35]. Based on the observation that testicular oocytes were found only in F1 mice that had an MRL father, it was suggested that this phenotype was dominant as long as the Y chromosome was derived from the MRL background, meaning that one of the genes responsible for the appearance of testicular oocytes is on the Y chromosome. However, the oocyte score of B6MRLF1 was much lower than that of MRL, indicating that multiple genes, probably on the autosomes, are also required for the development of this phenotype.
It is generally believed that fetal germ cells are intrinsically programmed to enter meiosis and initiate oogenesis unless specifically prevented by the existence of a meiosis-inhibiting factor [36]. Recently, it has been reported that retinoic acid induces meiosis, and CYP26B1, an enzyme involved in retinoid metabolism, has a key role in preventing meiosis in the fetal testis [37, 38]. As a result of sequestration within testis cords, germ cells are arrested in G0/G1 of the mitotic cell cycle by E13.5, and they do not enter meiosis until after birth in the gonad of the XY mouse [30]. However, there are two candidate cell types expressing Cyp26b1, myoid cells [37] and Sertoli cells [38]. Testicular oocytes of MRL mice always coexisted with normal spermatogonia that could undergo spermatogenesis after birth, indicating that the testicular cords have the ability to prevent germ cells from entering meiosis during the embryonic period. However, meiotic germ cells were observed in the testes of E14.5, E16.5, and E18.5 MRL mice, and in the testes of E14.5 B6 mice; these cells were located near the rete testis. Retinoic acid is produced and released by the mesonephros [38], indicating that the rete testis might be the place with the highest concentration of retinoic acid, which causes the initiation of meiosis in neighboring germ cells. Temporal existence of meiotic germ cells observed in B6 fetal testes has been also reported in CD-1 mice embryos [39]. Since male germ cells that prematurely enter meiosis were known to follow apoptotic fates [40], the meiotic germ cells in B6 mice might be eliminated by a meiosis-preventing mechanism present in the fetal testis. Taking all of the above into consideration, the following possibilities could explain the presence of germ cells that enter meiosis in the fetal testis: 1) genetic mutations in genes such as Sry and Cyp26b1; 2) the existence of germ cells with high sensitivity to retinoic acid; 3) the influence of unknown factors that impede the meiosis-preventing mechanism; 4) defects in the mechanism eliminating meiotic germ cells; or 5) a combination of the above. In any case, a unique meiotic progression mechanism exists in the fetal testes of MRL mice that may be involved in the appearance of testicular oocytes.
Where Will Testicular Oocytes Lead Us?
Although further research is needed, the testicular oocytes in MRL mice can provide more clues about the development of the reproductive system, such as the mechanisms of sex differentiation, sex-specific methylation patterns of germ cells, and the mechanism that prevents entry into meiosis in male embryos. The fertility of XY oocytes derived from XY sex-reversed females, B6.YDOM, undergo fertilization efficiently but cannot develop beyond the two-cell stage either in vivo or in vitro due to defects of both nuclear and cytoplasmic components in the oocytes [41, 42]. However, in the field mouse Akodon azarae, a proportion of fertile females have sex chromosomes cytogenetically indistinguishable from the XY chromosomes of males, and these females are known to produce both X and Y oocytes [43]. The present study showed that testicular oocytes could not develop follicles further than the early stages of the secondary follicle in the testicular environment. However, if the testicular oocytes can be made to mature properly in vitro, it may be possible to obtain an embryo with a genome derived entirely from male mice by in vitro fertilization and gamete intrafallopian transfer techniques. Because of their morphological similarity to ovarian oocytes, it is speculated that the testicular oocytes would become haploids with an X chromosome after maturation. This means that the sex ratio of the embryos obtained after in vitro fertilization would become one male to one female, and the existence of females with a genome originating only from males could be like deriving "Eve from Adam." Furthermore, if we obtain testicular oocytes from one side of the testes and sperm from the other side of the epididymis, an embryo with a genome derived from only one male will be produced. Thus, testicular oocytes in MRL mice can establish a new concept in reproductive biology.
FOOTNOTES
1Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (19380162) and the Novartis Foundation (Japan) for the Promotion of Science. 
Correspondence: 2Yasuhiro Kon, Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi9, Kita-ku, Sapporo 060-0818, Japan. FAX: 81 11 706 5189; e-mail: y-kon{at}vetmed.hokudai.ac.jp
Received: 22 July 2007.
First decision: 15 August 2007.
Accepted: 27 February 2008.
REFERENCES
XY chimeric testes. Proc Natl Acad Sci U S A 2005; 102:4039–4044.
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