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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022681
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BIOLOGY OF REPRODUCTION 70, 702–707 (2004)
DOI: 10.1095/biolreprod.103.022681
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Sex Differentiation and Germ Cell Production in Chimeric Pigs Produced by Inner Cell Mass Injection into Blastocysts

Hiroshi Nagashima1, Christopher Giannakis, Rodney J. Ashman, and Mark B. Nottle

Reproductive Biology Division, BresaGen Limited, Rundle Mall, Adelaide, South Australia 5000, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study aimed at collecting background knowledge for chimeric pig production. We analyzed the genetic sex of the chimeric pigs in relation to phenotypic sex as well as to functional germ cell formation. Chimeric pigs were produced by injecting Day 6 or Day 7 inner cell mass (ICM) cells into Day 6 blastocysts. Approximately 20% of the piglets born from the injected blastocysts showed overt coat color chimerism regardless of the embryonic stage of donor cells. The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICM cells, respectively, showing an obvious bias toward males. When XX donor cells were injected into XY blastocysts at the same embryonic stage, the phenotypic sex of the resulting chimera was male with no germ-line cells formed from the donor cell lineage. On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was male, and germ-line cells were derived only from the donor cells. The combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when it appeared in coat color. When the embryonic stage of the donor was advanced by 1 day in the XY-XY combination, 100% of the germ-line cells of the chimeras were derived from the donor cell lineage. These data showed that characteristics of sex differentiation and germ cell formation in chimeric pigs are similar to those in chimeric mice.

developmental biology, early development, gametogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An unbalanced sex ratio is one phenomenon of chimeric mice produced by embryo aggregation or blastocyst injection (for review, see [1]). When chimeras are produced from random combinations within a single population of embryos, the phenotypic male:female ratio is approximately 3:1, although embryonic combinations are theoretically male-male:male-female:female-female = 1:2:1 [1]. The sex ratio in chimeric mice is biased toward males because when male and female embryos are paired, the resulting chimeras tend to differentiate into males [2–4].

Functional germ cell chimerism in mice has also been studied in detail. In phenotypically male chimeras resulting from a combination of male and female embryos, it is highly unlikely that any of the functional gametes are of female lineage [4, 5].

Detailed analysis of factors affecting the production of chimeras is highly significant in the creation of genetically modified animals (for review, see [5, 6]). For instance, determining the optimal conditions for the production of chimeric mice using genetically modified embryonic stem cells is important for obtaining specially designed transgenic and gene-targeted mice [7–9]. Development of embryonic stem- or embryonic germ cell lines in pigs is ongoing in the hope that an established totipotent embryonic cell line would render advanced genetic modifications feasible and improve the efficiency of pig production and applications in biomedical research [10–15]. Since totipotent cells are used to produce individuals by means of chimera formation [10, 12], data regarding chimeric pig production are valuable and much needed. However, only a few reports [16, 17] describing methods of embryo micromanipulation and identification of chimerism have been published on chimera pig production. In these studies, aspects such as the sex ratio and frequency of germ-line chimerism have not been studied as intensively as has been done in mice [1–4].

We therefore collected background information for chimeric pig production. We studied the efficiency of chimera production using donor cells at various embryonic stages along with fertility, phenotypic sex, coat color chimerism, and characteristics in germ-line chimerism in the chimeras. In addition, the genetic sex of the chimeras was retrospectively determined by DNA analysis, and these chimeras were subjected to a progeny test. We then interpreted genetic sex in relation to phenotypic sex and functional germ cell formation in chimeras derived from the same or different sex combinations of donor and recipient embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Embryos

White crossbred (Large White x Landrace; LW) and brown Duroc gilts were used as the embryo donors. Pregnant gilts were aborted by an i.m. injection of 1 mg of prostaglandin F2{alpha} analog (Cloprostenol, Estrumate; Pitman-Moore, New South Wales, Australia) between 25 and 40 days after mating, followed by a second injection of 0.5 mg Cloprostenol 24 h later. Seven hundred and fifty international units (IU) of eCG (Pregnecol; Heriot AgVet, Victoria, Australia) were administered (i.m.) at the same time as the second Cloprostenol injection. Ovulation was induced by administering an i.m. injection of 500 IU hCG (Chorulon; Intervet, New South Wales, Australia) approximately 72 h after the eCG. Embryo donors were artificially inseminated with boar semen of the same breed on the afternoon (Day 0) following the hCG injection. Blastocysts were collected on Days 6 and 7 by surgically flushing the uterine horns under general anesthesia. Animal experiments were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC 1996) and approved by the University of Adelaide Animal Ethics Committee.

Isolation of Inner Cell Mass Cells and Blastocyst Injection

Inner cell mass (ICM) was isolated from Day 6 or Day 7 blastocysts by immunosurgery. Briefly, blastocysts were incubated at 37°C for 15 min with anti-pig spleen cell rabbit serum (given by courtesy of Dr. Deirdre Warnes, Medical School, Adelaide University, South Australia) diluted 1:8 with 21 mM Hepes-buffered Minimal Essential Medium (with Earle salts, L-glutamine and nonessential amino acids; Gibco-BRL, Invitrogen, Carlsbad, CA) supplemented with 5 mg/ml BSA (MEM-Hepes). Embryos were then incubated with guinea pig complement serum (S1639; Sigma, St. Louis, MO), diluted 1:8 with MEM-Hepes at 37°C for 15 min. Swollen trophectodermal cells were dissociated by vigorous pipetting (inner diameter, 100 µm). The ICM was washed thoroughly with MEM-Hepes, incubated with Dulbecco PBS (without Ca++/Mg++) containing 100 µM EDTA and 0.01% polyvinylalcohol for 10 min, and then disaggregated into two to four pieces by gentle pipetting. Before injection into blastocysts, small clumps of ICM cells (10–15 cells) were incubated with 7.5 µg/ml cytochalasin B in MEM-Hepes containing 10% fetal calf serum.

Small clumps of ICM cells were individually aspirated into an injection pipette with an outer diameter of 35 µm and a beveled tip with a sharp spike. The ICM clumps were then gently injected into the blastocoel of recipient blastocysts collected on Day 6 (Fig. 1). The ICM clumps isolated from LW blastocysts were injected into Duroc blastocysts and vice versa. The injected blastocysts were cultured in Whitten + 1.5% BSA + 10% fetal calf serum under an atmosphere of 5% CO2/5% O2/90% N2 at 38.5°C for 14–16 h.



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  		FIG. 1. Microinjection of inner cell mass cells into a recipient blastocyst Original magnification x200

Embryo Transfer

Blastocysts were surgically transferred to LW recipient gilts on Day 5 under general anesthesia by 1 g of thiopental sodium (Ravonal; Tanabe Seiyaku, Osaka, Japan) and 2%–4% halothane (Fluothane; Takeda Chemicals Industries, Osaka, Japan) in oxygen. Pregnant recipient gilts were treated in the same manner as the embryo donors, except that they were injected with 500 IU eCG to synchronize their estrous cycles. Embryos were transferred to the tip of uterine horn of the recipients using a 114 x 12-mm plastic catheter (Tom Cat Catheter, Kendoll, MA) with a small amount of MEM-Hepes.

Measurement of Coat Color Chimerism and Progeny Test of Chimeric Pigs

Coat color chimerism was visually determined in piglets after weaning. Proportion of the coat color derived from donor ICM cells was measured with the eye. After reaching sexual maturity (8–10 mo), coat color chimeras were mated with Duroc gilts or boars to examine fertility and germ-line chimerism. White piglets were judged to have derived from germ cells of LW origin because white coat color is dominant to brown in pigs [18]. On the other hand, brown piglets were judged to have resulted from Duroc germ cells.

Identification of Genetic Sex of Chimeric Pigs

To identify the genetic sex of the chimeric pigs, DNA extracted from white and brown hair roots was analyzed by polymerase chain reaction (PCR). We extracted DNA as described by Haegel et al. [19]. Briefly, the proximal 5-mm portion dissected from two to three hairs was placed in 30 µl of buffer (PBS:H2O = 1:1) and boiled at 100°C for 10 min. After cooling on ice, 1 µg of proteinase K was added and incubated at 37°C for 2 h, followed by 100°C for 10 min. The DNA extract was separated by centrifugation, and 6 µl of supernatants were amplified by PCR. The male-specific 236-base-pair (bp) sequence was amplified using porcine male specific primers [20]. The amplification profile comprised 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min.

Statistics

We used a t-test to analyze differences in the average proportion of donor ICM-derived coat color in chimeras between the two groups (Day 6 donor/Day 6 recipient vs. Day 7 donor/Day 6 recipient combinations).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Efficiency of Chimeric Pig Production

After ICM cells of a Day 6 blastocyst were injected into 48 blastocysts, 11 to 20 of these were transferred to each of three recipient gilts. Of those, two were cotransferred with five to seven noninjected blastocysts. All three recipients became pregnant, and 20 piglets were produced, including nine overt coat color chimeras. At least one coat color chimera was obtained from each recipient.

In experiments using Day 7 ICM cells as donors, 48 blastocysts were injected, and 13 to 18 of them were transferred to each of three recipients, all of which became pregnant. However, one transferred with 13 injected and two noninjected blastocysts had a miscarriage at Day 42, and 10 fetuses were collected, but chimerism was not analyzed.

The other two recipients that received a total of 35 injected blastocysts gave birth to 12 young and one stillbirth. Of these, seven were coat color chimeras.

Approximately 20% of the piglets born from the injected blastocysts were overt coat color chimeras regardless of the embryonic stage of donor cells (Table 1).


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  		TABLE 1. Production efficiency of chimeric pigs by ICM injection into blastocysts

Characteristics of Coat Color Chimerism and Sex Bias in Chimeric Pigs

Table 2 summarizes the characteristics of coat color and the phenotypic sex of the chimeras. Coat color patterns between individual chimeric pigs derived from Day 6 ICM injection were highly variable; the ratio of coat color from the donor cell was 5%–40% (Fig. 2) in males and 5%–100% in females. The coat color of these chimeras that was derived from donor cells appeared as either stripes or spots at approximately the same rates. Origin of the donor ICM (LW or Duroc) did not affect the pattern of coat color chimerism.


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  		TABLE 2. Sex bias in chimeric pigs produced by ICM injection into blastocysts.*



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  		FIG. 2. Chimeric pigs produced by microinjection of Day 6 inner cell mass cells into Day 6 blastocysts. a–d) Chimeric boars (C1–C4) subjected to the progeny test.

When Day 7 ICM cells were donors, the donor coat color appeared at 50%–95% in the males (Fig. 3), a ratio that was higher than that of chimeras derived from Day 6 ICM cells (mean: 70.8% ± 7.4% vs. 15.0% ± 6.5%, P < 0.05). As shown in Figure 3, b–f, most of these chimeric males had striped coats (six of seven individuals).



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  		FIG. 3. Chimeric pigs produced by microinjection of Day 7 inner cell mass cells into Day 6 blastocysts. Six chimeric boars (a, C5; b, C6, C7; c, C8; d, C9; e, C10) and a gilt (f, C11) subjected to the progeny test.

The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICMs, respectively, showing an obvious bias toward males.

Phenotypic Sex, Genetic Sex, and Germ-Line Chimerism in Chimeric Pigs

Tables 3 and 4 summarize the relationship between phenotypic and genotypic sexes in chimeric pigs and present the results of germ-line chimerism determined from the progeny test in which each of the male chimeras was mated with three to four females to obtain 24 to 42 offspring. Female chimeras also produced a litter of 10 in a single birth.


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  		TABLE 3. Phenotypic sex and germ-line chimerism in chimeric pigs produced by injection of Day 6 ICM into Day 6 blastocysts


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  		TABLE 4. Phenotypic sex and germ line chimerism in chimeric pigs produced by injection of Day 7 ICM into Day 6 blastocysts

When the donor cell and recipient blastocyst were at the same embryonic stage (Day 6) and the sexes of the donor and recipient were XX and XY, respectively, the phenotypic sex of the resulting chimera was male with no germ-line cells of donor cell lineage (C1, Fig. 2a). On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was also male, but germ-line cells in the chimera were derived only from the donor cell (C2 and C3, Fig. 2, b and c). In this genetic combination, despite lower ratios of coat color chimerism as seen in chimeric boar C3, the donor cell lineage was dominant in the germ-line formation of some chimeras. In contrast, the combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when apparent in coat color at a ratio of 50% (C4, Fig. 2d).

When the donor embryonic stage was 1 day ahead of that of the recipient (Day 7 donor and Day 6 recipient), 100% of the germ-line cells were derived from the donor cell lineage in five of five chimeras in the combinations XY-XX (C9, Fig. 3d) and XY-XY (C5–C8, Fig. 3, a–c). In a chimeric boar (C10, Fig. 3e) that resulted from an XX-XY combination, donor cell lineage did not contribute to germ-line formation even when it appeared in coat color.

In XX-XX chimera (C11, Fig. 3f), the donor cell lineage was not always dominant in the germ line, even when the donor cell was 1 day ahead of the recipient.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study showed that ICM cell injection into blastocysts efficiently produced chimeric pigs. The pregnancy rate of the recipients reached 100% after transfer of injected blastocysts. This may be attributable to the fact that Day 7 blastocysts were transplanted into Day 5 recipient gilts after overnight culture of Day 6 blastocysts. The pregnancy efficiency was about 30% in a preliminary test when Day 7 blastocysts were transferred to Day 6 recipients (data not shown). The results confirmed the significance of estrous synchronization on transferring blastocysts injected with cells. Transferring Day 7 embryos into Day 6 recipients is an accepted protocol in pig embryo transfer. However, the development of the injected blastocyst is likely to be delayed because of factors such as physical damage by injection, culture conditions, and interaction between the injected ICM cells and the ICM of the recipient blastocysts. Therefore, transfer into Day 5 recipients might favor subsequent embryonic development.

In this study, we injected ICM clumps into the recipient blastocysts. In the preliminary experiment, injecting ICM clumps instead of disaggregated single ICM cells was found to be much easier and less harmful to the recipient blastocysts. Injection of ICM clumps also helped minimize the amount of media injected into blastocoel with the donor cells compared with the injection of disaggregated single cells. Together, these might have caused an efficient production of chimeric pigs.

The developmental stage of the donor cell, regardless of whether it was 1 day ahead or the same as that of the recipient blastocyst, did not affect the production efficiency of coat color chimeras by blastocyst injection. However, when the donor cell was advanced by 1 day, the donor cell lineage contributed to coat color chimerism at a higher rate. In addition, the advanced development of the donor cell compared with the recipient blastocyst seems to determine the pattern of germ cell chimerism. This notion was supported by the finding that the chimeras obtained from combining Day 7 XY donors and Day 6 XY recipients produced only offspring derived from the donor cell lineage. It is known in chimera production by embryo aggregation that blastomeres that are more advanced in development tend to contribute to ICM formation of the chimeric embryos [21].

Furthermore, this study showed that only phenotypic males resulted from chimeric embryos consisting of Day 6 XY blastocysts and Day 7 XX ICM cells. This indicated that the sex determination of chimeras was dominated more by the sex of the donor or recipient cells in the chimeric embryos than by the developmental stages of the donors and recipients.

The male:female sex ratio of the chimeric pigs was 7:2 when the donor and recipient were at the same developmental stages and 6:1 when the donor cell was 1 day more advanced. The chimeric mouse population shows a 3:1 sex bias [1], and the sex ratio of the chimera pigs in this study was also unbalanced. When chimeric pigs were genetically XY/XX, (i.e., formed by XY-cell injection into XX blastocysts or vice versa), all of them (five of five) differentiated into males, which was consistent with the results obtained using XY/XX chimeric mice [1–4]. However, the reported abnormal sex differentiation in XY/XX chimera mice [2] was not observed in pig chimeras, and chimeric pigs obtained in this study were all fertile. Considering the low frequency of intersexual chimeras in mice obtained from the XY-XX combination [2, 5], abnormal sex differentiation may appear if the production of chimeric pigs is increased.

In conclusion, this study showed that the characteristics of sex differentiation and germ cell formation in chimeric pigs were similar to mice. These results are not only significant for the biology of mammalian chimeras but also useful in terms of porcine developmental technologies. When pluripotent cell lines are established in pigs, this study will provide significant background for the efficient production of germ-line chimeras.

"Rescuing chimeras" [5] is one application of chimeras; an embryo with perturbed developmental potency can be rescued by producing chimeras with normal embryos. This method was also used to support the development ability of cloned embryos in rabbits by forming a chimera with normal embryos [22]. When applying this method to pigs, our data will be significant in regulating sex differentiation and germ-line chimerism in chimeric embryos.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the support of Northfield Pig Research Unit, SARDI, South Australia.


   FOOTNOTES
 
1 Correspondence and current address: Hiroshi Nagashima, Laboratory of Developmental Engineering, Department of Life Science, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japan. FAX: 81 44 934 7824; hnagas{at}isc.meiji.ac.jp Back

Received: 28 August 2003.

First decision: 17 September 2003.

Accepted: 6 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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