| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reproductive Technology |
Center for Animal Transgenesis and Germ Cell Research,3 Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348
GTC Biotherapeutics, Inc.,4 Framingham, Massachusetts 01701
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
male reproductive tract, sperm, spermatogenesis, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The transgenic donor goats used in this study carried the human alpha-1 antitrypsin (AAT) gene, also known as alpha-1 protease inhibitor, under the control of the caprine beta-casein promoter to direct the expression of AAT to the lactating mammary gland. In humans, AAT is synthesized in the liver and immediately released into the blood, where it inhibits the action of proteases such as trypsin, collagenase, and elastase. AAT deficiency is an autosomal recessive disorder caused by defective alleles carried by up to 10% of European descendants [7]. The progression of the disease can be delayed by raising the AAT levels of affected individuals with regular infusions of concentrates that are now derived from pooled plasma [8]. Recombinant production of AAT in the milk of transgenic dairy animals will yield an unlimited supply of this factor, while affording better control of the source and reducing the risk of transmission of adventitious infectious agents. In the present study, however, the transgene was used primarily as a marker to unequivocally detect the success of germ-cell transplantation. Detection of the transgene in sperm from recipient goats and the somatic cells of kids resulting from mating of a recipient to wild-type does was used to demonstrate the occurrence of donor-derived spermatogenesis and germline transmission of the donor haplotype after germ-cell transplantation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Donor transgenic goats 872 and 892 used in this study were the F1 offspring of transgenic founders #472-94 and #463-95, respectively, previously derived by pronuclear microinjection of the beta casein-AAT expression construct BC30 (see Fig. 3c; Meade et al., unpublished data). At the time of castration, the donor goats were sexually mature and 3.5 and 3 yr of age, respectively. Five prepubertal male dairy goats (about 4 mo of age, see Table 1) were used as recipients. All goats were originally maintained at the GTC Biotherapeutics production and research facility in Charlton, MA. Prior to surgery, donor goats were transferred to the University of Pennsylvania School of Veterinary Medicine at New Bolton Center. Animals were handled and treated according to the guidelines of the Animal Care and Use Committee at the University of Pennsylvania.
|
|
Preparation of Cells
Two transgenic goats were castrated under general anesthesia and the testes were subjected to a two-step enzymatic digestion as previously described [5, 6]. The cells were washed and resuspended in Dulbecco modified Eagle medium and kept on ice until transplantation, which occurred within 3 h. Cell viability as assessed by trypan blue exclusion was >90%. The mixed population of testis cells obtained using this method consisted mostly of germ cells and Sertoli cells, along with some contaminating peritubular cells; no enrichment for germline stem cells was applied.
Injection of Cells into Recipient Testes
Transgenic donor cells were transferred into the seminiferous tubules of recipient goat testes (n = 5 goats) by injection into the rete testis using ultrasound-guided injection according to a technique we recently described for pigs [5] and goats [6]. On average 300 x 106 testis cells (Table 1) in a volume of about 5 ml were injected into each testis using minimal hydrostatic pressure.
Detection of the Transgene
Starting 103–159 days posttransplantation, semen samples were collected from recipient goats with an artificial vagina for subsequent analysis of the sperm DNA for the presence of transgenic sperm. Semen collection was performed every 1 or 2 wk for a period of 7 mo. Semen samples were processed immediately for cryopreservation as previously described [9]. For isolation of sperm DNA, sperm were incubated for 1 h at 37°C in the presence of 100 mM dithiothreitol (DTT) in lysis buffer followed by DNA extraction using a commercially available kit (QiAmp DNA Minikit; Quiagen Science, Valencia, CA). DNA was isolated from skin samples of the doe and goat kids using the same kit.
For polymerase chain reaction (PCR) detection of the AAT gene, the following primers were used: 5'-ACGACAATGCCGTCTTCTGTCTCGTG-3' (position 10, sense strand of the AAT gene), and 5'-GTGTGCCAGCTGGCGGTATAGGCTG-3' (position 220, antisense strand of the AAT gene), resulting in a 210-base pair (bp) fragment. The PCR reactions included serial dilutions of the BC30 vector for estimation of the sensitivity of the assay. Each PCR reaction contained DNA from about 500 sperm. As control, primers were used for the endogenous goat beta casein exon 7: 5'-CCAGGCACAGTCTCTAGTCTA-3' and 5'-GGACAGGACCAAGTACAGCT-3', resulting in a 440-bp fragment.
Southern blot analysis was performed with a human AAT probe. An aliquot of each of the genomic DNAs and of BC30, the vector used to create the AAT-expressing goats, were digested with BamHI (New England Biolabs, Beverly, MA) according to the manufacturer's directions. The digested DNAs were quantitated using a DyNA Quant 200 (Amersham Pharmacia, Piscataway, NJ), and 2 µg of each digested DNA were electrophoresed on a 0.7% agarose gel. Aliquots of BamHI-digested BC30 DNA were included on the gel as standards for determining the approximate number of copies of AAT per cell. Copy number calculations were based on the assumption that there are 6 pg of genomic DNA per somatic cell. After electrophoresis, the DNA was transferred to Duralon-UV membranes (Stratagene, Cedar Creek, TX) by capillary transfer in 10x SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0). After transfer, the membrane was rinsed in 2x SSC and cross-linked in a UV Stratalinker 2400 (Stratagene). Prehybridization was done for a minimum of 1 h at 65°C in a buffer modified from that developed by Church and Gilbert [10]. The buffer composition was 0.125 M Na2HPO4, pH 7.2; 0.25 M NaCl; 7% SDS; 1 mM EDTA; and 0.1 mg/ml Escherichia coli tRNA (Sigma). The probe used for blot hybridization was a 32P-labeled 1.5-kb human AAT genomic fragment, labeled using the Prime-It II Random Primer Labeling Kit (Stratagene) according to the manufacturer's instructions. Hybridization was at 65°C overnight. After hybridization, the membranes were washed twice in 2x SSC and 0.1% SDS at room temperature and twice in 0.2x SSC and 0.1% SDS at 65°C. For purposes of quantitation, the membranes were analyzed on a Storm 860 phosphoimager (Amersham). The membranes were also exposed to X-Omat AR film (Kodak, Rochester, NY) to provide a permanent record of the data.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
In sperm from two of five recipient goats, the transgene was detected starting from 3.5 or 6 mo after germ-cell transplantation until the end of the experiment (11 mo after transplantation). This indicated that donor-derived transgenic stem cells had integrated into the seminiferous epithelium of recipient tubules and contributed to recipients' spermatogenesis, producing transgenic sperm in a stable manner (Fig. 2). Based on the sensitivity of the PCR assay, as determined by dilution of transgenic donor cells with wild-type cells prior to DNA extraction, it was estimated that at least 1 in 50 sperm carried the transgene.
|
One of the two recipient goats with detectable levels of transgenic sperm was bred to eight wild-type female goats, resulting in the birth of 15 normal kids (7 sets of twins and 1 singleton). A small sample of skin was obtained from the ear of each offspring for DNA isolation. One male kid (Fig. 1B) was found to be transgenic for AAT by PCR (Fig. 3A) and Southern blot analysis (Fig. 3B). The pattern of bands observed for DNA from donor goat 892 (Fig. 3B, lane 9) represents the transgene and two higher molecular-weight bands that are most likely due to rearrangements of some copies of the transgene at the chromosomal integration site, a phenomenon fairly frequently observed in transgenic animals. The same pattern was observed for DNA isolated from one kid (Fig. 3B, lane 16). Comparable samples from the doe and the twin of this kid were negative for the transgene.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Sustained production of donor-derived sperm can only occur if donor spermatogonial stem cells colonize the recipient seminiferous tubules. In the present study, detectable levels of sperm originating from the transgenic donor germ cells were observed in two of five (40%) recipient goats for at least 11 mo after transplantation (end of study). This indicates that donor stem cells were stably engrafted in the recipient testes. Both of these animals received cell transplants from the donor animal carrying the higher number of transgene copies (see Figure 3 and Table 1). It is possible that testes in the other recipients were also engrafted with donor germ cells but, because only about 500–1000 sperm were analyzed per sample whereas an ejaculate contained about a billion sperm, the level of sperm produced that carried the donor transgene might have been too low to be detectable by PCR analysis of a random sample of ejaculated sperm. In a previous study, we demonstrated that the fluorescent-labeled donor cells were detectable in testes of all recipient goats for up to 3 mo after transplantation [6]. In the present study, mating of one recipient with detectable levels of transgenic sperm resulted in about 7% transgenesis of progeny. However, more reliable transmission rates will have to be determined by mating larger numbers of recipient animals to wild-type does. It is difficult to compare the success rate observed in the present study to results reported in mice because most mouse studies report only the percentage of seminiferous tubules colonized with donor germ cells, not the presence of the transgene in sperm or fertility. Previously infertile mouse recipients became fertile when more than 30% of tubules were colonized with donor germ cells [11, 12]. When recipient animals had some endogenous spermatogenesis, even colonization of less than 10% of tubules resulted in the transmission of the donor haplotype to the offspring, albeit at a low frequency, possibly because the recipient's endogenous sperm acted as carriers for the donor-derived sperm [2]. Therefore, the degree of colonization with donor cells does not have to be extensive to allow for the transgene to be transmitted to progeny [12]. After retroviral transfection of donor cells prior to transplantation, it was estimated that 9% of the sperm carried the transgene, resulting in 4.5% transgenic offspring [13]. In view of these reports, the results achieved in the present study are very promising, particularly because no attempts were made to reduce endogenous germ cells in the recipient testes or enrich the donor cell population for stem cells.
Following germ-cell transplantation, successful initiation of donor-derived spermatogenesis requires that a transplanted donor stem cell migrate from the lumen, where it is introduced, to the basal membrane of the epithelium lining the recipient seminiferous tubules. This will be facilitated if recipient spermatogenesis has been obliterated to allow thinning of the germ-cell layers; therefore, in rodents, recipients are frequently chosen from strains that inherently lack spermatogenesis or are prepared by pretreatment with cytotoxic agents to destroy endogenous spermatogenesis. In the present study, we did not use treatments to reduce endogenous germ cells in recipient animals but instead chose prepubertal goats as recipients. In prepubertal goats, there is only a single layer of germ cells and Sertoli cells in the tubules, facilitating access of transplanted donor cells to the basal membrane [14]. Furthermore, the immature testis not only exerts less resistance against the flow of cell suspension infused into the seminiferous tubules [15] but also provides a more favorable microenvironment for colonization of transplanted stem cells than the adult testis [16].
In future experiments, it might be possible to reduce competition from endogenous germ cells by depleting endogenous germ cells with selective cytotoxic treatment or irradiation of the testes prior to transplantation and to potentially enrich the donor cells for stem cells [17, 18]. This may further enhance colonization of recipient testes by transplanted donor cells and thereby increase the percentage of transgenic sperm in the ejaculate. Subsequently, transplantation of germ cells that have been genetically manipulated in vitro will provide a novel alternative for the production of transgenic sperm to be used for in vivo or in vitro fertilization. Selection and transfer of transgenic embryos [19] following in vitro fertilization can ensure a high prevalence of transgenic offspring.
For germ-cell transplantation in mice, recipients are often selected from strains that are either genetically compatible with the donor animals, are inherently immunodeficient, or have undergone immunosuppressive treatments. In contrast, recipient goats in this study were not related to the donor goats nor did they receive any immunosuppressive treatments. Although we had indications that, in pigs and goats, testis cells from unrelated donors are tolerated in immunologically competent recipients [5, 6], the present study demonstrated that not only are such transplanted cells tolerated but they also are allowed to initiate and maintain normal spermatogenesis. It is a surprising and significant finding that, at least in the immature goat, the basolateral compartment of the seminiferous tubules in the testis can tolerate allogeneic germ cells and therefore appears to represent an immunoprivileged site, a characteristic that was previously only attributed to the adluminal compartment of the seminiferous tubules. In contrast, in a recent report in mice, allogeneic transplantation of male germ cells into the testes of immunocompetent recipients failed to result in sustained spermatogenesis or fertility [3]. From a practical point of view, abolishing the need for immunosuppressive treatments or genetically matched donor and recipient animals makes germ-cell transplantation significantly more applicable to the commercial production of transgenic animals. In addition, normal ejaculates were collected from all recipient animals, indicating that the transplantation technique, involving insertion of the infusion catheter through the epididymis and testis tissue, did not result in blockage of the epididymis or obstruction of the rete testis. Examination of the testes after castration revealed none or only minor signs of fibrotic changes at the site of needle passage that obviously did not hinder the function of the testes in these animals.
Currently, a major limitation for generating transgenic domestic animals is that, unlike in the mouse, there are no embryonic stem-cell lines available for farm animal species. Until recently, the production of transgenic animals has relied almost exclusively on the microinjection technique, in which the gene of interest is introduced into the male pronucleus of the fertilized egg [20]. The emerging use of cultured somatic cells as karyoplast donors for nuclear transfer (cloning) has facilitated the germline modification of goats [21, 22]. However, the success rate of these techniques has been low, with typically only 0.5%–3% of manipulated embryos giving rise to transgenic offspring [20–22]. Furthermore, developmental abnormalities inherent in cloned embryos are frequently associated with aborted pregnancies and serious health complications after birth. Because of low efficiency and high cost of generating transgenic animals by the current methods, the application of transfected germ-cell transplantation may provide a cheaper way of introducing heterologous DNA for production of transgenic animals. In this study, the time from transplantation of germ cells to first detection of transgenic sperm in the ejaculate was about 4 mo. Therefore, by inducing transgenesis in the testes of prepubertal animals, the time required to start collecting transgenic sperm may be reduced by one generation to about one half of that required by current methods. This can significantly reduce the maintenance costs and accelerate the generation of a transgenic herd needed for large-scale production. In vitro manipulation of germline stem cells to introduce genes of interest has been achieved in mice [13, 23] and rats [24, 25] and more recently in farm animals [26]. Therefore, it is expected that transplantation of genetically modified germ cells can become a viable alternative for the production of transgenic animals in the near future. The present study represents the important first step toward achieving this goal and provides proof of the principle that germ-cell transplantation in immunocompetent animals can result in sperm production, fertility, and germline transmission of the donor haplotype to the next generation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA 19348. FAX: 610 925 8121; dobrinsk{at}vet.upenn.edu
Received: 29 April 2003.
First decision: 21 May 2003.
Accepted: 28 May 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Z. Zhang, J. Hill, M. Holland, Y. Kurihara, and K. L. Loveland Bovine Sertoli Cells Colonize and Form Tubules in Murine Hosts Following Transplantation and Grafting Procedures J Androl, July 1, 2008; 29(4): 418 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Honaramooz, S. Megee, W. Zeng, M. M. Destrempes, S. A. Overton, J. Luo, H. Galantino-Homer, M. Modelski, F. Chen, S. Blash, et al. Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation FASEB J, February 1, 2008; 22(2): 374 - 382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wyns, M. Curaba, B. Martinez-Madrid, A. Van Langendonckt, W. Francois-Xavier, and J. Donnez Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice Hum. Reprod., June 1, 2007; 22(6): 1603 - 1611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Herrid, S. Vignarajan, R. Davey, I. Dobrinski, and J. R Hill Successful transplantation of bovine testicular cells to heterologous recipients. Reproduction, October 1, 2006; 132(4): 617 - 624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Trefil, A. Micakova, J. Mucksova, J. Hejnar, M. Poplstein, M. R. Bakst, J. Kalina, and J.-P. Brillard Restoration of Spermatogenesis and Male Fertility by Transplantation of Dispersed Testicular Cells in the Chicken Biol Reprod, October 1, 2006; 75(4): 575 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Shinohara, M. Kato, M. Takehashi, J. Lee, S. Chuma, N. Nakatsuji, M. Kanatsu-Shinohara, and M. Hirabayashi Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination PNAS, September 12, 2006; 103(37): 13624 - 13628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, S. Shao, and M. L. Meistrich Irradiated Mouse Testes Efficiently Support Spermatogenesis Derived From Donor Germ Cells of Mice and Rats J Androl, May 1, 2006; 27(3): 365 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Honaramooz, E. Behboodi, C. L. Hausler, S. Blash, S. Ayres, C. Azuma, Y. Echelard, and I. Dobrinski Depletion of Endogenous Germ Cells in Male Pigs and Goats in Preparation for Germ Cell Transplantation J Androl, November 1, 2005; 26(6): 698 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Khaira, D. McLean, D. A. Ohl, and G. D. Smith Spermatogonial Stem Cell Isolation, Storage, and Transplantation J Androl, July 1, 2005; 26(4): 442 - 450. [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Oatley, A. Tibary, D. M. de Avila, J. E. Wheaton, D. J. McLean, and J. J. Reeves Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone J Anim Sci, March 1, 2005; 83(3): 604 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Shin, K. C. Lo, and L. I. Lipshultz Treatment Options for the Infertile Male With Cancer J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 48 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Orwig and S. Schlatt Cryopreservation and Transplantation of Spermatogonia and Testicular Tissue for Preservation of Male Fertility J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 51 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo Biol Reprod, October 1, 2004; 71(4): 1202 - 1207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Oatley, J. J. Reeves, and D. J. McLean Biological Activity of Cryopreserved Bovine Spermatogonial Stem Cells During In Vitro Culture Biol Reprod, September 1, 2004; 71(3): 942 - 947. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
