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Testis |
Institute of Animal Sciences,3 Swiss Federal Institute of Technology Zurich, CH-8092 Zurich, Switzerland
Department of Farm Animals4
Institute of Zoology,5 University of Zurich, CH-8057 Zurich, Switzerland
Institute of Reproductive Medicine,6 University of Munster, D-48149 Munster, Germany
Department of Animal Husbandry,7 Chulalongkorn University, Bangkok 10330, Thailand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, male reproductive technology, spermatogenesis, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Until today, most experiments in the field of germ cell transfer have been performed in mice and rats. Bovine testes have a large surface:volume ratio, a resistant lamina propria, and seminiferous tubules that are not as accessible as those in mice. Therefore, an injection technique into the rete testis optimized by use of ultrasonography was established [4]. With this new technique, germ cell transplantation in pigs was successful [5].
Spermatogonial germ cells can be enriched by magnetic cell sorting for c-kit-positive cells [6]. This method guarantees isolation of viable spermatogonia from rodent and primate testis. ß1- and 
1-integrin surface markers can be used for detecting spermatogonial stem cells with a high ability to colonize recipient testes of mice [7]. In cattle, spermatogonial enrichment is done using enzymatic digestion of 3-mo-old testes and sedimentation of single cells according to the procedure of Brinster and Zimmermann [1].
Many different techniques have been established to detect the donor-derived cells in the recipient testis. Fluorescent staining of donor cells [5], donor cells carrying the lac-Z transgene [1], morphological differences of sperm cells in the hamster-to-mouse transplantation [2], or detection of donor-derived sperm by species-specific polyclonal antibodies with immunohistochemistry [8] have been described. An alternative approach is extracting genomic DNA at different time points after transplantation and then amplifying the DNA by polymerase chain reaction (PCR) with species-specific primers.
In the present study, a Klinefelter bull was used as a recipient of donor cells, because this bull had no endogenous spermatogenesis. The Klinefelter bull was found through routine chromosomal examination by investigating its mother as a cow with sex-chromosome trisomy (XXX). Several examples of viable sex-chromosome trisomics exist in cattle. Two types of Klinefelter syndrome are known: the nonmosaic Klinefelter (XXY), which is caused by nondisjunction in meiosis, and the mosaic Klinefelter (XY/XXY), which arises through nondisjunction in mitotic division. The typical phenotype is azoospermia for a nonmosaic Klinefelter and oligospermia for a mosaic Klinefelter. Most Klinefelters also have a low testosterone level, and their LH and FSH levels are higher than normal [3]. To quantify the ratio of donor to recipient cells in the biopsy specimens or ejaculates, we employed an allele-specific, quantitative PCR using a genetic mutation, which is associated with the phenotype of coat color and, therefore, with the breed [9]. The molecular approach based on establishing a quantitative PCR with allele discrimination was used to quantify the ratio of donor or recipient cells in the biopsy specimens or ejaculates. Analyzing a weekly ejaculate, we discovered a time-dependent course of development for the donor-derived cells. To detect the stage of the cells, the ejaculates were analyzed under the microscope.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatogonial enrichment was done according to the method of Brinster and Zimmermann [1]. Small pieces of the testis from slaughtered bulls were cut away with fine scissors. The pieces of testis were incubated with collagenase type 1 (1 mg/ml) and DNase (5 µg/ml) in Dulbecco modified Eagle medium (DMEM; Invitrogen, San Diego, CA) supplemented with antibiotic and antimycotic (ABAM; Invitrogen) for 5 min at 37°C in a shaking water bath (90 cycles/min). During digestion, the pieces of tissue were mixed six times by repetitive pipetting with a 10-ml pipette. The sample was sedimented for 2 min, the supernatant discarded, and a second incubation done with fresh medium supplemented as described above and with hyaluronidase type 1 S (0.5 mg/ml) for 20 min. Every 5 min, the sample was flushed with medium using a 10-ml pipette. The cells were centrifuged for 5 min at 500 x g and then resuspended in PBS (Seromed, Berlin, Germany). The enzymatic digestion and the enrichment of stem cells were controlled under the microscope by searching for cell clusters and spermatogonial stem cell-like cells. The cell solution was accepted for transfer if 80% of the cells were single and 80% of the cells showed spermatogonial stem cell morphology.
Before transplantation, ultrasound-guided puncture was practiced with 40 testes obtained from a slaughterhouse. A trypan blue solution was injected, and a section was performed immediately to control the distribution of the solution. When the Klinefelter bull was 12 mo old, the cell solutions from two slaughtered bulls were injected. The suspension of 5 ml (
5 million cells) was successfully injected directly into the rete testis controlled by ultrasound and by the low pressure used for injection. Postoperatively, the bull was treated with antibiotics and analgesics for 3 days (Permission 36/2002; Katonales Veterinäramt, Zürich, Switzerland).
Two weeks after germ cell transfer, the first biopsy was performed. The testis biopsy specimens were taken under sedation and local anesthesia. The bull was placed in lateral recumbency, and two random biopsies were taken with an automatic, single-use biopsy device (BARD MaxCore; Bard, Covington, GA). A small stab incision was made in the scrotal skin, and a 20-gauge, side-notch needle was pushed into the testicular parenchyma. After shooting the needle into the parenchyma, the needle was removed from the testis, and the tissue sample was immediately placed in the culture medium. Testicular biopsies were performed three times, every other week, from each testis. Thereafter, ejaculates were collected weekly with an artificial vagina.
For testing the endocrinological function of the testis, the GnRH analog, busereline acetate (Receptal; Veterinaria AG, Zurich, Switzerland), was given intravenously, and blood samples were taken every 30 min before (n = 3) and after (n = 7) GnRH application. This test was performed before and 1 mo after hormone treatment. To stimulate spermatogenesis, gonadorelin acetate (Lutrelef; Ferring AG, Wallisellen, Switzerland) was administrated subcutaneously through a battery-operated minipump (Zyklomat pulse; Ferring AG). The hormone (10 µg of gonadorelin) was given every 2 h over a period of 2 mo. Blood plasma testosterone quantification was done with the testosterone direct radioimmunoassay kit TESTO-CTK (P3090; DiaSorin, Saluggia, Italy) in duplicate.
Chromosome Preparation
Leukocytes of peripheral blood were cultured in RPMI 1640 growth medium (Seromed, Berlin, Germany) supplemented with 15% fetal calf serum, 2 mM L-glutamine, 0.8 µg/ml of pokeweed, and 1% ABAM for 3 days at 37°C. Six hours before harvesting the cells, 100 µg/ml of bromodeoxyuridine were added. Forty-five minutes before harvesting, 0.025 µg/ml of Colcemid (Seromed) was added. After a hypotonic treatment of 0.075 M KCl, the cells were washed in fixative (acetic acid and methanol, 1:3) and dropped on clean slides. Air-dried slides were dipped into a 0.2% acridine orange solution for 45 sec and then rinsed with water. The slides for G banding were dipped in 0.025% trypsin solution for 2 min, briefly rinsed with water, and stained with Giemsa solution. They were mounted with Sørensen solution (pH 6.8) and observed under ultraviolet light on an Axioplan II microscope (Zeiss, Jena, Germany). Meiotic preparations were performed in the same way, starting with hypotonic treatment.
Fragment Length Analysis
The restriction fragment length polymorphism (RFLP) test for a single nucleotide mutation in the MSHR gene was used to genotype both donor and recipient [10]. Red animals, like the recipient bull, normally have a deletion of one of the two Gs at position 310 or 311 compared to the wild-type allele, as in the donor cells. The PCR was done on a thermal cycler (Hybaid, Teddington, UK) in a reaction volume of 25 µl containing 50–100 ng of genomic DNA, 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2), 0.2 mM dNTP, 0.4 µM each of Fam-labeled forward (P6 in Fig. 3) and reverse primer (P12 in Fig. 3), and 2.5 U of Taq DNA Polymerase (Expand TM High Fidelity PCR System; Amersham Pharmacia Biotech, Buckinghamshire, U.K.). After an initial denaturation at 95°C for 5 min, the PCR profile consisted of a denaturation step at 95°C for 30 sec, an annealing step at 55°C for 30 sec, and an elongation step at 72°C for 30 sec for a total of 30 cycles, followed by a final extension at 72°C for 7 min. Ten microliters of the PCR reaction were digested with 5 U of MSP1 restriction enzyme for 2 h in a 37°C water bath. With the ABI PRISM 337 DNA Sequencer (Applied Biosystems, Foster City, CA), the DNA fragments were analyzed using the GeneScan Analysis Software (Applied Biosystems).
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Quantitative PCR
For all DNA extractions, the High Pure PCR Template Preparation Kit (Roche Applied Science, Basel, Switzerland) was used. The DNA was extracted either from blood, germ cells, or muscle tissue. The DNA from blood (200 µl) and tissue (25–50 mg) was extracted following the protocol of the kit. Because no sperm cells were in the ejaculates, DNA from ejaculates was extracted following the protocol of the same kit for blood. The concentration of the extracted DNA was determined by measuring the light absorption of the DNA using a photospectrometer (Lambda Bio UV/Vis; Perkin Elmer, Foster City, CA). Real-time PCR was done in a thermal cycler ABI Prism 7700 Sequence Detector (Applied Biosystems) using Micro Amp optical tubes (Applied Biosystems). The PCR was carried out in a reaction volume of 25 µl containing 100 ng of DNA, 300 nM of forward and reverse primer, 250 nM TaqMan probe, and 12.5 µl of Universal PCR Master Mix (Applied Biosystems) with AmpErase Uracil N-glycosylase (UNG). The following parameters were used: 2 min at 50°C (for optimal AmpErase UNG enzyme activity), 10 min at 95°C (for activation AmpliTaq Gold DNA Polymerase), followed by 40 cycles of denaturation for 15 sec at 95°C and annealing and extension for 1 min at 60°C. The DNA template was diluted 1:10, 1:100, and 1:1000 to test the stability of the system. A system was defined as stable when the differences between cycle of threshold (Ct) values of a 1:10 dilution and the Ct of the undiluted samples were more than 2.65 (80% of an exponential amplification). The two ejaculates from the same day were pooled and analyzed together. Three runs were carried out for each DNA sample of three different extractions from the pooled ejaculate. From the nine results of the pooled ejaculate, the mean was calculated, and the values that differed more than one Ct from the mean were eliminated. With the remaining values, the average was calculated again. The ratio of the donor cells to the recipient was calculated under the assumption that the Ct values are from the range in which the PCRs are in a complete exponential phase.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of two X chromosomes and, therefore, 61 chromosomes in all metaphases from the peripheral blood proved that the recipient bull was a Klinefelter (Fig. 1A). The metaphase spread of the bull's dam showed 61 chromosomes, with the presence of three X chromosomes (Fig. 1B). To control the segregation of the X chromosome, nine microsatellite markers on bovine X chromosome were typed for the bull, the bull's parents, and the bull's maternal grandparents. The mother has three different alleles of marker BMS911, and the bull has two alleles with the same length as in the mother (Fig. 2). The marker analysis confirms that the bull received two X chromosomes from the mother. Therefore, all the bull's cells contained three sex chromosomes, and the bull is a real nonmosaic Klinefelter.
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In terms of the hormone stimulation test, we found that the blood testosterone concentrations of the three samples taken before Busereline acetate stimulation, were between 0.8 and 1.8 ng/ml. Thirty minutes after stimulation, an increase in testosterone level took place. After both stimulations, the highest peak was after 2 h. The highest values were between 3.8 and 5 ng/ml. The values of the random sample, taken around every 2 wk over the course of 5 mo, were found to be between 0.5 and 4.7 ng/ml. These values are typical for a reaction after a stimulation and for a normal episodic release in an adult bull. Hypogonadism was phenotypically noticeable, and in a histological examination of the testes, hyalinizing fibrosis of the seminiferous tubules was detected.
Donor Cells Detected
The donor bulls belong to the Brown Swiss breed and the recipient to the Simmental breed; this allowed us to use a known genetic mutation for an RFLP assay [10]. With fragments of 203 base pairs (bp), wild-type cells (donor) can be identified (see Fig. 4). With a peak at 240 bp, the cells can be identified as being from the recipient. All biopsy specimens had the two fragments (203 and 240 bp). Therefore, in all the biopsy specimens, cells from the donor as well as cells from the recipient are found. The cell ratios were not quantified with this method, however, because repeatability was low.
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For quantification, an allele-specific, real-time PCR was performed. The best primer for exclusive donor-specific amplification and high amplification efficiency was the reverse primer with the allele-specific base pair at the 3' end and a mismatch at position -5 from the 3' end (p25-5 in Fig. 3). The best amplification of DNA from the recipient bull and no amplification of donor DNA were achieved with reverse primer p26-5 (allele-specific base pair at the 3' end and mismatch at the fifth position) (Fig. 3). According to the result from this experiment, p25-5 and p26-5 were selected as being specific and the most efficient primers for wild-type and donor-specific amplifications, respectively.
The stable range of the PCR was defined using the DNA dilutions. With recipient DNA, the Ct differences between the four dilution steps were more than 2.6 (Fig. 5A). Therefore, the system is stable between Ct 25.7 and Ct 36.0 for the recipient DNA. The differences between the Ct values from undiluted and 1:10-diluted and between 1:10- and 1:100-diluted DNA of donor cells were 2.85 and 2.9, respectively. The difference between the Ct values from 1:100- and 1:1000-diluted DNA of donor cells was only 1.8, and the Ct values of the 1:1000 dilution showed differences up to 1.9. These two results indicated that the system is not stable in this range. Therefore, the stable range of donor DNA amplification was between Ct 26.6 and Ct 32.4.
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In the first 6 wk after germ cell transplantation, biopsy samples were taken. The highest fraction (30.3% of donor cells) was present in a sample taken 4 wk after germ cell transplantation (Fig. 6). The ejaculate analysis started 2 mo after germ cell transplantation. For 4 mo, donor cells were present in the ejaculates. The fraction of donor cells fluctuated from 5% to 67% (Fig. 6). Twenty-three weeks after germ cell transfer, donor cells were no longer present. Ejaculate analysis was carried out until 9 mo after germ cell transfer, even if donor cells were no longer present. Sperm cells were never present in the ejaculate. According to morphological criteria, the cells present in the ejaculate were epithelial cells, medusa formations, and germ cells. After the recipient was slaughtered, conventional meiotic preparation of the testis was performed. No nuclei in stages of meiotic division were observed in the preparations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In terms of donor cells, as we noted in Results, no donor cells were present in the ejaculate 6 mo after germ cell transplantation. The amount of donor cells did not decrease slowly. The rejection after 6 mo suggests that the donor cells could not stably incorporate in the testis and could not undergo spermatogenesis. If the rejection was caused by immunological reasons, then rejection would have happened earlier. This suggests that no immunological reaction occurred, which has already been suggested in other studies [1, 2, 5, 10].
Another important finding of the present study is that the 6 mo during which the germ cells were present in the ejaculate demonstrate that the transfer technique of ultrasound-guided puncture of rete testis could be adapted for living cattle. With this technique, it is already possible to colonize 50% of seminiferous tubules from premature pigs with donor germ cells. In this case, donor cells were present up to 4 weeks in the tubules [5]. It is difficult to compare the results from the premature pig study to those of the present study, because the analysis of donor cells was done in the testis and not in the ejaculates. In the present study, germ cells, medusa formations, and epithelial cells were present in the ejaculates; no sperm cells were seen. Furthermore, neither stages of meiotic division nor sperm cells were found on meiotic preparations. This showed that despite hormone therapy, neither recipient nor donor germ cells could ever undergo spermatogenesis.
It is also significant that the results of these experiments excluded some causes of infertility of the Klinefelter recipient. The endocrine function was comparable to that of a normal XY bull. However, the Leydig cells were functionally normal, and the germ cells cannot be the only cause for infertility. Thus, these results challenge previous work that argued Klinefelters have an altered endocrine function [11]. The microenvironment was not able to support spermatogenesis. In the literature, all nonmosaic Klinefelters show azoospermia. Very rarely, sperm can be observed, and the literature reports some exceptional cases of spontaneous paternity [12]. Sperm from a patient with hypergonadotropic nonmosaic Klinefelter syndrome, when used for intracytoplasmic sperm injection, can lead to a pregnancy [13]. These two examples show that germ cells from a Klinefelter can lead to fertilization of an oocyte. A further attempt to get a donor-derived spermatogenesis in a nonmosaic Klinefelter bull is the simultaneous transplantation of Sertoli cells [14]; an alternative recipient could be a mosaic Klinefelter. In a mosaic Klinefelter, the subfertility indicates that the endogenous spermatogenesis is, to a certain extent, functional. If fertility is only reduced slightly, problems could arise where the endogenous spermatogenesis could displace the donor-derived germ cells.
Another candidate recipient for germ cells is a bull with Sertoli cell-only (SCO) syndrome. Two types of SCO syndrome are known: complete germ cell aplasia and focal SCO. The complete germ cell aplasia has a characteristic histology in which tubules are reduced and only contain Sertoli cells (and no other cells) involved in spermatogenesis. The focal SCO syndrome, with a percentage of tubules containing germ cells but with limited spermatogenesis, is a model for the recipient in the present study. Alternatively, Khainag, a sterile hybrid between cattle and yak, could be considered as a model. Female Khainags are fertile, and male Khainags are sterile. Reduced numbers of spermatogonia were reported in the testicular tubules of the Khainag, and despite the identical chromosome number of the two parental karyotypes, synaptic anomalies were described at meiotic prophase in primary spermatocytes [15].
A further approach could be the use of RNA interference. The idea is to insert homologous, double-stranded RNA and disrupt germline development by targeting a specific gene. A possibility would be to inject double-stranded RNA in the time frame when germ cells start to migrate to prevent their migration into the genital ridge. In this way, an animal without germ cells will be generated. Thus, further studies on these models will lead to the most suitable model for donor-derived spermatogenesis in cattle.
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FOOTNOTES |
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2 Correspondence: Hannes Joerg, Institute of Animal Sciences, ETH Zurich, Tannenstr. 1, CH-8092 Zurich, Switzerland. FAX: 41 1 632 11 67; hannes.joerg{at}inw.agrl.ethz.ch
Received: 17 June 2003.
First decision: 14 July 2003.
Accepted: 31 July 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA 1999 96:5504-5509This article has been cited by other articles:
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  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]
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 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]
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 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]
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