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Testis |
Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous reports of successful sperm production in ectopic grafting, the use of neonatal donors and castration of recipient mice were necessary [1–3]. This success was attributed to an immature testicular cell population, consisting of gonocytes as the only germ cells present and a recipient host environment that resembled a neonatal phase in terms of the concentrations of circulating hormones. In contrast with published reports with several other species, the only report using the bovine as a donor has shown the capability of germ cell differentiation using a 19-wk-old prepubertal donor animal and intact recipient nude mouse host [4].
In the bovine testis, postnatal developmental assembly of the seminiferous epithelium and establishment of spermatogenesis occur over a period of months. Postnatally, at 2 wk of age, gonocytes are found within the center of seminiferous cords and have begun migration to the basement membrane [5]. At 4 wk of age, the majority of gonocytes have completely migrated, and differentiating spermatogonia first appear at 12 wk of age [5, 6]. Within this 4- to 12-wk period, gonocyte conversion to spermatogonial stem cells and the establishment of stem cell niches must occur. It is not until 16 wk of age that meiotic spermatocytes first appear in the seminiferous epithelium, and elongating spermatids can be seen at 28 wk [6]. Access to a germ cell population enriched for undifferentiated spermatogonial stem cells is beneficial for genetic manipulation of male germ cells because one genetically altered spermatogonial stem cell could yield thousands of transgenic spermatozoa [7]. At 19 wk of age in bulls, differentiated spermatogonia are clearly present along with primary spermatocytes and the ratio of differentiated germ cells to spermatogonial stem cells is higher compared with earlier age points [6]. Therefore, use of neonatal tissue has the advantage of containing fewer differentiated germ cells and a more enriched spermatogonial stem cell population.
Previous attempts to transduce germ cells in the mammalian testis with a transgene have been met with challenges [8–12]. Electroporation of mice testes following injection of a transgene-containing vector into the seminiferous tubules has resulted in somatic Sertoli and Leydig cell expression or transient germ cell expression [12]. Direct transfection of spermatozoa in vitro often results in transient rather than stable chromosomal incorporation [8, 9]. The most successful method to date for stable transduction of male germ cells has been through the use of retroviral infections of rodent spermatogonial stem cells [13– 15]. This technique has resulted in the in vivo production of transgenic spermatozoa that are capable of fertilization and vertical transmission of the transgene to offspring [15]. Production of transgenic spermatozoa using retroviral transfection of spermatogonial stem cells has thus far required the use of a spermatogonial transplantation technique, which at this time is only applicable to rodents and is limited in domestic livestock.
The objectives of this study were to evaluate the development of spermatogenesis in ectopically grafted neonatal bovine testicular tissue and investigate the utility of using electroporation to stably transfect spermatogonial stem cells within grafts with a foreign ß-galactosidase transgene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Washington State University Animal Care and Use Committee approved all animal procedures. Donor testicular parenchyma was collected from bulls at 4 wk of postnatal age (n = 6). Sections of donor testicular parenchyma approximately 10 mg in weight were collected into Hanks balanced salt solution (HBSS) and kept on ice until grafting. Six pieces of donor tissue were ectopically grafted onto the backs of each castrated adult immunodeficient recipient nude mouse (n = 18 mice/donor) (Taconic, Germantown, NY). Recipient mice were anesthetized with a combination of ketamine (0.1 mg/kg body weight [bw]) and xylazine (0.05 mg/kg bw) in sterile physiological saline. Mice were castrated before small incisions were made through the skin on the backs of the animal where the muscle was scored in individual areas, and six 10-mg pieces of donor bull testis were placed individually within the scored muscle areas of each recipient mouse. The incisions were closed with suture and the animals allowed to recover.
Electroporation
Prior to grafting, three of the six pieces of donor testicular tissue were electroporated with a linearized ß-galactosidase expression vector. The construct was created by restriction enzyme digestion and ligation of a 4.5-kilobase fragment of LacZ into a pMSCV vector (BD Biosciences Clontech, Palo Alto, CA). The vector was suspended at a concentration of 1 µg/µl in Opti MEM (100 µl) and pipetted into the center of each piece of testicular tissue. Tweezer trodes were used to electroporate each of the three pieces with one of three different voltages, 25, 50, or 75 mV (BTX Harvard Apparatus, Holliston, MA). The tissues were then placed on ice in HBSS until the time of grafting. Testicular tissue from the donor testis before grafting (T0) was used as a representation of the starting material.
Donor Graft Analysis
Recipient mice were killed at 1, 4, 12, or 24 wk after grafting by CO2 inhalation followed by cervical dislocation (n = 9 mice/time point). Grafts were removed, fixed in Bouin solution for 4 h at 4°C and subsequently dehydrated and stored at 4°C in 70% ethanol. Each donor graft was weighed individually following fixation and dehydration. Samples were then embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin. Electroporated grafts were fixed in 4% paraformaldehyde for 2 h at 4°C followed by incubation with X-Gal to detect any ß-galactosidase expression. Samples were subsequently dehydrated, embedded in paraffin, sectioned at 10 µm, and counterstained with eosin.
Samples were evaluated using light microscopy and digital images captured with a CoolSnap digital camera (Media Cybernetics, Silver Spring, MD). Analysis of nonelectroporated cross-sections included assessment of the average seminiferous tubule diameter, which was calculated by measuring 20 round tubules per sample at each time point using a slide micrometer as a reference. The average percentage of tubules containing spermatogonia, pachytene spermatocytes, round spermatids, and elongating spermatids was also calculated for each nonelectroporated sample by counting the total number of round tubules/three microscopic fields and the number of round tubules containing each germ cell type within the same three fields. Analysis of electroporated samples included assessment of the average number of seminiferous tubules with ß-galactosidase expression, which was calculated by counting the number of tubules/three microscopic fields and the number of tubules containing any blue staining within these fields. The average number of tubules with germ cell ß-galactosidase expression was also determined by counting the number of tubules with blue-stained germ cells within the three microscopic fields.
Recipient Mouse Analysis
Blood was collected from recipient mice at the time of sacrifice by cardiac puncture and serum was subsequently collected by centrifugation. Two populations of control mice were also used, intact adult males and adult males castrated 2 days before sacrifice. Serum samples were then analyzed for testosterone concentration using a commercial kit (DSL-400; Diagnostic Systems Laboratory Inc., Webster, TX).
Statistical Analyses
Data were analyzed using the SAS systems software (SAS Institute, Cary, NC) with the Proc GLM function. Differences between means were determined for tubule diameter, graft weight, gonocyte migration, percentage of tubules with different germ cell types, recipient testosterone concentration, percentage of tubules with blue staining, and percentage of tubules with blue-stained germ cells using Duncan test for significance. Data were considered significantly different at P 
0.05. In all figures, data are presented as the mean ± SEM and bars with different letters are significantly different.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine testis tissue grafts were weighed at each time point after removal from nude mice to provide an indication of the proliferation of testicular somatic cells and progression of spermatogenesis in the bovine tissue. Average graft weight showed a significant increase in a time-dependent manner beginning at 4 wk after grafting (Fig. 1A). The average weight of 1-wk grafts compared with T0 was not different (P 
0.05), whereas the average weight of grafts at 4 and 12 wk were significantly (P 0.05) higher than T0 and 1 wk but not different from each other. Graft weights at 24 wk were significantly higher than at any other time point. Seminiferous tubule diameter was measured to assess Sertoli and germ cell proliferation. Average seminiferous tubule diameter showed a significant increase with increasing time after grafting, beginning between 1 and 4 wk (Fig. 1B).
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Bovine testicular tissue removed at 1, 4, 12, and 24 wk after grafting was examined histologically to evaluate the establishment of spermatogenesis. Tubular morphology was maintained in all grafts throughout the 24-wk grafting period (Fig. 2). At T0, the tubules resembled seminiferous cords with apparent gonocytes in the center of the cords (Fig. 2A). Testis tissue grafts 1 wk after grafting had seminiferous tubules very similar in diameter to T0 (Fig. 1B). However, the percentage of tubules with complete gonocyte migration to the basement membrane was significantly increased in 1-wk grafted tissue compared with T0 (Fig. 3). The majority of cords at T0 had gonocytes still present in the center of the cords, whereas at 1 wk after grafting, gonocyte migration to the basement membrane was complete in the majority of cords (Fig. 3).
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Bovine testis tissue 4 wk after grafting was characterized by a germ cell population predominated by gonocytes without the presence of any differentiated germ cell types (Fig. 2C, Table 1). Transition of gonocytes to spermatogonia was apparent at 12 wk after grafting and the structurally intact seminiferous tubules were present (Fig. 2D). Meiotic germ cell development, as determined by the presence of pachytene spermatocytes, was not present until 24 wk after grafting (Fig. 2E, Table 1). At 24 wk after grafting, the germ cell population consisted of spermatogonia, pachytene spermatocytes, round spermatids, and elongate spermatids (Fig. 4, Table 1). The average percentage of tubules containing pachytene spermatocytes was 49.9%, whereas 92.9% of the total tubules contained spermatogonia (Fig. 5). Germ cell progression to the round spermatid stage was seen in an average of 18.3% of the tubules (Fig. 5). The average percentage of tubules containing elongate spermatids was low, comprising only 10.9% of the total tubules (Fig. 5). The appearance of tubules containing Sertoli cells only was evident at 24 wk after grafting (Fig. 2F), comprising 7.1% of the total tubules (Fig. 5).
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Recipient Mouse Testosterone Analysis
Serum testosterone concentration was measured to evaluate the Leydig cell function in grafted bovine testicular tissues. Testosterone concentration was significantly increased in recipient serum at 4, 12, and 24 wk after grafting compared with 1 wk (Fig. 1C). The serum concentrations of testosterone at 4, 12, and 24 wk after grafting were at physiological concentrations consistent with what was present in intact adult male mouse controls (1–4 ng/ml). Serum from control male mice 2 days after castration had a testosterone concentration below the low standard of the assay (<0.05 ng/ml).
Electroporated Graft Analysis
Donor bovine testicular tissue was electroporated with a vector containing the ß-galactosidase gene before grafting to investigate the ability to stably transfect spermatogonial stem cells with a transgene and possibly generate transgenic differentiated germ cells. Because the efficiency of electroporation to transduce a cell can be variable at different voltages, three different voltages of 25, 50, and 75 mV were tested. All grafts removed at 4, 12, and 24 wk after grafting showed positive staining for ß-glactosidase (Fig. 6A). Based on whole-mount observations of the grafts, expression appeared to be seminiferous tubule specific and no discernible differences could be detected between the different voltages before histological analysis. Because the most significant germ cell development was not observed until 24 wk after grafting, only those results will be presented. Cross-sections of 24-wk electroporated grafts showed seminiferous tubule-specific ß-galactosidase expression within both Sertoli and germ cells, whereas no interstitial cell expression was observed (Fig. 6, B–D). The 50-mV tissues showed a significantly higher average number of tubules with ß-galactosidase expression than the 25- and 75-mV tissues (Fig. 7A). Likewise, the 50-mV tissues had a significantly higher number of tubules with germ cell ß-galactosidase expression compared with the other voltages (Fig. 7B). The 25- and 75-mV tissues were significantly different from the 50-mV tissues based on both the average number of tubules with any ß-galactosidase expression and average number of tubules with germ cell expression. Transgene expression was observed in differentiated germ cells within the 25- and 50-mV electroporated tissues (Fig. 6). The most advanced germ cell type observed in the 75-mV electroporated tissues were spermatogonia and nearly all ß-galactosidase expression was within Sertoli cells. It appeared that the 75-mV electroporation had a negative effect on germ cell development within the grafts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Survival and development of grafted testis tissue was monitored by histological analysis and measuring the weight of grafts from 1 to 24 wk after transplantation. A significant increase in tissue weight occurred 4 wk after grafting. The weight increase and histological examination of tissue at 4 wk indicted somatic cell proliferation. In addition, graft weight increased throughout the 24-wk period, including a significant increase between 12 and 24 wk after grafting. Tubule diameter was also significantly different between 12 and 24 wk after grafting, which was most likely due to germ cell development and changes in Sertoli cell morphology. It is well established that Sertoli cell proliferation in the testis occurs neonatally in a variety of mammalian species. In rats, Sertoli cell proliferation occurs at the start of seminiferous cord formation and ceases at approximately Day 16 of postnatal life with first appearance of meiotic cells [16, 17]. In bulls, the time frame of postnatal Sertoli cell proliferation is not firmly established, but the data available indicate that it ceases around 28 wk of postnatal age [6]. Previous reports have demonstrated changes in Sertoli cell size and morphology with advancing germ cell development in the rat testis [18, 19]. Graft weight and seminiferous tubule diameter data collected in this study demonstrate growth and proliferation of neonatal bovine testicular tissue and cells when ectopically xenografted onto recipient mice in a pattern that is similar to normal maturation of the intact bull calf testis.
The average percentage of total tubules before grafting at T0 (4-wk-old donor) that contained gonocytes in the center of the seminiferous cords was 62.9%, whereas only 37.1% of the cords had complete gonocyte migration. In contrast, at 1 wk after grafting, comparable with a 5-wk-old bull calf, only 20.1% of cords had gonocytes still present in the center and 79.9% had complete migration. In the developing postnatal mammalian testis, gonocytes migrate from the center of seminiferous cords to the basement membrane for the differentiation to spermatogonia or spermatogonial stem cells and the establishment of stem cell niches [20]. In the mouse testis, this occurs from Postnatal Days 0–5 [21, 22]; however, in the bovine, this process is much longer. Gonocyte migration begins at 2 wk of postnatal life and is complete by 8 wk in the bovine testis [5]. Within the window of 2–12 wk of postnatal life, spermatogonial stem cell conversion and niche assembly must occur. The data in this study indicate that gonocyte migration is accelerated in grafted neonatal bovine testicular tissue. This initial period during 1 wk after grafting could potentially be used for manipulation to increase the efficiency of spermatogonial stem cell conversion and niche assembly, which may correlate to increased and accelerated sperm production in grafted testicular tissues.
In this study, castrated adult nude mice were used as recipients to provide an environment for the neonatal donor testicular tissue that was deficient in testosterone. In an in vivo situation, testosterone concentration in the serum of a neonatal male is low and does not become elevated until later in germ cell development. Therefore, providing grafted neonatal donor testicular tissue with an environment that has low testosterone concentration after grafting has similarities to the neonatal period and may be beneficial for establishment of that tissue in a recipient host. Likewise, spermatogonial stem cell functionality has been shown to be enhanced in a low testosterone environment [23, 24]. Therefore, for gonocyte-to-stem cell conversion and niche assembly to occur in grafted neonatal testicular tissue, a low testosterone environment may be essential. In a previous report, differentiated germ cell production occurred using 19-wk-old pubertal bulls as donors and intact recipient mice [4]. This approach thus provided a recipient host environment that had high concentrations of circulating testosterone, which mimicked the environment present in an intact bull at the same developmental age. In the work reported here, testosterone concentration in the serum of castrated recipient mice 4–24 wk after grafting was found to be at a level similar to intact adult male mouse controls (1– 4 ng/ml), demonstrating vascularization and functionality of the donor bovine Leydig cells. However, at 1 wk after grafting, the recipient testosterone concentration was low (0.2 ± 0.04 ng/ml), possibly due to inability of the immature Leydig cells to initially respond to LH. In the intact bull calf, testosterone concentration in the serum begins to rise at 18 wk of age [25]. Our data suggest that Leydig cell differentiation is accelerated in grafted testis tissue. It is also possible that the high LH concentration in recipient mouse serum initiated functionality of the bovine Leydig cells earlier than would be normally seen in an intact bull at the same developmental stage.
In vivo, meiotic germ cells are first apparent in the developing bovine testis at 16 wk of age [6]. In this study, pachytene spermatocytes were first seen in the grafted testicular tissue at 24 wk after grafting, which is comparable with a 28-wk-old intact bull calf testis. These data indicate that spermatogenesis is not accelerated to the point of meiotic germ cell development when using a 4-wk-old donor bull. However, the exact time point between 12 and 24 wk after grafting when meiotic cells were present cannot be determined from this study. At 24 wk after grafting, the average percentage of the total tubules that contained pachytene spermatocytes was only 49.9%, unlike in the in vivo situation, where a much higher percentage of tubules containing meiotic germ cells at a comparable developmental age would be expected. No acceleration of spermatogenesis in the bovine testis is of practical importance when using bovine sperm generated from grafts. The time to remove the graft to obtain the maximal number of sperm needs to be determined, but our research suggests a minimum of 24 wk is required.
The presence of elongating spermatids was used to indicate production of differentiated germ cells in the grafted bovine testicular tissues. Spermatids were observed at 24 wk after grafting, which was comparable with a 28-wk-old intact bull. In an intact bull, elongating spermatids are not seen until 28-wk-of-age [6]. Only an average of 11% of the total tubules contained elongate spermatids at 24 wk after grafting, indicating a low number of tubules had supported germ cell differentiation in grafted neonatal bovine testicular tissue. In contrast with reports of accelerated germ cell development in ectopically grafted neonatal mouse, pig, goat, and primate testis, no comparable acceleration of germ cell differentiation was seen with neonatal bovine testis in this study.
This is the first report of transducing spermatogonial stem cells of bulls with a foreign ß-galactosidase transgene through electroporation of neonatal bovine testis followed by grafting onto recipient mice for further growth, maturation, and evaluation. This technique could potentially be an alternative to sperm-mediated transgenesis and pronuclear injection because it could result in a high success rate of stable transgene chromosomal incorporation. The multiple mitotic and meiotic cell divisions and checkpoints that a male germ cell undergoes before becoming mature spermatozoa would ensure that expression of a transgene in spermatozoa would likely be stable if it was incorporated at the spermatogonial stem cell stage. With sperm-mediated delivery of transgenes into oocytes, the success of stable chromosomal incorporation is low, usually resulting in a mean range of 0–8% of the embryos expressing the transgene [26–28]. When using methods applied to the embryo, such as pronuclear injection or embryonic stem cell manipulation, the success rate is also low, at 1–8% [29–31]. Coinjection of pretreated mouse sperm with a reporter gene into oocytes has been more successful, resulting in 64–94% of embryos expressing the transgene, but only an average of 40% of the offspring expressed the transgene [32]. Use of this method is not optimal for stable transgene insertion because the first developmental checkpoint occurs after fertilization, where the expression of transgenes with unstable incorporation are suppressed later in development or embryo viability is lost. If successful, these methods are still limited to the number of stably modified offspring they could produce because the dependent factor is the embryo, which most likely would only result in one offspring. The potential exists to use genetic modification of germ cells in grafted testicular tissue along with intracytoplasmic sperm injection (ICSI) to generate substantial numbers of transgenic offspring. Genetic manipulation of one spermatogonial stem cell in grafted testicular tissue has the potential to yield hundreds of stably modified spermatozoa for use in ICSI. Thus, the efficiency of generating transgenic embryos could be enhanced compared with current methods of sperm-mediated transgenesis, pronuclear injection, or coinjection of pretreated sperm and transgene DNA.
Over the 24-wk period, electroporated grafts grew and developed in a similar pattern to the nonelectroporated grafts. A significant observation in this study was the localized expression of ß-galactosidase to the seminiferous tubules without interstitial cell expression. The pMSCV-ß-galactosidase vector used in this study contains retroviral murine stem cell virus (MSCV) LTR regions that are designed for high stem cell expression. In order for chromosomal integration to occur following electroporation, a cell must be mitotically active. Histological evaluation of nonelectroporated grafts indicates that interstitial cells are not mitotically active shortly after grafting. Therefore, Sertoli and germ cell mitosis shortly after grafting could explain the tubule-specific incorporation of the vector.
At 4 wk in the bovine male, gonocytes or spermatogonial stem cells are the only germ cell types histologically detectable in the testis. Therefore, these data demonstrate the ability to transduce spermatogonial stem cells with a ß-galactosidase transgene for later expression in differentiated germ cell types 24 wk later. Based on the observation of differentiated germ cell expression of the transgene, both 25 and 50 mV were capable of stable transduction of the donor spermatogonial stem cells. In contrast, differentiated germ cells were not observed in any of the 75-mV tissues. Based on the average percentage of total tubules with germ cell expression of the transgene, 50 mV was significantly better than the 25- or 75-mV samples. These data indicate that a range between 25 and 50 mV is the most efficient means for transfection of spermatogonial stem cells with a foreign transgene in neonatal bovine testicular tissue by electroporation.
As an application tool in the livestock industry, ectopic testicular grafting could be used as part of a novel technique for generation of transgenic livestock. Genetic manipulation of undifferentiated germ cells in donor testicular tissue before grafting has the potential to result in production of transgenic spermatozoa that could be used for in vitro fertilization (IVF) or ICSI to generate transgenic embryos. As concern over food safety increases, interest in transgenic livestock animal production has been suggested as a means for production of food animals resistant to particular pathogens that result in harmful diseases. As a research tool, genetic manipulation of germ or somatic cells in donor testicular tissue before grafting has the potential for use in increasing the understanding of spermatogenesis at the molecular level in gene-knockout and overexpression studies. The systems demonstrated in this study can now be tested for their ability to produce transgenic spermatozoa that can be used for IVF or ICSI as an alternative means of generating transgenic embryos.
In this study, fully developed spermatozoa were not observed, but elongated spermatids were present. Allowing the donor testis more time after grafting could yield fully developed sperm capable of fertilization using ICSI. Even with the production of spermatids in the grafted tissue, oocyte fertilization could occur through procedures such as round spermatid injection [33]. Manipulation of the donor tissue or the recipient animal could enhance the efficacy of this procedure by increasing the percentage of tubules in which complete spermatogenesis occurs or by accelerating and enhancing germ cell development in tubules. Transgenic animal production has many applications in the livestock industries, and the generation of transgenic animals may be accelerated through the male germ line stem cell.
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FOOTNOTES |
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2 Correspondence: Derek J. McLean, Department of Animal Sciences, Washington State University, Pullman, WA 99164. FAX: 509 335 4246; dmclean{at}wsu.edu
Received: 28 January 2004.
First decision: 9 February 2004.
Accepted: 25 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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