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Department of Animals Sciences and Center of 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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bovine, gamete biology, spermatogenesis, testis, xenografting
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first round of postnatal spermatogenesis can occur in testicular tissue even when it has been removed and placed at locations distal from the testis [1–4]. Recently, postnatal spermatogenesis has been demonstrated in testicular tissue from several species xenografted onto the backs of recipient mice [1–4]. In mice, pigs, goats, and primates, the first round of spermatogenesis in testicular tissue ectopically xenografted onto mice is accelerated [1–3]; however, in the bovine, a comparable acceleration has not been reported [4]. In previous studies, successful establishment of spermatogenesis required neonatal donors as a source of testicular tissue [1–4]. This necessity has been attributed to donor germ and somatic cell populations that are immature, thus allowing the progression of the first round of spermatogenesis to occur following grafting.
During the time of prepubertal testis development, both the somatic and germ cells undergo stages of developmental change. In the early period of Postnatal Days 1–5 in the mouse, the gonocytes undergo translocation to the basement membrane followed by conversion to spermatogonial stem cells, eventually producing differentiating spermatogonia [5–7]. In bulls, this period is much longer, lasting from Day 0 to 16 wk of life, when differentiating spermatogonia are first seen [8]. During this early stage of postnatal testicular development, the somatic cell population consists of immature proliferative Sertoli and Leydig cells that become differentiated to function in their supporting roles of spermatogenesis. In the postnatal mouse testis, Sertoli cell proliferation occurs from Day 0 to Day 10, at which time differentiation occurs [9].
A major application of testicular grafting could be generation of transgenic spermatozoa. Production of stably transfected spermatozoa could be accomplished by transduction of spermatogonial stem cells before grafting. Use of a neonatal donor would be ideal because the germ cell population would be enriched for spermatogonial stem cells compared with other differentiated germ cell types. In previous experiments, the percentage of seminiferous tubules in which differentiated germ cells were produced was low following ectopic xenografting of neonatal or prepubertal testicular tissue [3, 4]. Increasing the number of tubules containing haploid germ cells and therefore sperm would be beneficial for enhancing the utility of this technique as a reproductive technology. In all species studied, donors at a single neonatal age have been used as a source of testicular tissue. The extent of proliferation and differentiation of somatic and germ cells in testicular tissue during different periods of development in the postnatal mammalian testis is likely different and may correlate into different levels of sperm production following ectopic testicular xenografting. The objective of the present study was to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls at developmental stages between 0 and 16 wk of life. We hypothesized that testicular tissue from 12-wk-old bulls would result in the most effective establishment of spermatogenesis following grafting because this is the age just before germ cell maturation into differentiating spermatogonia initiates.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal procedures were approved by the Washington State University Animal Care and Use Committee. Testicular tissue was collected from donor bulls at the postnatal ages of 2, 4, 8, 12, and 16 wk (n =3 calves/age). Donor tissue was then diced into approximately 10-mg pieces and maintained in Hanks balanced salt solution on ice until the time of grafting. Four pieces of donor tissue were ectopically grafted onto the backs of each castrated adult immunodeficient recipient nude mouse (n = 3 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 just before small incisions were made through the skin on the backs of the animal where the muscle was roughened in individual areas, and four 10-mg pieces of donor bull testis placed individually within the scored muscle areas of each recipient mouse. The incisions were closed with suture and the animals were allowed to recover. Pieces of nongrafted testicular tissue from each donor were also fixed in Bouin solution for 4 h at 4°C followed by dehydration and storage in 70% ethanol.
Histological Analysis of Donor Grafts
Recipient mice were killed 24 wk after grafting by CO2 inhalation followed by cervical dislocation. Donor bovine grafts and recipient mouse seminal vesicles were removed, weighed, and fixed in Bouin solution at 4°C for 4 h, followed by dehydration in 70% ethanol. All grafts were subsequently blocked in paraffin, cut into 6-µm-thick sections, and stained with hematoxylin. Sections were evaluated using light microscopy, and digital images were captured with a Cool Snap cf. digital camera (Media Cybernetics; Silver Spring, MD) at 100–200x magnification. The average percentage of tubules containing spermatogonia only, Sertoli cells only, pachytene spermatocytes, and elongating spermatocytes were calculated for each sample by dividing the number of round tubules containing each germ cell type/three microscopic fields by the total number of round tubules within the same three fields.
Immunohistochemistry of Transition Protein-2
Representative cross-sections of grafts from each aged donor after grafting were processed for detection of transition protein 2 (TP-2) by immunohistochemistry to identify late meiotic germ cells. Cross-sections were deparaffinized and rehydrated followed by boiling in sodium citrate (pH 6.0). Endogenous peroxidase activity was then blocked by incubating the samples in 3% H2O2 in methanol for 10 min, followed by washing in PBS. Nonspecific antibody binding was then blocked by incubation of samples in 10% nonimmune rabbit serum for 15 min at room temperature. Primary antibody (goat anti-mouse TNP2; Santa Cruz Biotechnology, Santa Cruz, CA) diluted to 1:100 was added to samples and incubated at 4°C overnight in a humidified chamber. The next day, samples were washed in PBS three times for 2 min each and biotinylated secondary antibody (rabbit anti-goat IgG; Santa Cruz Biotechnology) was added and incubated at room temperature for 1 h, followed by extensive washing in PBS. Streptavidin-horse radish peroxidase was then added and samples were incubated for 10 min at room temperature followed by extensive washing in PBS. Samples were developed with aminoethyl carbazole (AEC) following manufacturer instructions (AEC substrate kit; Santa Cruz Biotechnology), counterstained with hematoxylin, and mounted with a coverslip. The morphology of stained sections was evaluated using light microscopy, and digital images were captured.
Recipient Mouse Analysis
At the time of killing after grafting, recipient mice were bled by cardiac puncture and seminal vesicles were weighed. Serum was subsequently collected by centrifugation and analyzed for testosterone concentration by RIA using a commercial kit (DSL-400; Diagnostic Systems Laboratory Inc., Webster, TX).
Statistical Analyses
Data were analyzed using the Proc GLM function of SAS systems software. Differences between means were determined for graft weight, percentage tubules with germ cell types, and recipient-mouse testosterone concentration using Duncan test for significance. Differences between means were considered significantly different at P 
0.05. Data are presented as the mean ± SEM in all figures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The average weight of individual grafts for each donor age was measured at removal to provide an evaluation of differences in growth of different aged donor bovine testicular tissue over the 24-wk grafting period. Growth of donor testicular tissue under the skin on the backs of recipient mice could be visually identified over the 24-wk grafting period. Analysis of testis graft weights at removal indicated the growth potential varied depending on donor age. Average weights of grafts from 2-wk-old donor bull calves were significantly higher (P 0.05) compared with both 12- and 16-wk-old donors, but not significantly different (P 
0.05) from 4- and 8-wk donors (Fig. 1A). The 4- and 8-wk donor average graft weights were significantly higher than that of 16-wk donors, but not different from each other or 12-wk donor weights (Fig. 1A). The average weight of 12- and 16-wk donor graft weights were not significantly different from each other (Fig. 1A). Overall, testis tissue from 2-wk bull calves had the greatest growth potential.
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Castrated recipient mouse serum testosterone concentration was measured to evaluate Leydig cell differentiation and function in grafted neonatal bovine testicular tissues. The average testosterone concentration in recipient mice was found to be at a physiological level of intact control mice (
1–5 ng/ml), regardless of the age of the donor testicular tissue (Fig. 1B). No significant differences could be found between different donor ages.
Recipient mouse seminal vesicle weights were measured to further assess testosterone production by Leydig cells in grafted bovine testicular tissues. Only average seminal vesicle weights from 12- and 16-wk donor bulls were significantly different from each other (Fig. 1C). There was no significant difference between the donor ages of 2, 4, 8, and 12 wk or 2, 4, 8, and 16 wk. Overall, analysis of recipient seminal vesicle weights indicated testosterone production by grafted bovine testis tissue correlated with seminal vesicle size.
Histological Analysis
Histological examination of donor testicular tissue at the time of grafting was conducted to evaluate differences in germ cell populations between donor ages. The ability of germ cell differentiation to commence and the extent of differentiation that occurs following grafting may be influenced by the stage of germ cell development at the time of grafting. Evaluation of both 2- and 4-wk-old donors revealed seminiferous cords that contained only gonocytes (Fig. 2, A and B). Eight- and 12-wk-old donor seminiferous tubules contained spermatogonia as the most advanced germ cell type (Fig. 2, C and D). Sixteen-week-old donor tissue contained spermatogonia and early meiotic germ cells (Fig. 2E).
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Donor testicular tissue 24 wk after grafting was investigated histologically for evaluation of establishment of spermatogenesis. Germ cell differentiation to the elongate-spermatid stage has been demonstrated in neonatal testicular tissue from several species, including bulls; however, the percentage of tubules with germ cell differentiation was low [1–4]. The extent of spermatogenesis in grafted bovine testicular tissue from different bull-calf ages was determined. Evaluation of all grafts revealed the presence of intact seminiferous tubules and advanced stages of spermatogenesis in some tubules throughout the donor testicular tissue (Fig. 3). Intact seminiferous tubules containing spermatogonia only (Fig. 3A) and Sertoli cells only (Fig. 3A) were identified. More advanced stages of spermatogenesis containing meiotic germ cells (Fig. 3B) and elongated spermatids (Fig. 3C) were also present within the grafts. The extent of germ cell differentiation in donor testis tissue following grafting varied with age of the donor bull calf.
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Seminiferous tubules with Sertoli cells only could be observed in grafts from all the different donor ages. Upon comparison of donor ages for prevalence of Sertoli cell-only tubules, grafts from 12- and 16-wk donors were significantly different from each other (Fig. 4A). The average percentage of Sertoli cell-only tubules in the 2-, 4-, and 8-wk donor grafts were not significantly different from each other.
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The appearance of tubules containing spermatogonia only without any advancing germ cell types was seen in all grafts; however, the percentages of tubules with this phenotype varied by age of the donor (Fig. 4B). Grafted testicular tissue from 2- and 4-wk donors had a significantly higher percentage of tubules containing only spermatogonia than grafts from both 8- and 16-wk donors. The percentage of tubules with spermatogonia only from 12-wk-old donor calves did not differ from any of the other donor ages.
Maturation of germ cells to meiosis was seen in grafts from all donor ages. Grafts from 8- and 16-wk-old donor calves contained significantly higher percentages of tubules with meiotic germ cells than grafts from 2-, 4-, and 12-wk donors (Fig. 4C). The average percentage of tubules containing meiotic cells in 8- and 16-wk grafts was approximately 60%, whereas, meiotic cells were present in an average range of 37–43% of the tubules in grafts from 2-, 4-, and 12-wk donor testes.
Differentiation of germ cells to the elongate spermatid stage was observed in grafts from 4-, 8-, 12-, and 16-wk-old donors but not 2-wk-old donors. Comparison of all the donor ages revealed a significantly higher average percentage of tubules containing elongated spermatids in grafts from 8-wk donors (Fig. 4D). The average percentage of tubules containing elongated spermatids from 4-, 8-, 12-, and 16-wk donors was low at only approximately 0.5%, whereas the average percentage of 8-wk donors was approximately 10-fold higher, at 5%.
Transition Protein 2 Immunohistochemistry
Identification of late meiotic germ cells in grafted bovine testicular tissue were identified by immunohistochemical staining of TP-2. In grafts from all donor ages, cells positive for TP-2 were detected within some tubules; however, the distribution and abundance of these cells varied by age of the donor. Grafts from 2-, 4-, 12-, and 16-wk donor bull calves contained tubules that had one or two positively stained cells. In comparison, grafts from 8-wk donors had multiple positively stained cells, with a distinct location toward the lumen of the tubules (Fig. 3D). Based on the observations of morphology of seminiferous tubules, 8-wk donor testicular tissue contained the most advanced stages of spermatogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Average weight of donor testicular tissue 24-wk after grafting was higher with younger donor bull calves. The largest increase in weight occurred in grafted testicular tissues from 2-wk-old donors with a sixfold increase from the weight of the starting tissue. Both 4- and 8-wk average donor graft weights increased approximately fourfold compared with starting weight and were significantly higher than that of 16-wk donors, which did not increase in weight compared with the starting tissue. An increase in weight of grafted testicular tissue could be attributed to several things but was most likely a result of somatic cell proliferation and progression of spermatogenesis. In the bull, the time frame of postnatal Sertoli cell proliferation is not well established. There are available data to suggest that proliferation of immature Sertoli cells occurs between birth and 20 wk of life [8]. At the cessation of proliferation, Sertoli cell differentiation begins at 20 wk and is completed at 28 wk, when the maximal number of mature differentiated Sertoli cells is present [8]. Grafting of testicular tissue at different developmental stages, as was done in the present study, could have resulted in different abilities of Sertoli cells to undergo proliferation and differentiation following grafting, which would thus result in larger weight increases following grafting.
Use of castrated recipient mice for grafting provides an endocrine environment containing high FSH following grafting of the donor bovine testicular tissues [2]. In rodents, Sertoli cell proliferation has been shown to be regulated by FSH [10–14]. If the same principle holds true in the bovine, then the high FSH environment to which the Sertoli cells were exposed following grafting would have supported proliferation. FSH levels in bull calves from 4 to 16 wk of life gradually increase from 4 ng/ml to 6 ng/ml [15]. The level of FSH in adult rodents is generally higher than bulls [16] and castration of rodents leads to FSH levels that are five times higher than controls [17]. Differences in the proliferation potential of the Sertoli cells in the grafted bovine testicular tissues at different developmental ages, based on FSH receptor expression or other factors, could have resulted in different responses to the high FSH. The lack of weight increase in 16-wk donor tissues may have been a result of the inability of Sertoli cells to respond to recipient mouse FSH. Testis tissue from younger donor animals contained a more immature Sertoli cell population with greater potential for response to high FSH following grafting.
In order for a tissue to become established following grafting, vascularization must occur to provide nutrients for survival and function. In order for Sertoli cells in grafted testicular tissue to respond to recipient mouse pituitary-released FSH, vascularization must occur. Differing degrees of vascularization in testicular tissue from donor bull calves may have contributed to differences in the abilities of the Sertoli cells to respond to FSH for proliferation and thus increase in weight over the 24-wk period. Investigation of the development of vascularization in the testis has focused on prenatal events [18]. Information on continued blood vessel growth in the testis after birth in hamsters indicates this is a regulated process and the testis vasculature is mature by Day 35 after birth [19]. A better understanding of the timing and development of the testis vasculature in bulls and how blood vessel growth from or to the bovine testis grafts occurs may lead to improved production of sperm by grafts due to hormonal regulation of this process.
Evaluation of recipient serum testosterone concentration revealed the ability of Leydig cells in the grafted testicular tissues to respond to recipient mouse LH for production of testosterone at physiological concentrations normally found in intact adult male mice. Differences between the testis tissues from different donor ages in the ability of Leydig cells to produce testosterone could not be detected. This observation suggests that Leydig cell maturation does not vary based on stage of the postnatal testis. Like Sertoli cell responsiveness to FSH following grafting onto castrated recipients, Leydig cells in the bovine testicular tissue were exposed to high LH concentrations following grafting. In the intact bull, LH concentration in the serum does not become elevated until 18 wk of age, at which time Leydig cell maturation occurs and testosterone production commences [20]. It appears that increasing LH concentration in the serum is a key component to Leydig cell maturation. In neonatal bovine testicular tissue grafted onto castrated recipients, the immature Leydig cells appear to undergo spontaneous maturation and begin to produce physiological concentrations of testosterone in response to a high-LH environment.
The appearance of seminiferous tubules containing a Sertoli cell-only phenotype was observed in all testicular grafts, with only marginal variation between donors of different developmental ages. The only significant difference in the average percentage of seminiferous tubules containing Sertoli cells only was found between 12- and 16-wk donors, which also constituted the highest and lowest percentages, respectively. The overall average percentage of total tubules from all grafts was approximately 25%. Other studies have also reported the occurrence of seminiferous tubules containing this phenotype in ectopically grafted testicular tissue [1–4]. Like the incidence of Sertoli cell-only syndromes in men, the causative reason for the formation of this phenotype remains elusive [21–22]. The highest percentage of tubules with Sertoli cells only occurred in grafted testicular tissue from 12-wk donor bull calves in this study. This age could represent a development stage in bulls in which the Sertoli cells are most susceptible to detrimental influences of endocrine disruption.
The appearance of seminiferous tubules containing spermatogonia only was also evident in grafted tissues from all donor bulls. Testicular grafts from 2- and 4-wk-old donor bulls had significantly higher averages of seminiferous tubules with this phenotype than grafts from both 8- and 16-wk donors. The causative reason for the establishment of these tubules is not clearly evident. Defects in proliferation and/or maturation of both Sertoli and germ cells may have been contributing factors. Lack of Sertoli cell maturation and attainment of normal differentiated function would result in the inability for supporting germ cell maturation. Differences in the germ cell population at the developmental stages of testicular development in the different aged bulls used in this study may have had an impact on the incidence of spermatogonia-only tubules.
Progression of germ cells to meiotic stages was evident in grafted testicular tissue from donor bulls of all ages used in this study. Like the occurrence of spermatogonial-only tubules, both 8- and 16-wk donor grafts resulted in significantly higher average percentages of total tubules containing meiotic germ cells. The lower average percentage of tubules containing meiotic cells in grafts from 2-, 4-, and 12-wk donors was most likely a result of higher percentages of tubules containing Sertoli cell-only and spermatogonia-only phenotypes within the same grafts. Detection of late meiotic germ cells by immunohistochemical staining for TP-2 showed the greatest advancement of germ cell maturation in seminiferous tubules from 8-wk donors. These data indicate that testicular tissue from bulls at the postnatal developmental stage of 8 wk is most amenable to establishment of spermatogenesis following ectopic xenografting.
The presence of elongated spermatids in grafted tissues marked the differentiation of germ cells through meiosis and into spermiogenesis. The average percentage of total tubules containing this cell type was significantly higher in 8-wk donor tissues, with approximately a 10-fold increase compared with all other donor ages. Advancement of germ cells to the elongate spermatid stage could not be detected in testicular grafts from 2-wk-old donor bull calves. Prior studies have demonstrated an acceleration of the first round of postnatal spermatogenesis in neonatal testicular tissue of pigs, goats, mice, and primates ectopically xenografted onto castrated recipient mice [1–3]. Lack of observed elongated spermatids in 2-wk-old neonatal bovine testicular tissue 24 wk after grafting suggests that a comparable acceleration does not occur with the bovine. In the intact bull, elongated spermatids can be first observed at 28 wk of postnatal age [8]. Allowing testicular tissue from 2-wk donors more time after grafting that is relative to the donor age (i.e., 26 wk) could yield elongated spermatids. The lower average percentage of tubules containing elongated spermatids in 4-wk donors was most likely a function of higher Sertoli and spermatogonia-only tubules. Testicular tissue at the developmental ages of 12 and 16 wk possibly contains a high percentage of seminiferous tubules with somatic and germ cell populations that are at a differentiated stage of development where the setup of the seminiferous epithelium is inhibited in the majority of tubules. Due to the bull donor age and the differentiation of germ cells in grafts at removal, the number of seminiferous tubules counted to determine the percentage of tubules containing differentiating germ cells varied slightly between groups. These differences could impact the interpretation of these data because of variability. However, the standard error within groups was low, indicating the differentiation of bovine testis tissue after grafting was consistent between donors. Indeed, the 8-wk bull calf donors were consistently and clearly superior for the production of elongated spermatids.
Ectopic testicular xenografting is a method that has potential use for investigating spermatogenesis in nonrodent mammalian species where experimentation in the target species is difficult. As an applied technology, testicular xenografting could be used as an alternative means to generate stably modified transgenic spermatozoa through manipulation of spermatogonial stem cells before grafting. Use of neonatal donors is of benefit because undifferentiated spermatogonia are the only germ cell type present and the population is enriched for spermatogonial stem cells. Understanding the establishment of spermatogenesis in grafted neonatal testicular tissue can lead to enhancement of sperm production. In the present study, we demonstrate differences in the establishment of spermatogenesis in testicular tissue from bull calves at different neonatal stages of testicular differentiation ectopically xenografted onto recipient mice. The data indicates that 8 wk of postnatal life is the developmental stage in the bovine testis when the Sertoli and germ cells are most amenable to establishing spermatogenesis in ectopically xenografted testicular tissue.
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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: 7 April 2004.
First decision: 22 April 2004.
Accepted: 14 September 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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