Spermatogonial stem cell biology in the bull: development of isolation, culture, and transplantation methodologies and their potential impacts on cattle production.

Widespread adoption of artificial insemination as a breeding practice has allowed for expanded use of desirable genetics from specific sires and greatly influenced production traits in dairy cattle populations worldwide. In fact, the average dairy cow in the US in 2009 produced 4.5 times more milk than in 1940 when commercialization of artificial insemination began. While many factors have contributed to this rapid increase in levels of milk production, genetic gain through expanded utilization of germlines from specific sires has been a major contribution. In comparison, use of artificial insemination in beef cattle populations has been limited due to challenges with implementing intensive management strategies required for success. Thus, there is need for alternative reproductive tools to expand use of desirable male genetics in the beef cattle industry. The process of sperm production, termed spermatogenesis, is supported by a tissue-specific stem cell population referred to as spermatogonial stem cells (SSCs). These unique cells have the capacity for infinite self-renewal and long-term regeneration of spermatogenesis following transplantation. In rodents, methods for isolating, culturing, and transplanting SSCs have been devised. For beef cattle, transplanting SSCs isolated from a donor male into the testes of recipient males in which donor-derived spermatogenesis occurs and offspring with donor genetics are produced from natural breeding has great potential as an alternative to artificial insemination. This potential reproductive strategy would allow for expansive use of genetics from desirable sires that overcomes the logistical challenges of artificial insemination. Translation of the methods devised for rodents to cattle is at the forefront of development. Devising means for isolating an SSC-enriched cell fraction from donor testes and identifying conditions that support long-term maintenance and proliferation of bovine SSCs in vitro are two tools that would greatly accelerate the pace at which transplantation will become a commercially viable option for cattle industries. Recent studies showed that expression of THY1 by SSCs is a conserved phenotype between rodents and cattle, and selection of the THY1 + fraction from donor testes can be used for isolating an SSC-enriched germ cell population. In addition, the conditions devised for expanding the number of rodent SSCs in vitro continues to serve as the basis for developing conditions that support bovine SSCs. With these tools in hand major advances in developing implementable reproductive tools with SSCs for commercial cattle production will be made in the coming decade.

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone.

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.

Luteinizing hormone-releasing hormone fusion protein vaccines block estrous cycle activity in beef heifers.

Two LHRH fusion proteins, thioredoxin and ovalbumin, each containing seven LHRH inserts were tested for their ability to inhibit estrous cycle activity. The objective was to evaluate immune and biological responses from alternating the two fusion proteins in an immunization schedule. One hundred ten heifers were divided equally into 11 groups. Two control groups consisted of either spayed or intact, untreated heifers. Heifers in the other nine groups were immunized on wk 0, 4, and 9. Treatments were immunizations of the same protein throughout or alternating the proteins in different booster sequences. Blood was collected weekly for 22 wk, and serum was assayed for concentrations of progesterone and titers of anti-LHRH. At slaughter, reproductive tracts were removed from each heifer and weighed. Heifers with >or=1 ng/mL of progesterone were considered to have a functional corpus luteum and thus to have estrous cycle activity. All LHRH-immunized groups of heifers had a smaller (P < 0.05) proportion of heifers showing estrous cycle activity after 6 wk than the intact, untreated control group. There was no difference in number of heifers cycling between the immunized groups and the spayed heifers during wk 9 to 22. Anti-LHRH did not differ among immunized groups during wk 1 to 9. Starting at wk 10 and continuing through the conclusion of the study, there was an overall difference among treatment groups for anti-LHRH (P < 0.05). Uterine weights differed among treatments (P < 0.05), with intact control animals having heavier uteri than all other groups (P < 0.05). Uterine weights were negatively correlated with maximum LHRH antibody binding (r = -0.44). In summary, the LHRH fusion proteins were as effective as surgical spaying in suppression of estrous cycle activity, but alternating the two proteins in an immunization schedule did not enhance the immunological or biological effectiveness of the vaccine.

Transplantation of bovine germinal cells into mouse testes.

To develop techniques for spermatogonial transplantation in bulls, it is essential to have an effective bioassay procedure to evaluate the transplantation efficiency of spermatogonial stem cell collection, purification, and culture techniques. The objective of the present study was to develop a mouse bioassay model to evaluate transplantation efficiency of fresh and cultured bovine germ cells. Bull calves of four ages (1, 2, 3, and 4 mo) were used as a source of donor testes cells. Two calves were used for each age point, one calf was experimentally made cryptorchidistic at 1 wk of age and the other left normal. A STO (mouse fibroblast) feeder cell line was used to culture bovine testes cells for 2 wk preceding transfer into recipient testes. Immunodeficient nude mice (nu/nu) in which endogenous spermatogenesis had been abolished by busulfan treatment served as recipient animals for transplantation. Donor bovine germ cells were microinjected into mouse seminiferous tubules. Mouse testes were analyzed 2 wk after transplant with the use of a bovine-specific antibody and whole-mount immunohistochemistry for the presence of bovine donor germ cells. Bovine testis cells were present in all recipient mouse testes analyzed. Fresh bovine testes cells were observed as colonies of round cells within mouse seminiferous tubules, indicating spermatogonial expansion and colonization; however, cultured bovine testes cells appeared as fibrous tissue and not as spermatogenic colonies. The average number of colonies resulting from donor cryptorchid testes was not different (P > 0.05) from noncryptorchid, 56+/-4 and 78+/-7, respectively. Fresh donor cells from calves older than 1 mo gave rise to a greater average number of colonies within recipient testes (P <0.05) (1 mo, 33+/-4; 2 mo, 70+/-8; 3 mo, 63+/-6; 4 mo, 87+/-9). Fresh bovine germ cells are capable of colonization in the busulfan-treated nude mouse testis, making it a suitable model for evaluation and development of spermatogonial transplant techniques in bulls.