Primary cilia in the developing pig testis.

In vertebrates, a variety of cell types generate a primary cilium. Cilia are implicated in determination and differentiation of a wide variety of organs and during embryonic development. However, there is little information on the presence or function of primary cilia in the mammalian testis. Therefore, the objective of this study was to characterize expression of primary cilia in the developing pig testis. Testicular tissue from pigs at 2-10 weeks of age was analyzed for primary cilia by immunocytochemistry. Expression of primary cilia was also analyzed in testicular tissue formed de novo from a single cell suspension ectopically grafted into a mouse host. Functionality of primary cilia was monitored based on cilia elongation after exposure to lithium. Analysis showed that the primary cilium is present in testis cords as well as in the interstitium of the developing pig testis. Germ cells did not express primary cilia. However, we identified Sertoli cells as one of the somatic cell types that produce a primary cilium within the developing testis. Primary cilium expression was reduced from the second to the third week of pig testis development in situ and during de novo morphogenesis of testis tissue from a single cell suspension after xenotransplantation. In vitro, primary cilia were elongated in response to lithium treatment. These results indicate that primary cilia on Sertoli cells may function during testicular development. De novo morphogenesis of testis tissue from single cell suspensions may provide an accessible platform to study and manipulate expression and function of primary cilia.

Viral transduction of male germline stem cells results in transgene transmission after germ cell transplantation in pigs.

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

Germ cell transplantation and testis tissue xenografting in mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994(1,2). These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal(2). The use of donor males carrying the bacterial ß-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals(1). Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located(3). It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation(4,5), to study Sertoli cell-germ cell interaction(6,7), SSC homing and colonization(3,8), as well as SSC self-renewal and differentiation(9,10). Germ cell transplantation is also feasible in large species(11). In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species(12). An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm(13). Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice(14).

Expression pattern of acetylated alpha-tubulin in porcine spermatogonia.

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated alpha-tubulin (Ac-alpha-Tu) is a component of long-lived, stable microtubules and deacetylation of alpha-tubulin enhances cell motility, we hypothesized that acetylation of alpha-tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac-alpha-Tu at different stages of testis development was characterized by immunohistochemistry for Ac-alpha-Tu and spermatogonia-specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac-alpha-Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac-alpha-Tu appeared polarized toward the basement membrane. In adult pig testes, Ac-alpha-Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL-), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac-alpha-Tu. Comparison with the expression pattern of beta-tubulin and tyrosinated alpha-tubulin confirmed that only Ac-alpha-Tu is specific to germ cells. The unique pattern of Ac-alpha-Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348-352, 2010. (c) 2009 Wiley-Liss, Inc.

A study of morphological and haemodynamic determinants of testicular echotexture characteristics in the ram.

The ultrasonographic image of an organ is a product of scattering and reflection of high-frequency ultrasound beams by discrete units of tissue. The number of acoustic tissue interfaces and vascularity affects the quantitative characteristics of grey-scale ultrasonographic images. This study was undertaken to examine the influences of scrotal/testicular integument and blood flow on testicular echotexture parameters in the ram. Serial ultrasonographic images were obtained during surgical castration of 7 Rideau Arcott rams aged 20-22 weeks. The first 2 sets of images were taken through the scrotum, prior to and after induction of anaesthesia. The third set was taken through the tunica vaginalis, the fourth set was obtained through the tunica albuginea, the fifth set was taken when the testicular cord and internal blood vessels were clamped, and the final set of images was recorded after allowing the blood to drain from dissected testicles (5 min). All images were then subjected to computerized image analyses and the testicles were processed for histology. The removal of the scrotal skin and tunica vaginalis both resulted in significant (P < 0.05) increments in numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of pixel values) of the testicular parenchyma. There were no differences (P > 0.05) in testicular echotexture between images taken just before or after clamping the testicular cord vessels, or after draining. At all stages, NPVs were correlated (P

Isolation and transplantation of spermatogonia in sheep.

Studies in rodents show that spermatogonial transplantation is an excellent new tool for studying spermatogenesis and for preservation and dissemination of genetics. The aim of this study was to adapt the technique to rams. Two issues were addressed: purification of stem cell spermatogonia, and efficient injection of donor spermatogonia into the seminiferous tubules of rams. We compared differential plating and Percoll gradient methods for purifying donor spermatogonia from ram lamb testes. Spermatogonia were identified with an antibody against PGP 9.5, a ubiquitin C-terminal hydrolase. Both purity and total number of spermatogonia recovered were higher after purification by Percoll gradient than by differential plating. Four approaches for injecting cells into the seminiferous tubules of ram testes were compared ex vivo: insertion of a needle into the extra-testicular rete testis after reflection of the head of the epididymis ('surgical' approach), and ultrasound-guided insertion of a needle into the extra-testicular rete, and the proximal and distal parts of the intra-testicular rete testis. 'Surgical' and ultrasound-guided approaches into the extra-testicular rete resulted in highest success rates and best filling of the seminiferous tubules. Finally, the ultrasound guided approach into the extra-testicular rete testis was validated in vivo by transplanting purified spermatogonia previously labeled with a fluorescent molecule (CFDA-SE). In seven of eight testes injected, donor cells were identified within the seminiferous epithelium for up to 2wk after transplantation, indicating the integration of donor cells.