No abstract

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.

Development of the mammalian testis and spermatogenesis involve complex processes of cell migration, proliferation, differentiation, and cell-cell interactions. Although our knowledge of these processes has increased in the last few decades, many aspects still remain unclear. The lack of suitable systems that allow to recapitulate and manipulate both testis development and spermatogenesis ex situ has limited our ability to study these processes. In the last few years, two observations suggested novel strategies that will improve our ability to study and manipulate mammalian spermatogenesis: i) testis tissue from immature animals transplanted ectopically into immunodeficient mice is able to respond to mouse gonadotropins and to initiate and complete differentiation to the level where fertilization-competent sperm are obtained, and ii) isolated testis cells are able to organize and rearrange into seminiferous cords that subsequently undergo complete development, including production of viable sperm. The current paper reviews recent advances that have been obtained with both techniques that represent novel opportunities to explore testis development and spermatogenesis in diverse mammalian species.

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