Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan.

Germ cell transplantation has facilitated spermatogonial stem cell (SSC) and spermatogenesis research and shown great potential in the seed-breeding of domestic livestock. However, little progress has been made in large animals, primarily reflecting the difficulties in preparing sterile recipients. Here, we developed a novel protocol to prepare recipient pigs through the direct injection of busulfan into the cavum vaginale of the scrotums of Landrace-Large bi-crossbreeding male pigs and Seghers male pigs, two economically-important types of pigs, to eliminate endogenous spermatogonia. No severe diseases or weight loss was observed in either pig type after the injection with busulfan. Histologic analysis showed an advanced and dose-dependent germ cell loss, with complete germ cell loss observed in the highest dose group, 3.0 mg/kg in the Landrace-Large bi-crossbreeding pigs and 2.0 mg/kg in the Seghers pigs. A smaller seminiferous tubule diameter, a vacuolized seminiferous epithelium and the overproliferation interstitial cells, frequently observed in mouse germ cell deficiency models, were present in the most of the high-dose busulfan-treated groups. Molecular markers detected in Seghers pigs further confirmed the depletion of endogenous germ cells, providing an accessible niche for exogenous SSCs. This study provides a basis to prepare the transplantation recipients of SSCs in pigs.

Long-term culture and analysis of cashmere goat Sertoli cells.

Sertoli cells have important functions in the testis for spermatogenesis. Thus, Sertoli cell culture systems have been established in many animals, such as rat, mouse, human, dog, cow, and pig, but a goat culture has not been reported. This study describes the isolation and culture of Sertoli cells from 3- to 4-month-old cashmere goat (Capra hircus) testes. These proliferative cells were expanded for 20 passages and repeatedly cryopreserved in vitro, in contrast to previous study in human, of which maintain steady growth for up to seven passages and only passages 1 to 5 could be refrozen. The microstructure and ultrastructure of the culture were typical of Sertoli cells, bearing irregular nuclei and a cytoplasm that was rich in smooth and rough endoplasmic reticulum, mitochondria, Golgi, lysosomes, lipid drops, and glycogenosomes. By immunofluorescence analysis, the all cells expressed SRY-related HMG box gene 9 (Sox9). Growth curves and 5-bromo-2'-deoxyuridine (BrdU) incorporation were used to analyze the proliferation of the cultured cells. With increasing passage times, the proliferation of the Sertoli cells declined, but the transcription of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF), and ß1-integrin was constant. By flow cytometry, the cells retained the ability to proliferate after 5 yr of cryopreservation. Thus, cashmere goat Sertoli cells have significant proliferative potential in vitro, expressing germ cell regulatory factors and have important applications in studying Sertoli cell-germ cell interactions, spermatogenesis, reproductive toxicology, and male infertility.

Tribbles homolog 3 expression in spermatogonial stem cells of rat testes.

Tribbles are a family of signal-regulating proteins shown to coordinate the action and the suppression of different pathways. Tribbles homolog 3 (Trib3), the best-studied member of the mammalian tribble family, has a key function in determining cell fate when responding to environmental challenges. Trib3 effects are also modulated by its direct interaction with other signaling molecules. We found that Trib3 is highly expressed in the early development of rat testis, at just the time when the gonocytes resume proliferation to give rise to A spermatogonia. Immunofluorescence staining of cross-sections of rat testis and cultured spermatogonial stem cells (SSCs) also confirmed that Trib3 is expressed in rat SSCs.

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro.

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.

Construction of targeting vector for expressing human GDNF in cattle mammary gland.

Glial cell line-derived neurotrophic factor (GDNF) is a type of neurotrophic factor with significant potential in treatment of Parkinson's disease. Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The aim of this study was to construct a gene-targeting vector for the human gdnf gene knockin at the bovine beta-casein gene locus so that human GDNF protein can be produced in the mammary gland of the gene-targeted bovine. The constructed vector contains the 2.2 kb 5' homologous arm and the 5.7 kb 3' homologous arm. The human gdnf cDNA was located at the downstream of the 5' homologous arm. The neo gene placed between the 5' and 3' homologous arms as positive selection marker gene. The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes, respectively. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the plasmid DNA was transfected into human mammary tumor cell line Bcap-37 by lipofectamine. Reverse transcription polymerase chain reaction and Western-blotting analysis showed that the transfected cells produced human GDNF mRNA and protein. The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells. At the same time, it is first time to confirm that human mammary tumor cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.