Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs.

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified.

In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function.

The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed.

Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules.

In the human testis, beginning at approximately 2 months of age, gonocytes are replaced by adult dark (Ad) and pale (Ap) spermatogonia that make up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool arises from gonocytes approximately 6 days after birth. During puberty in both species, complete spermatogenesis is established by cells that differentiate from SSCs.

Spermatogonial stem cells derived from infertile Wv/Wv mice self-renew in vitro and generate progeny following transplantation.

Loss-of-function mutation of the Kit gene causes a severe defect in spermatogenesis that results in infertility due to the inability of its cognate ligand, KIT ligand (KITL), to stimulate spermatogonial proliferation and differentiation. Although self-renewal of mouse spermatogonial stem cells (SSCs) depends on glial cell line-derived neurotrophic factor (GDNF), there is no unequivocal evidence that SSCs with a KIT deficiency can self-renew in vivo or in vitro. In the testis of W(v)/W(v) mice, in which the KIT tyrosine kinase activity is impaired, spermatogonia with SSC phenotype were identified.

Identification of glial cell line-derived neurotrophic factor-regulated genes important for spermatogonial stem cell self-renewal in the rat.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis throughout the life of a male. Because SSCs of many species can colonize the mouse testis, and glial cell line-derived neurotrophic factor (GDNF) is responsible for stimulating SSC self-renewal in rodents, we reasoned that molecular mechanisms of SSC self-renewal are similar across species. GDNF-regulated genes have been identified in mouse SSCs; however, downstream targets of GDNF are unknown in other species.

Female mice delay reproductive aging in males.

Reproductive aging of the male is characterized by decreasing fertility; however, factors that protect against reproductive aging in the male are largely unknown. Previous work has demonstrated that both female presence and aging have a dramatic effect on fertility in the male; yet, the effect of female presence on fertility in the aging male mouse is unknown. The objective of this work was to determine the effect of long-term isolation or cohabitation with females on fertility in aged male mice.

Analysis of gene expression in bovine testis tissue prior to ectopic testis tissue xenografting and during the grafting period.

The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately.

Mycoplasma hyopneumoniae mhp379 is a Ca2+-dependent, sugar-nonspecific exonuclease exposed on the cell surface.

Mycoplasma hyopneumoniae mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in Escherichia coli following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein.

Grafting period and donor age affect the potential for spermatogenesis in bovine ectopic testis xenografts.

Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life.

Effect of vascular endothelial growth factor and testis tissue culture on spermatogenesis in bovine ectopic testis tissue xenografts.

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation.