BMP4-induced differentiation of a rat spermatogonial stem cell line causes changes in its cell adhesion properties.

Spermatogonial stem cells (SSCs) are at the basis of the spermatogenic process and are essential for the continuous lifelong production of spermatozoa. Although several factors that govern SSC self-renewal and differentiation have been investigated, the direct effect of such factors on SSCs has not yet been studied, mainly because of the absence of markers to identify SSCs and the lack of effective methods to obtain and culture a pure population of SSCs. We now have used a previously established rat SSC cell line (GC-6spg) to elucidate the role of BMP4 in SSC differentiation. We found that GC-6spg cells cultured in the presence of BMP4 upregulate KIT expression, which is an early marker for differentiating spermatogonia. GC-6spg cells were found to express three BMP4 receptors and the downstream SMAD1/5/8 proteins were phosphorylated during BMP4-induced differentiation. A time-course DNA micro-array analysis revealed a total of 529 differentially regulated transcripts (≥2-fold), including several known downstream targets of BMP4 such as Id2 and Gata2. Pathway analysis revealed that the most affected pathways were those involved in adherens junctions, focal junctions, gap junctions, cell adhesion molecules, and regulation of actin cytoskeleton. Interestingly, among the genes belonging to the most strongly affected adhesion pathways was Cdh1 (known as E-cadherin), an adhesion molecule known to be expressed by a subpopulation of spermatogonia including SSCs. Overall, our results suggest that BMP4 induces early differentiation of SSCs in a direct manner by affecting cell adhesion pathways.

Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis.

The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated embryonic cell transcription factor 1 (UTF1) is restricted to distinct germ cells within the testis. In embryonic and neonatal testes, all gonocytes were found to strongly express UTF1. During further testicular development, expression of UTF1 was restricted to a subset of A spermatogonia and with the increase in age the number of cells expressing UTF1 decreased even more. Ultimately, in the adult rat testis, only a small subset of the A spermatogonia expressed UTF1. Remarkably, even in testes of vitamin A-deficient rats, in which the early A spermatogonia (A(s), A(pr), and A(al)) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is restricted to a subpopulation of the ZBTB16 (PLZF)-positive early A spermatogonia. Furthermore, the observed distribution pattern of UTF1-expressing cells over the different stages of the cycle of the seminiferous epithelium suggests that the expression of UTF1 is restricted to those A(s), A(pr), and short chains of A(al) spermatogonia that are in the undifferentiated state and therefore maintain the ability to differentiate into A1 spermatogonia in the next round of the epithelial cycle or possibly even in other directions when they are taken out of their testicular niche.

Transgenic mice with mammary gland targeted expression of human cortactin do not develop (pre-malignant) breast tumors: studies in MMTV-cortactin and MMTV-cortactin/-cyclin D1 bitransgenic mice.

BACKGROUND: In human breast cancers, amplification of chromosome 11q13 correlates with lymph node metastasis and increased mortality. To date, two genes located within this amplicon, CCND1 and EMS1, were considered to act as oncogenes, because overexpression of both proteins, respectively cyclin D1 and cortactin, correlated well with 11q13 amplification. Cyclin D1 is involved in cell cycle regulation and the F-actin-binding protein cortactin in cytoskeletal dynamics and cell migration. To study the role of cortactin in mammary gland tumorigenesis, we examined mouse mammary tumor virus (MMTV)-cortactin transgenic mice and MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice. METHODS: MMTV-cortactin transgenic mice were generated and intercrossed with previously described MMTV-cyclin D1 transgenic mice. Immunohistochemical, Northern and Western blot analyses were performed to study the expression of human transgene cortactin during mammary gland development and in mammary tumors. For tumor incidence studies, forced-bred, multiparous mice were used to enhance transgene expression in the mammary gland. Microscopical examination was performed using haematoxylin and eosin staining. RESULTS: Mammary gland tumors arose stochastically (incidence 21%) with a mean age of onset at 100 weeks. This incidence, however, did not exceed that of aged-matched control FVB/N mice (38%), which unexpectedly, also developed spontaneous mammary gland tumors. We mimicked 11q13 amplification by generating MMTV-cortactin/-MMTV-cyclin D1 bitransgenic mice but did not observe any synergistic effect of cortactin on cyclin D1-induced mammary hyperplasias or carcinomas, nor development of distant metastasis. CONCLUSION: From this study, we conclude that development of (pre-malignant) breast tumors in either wild type or MMTV-cyclin D1 mice was not augmented due to mammary gland targeted overexpression of human cortactin.

Spermatogonial stem cells: characteristics and experimental possibilities.

The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFRalpha-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers.

A Long-Lived Luminal Subpopulation Enriched with Alveolar Progenitors Serves as Cellular Origin of Heterogeneous Mammary Tumors.

It has been shown that the mammary luminal lineage could be maintained by luminal stem cells or long-lived progenitors, but their identity and role in breast cancer remain largely elusive. By lineage analysis using Wap-Cre mice, we found that, in nulliparous females, mammary epithelial cells (MECs) genetically marked by Wap-Cre represented a subpopulation of CD61+ luminal progenitors independent of ovarian hormones for their maintenance. Using a pulse-chase lineage-tracing approach based on Wap-Cre adenovirus (Ad-Wap-Cre), we found that Ad-Wap-Cre-marked nulliparous MECs were enriched with CD61+ alveolar progenitors (APs) that gave rise to CD61- alveolar luminal cells during pregnancy/lactation and could maintain themselves long term. When transformed by different oncogenes, they could serve as cells of origin of heterogeneous mammary tumors. Thus, our study revealed a type of long-lived AP within the luminal lineage that may serve as the cellular origin of multiple breast cancer subtypes.

RUNX1, a transcription factor mutated in breast cancer, controls the fate of ER-positive mammary luminal cells.

RUNX1 encodes a RUNX family transcription factor (TF) and was recently identified as a novel mutated gene in human luminal breast cancers. We found that Runx1 is expressed in all subpopulations of murine mammary epithelial cells (MECs) except the secretory alveolar luminal cells. Conditional knockout of Runx1 in MECs by MMTV-Cre led to a decrease in luminal MECs, largely due to a profound reduction in the estrogen receptor (ER)-positive mature luminal subpopulation, a phenotype that could be rescued by the loss of either Trp53 or Rb1. Mechanistically RUNX1 represses Elf5, a master regulatory TF gene for alveolar cells, and regulates mature luminal TF/co-factor genes (e.g., Foxa1 and Cited1) involved in the ER program. Collectively, our data identified a key regulator of the ER⁺ luminal lineage whose disruption may contribute to the development of ER⁺ luminal breast cancer when under the background of either TP53 or RB1 loss.

Lineage tracing of mammary epithelial cells using cell-type-specific cre-expressing adenoviruses.

Lineage tracing using Cre/lox transgenic mice provides a powerful tool for studying normal mammary epithelial cell (MEC) development and the cellular origins of mammary tumors under physiological settings. However, generation of new transgenic mice for lineage-tracing purposes is often time consuming. Here, we report a lineage-tracing tool for MECs based on intraductal injection of lineage-specific Cre-expressing adenovirus (Ad-Cre). Using well-characterized promoters for Keratin 8 and Keratin 14, we generated lineage-specific Ad-Cre lines for luminal and basal MECs, respectively. By pulse-chase lineage tracing using these Ad-Cre lines, we showed that luminal and basal lineages are largely self-sustained and that IRS1 and IRS2 are essential for maintaining the basal lineage; we also showed that heterogeneous mammary tumors can be induced from luminal MECs in mice carrying the Etv6-NTRK3 fusion gene. Overall, we validated the Ad-Cre system as a promising and efficient tool for fate mapping of normal and malignant cells in adult tissues.

LY6A/E (SCA-1) expression in the mouse testis.

Recently, it was found by two research groups that LY6A, known widely in the stem cell community as stem cell antigen-1 or SCA-1, is expressed on testicular side population (SP) cells. Whether these SP cells are spermatogonial stem cells is a point of disagreement and, therefore, the identity of the LY6A-positive cells as well. We studied the expression pattern of LY6A in testis by immunohistochemistry and found it to be expressed in the interstitial tissue on peritubular myoid, endothelial, and spherical-shaped peritubular mesenchymal cells. To address the question whether LY6A has a function in spermatogenesis or testis development, we studied the testis of Ly6a(-/-) mice (allele Ly6a(tm1Pmf)). We found no morphological abnormalities or differences in numbers of spermatogonia, spermatocytes, Leydig cells, or macrophages in relation to the number of Sertoli cells. Therefore, we conclude that LY6A expression does not influence testis development or spermatogenesis and that spermatogonial stem cells are LY6A negative.