Luis Dettin

Immortalization of Mouse Germ Line Stem Cells

In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T‐antigen gene (LTAg) under the control of an ecdysone‐inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct‐4, a transcription factor, and GFRα‐1, the receptor for glial cell line–derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology.

Morphological Characterization of the Spermatogonial Subtypes in the Neonatal Mouse Testis

Abstract Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (As), A-paired (Apr), A-aligned (Aal), and A1–A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (∼1 µm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRα-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially As, Apr, and possibly Aal spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.

Regulated Expression and Ultrastructural Localization of Galectin-1, a Proapoptotic β-Galactoside-Binding Lectin, During Spermatogenesis in Rat Testis

Abstract Galectin-1, a highly conserved β-galactoside-binding protein, induces apoptosis of activated T cells and suppresses the development of autoimmunity and chronic inflammation. To gain insight regarding the potential role of galectin-1 as a novel mechanism of immune privilege, we investigated expression and ultrastructural localization of galectin-1 in rat testis. Galectin-1 expression was assessed by Western blot analysis and immunocytochemical localization in testes obtained from rats aged from 9 to 60 days. Expression of this carbohydrate-binding protein was developmentally regulated, and its immunolabeling exhibited a stage-specific pattern throughout the spermatogenic process. Immunogold staining using the anti-galectin-1 antibody revealed the typical Sertoli cell profile in the seminiferous epithelium, mainly at stages X–II. During spermiation (stages VI–VIII), a strong labeling was observed at the luminal pole of seminiferous epithelium, localized on apical stalks of Sertoli cells, on heads of mature spermatids, and on bodies of residual cytoplasm. Moreover, spermatozoa released into the lumen showed a strong immunostaining. Following spermiation (stage VIII), galectin-1 expression was restored at the basal portion of Sertoli cells and progressively spread out through the whole cells as differentiation of germinal cells proceeded. Immunoelectron microscopy confirmed distribution of galectin-1 in nuclei and cytoplasmic projections of Sertoli cells and on heads and tails of late spermatids and residual bodies. Surface localization of galectin-1 was evidenced in spermatozoa from caput epididymis. Thus, the regulated expression of galectin-1 during the spermatogenic cycle suggests a novel role for this immunosuppressive lectin in reproductive biology.

Expression of Vascular Endothelial Growth Factor Receptors During Male Germ Cell Differentiation in the Mouse

Abstract Overexpression of vascular endothelial growth factor (VEGF) in the testis of transgenic mice induces infertility, suggesting a potential role for VEGF in the process of spermatogenesis. Spermatogenesis occurs within the confines of the seminiferous tubules, and the seminiferous epithelium lining these tubules consists of Sertoli cells and germ cells in various stages of maturation. We investigated the source of VEGF and VEGF-target cells within the seminiferous tubules of the normal mouse testis. Sections of testes fixed in Bouin solution and embedded in paraffin were subjected to immunofluorescent staining with specific antibodies against VEGF, and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Total RNA was extracted from isolated populations of Sertoli cells, type A spermatogonia, pachytene spermatocytes, and spermatids. Primer pairs specific for VEGF and its receptors were designed and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. Immunofluorescent studies indicated that VEGF is strongly expressed in the cytoplasm of Sertoli cells. VEGFR-1 and VEGFR-2 were not expressed by the Sertoli cell. In contrast, a differential expression of VEGF receptors was observed in germ cells. Although VEGFR-2 was expressed in the cytoplasm of type A spermatogonia, VEGFR-1 was expressed in the acrosomal region of spermatids and spermatozoa. Pachytene spermatocytes did not exhibit any staining. Further, we examined the transcription of VEGF and its receptors by RT-PCR. VEGF was actively transcribed only in Sertoli cells. The transcription of VEGFR-2 was confined to type A spermatogonia. Interestingly, VEGFR-1 was transcribed both in pachytene spermatocytes and round spermatids. The mRNA expression of VEGFR-1 and VEGFR-2 in germ cells was inversely correlated during postnatal development of the mouse testis. Thus, VEGF may play a potential role in regulating the initial stages of the process of spermatogonial proliferation through VEGFR-2 and spermiogenesis through VEGFR-1.

Contrast-enhanced in vivo imaging of breast and prostate cancer cells by MRI

The development of effective cancer therapies has been hampered, in part, by the inability to non-invasively follow tumor progression from the initial cancerous lesion through to metastasis. We have previously shown that superparamagnetic iron oxide particles can be used as magnetic resonance imaging contrast agents to label embryonic, mesenchymal and hematopoietic stem cells in vivo. Improving the capacity to non-invasively image cancer progression is an appealing method that could be useful for assessing the efficacy of anticancer therapies. We have established that human prostate (LNCaP, DU145, PC3), rodent prostate (TRAMPC1, YPEN-1), human breast (MDA-MB-213) and mouse mammary (Myc/VEGF) cancer cell lines were readily labeled by fluorescent superparamagnetic sub-micron particles of iron oxide (MPIOs). The MPIOs were essentially inert with respect to cell proliferation and tumor formation. Fluorescence stereomicroscopy and three dimensional magnetic resonance imaging (MRI) determined that subcutaneous, intramuscular or orthotopically implanted labeled cancer cells could be imaged, in vivo, despite in some cases being undetectable by manual palpation. The MPIO-labeled cancer cells could also be imaged, in vivo, at least 6 weeks after implantation. The fluorescent MPIOs further allowed for the ex vivo identification of tumors cells from histological sections. This study demonstrates the feasibility of using fluorescent MPIOs in prostate and breast cancer cell lines as both a negative contrast agent for in vivo MRI as well as a fluorescent tumor marker for optical imaging in vivo and ex vivo.

C-jun Inhibits Mammary Apoptosis In Vivo

c-Jun mediates ROS production and apoptosis.

Mammalian Testes: Structure and Function

The male reproductive system consists of the primary sex organs, the two testes and a set of accessory sexual structures. The adult mammalian testis performs two important functions, spermatogenesis and male sex hormone production. It is an organ structurally designed to produce the haploid male gametes from diploid postnatal germ-line stem cells, i.e. type A spermatogonia. The process of morphological and functional differentiation of type A spermatogonia into the haploid male gamete, the spermatozoon, is termed spermatogenesis. In addition, the testis elaborates a steroid hormone, testosterone, that is responsible for maintaining the spermatogenic process as well as the secondary male sexual characteristics. Furthermore, testosterone is important for several different functions in various organ systems including the maintainance of muscle mass and bone density. The process of testosterone formation from its precursor, cholesterol, is termed steroidogenesis. In this chapter, we will discuss how the structure and form of the testis contributes to the processes of spermatogenesis and steroidogenesis.