Federica M.F. van Dissel-Emiliani

Identification of Distinct Populations of Prostasomes That Differentially Express Prostate Stem Cell Antigen, Annexin A1 and GLIPR2 in Humans

ABSTRACT In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (∼1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.

Spermatogonial stem cell transplantation

The development of the spermatogonial transplantation technique has given new impetus to research on spermatogonial stem cells. Possibilities opened by this technique include: (a) New ways to study fundamental aspects of spermatogenesis; (b) Generation of transgenic large domestic animals; (c) Protection of (young) male cancer patients from infertility due to chemotherapy or radiotherapy. Spermatogonial stem cell transplantation for the above purposes encompasses a number of steps. First, the stem cells have to be isolated and possibly purified. Second, it should be possible to cryopreserve the stem cells, for example till the children have reached puberty. Third. it should be possible to culture spermatogonial stem cells for a prolonged period of time which would also allow transfection and subsequent selection of stably transfected cells. Fourth, in case of animal studies. the host testis should be emptied from endogenous stem cells. This is probably best done by local irradiation. Finally, the stem cells will have to be transplanted.

Transient receptor potential vanilloid receptor-1 confers heat resistance to male germ cells

Testicular hyperthermia in mice lacking transient receptor potential vanilloid receptor-1 results in a much more rapid and massive germ cell depletion from the seminiferous tubules than in wild-type animals, indicating that this receptor protects germ cells against heat stress.

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

BackgroundConflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.MethodsThe rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.ResultsInitial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.ConclusionCAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro

BackgroundThe initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential.Oncostatin M (OSM) is a multifunctional cytokine and member of the interleukin (IL)-6 family that also includes other cytokines such as LIF. In the rat OSM is highly expressed in the late fetal and neonatal testis, and may thus be a candidate factor involved in Leydig cell progenitor formation.MethodsInterstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days in the presence of different doses of OSM (range: 0.01 to 10 ng/ml) alone or in combination with LH (1 ng/ml). The effects of OSM and LH on cell proliferation were determined by incubating the cultures with [3H]thymidine or bromodeoxyuridine (BrdU). Developing progenitor cells were identified histochemically by the presence of the marker enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD).ResultsOSM, when added at a dose of 10 ng/ml, caused a nearly 2-fold increase in the percentage of Leydig cell progenitors after 8 days of culture. Immunohistochemical double labelling experiments with 3beta-HSD and BrdU antibodies showed that this increase was the result of differentiation of stem Leydig cells/precursor cells and not caused by proliferation of progenitor cells themselves. The addition of LH to the cultures consistently resulted in an increase in progenitor formation throughout the culture period. Surprisingly, when OSM and LH were added together, the LH induced rise in progenitor cells was significantly inhibited after 3 and 8 days of culture.ConclusionTaken together, the results of the present study suggest that locally produced OSM may not only play a role in the regulation of Sertoli cell proliferation and the initiation of spermatogenesis but may also play a role in the regulation of Leydig cell progenitor formation by keeping the augmenting effects of LH on this process in abeyance.