Background Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. Methods Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. Results Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. Conclusions Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis. © 2005 Wiley‐Liss, Inc.

Stem cells in various somatic tissues (bone marrow, skeletal muscle) can be identified by the `Side Population' marker based on Hoechst 33342 efflux. We show that mouse testicular cells also display a `Side Population' that express Bcrp1 mRNA, the ABC transporter responsible for Hoechst efflux in hematopoietic cells. Inhibition of Hoechst efflux by specific BCRP1 inhibitor Ko143 show that germinal `Side Population' phenotype is dependent on BCRP1 activity. Analysis of two well-defined models of altered spermatogenesis(W/Wv mutants and cryptorchid male mice) and RNA expression studies of differentiation markers demonstrate that germinal `Side Population' contains spermatogonial cells. In addition,α 6-integrin and Stra8 germinal stem cell markers, are expressed in the `Side Population'. In vivo repopulation assay clearly establishes that testis `Side Population' in adult mice is highly enriched in male germ stem cells.

In adults, stem cells are responsible for the maintenance of many actively renewing tissues, such as haematopoietic, skin, gut and germinal tissues. These stem cells can self-renew or be committed to becoming progenitors. Stem-cell commitment is thought to be irreversible but in male and female Drosophila melanogaster, it was shown recently that differentiating germ cells can revert to functional stem cells that can restore germinal lineage. Whether progenitors are also able to generate stem cells in mammals remains unknown. Here we show that purified mouse spermatogonial progenitors committed to differentiation can generate functional germinal stem cells that can repopulate germ-cell-depleted testes when transplanted into adult mice. We found that GDNF, a key regulator of the stem-cell niche, and FGF2 are able to reprogram in vitro spermatogonial progenitors for reverse differentiation. This study supports the emerging concept that the stem-cell identity is not restricted in adults to a definite pool of cells that self-renew, but that stemness could be acquired by differentiating progenitors after tissue injury and throughout life.

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Oocyte activation is a series of events triggered by the fertilizing spermatozoon and necessary for the beginning of the embryonic development. Calcium plays a pivotal role in this process. Here we used confocal laser scanning microscopy to examine the changes in the concentration of intracellular free calcium ([Ca2+]i) in human oocytes after intracytoplasmic sperm injection (ICSI). The first considerable but short (< 2 min) increase in [Ca2+]i was detected immediately after the penetration of the microinjection needle into the ooplasm. This rise by itself did not provoke oocyte activation and was also obtained after the injection of medium without spermatozoa. After a lag period of 4-12 h, oocytes that were subsequently activated initiated a second period of [Ca2+]i changes. These changes were sperm-dependent and followed one of two alternative patterns, a non-oscillatory one and an oscillatory one. The non-oscillatory pattern resembled the changes described previously during parthenogenetic activation of mammalian oocytes. The oscillatory pattern was similar to the changes accompanying normal fertilization in different mammalian species. It is concluded that the initial [Ca2+]i rise provoked by the ICSI procedure is not responsible for oocyte activation, and that a release of a sperm factor(s) is required to initiate this process.

Preliminary evidence has suggested that the phosphodiesterase inhibitor pentoxifylline augments the fertilizing potential of asthenozoospermic sperm samples, presumably by improving sperm movement. Here, we used computer-assisted sperm movement analysis to compare the effects of pentoxifylline in normozoospermic and asthenozoospermic specimens. The study focused on the following issues: the changes in individual movement characteristics in response to pentoxifylline, the rapidity of the response, the effect of sperm capacitation on the response, the persistence of the response after drug removal and the variability of responses among asthenozoospermic individuals. Data obtained show that (i) pentoxifylline increases the curvilinear velocity, path velocity, straight-line velocity, lateral head displacement, beat cross frequency and sperm hyperactivation in both normozoospermic and asthenozoospermic specimens, (ii) pentoxifylline does not modify the percentage of motile spermatozoa, (iii) the pentoxifylline effect reaches a maximum within 10 min of treatment in fresh semen as well as in capacitated sperm suspensions and persists for at least 2 h after drug removal and (iv) pentoxifylline improves the movement characteristics in most asthenozoospermic individuals. Results are discussed with regard to methods of therapeutic application of pentoxifylline as an enhancer of sperm movement in assisted reproductive technology.

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