Comparative analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias.

Testicular biopsy is needed to confirm diagnosis in azoospermic patients and to recover spermatozoa, if possible. This report aims to quantitatively analyse the germline markers stage-specific embryonic antigen (SSEA-1), c-KIT and VASA in testicular biopsies with distinct azoospermic aetiologies. Twenty-three testicular biopsies were analysed by flow cytometry and RT-qPCR for c-KIT, SSEA-1 and VASA. In all the Sertoli cell-only (SCO) samples, significantly lower VASA mRNA expression and fewer VASA+ cells were found compared with obstructive controls. Maturation arrest (MA) cases showed significant differences only with the non-mosaic SCO samples when compared for VASA mRNA expression and percentage of VASA+ cells, but not with the mosaics. However, the normalized VASA-KIT parameter obtained by subtracting the percentage of c-KIT+ cells from the percentage of VASA+ cells showed significant differences between the MA and all the SCO samples. RT-qPCR consistently found differences for the VASA expression between SCO mosaic and non-mosaic samples. However, by flow cytometry, only VASA-KIT showed significant differences between them. Conversely, the percentage of SSEA-1+ cells revealed no inter-group differences. In conclusion, testicular biopsies display different expression profiles for c-KIT and VASA depending on the azoospermic aetiology. These results can be used as a complementary tool to create new molecular categories for diagnoses in azoospermic patients, particularly useful to discriminate between mosaic and non-mosaic SCO patients.

Differentiation of germ cells and gametes from stem cells.

BACKGROUND: Advances in stem cell research have opened new perspectives for regenerative and reproductive medicine. Stem cells (SC) can differentiate under appropriate in vitro and in vivo conditions into different cell types. Several groups have reported their ability to differentiate SCs into germline cells, and some of them have been successful in obtaining male and female gamete-like cells by using different methodologies. METHODS: This review summarizes the current knowledge in this field and emphasizes significant embryological, genetic and epigenetic aspects of germ cells and gametes in vitro differentiation in humans and other species, highlighting major obstacles that need to be overcome for successful gametogenesis in culture: studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and somatic SCs are critically reviewed. RESULTS: Published studies indicate that germ cells can be consistently differentiated from mouse and human ESC. However, further differentiation of germ cells through gametogenesis still has important genetic and epigenetic obstacles to be efficient. CONCLUSIONS: Differentiation of germ cells from SCs has the potential of becoming a future source of gametes for research use, although further investigation is needed to understand and develop the appropriate niches and culture conditions. Additionally, if genetic and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered.

Zinc chelation during non-lesioning overexcitation results in neuronal death in the mouse hippocampus.

In the hippocampus, chelatable zinc is accumulated in vesicles of glutamatergic presynaptic terminals, abounding specially in the mossy fibers, from where it is released with activity and can exert a powerful inhibitory action upon N-methyl-D-aspartate receptors. Zinc is therefore in a strategic situation to control overexcitation at the zinc-rich excitatory synapses, and consequently zinc removal during high activity might result in excitotoxic neuronal damage. We analyzed the effect of zinc chelation with sodium dietyldithiocarbamate under overexcitation conditions induced by non-lesioning doses of kainic acid in the mouse hippocampus, to get insight into the role of zinc under overexcitation. Swiss male mice were injected with kainic acid (15 mg/kg, i.p.) 15 min prior to sodium dietyldithiocarbamate (150 mg/kg, i.p.), and left to survive for 6 h, 1 day, 4 days, or 7 days after the treatment. Cell damage was analyzed with the hematoxylin-eosin and acid fuchsin stainings. Neither control animals treated only with kainic acid nor those treated only with sodium dietyldithiocarbamate suffered seizures or neuronal damage. By contrast, the kainic acid+sodium dietyldithiocarbamate-treated animals showed convulsive behavior and cell death involving the hilus, CA3, and CA1 regions. Pretreatment with the N-methyl-D-aspartate receptor antagonist MK801 (1 mg/kg, i.p.) completely prevented neuronal damage. Experiments combining different doses of sodium dietyldithiocarbamate and kainic acid with different administration schedules demonstrated that the overlap of zinc chelation and overexcitation is necessary to trigger the observed effects. Moreover, the treatment with a high dose of sodium dietyldithiocarbamate (1000 mg/kg), which produced a complete bleaching of the Timm staining for approximately 12 h, highly increased the sensitivity of animals to kainic acid. Altogether, our results indicate that the actions of sodium dietyldithiocarbamate are based on a reduction of zinc levels, which under overexcitation conditions induce seizures and neuronal damage. These findings fully support a protective role for synaptically released zinc during high neuronal activity, most probably mediated by its inhibitory actions on N-methyl-D-aspartate receptors, and argue against a direct action of synaptic zinc on the observed neuronal damage.

Parvalbumin-containing interneurons do not innervate granule cells in the olfactory bulb.

Combining pre-embedding parvalbumin immunostaining and post-embedding immunogold detection of GABA in the olfactory bulb, we investigated whether the parvalbumin-containing GABAergic interneurons of the external plexiform layer exclusively innervate principal cells, or whether they also establish inhibitory synapses upon GABAergic local neurons such as granule cells. Our results demonstrate that the parvalbumin-containing cells do not contact GABAergic interneurons in the neuropil of the external plexiform layer. On the contrary, their postsynaptic elements were always non-GABAergic principal cells. Although classically it has been accepted that the interneurons of the external plexiform layer could exert a disinhibitory action upon principal cells, via inhibition of GABAergic granule cells, we conclude that they exert a feedback inhibitory action directly and exclusively upon principal cells.