Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).

Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.