Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1-/- mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1-/- mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1-/- compared to wild-type mice. Furthermore, cultured Uch-l1-/- SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

PNKP is required for maintaining the integrity of progenitor cell populations in adult mice.

DNA repair proteins are critical to the maintenance of genomic integrity. Specific types of genotoxic factors, including reactive oxygen species generated during normal cellular metabolism or as a result of exposure to exogenous oxidative agents, frequently leads to "ragged" single-strand DNA breaks. The latter exhibits abnormal free DNA ends containing either a 5'-hydroxyl or 3'-phosphate requiring correction by the dual function enzyme, polynucleotide kinase phosphatase (PNKP), before DNA polymerase and ligation reactions can occur to seal the break. Pnkp gene deletion during early murine development leads to lethality; in contrast, the role of PNKP in adult mice is unknown. To investigate the latter, we used an inducible conditional mutagenesis approach to cause global disruption of the Pnkp gene in adult mice. This resulted in a premature aging-like phenotype, characterized by impaired growth of hair follicles, seminiferous tubules, and neural progenitor cell populations. These results point to an important role for PNKP in maintaining the normal growth and survival of these murine progenitor populations.

Transcriptional Profiling of the Adult Hair Follicle Mesenchyme Reveals R-spondin as a Novel Regulator of Dermal Progenitor Function.

The adult hair follicle (HF) undergoes successive regeneration driven by resident epithelial stem cells and neighboring mesenchyme. Recent work described the existence of HF dermal stem cells (hfDSCs), but the genetic regulation of hfDSCs and their daughter cell lineages in HF regeneration remains unknown. Here we prospectively isolate functionally distinct mesenchymal compartment in the HF (dermal cup [DC; includes hfDSCs] and dermal papilla) and define the transcriptional programs involved in hfDSC function and acquisition of divergent mesenchymal fates. From this, we demonstrate cross-compartment mesenchymal signaling within the HF niche, whereby DP-derived R-spondins act to stimulate proliferation of both hfDSCs and epithelial progenitors during HF regeneration. Our findings describe unique transcriptional programs that underlie the functional heterogeneity among specialized fibroblasts within the adult HF and identify a novel regulator of mesenchymal progenitor function during tissue regeneration.

Primary cilia on porcine testicular somatic cells and their role in hedgehog signaling and tubular morphogenesis in vitro.

The primary cilium is a microtubule-based sensory organelle found on nearly all eukaryotic cells but little is understood about its function in the testis. We investigate the role of primary cilia on testis cells in vitro by inhibiting formation of the primary cilium with Ciliobrevin D, a cell-permeable, reversible chemical inhibitor of ATPase motor cytoplasmic dynein. We analyzed cultured cells for the presence of primary cilia and their involvement in hedgehog signaling. Primary cilia were present on 89.3 ± 2.3 % of untreated testicular somatic cells compared to 3.1 ± 2.5 % cells with primary cilia for Ciliobrevin D-treated cells. Protein levels of Gli-2 and Smoothened were lower on Western blots after suppression of cilia with Ciliobrevin D. The inhibitor did not affect centrosome localization or cell proliferation, indicating that changes were due to ablation of the primary cilium. Testicular somatic cells have the ability to form three-dimensional tubules in vitro. In vitro-formed tubules were significantly longer and wider in the control group than in the Ciliobrevin D-treated group (9.91 ± 0.35 vs. 5.540 ± 1.08 mm and 339.8 ± 55.78 vs. 127.2 ± 11.9 µm, respectively) indicating that primary cilia play a role in tubule formation. Our results establish that the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, affects the hedgehog signaling pathway and impairs tubule formation in vitro. These findings provide evidence for a role of cilia in the testis in cell signaling and tubular morphogenesis in vitro.

Phthalate esters affect maturation and function of primate testis tissue ectopically grafted in mice.

Di-n-Butyl (DBP) and Di-(2-EthylHexyl) (DEHP) phthalates can leach from daily-use products resulting in environmental exposure. In male rodents, phthalate exposure results in reproductive effects. To evaluate effects on the immature primate testis, testis fragments from 6-month-old rhesus macaques were grafted subcutaneously to immune-deficient mice, which were exposed to 0, 10, or 500 mg/kg of DBP or DEHP for 14 weeks or 28 weeks (DBP only). DBP exposure reduced the expression of key steroidogenic genes, indicating that Leydig cell function was compromised. Exposure to 500 mg/kg impaired tubule formation and germ cell differentiation and reduced numbers of spermatogonia. Exposure to 10 mg/kg did not affect development, but reduced Sertoli cell number and resulted in increased expression of inhibin B. Exposure to DEHP for 14 week also affected steroidogenic genes expression. Therefore, long-term exposure to phthalate esters affected development and function of the primate testis in a time and dosage dependent manner.

From in vitro culture to in vivo models to study testis development and spermatogenesis.

The testis is a complex organ playing host to one of the most intricate mass cell divisions occurring in postnatal life. Since the beginning of the 20th century, great efforts have been made to recapitulate spermatogenesis and elucidate spermatogonial stem cell function. These efforts have resulted in the development of a variety of model systems that provide invaluable knowledge regarding testis organogenesis, key cell types and their interactions, and signaling pathways controlling testis function. The goal of this review is to elaborate on the evolution of the techniques available from in vitro culture systems to in vivo bioassays by providing up to date information and weighing their particular strengths and weaknesses. Each technique offers a different approach to the elucidation of male reproduction, the enhancement of germ-lineage genetic manipulation, the preservation of gametes, the restoration of fertility, and the improvement in our understanding of stem cell biology.