Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

BACKGROUND: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. OBJECTIVE: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. MATERIALS AND METHODS: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. RESULTS: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. CONCLUSIONS: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

EPAS1 expression contributes to maintenance of the primordial follicle pool in the mouse ovary.

Oxygen availability can have profound effects on cell fate decisions and survival, in part by regulating expression of hypoxia-inducible factors (HIFs). In the ovary, HIF expression has been characterised in granulosa cells, however, any requirement in oocytes remains relatively undefined. Here we developed a Hif2a/Epas1 germline-specific knockout mouse line in which females were fertile, however produced 40% fewer pups than controls. No defects in follicle development were detected, and quality of MII oocytes was normal, as per assessments of viability, intracellular reactive oxygen species, and spindle parameters. However, a significant diminishment of the primordial follicle pool was evident in cKO females that was attributed to accelerated follicle loss from postnatal day 6 onwards, potentially via disruption of the autophagy pathway. These data demonstrate the importance of HIF signalling in oocytes, particularly at the primordial follicle stage, and lend to the importance of controlling oxygen tension in the development of in vitro growth and maturation approaches for assisted reproduction.

Assessment of the impact of direct in vitro PFAS treatment on mouse spermatozoa.

Abstract: Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary: PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.

Immortalized mouse caput epididymal epithelial (mECap18) cell line recapitulates the in-vivo environment.

Residing between the testes and the vas deferens, the epididymis is a highly convoluted tubule whose unique luminal microenvironment is crucial for the functional maturation of spermatozoa. This microenvironment is created by the combined secretory and resorptive activity of the lining epididymal epithelium, including the release of extracellular vesicles (epididymosomes), which encapsulate fertility modulating proteins and a myriad of small non-coding RNAs (sncRNAs) that are destined for delivery to recipient sperm cells. To enable investigation of this intercellular communication nexus, we have previously developed an immortalized mouse caput epididymal epithelial cell line (mECap18). Here, we describe the application of label-free mass spectrometry to characterize the mECap18 cell proteome and compare this to the proteome of native mouse caput epididymal epithelial cells. We report the identification of 5,313 mECap18 proteins, as many as 75.8% of which were also identified in caput epithelial cells wherein they mapped to broadly similar protein classification groupings. Furthermore, key pathways associated with protein synthesis (e.g., EIF2 signaling) and cellular protection in the male reproductive tract (e.g., sirtuin signaling) were enriched in both proteomes. This comparison supports the utility of the mECap18 cell line as a tractable in-vitro model for studying caput epididymal epithelial cell function.

The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions.

In this study we explored the role of hypoxia and the hypoxia-inducible transcription factor EPAS1 in regulating spermatogonial stem cell (SSC) function in the mouse testis. We have demonstrated that SSCs reside in hypoxic microenvironments in the testis through utilization of the oxygen-sensing probe pimonidazole, and by confirming the stable presence of EPAS1, which is degraded at >5% O2. Through the generation of a germline-specific Epas1 knockout mouse line, and through modulation of EPAS1 levels in primary cultures of spermatogonia with the small drug molecule Daprodustat, we have demonstrated that EPAS1 is required for robust SSC function in regenerative conditions (post-transplantation and post-chemotherapy), via the regulation of key cellular processes such as metabolism. These findings shed light on the relationship between hypoxia and male fertility and will potentially facilitate optimization of in vitro culture conditions for infertility treatment pipelines using SSCs, such as those directed at pediatric cancer survivors.

Global profiling of the proteomic changes associated with the post-testicular maturation of mouse spermatozoa.

Spermatozoa acquire fertilization potential during passage through a highly specialized region of the extratesticular ductal system known as the epididymis. In the absence of de novo gene transcription or protein translation, this functional transformation is extrinsically driven via the exchange of varied macromolecular cargo between spermatozoa and the surrounding luminal plasma. Key among these changes is a substantive remodeling of the sperm proteomic architecture, the scale of which has yet to be fully resolved. Here, we have exploited quantitative mass spectrometry-based proteomics to define the extent of changes associated with the maturation of mouse spermatozoa; reporting the identity of >6,000 proteins, encompassing the selective loss and gain of several hundred proteins. Further, we demonstrate epididymal-driven activation of RHOA-mediated signaling pathways is an important component of sperm maturation. These data contribute molecular insights into the complexity of proteomic changes associated with epididymal sperm maturation.

Transcriptomic analysis of the seminal vesicle response to the reproductive toxicant acrylamide.

BACKGROUND: The seminal vesicles synthesise bioactive factors that support gamete function, modulate the female reproductive tract to promote implantation, and influence developmental programming of offspring phenotype. Despite the significance of the seminal vesicles in reproduction, their biology remains poorly defined. Here, to advance understanding of seminal vesicle biology, we analyse the mouse seminal vesicle transcriptome under normal physiological conditions and in response to acute exposure to the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or vehicle control daily for five consecutive days prior to collecting seminal vesicle tissue 72 h following the final injection. RESULTS: A total of 15,304 genes were identified in the seminal vesicles with those encoding secreted proteins amongst the most abundant. In addition to reproductive hormone pathways, functional annotation of the seminal vesicle transcriptome identified cell proliferation, protein synthesis, and cellular death and survival pathways as prominent biological processes. Administration of acrylamide elicited 70 differentially regulated (fold-change ≥1.5 or ≤ 0.67) genes, several of which were orthogonally validated using quantitative PCR. Pathways that initiate gene and protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways. Inflammation was also a key transcriptomic response to acrylamide, with the cytokine, Colony stimulating factor 2 (Csf2) identified as a top-ranked upstream driver and inflammatory mediator associated with recovery of homeostasis. Early growth response (Egr1), C-C motif chemokine ligand 8 (Ccl8), and Collagen, type V, alpha 1 (Col5a1) were also identified amongst the dysregulated genes. Additionally, acrylamide treatment led to subtle changes in the expression of genes that encode proteins secreted by the seminal vesicle, including the complement regulator, Complement factor b (Cfb). CONCLUSIONS: These data add to emerging evidence demonstrating that the seminal vesicles, like other male reproductive tract tissues, are sensitive to environmental insults, and respond in a manner with potential to exert impact on fetal development and later offspring health.

Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.

Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

Oxidative Stress Dysregulates Protein Homeostasis Within the Male Germ Line.

Aims: Oxidative stress is causally linked to male reproductive pathologies, driven primarily by lipid peroxidation and an attendant production of highly reactive lipid aldehydes, such as 4-hydroxynonenal (4HNE) within the male germ line. In somatic cells, 4HNE dysregulates proteostasis via targeting of vulnerable proteins for adduction, causing protein misfolding and eventually aggregation. The aims of this study were to explore whether oxidative stress precipitates an equivalent response in the male germ line and determine the protective mechanisms used by germ cells to prevent this cascade of protein damage. Results: We reveal a causative role for oxidative stress in the accumulation of protein deposits in male germ cells. Specifically, 4HNE treatment resulted in a significant increase in cytosolic protein aggregation within pre- and post-meiotic germ cells as measured by the aggregate-detecting fluorophores ProteoStat and Thioflavin T, and the amyloid-specific anti-A11 and anti-OC antibodies. Our data implicate nucleocytoplasmic transport machinery and molecular chaperones as potential mechanisms for the subcellular compartmentalization and/or suppression of aggregating proteins. Thus, the inhibition of karyopherin transport proteins and molecular chaperones resulted in a significant increase in the accumulation of aggregated cellular protein. Innovation: These data establish the novel paradigm that lipid peroxidation is a key contributor to a decline in proteostasis in developing germ cells. These findings will inform the development of novel strategies to protect germ cells from oxidative stress. Conclusion: Together, these results shed light on proteostasis mechanisms that may assist in the management of misfolded proteins in the male germ line under conditions of acute oxidative stress.

Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions.

BACKGROUND: The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. RESULTS: Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. CONCLUSION: Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation.

Differential cell death decisions in the testis: evidence for an exclusive window of ferroptosis in round spermatids.

Oxidative stress is a major aetiology in many pathologies, including that of male infertility. Recent evidence in somatic cells has linked oxidative stress to the induction of a novel cell death modality termed ferroptosis. However, the induction of this iron-regulated, caspase-independent cell death pathway has never been explored outside of the soma. Ferroptosis is initiated through the inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and is exacerbated by the activity of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that facilitates lipid degradation. Here, we demonstrate that male germ cells of the mouse exhibit hallmarks of ferroptosis including; a caspase-independent decline in viability following exposure to oxidative stress conditions induced by the electrophile 4-hydroxynonenal or the ferroptosis activators (erastin and RSL3), as well as a reciprocal upregulation of ALOX15 and down regulation of GPX4 protein expression. Moreover, the round spermatid developmental stage may be sensitized to ferroptosis via the action of acyl-CoA synthetase long-chain family member 4 (ACSL4), which modifies membrane lipid composition in a manner favourable to lipid peroxidation. This work provides a clear impetus to explore the contribution of ferroptosis to the demise of germline cells during periods of acute stress in in vivo models.

A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells.

Oxidative stress is a leading causative agent in the defective sperm function associated with male infertility. Such stress commonly manifests via the accumulation of pathological levels of the electrophilic aldehyde, 4-hydroxynonenal (4HNE), generated as a result of lipid peroxidation. This highly reactive lipid aldehyde elicits a spectrum of cytotoxic lesions owing to its propensity to form stable adducts with biomolecules. Notably however, not all elements of the sperm proteome appear to display an equivalent vulnerability to 4HNE modification, with only a small number of putative targets having been identified to date. Here, we validate one such target of 4HNE adduction, A-Kinase Anchor Protein 4 (AKAP4); a major component of the sperm fibrous sheath responsible for regulating the signal transduction and metabolic pathways that support sperm motility and capacitation. Our data confirm that both the precursor (proAKAP4), and mature form of AKAP4, are conserved targets of 4HNE adduction in primary cultures of post-meiotic male germ cells (round spermatids) and in mature mouse and human spermatozoa. We further demonstrate that 4HNE treatment of round spermatids and mature spermatozoa results in a substantial reduction in the levels of both proAKAP4 and AKAP4 proteins. This response proved refractory to pharmacological inhibition of proteolysis, but coincided with an apparent increase in the degree of protein aggregation. Further, we demonstrate that 4HNE-mediated protein degradation and/or aggregation culminates in reduced levels of capacitation-associated phosphorylation in mature human spermatozoa, possibly due to dysregulation of the signaling framework assembled around the AKAP4 scaffold. Together, these findings suggest that AKAP4 plays an important role in the pathophysiological responses to 4HNE, thus strengthening the importance of AKAP4 as a biomarker of sperm quality, and providing the impetus for the design of an efficacious antioxidant-based intervention strategy to alleviate sperm dysfunction.

Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation.

The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ -1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa.

Post-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29-31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.

Small molecule Hedgehog pathway antagonists.

Leveraging our quinolone-1-(2H)-one based Hedgehog signalling pathway (HSP) inhibitors we have developed two new classes of HSP inhibitors based on: l-tryptophan and benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amine. Synthesis of focused compound libraries identified six l-tryptophan based inhibitors, and two stimulators, of Gli at 10 µM compound concentration. 2,4-Dichloro-13 and indole 16 suppressed mRNA expression of Ptch1 in Shh LIGHT2 cells, with 13 suppressing and 16 stimulating Gli2 mRNA expression. Focused library development of the benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amine scaffold afforded two sub-micro molar potent inhibitors of Gli expression with 5-methoxy-1H-indole-2-carboxylic acid benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amide 29 and 5-chloro-1H-indole-2-carboxylic acid benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amide 30 returning IC50 values of 0.5 and 0.24 µM, respectively. Neither 29 nor 30 acted directly on Smo with our data supporting inhibition of the HSP downstream of Smo.

Dynamin 2 is essential for mammalian spermatogenesis.

The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

Quinolone-1-(2H)-ones as hedgehog signalling pathway inhibitors.

A series of quinolone-2-(1H)-ones derived from the Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line. A focused SAR study was conducted and revealed core characteristics of the quinolone-2-(1H)-ones required for cytotoxicity. These requirements included a C3-tethered indole moiety, an indole C5-methyl moiety, an aliphatic tail or an ester, as well as an additional aromatic moiety. Further investigation in the SAG-activated Shh-LIGHT2 cell line with the most active analogues: 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(1-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (5), 2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)-N-(pentan-2-yl)acetamide (23) and ethyl (2-(3-cyano-2-oxo-4-phenylquinolin-1(2H)-yl)-2-(5-methyl-1H-indol-3-yl)acetyl)glycinate (24) demonstrated a down regulation of the HSP via a reduction in Gli expression, and in the mRNA levels of Ptch1 and Gli2. Analogues 5, 23 and 24 returned in cell inhibition values of 11.6, 2.9 and 3.1 µM, respectively, making this new HSP-inhibitor pharmacophore amongst the most potent non-Smo targeted inhibitors thus far reported.

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis.

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development.

Queuosine deficiency in eukaryotes compromises tyrosine production through increased tetrahydrobiopterin oxidation.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.