The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The ...luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.
Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal ...connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1
mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.
Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not ...been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography–tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.
Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. ...However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent 'third'-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.
•Exogenous hormone use alters ovine cervicovaginal mucus characteristics.•Mucus is most abundant, clear in colour and less proteinaceous at oestrus.•Superovulation increases mucus production and ...protein concentration in Merino ewes.•Synchronisation of oestrus using PGF2α reduced volume of mucus produced.
Controlled breeding programmes utilising exogenous hormones are common in the Australian sheep industry, however the effects of such programmes on cervicovaginal mucus properties are lacking. As such, the aim of this study was to investigate cervicovaginal (CV) mucus from naturally cycling (NAT), progesterone synchronised (P4), prostaglandin synchronised (PGF2α), and superovulated (SOV) Merino ewes. Experiment 1; volume, colour, spinnbarkeit, chemical profile and protein concentration of mucus (NAT, P4, PGF2α and SOV; n=5 ewes/treatment) during the follicular (5 d) and luteal phases (8 d) was investigated. Experiment 2; in vivo mucus pH and in vitro mucus penetration by frozen-thawed spermatozoa (NAT, P4 and SOV; n=11 ewes/treatment) was investigated over oestrus (2 d) and the mid-luteal phase (pH only, 2 d). Oestrus mucus was more abundant, clearer in colour and less proteinaceous than luteal phase mucus (p<0.05). SOV increased mucus production and protein concentration (p<0.05) while PGF2α reduced mucus volume (p<0.05). Mucus pH (oestrus 6.2–6.5), chemical profile and mucus penetration by sperm were unchanged (p>0.05). Results indicate that exogenous hormones used for controlled breeding affect cervicovaginal mucus production, but few other tested characteristics. Further research is required to explain fertility differences between synchronised and naturally cycling animals following cervical AI.
Although essential for artificial insemination (AI) and MOET (multiple ovulation and embryo transfer), oestrus synchronisation and superovulation are associated with increased female reproductive ...tract mucus production and altered sperm transport. The effects of such breeding practices on the ovine cervicovaginal (CV) mucus proteome have not been detailed. The aim of this study was to qualitatively and quantitatively investigate the Merino CV mucus proteome in naturally cycling (NAT) ewes at oestrus and mid-luteal phase, and quantitatively compare CV oestrus mucus proteomes of NAT, progesterone synchronised (P4) and superovulated (SOV) ewes. Quantitative analysis revealed 60 proteins were more abundant during oestrus and 127 were more abundant during the luteal phase, with 27 oestrus specific and 40 luteal specific proteins identified. The oestrus proteins most disparate in abundance compared to mid-luteal phase were ceruloplasmin (CP), chitinase-3-like protein 1 (CHI3L1), clusterin (CLU), alkaline phosphatase (ALPL) and mucin-16 (MUC16). Exogenous hormones greatly altered the proteome with 51 and 32 proteins more abundant and 98 and 53 proteins less abundant, in P4 and SOV mucus, respectively when compared to NAT mucus. Investigation of the impact of these proteomic changes on sperm motility and longevity within mucus may help improve sperm transport and fertility following cervical AI.
This manuscript is the first to detail the proteome of ovine cervicovaginal mucus using qualitative and quantitative proteomic methods over the oestrous cycle in naturally cycling ewes, and also after application of common oestrus synchronisation and superovulation practices. The investigation of the mucus proteome throughout both the follicular and luteal periods of the oestrous cycle, and also after oestrous synchronisation and superovulation provides information about the endocrine control and the effects that exogenous hormones have on protein expression in the female reproductive tract. This information contributes to the field by providing important information on the changes that occur to the cervicovaginal mucus proteome after use of exogenous hormones in controlled breeding programs, which are commonly used on farm and also in a research setting.
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•Cervicovaginal mucus proteome alters over the oestrus cycle.•Exogenous hormones used for oestrus synchronisation affects the mucus proteome.•Progesterone synchronisation and superovulation alter the mucus proteome.
In Parkinson’s disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a ...methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery.
•Derivation of distinct dopaminergic subpopulations from pluripotent stem cells•Wnt signaling inhibitors promote SN dopaminergic neuron specification•Modeling selective vulnerability of SN dopaminergic neurons in vitro•Proteomics reveals pathways that promote SN dopaminergic neuron survival
In this article, Panman and colleagues show that by modulating Wnt signaling levels, distinct subpopulations of dopaminergic neurons can be derived from mouse and human pluripotent stem cells. Cultures enriched for substantia nigra dopaminergic neurons recapitulate the selective sensitivity to mitochondrial toxins as observed in Parkinson’s disease patients. The developed strategy facilitates disease modeling and drug discovery.
Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is ...important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid.
Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT.
Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure.
These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.
The source and composition of seminal plasma has previously been shown to alter the ability of spermatozoa to survive cryopreservation. In the present study, the ionic and proteomic composition of ...seminal plasma from rams with high (HSP; n=3) or low (LSP; n=3) freezing resilient spermatozoa was assessed. 75 proteins were identified to be more abundant in HSP and 48 proteins were identified to be more abundant in LSP. Individual seminal plasma proteomes were established for each of the six rams examined. For each ram, correlations were conducted between previously recorded freezing resilience 1 and individual spectral counts in order to identify markers of freezing resilience. 26S proteasome complex, acylamino acid releasing enzyme, alpha mannosidase class 2C, heat shock protein 90, tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase and transitional endoplasmic reticulum ATPase were found to be positively correlated (r2>0.7) with freezing resilience. Cystatin, zinc-2-alpha glycoprotein, angiogenin-2-like protein, cartilage acidic protein-1, cathepsin B and ribonuclease 4 isoform 1 were found to be negatively correlated (r2>0.7) with freezing resilience. Several negative markers were found to originate from the accessory sex glands, whereas many positive markers originated from spermatozoa and were part of or associated with the 26S proteasome or CCT complex.
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•Markers of sperm freezing resilience were identified within ram seminal plasma.•PSMD2, APEH, MAN2C1, HSP90AA1, TPP2, VCP, SORD and CCT were identified as positive markers.•CST3, AZGP1, ANG, CRTAC1, CTSB, RNASE4 were identified as negative markers.
Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by ...GeLC–MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation.
Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.
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•The seminal plasma proteome can be associated with the sperm preservation ability.•Several seminal proteins from sperm origin are biomarkers of sperm preservation.•The ZA2G increases mobility of fresh spermatozoa but decreases liquid preservation.
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