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AKR1B7 (mouse vas deferens protein) is dispensable for mouse development and reproductive success.
AKR1B7 (aldo-keto reductase family 1, member 7; also known as mouse vas deferens protein) is a member of the AKR superfamily, and has been suggested to play a role in detoxifying processes on account of its preferred substrates, 4-hydroxynonenal and isocaproaldehyde. High levels of protein expression were found in the vas deferens and the adrenal gland, where sustained expression is dependent on androgen or ACTH respectively. Recently, a remarkable induction of AKR1B7 expression has been reported in the ovary following exogenous injections of LH. In the present study, we confirm this regulation physiologically during the estrous cycle, observing Akr1b7 expression to be restricted to the theca and stromal cells of the proestrus ovary. To further investigate the role of this detoxifying enzyme in both male and female reproduction, we generated knockout mice deficient in AKR1B7. Although AKR1B7 expression in the vas deferens is considerable and tightly regulated in the ovary of wild-type animals, homozygous mutant animals were found to be viable and no reproductive phenotype was observed. Ovarian follicle maturation and spermatozoa parameters remained normal in the absence of this protein. The determination of serum progesterone revealed an increase in hormone concentration in metestrus, while progesterone was found to be decreased in the estrus phase of the cycle in knockout females.
Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption.
Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates.
Role of epididymal receptor HE6 in the regulation of sperm microenvironment.
HE6 (GPR64) is a highly conserved, tissue-specific heptahelical receptor of the human epididymis. The seven transmembrane (TM7) domains are a hallmark of G-protein-coupled receptors (GPCRs) which have a proven history of being excellent therapeutic targets. Of all currently marketed drugs, >30% are modulators of specific heptahelical receptors, emphasizing the potential of HE6 as a target for pharmaceutical intervention. Targeted mutation of the mouse HE6 counterpart resulted in male infertility, further emphasizing its role as a candidate target for male contraception. However, the precise function of HE6, together with its potential ligand(s), and signal transduction pathways have remained largely unknown. On the basis of shared sequence motifs within the TM7 region, HE6 has been grouped into the B class of GPCRs. Within this class, HE6 belongs to the 'large N-termini' family-B seven-transmembrane (LNB-TM7) receptors, also termed the adhesion-GPCRs. Members of this subgroup are 'orphan' receptors, and they all seem to be cleaved within a conserved GPCR proteolytic site (GPS) domain. The biological significance of the two-subunit architecture is still unknown. Clues to the function of HE6 within the epithelium of male excurrent ducts may come from its co-localisation with the apical actin cytoskeleton and from the down-regulation in "knockout" male mice of various proteins specific to the initial segment.
New approaches for male fertility control: HE6 as an example of a putative target.
Reversible contraceptive methods for males are still not available. During the last few years several marketing studies have clearly shown that men and women would welcome a situation where men could assume responsibility for family planning. Schering AG and Organon are currently collaborating to develop a hormonal method for male fertility control based on the combination of etonogestrel as gestagenic component and testosterone undecanoate. To further optimize male contraceptives in terms of improved efficiency, rapid onset, reversibility, fewer side effects and a convenient method of application, a search for innovative non-hormonal approaches was started. During the last few years, numerous proteins were identified which play a specific role in male fertility. These proteins have first to fulfil a set of indication-specific criteria before a drug discovery process can be initiated. The most important criteria for a putative target protein are tissue-selective expression, crucial biological function in fertility, drugable properties and feasibility of assay development for high-throughput-screening and lead optimization. The G-protein-coupled receptor HE6 was selected as target and the above selection criteria were applied. HE6 displays a preferred epididymis-specific expression pattern and belongs to the superfamily of GPCRs, which are well known to be drugable with small molecules. A knockout mouse was generated which revealed an infertility phenotype with the onset occurring 6 weeks after initiation of spermatogenesis at the latest. Surprisingly, no epididymis-specific phenotype was observed. Instead, the reabsorption of testicular fluid along the efferent ducts was strongly affected. No further obvious side effects were observed in male or female mice. This study with HE6 exemplifies how targets for male contraception have to be validated before drug development can start.
Mice over-expressing the 5-HT(1A) receptor in cortex and dentate gyrus display exaggerated locomotor and hypothermic response to 8-OH-DPAT.
The serotonin 1A (5-HT(1A)) receptor is one of the best described receptor subtypes of the serotonergic system. Due to the complex distribution pattern, the pre- and postsynaptic localisation, the impact on various monoamines, as well as the influence on a wide range of physiological functions, the contribution of 5-HT(1A) receptors to behavioural outcomes is difficult to define. In this study, we present a new transgenic mouse model with a prominent over-expression of the 5-HT(1A) receptor in the outer cortical layers (I-III) and the dentate gyrus. Behavioural studies revealed a slight decrease in baseline motor activity of homozygous mice during the open field test. Moreover, core body temperature of male transgenic mice was significantly lower than that of wild-type mice. Pharmacological studies with the 5-HT(1A) receptor agonist 8-OH-DPAT (0.1-2.5 mg/kg, i.p.) revealed an exaggerated drug response in mutant mice. 8-OH-DPAT led to a drastic decrease in motor activity in the open field and elevated plus maze test. This significant effect on motor activity became more apparent by investigating the serotonergic syndrome induced by 8-OH-DPAT. Concentration as low as 0.5 mg/kg 8-OH-DPAT caused immobility in transgenic mice for 30 min, head weaving behaviour, and backward walking, whereas in wild-type animals, typical behaviours of the serotonin syndrome were first observed at concentrations of 1.5 mg/kg and more. In addition, the 8-OH-DPAT induced hypothermia was more pronounced in mutant mice than in wild-type animals. Therefore, these genetically modified mice represent a promising model for further investigations of the role of 5-HT(1A) receptors.
Targeted deletion of the epididymal receptor HE6 results in fluid dysregulation and male infertility.
Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B(2)) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family.
Reduced anxiety-related behaviour in transgenic mice overexpressing serotonin 1A receptors.
Serotonergic neurons play a major role in the modulation of emotion and behaviour. Especially knockout studies have revealed a role for the serotonin(1A) (5-HT(1A)) receptor in anxiety related behaviour. Mutant animals exhibit enhanced anxiety-like responses, possibly resulting from impaired autoinhibitory control of midbrain serotonergic neurons. To further elucidate the role of the 5-HT(1A) receptors in affective behaviour, a complementary approach has been used and transgenic mice overexpressing this receptor subtype have been generated. The expression of the active 5-HT(1A) receptor protein as indicated by autoradiography was transiently increased during early postnatal development (P1.5) as compared to wild-type mice. Within the next 2 weeks, the increase in receptor binding vanished and was also not apparent in adult animals indicating adaptive changes in the regulation of 5-HT(1A) receptor expression. Although no evidence for increased receptor binding in the brains of adult homozygous mice was found by autoradiography, typical phenotypic changes indicative of 5-HT(1A) receptor overactivity were apparent. Transgenic mice revealed a reduced molar ratio of 5-hydroxyindoleacetic acid to serotonin in several brain areas and elevated serotonin values in the hippocampus and striatum. Moreover, anxiety-like behaviour was decreased in male and female transgenic mice and body temperature was lowered in male transgenic mice in comparison with heterozygous and wild-type mice. These findings further underline the pivotal role of 5-HT(1A) receptors in the homeostasis of anxiety-like behaviour and the crucial importance of stimulation of the 5-HT(1A) receptor during the early postnatal development for normal anxiety-like behaviour throughout life.
Analysis of the murine 5-HT receptor gene promoter in vitro and in vivo.
The expression level of the 5-HT(1A) receptor gene (htr1a) in the central nervous system (CNS) is implicated in the aetiology and treatment of anxiety disorders and depression. Previous studies of the murine htr1a have revealed that its proximal promoter is GC rich and TATA-less. Several functional transcription factor binding sites, including MAZ and SP1 recognition sequences, have been identified. To further analyse the promoter of this receptor gene, additional upstream sequence information extending to -5.5 kb of the murine htr1a was generated and promoter fragments extending to -20 kb were analysed for activity in cell culture and transgenic animals. Promoter fragments greater than 4.5 kb in length were active in 5-HT(1A) receptor mRNA positive cells and inactive in 5-HT(1A) receptor mRNA negative cells. Smaller fragments were not able to confer this specificity. In agreement, using additive transgenesis to drive LacZ expression in vivo, CNS specific reporter gene expression was found with these longer constructs. Transgene expression in the 4.5- and 20-kb mouse lines resembled the endogenous htr1a expression pattern, whereas the 5.5-kb mouse lines surprisingly revealed strongly reduced expression. None of the three constructs was prone to confer ectopic expression, however, variation of expression between the transgenic lines was observed. Using colocalization studies we analysed the degree of concurrence of transgenic and endogenous htr1a expression brought about by these three different constructs. The highest degrees of colocalization were observed in mice harbouring the 20-kb construct, suggesting a large promoter fragment is required to faithfully direct transgene expression in a 5-HT(1A) receptor like pattern.
Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity.
Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult.
Serine proteases in rodent hippocampus.
Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.
Association of walking volume and intensity with incident gout: a population-based cohort study.
Previous studies have reported that walking is associated with a lower risk of several metabolic diseases; however, evidence of its association with gout, a metabolic condition, is lacking. We conducted cohort studies using data from the UK Biobank (2013-2021) to examine the relationship of walking volume and intensity with the risk of incident gout. We included 92,066 participants who were free of gout and had valid baseline 7-day accelerometer data, which allowed us to derive walking volume (total steps), walking intensity (peak 30-minute cadence), and their combination (purposeful steps). Hazard ratios (HRs) for incident gout were estimated across different walking activity metrics groups using Cox proportional hazard models. During a mean follow-up period of 6.93 years, 706 participants developed incident gout. Walking volume and intensity alone showed no significant relationship to the risk of incident gout. However, compared with the low group (<5,000 steps/day) of purposeful steps, the risk of incident gout was lower in the middle group (5,000-7,999 steps/day) and high (≥8,000 steps/day) group, with adjusted HRs and 95% confidence intervals of 0.78 (0.66-0.94) and 0.72 (0.58-0.90), respectively (P for trend =0.003). These findings offer valuable insights for developing walking-based recommendations to prevent incident gout.
An essential role for the Zn2+ transporter ZIP7 in B cell development.
Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.
In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.
In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.
Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss.
The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.
Exploring the potential of laser-textured metal alloys: Fine-tuning vascular cells responses through in vitro and ex vivo analysis.
Medical stents are vital for treating vascular complications and restoring blood flow in millions of patients. Despite its widespread effectiveness, restenosis, driven by the complex interplay of cellular responses, remains a concern. This study investigated the reactions of vascular cells to nano/microscale wrinkle (nano-W and micro-W) patterns created on laser-textured nitinol (NiTi) surfaces by adjusting laser processing parameters, such as spot overlap ratio and line overlap ratio. Evaluation of topographical effects on endothelial and smooth muscle cells (SMCs) revealed diverse morphologies, proliferation rates, and gene expressions. Notably, microscale wrinkle patterns exhibited reduced monocyte adhesion and inflammation-related gene expression, demonstrating their potential applications in mitigating vascular complications after stent insertion. Additionally, an ex vivo metatarsal assay was utilized to bridge the gap between in vitro and in vivo studies, demonstrating enhanced angiogenesis on laser-textured NiTi surfaces. Laser-textured NiTi exhibits a guided formation process, emphasizing their potential to promote swift endothelialization. These findings underscore the efficacy of laser texturing for tailored cellular interactions on metallic surfaces and offer valuable insights into optimizing biocompatibility and controlling cellular responses, which may pave the way for innovative advances in vascular care and contribute to the ongoing improvement of stent insertion.
The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse.
The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.
A detailed methodology for a three-dimensional, self-structuring bone model that supports the differentiation of osteoblasts towards osteocytes and the production of a complex collagen-rich mineralised matrix.
BACKGROUND: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). METHODS: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. RESULTS: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. CONCLUSIONS: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.