The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction ...with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.
Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, ...and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.
In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our ...knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.
Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron ...density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells.
In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model.
NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.
Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be ...translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.
Primary ciliary dyskinesia (PCD) is an inherited autosomal-recessive disorder characterized by abnormal ciliary motion, due to a defect in ciliary structure and/or function. This genetic condition ...leads to recurrent upper and lower respiratory infections, bronchiectasis, laterality defect, and subfertility. Male infertility is often associated with PCD, since the ultrastructure of the axoneme in the sperm tail is similar to that of the motile cilia of respiratory cells. We present the first reported case of a male patient from a non-consanguineous Italian family who exhibited a severe form of asthenozoospermia factor infertility but no situs inversus and absolutely no signs of the clinical respiratory phenotype, the proband being a professional basketball player. Whole-exome sequencing (WES) has identified a homozygote mutation (CCDC103 c.461 A>C, p.His154Pro) in the proband, while his brother was a heterozygous carrier for this mutation. Morphological and ultrastructural analyses of the axoneme in the sperm flagellum demonstrated the complete loss of both the inner and outer dynein arms (IDA and ODA, respectively). Moreover, immunofluorescence of DNAH1, which is used to check the assembly of IDA, and DNAH5, which labels ODA, demonstrated that these complexes are absent along the full length of the flagella in the spermatozoa from the proband, which was consistent with the IDA and ODA defects observed. Noteworthy, TEM analysis of the axoneme from respiratory cilia showed that dynein arms, although either IDAs and/or ODAs seldom missing on some doublets, are still partly present in each observed section. This case reports the total sperm immotility associated with the CCDC103 p.His154Pro mutation in a man with a normal respiratory phenotype and enriches the variant spectrum of ccdc103 variants and the associated clinical phenotypes in PCD, thus improving counseling of patients about their fertility and possible targeted treatments.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may affect female reproductive health. Here, we investigated the potential of SARS-CoV-2 to infect the follicular microenvironment, in ...particular granulosa (GCs) and cumulus cells (CCs), thus providing evidence for a productive infection. GCs and CCs were recovered from women (n = 25) who underwent in vitro fertilization at the Assisted Reproductive Unit, Siena University Hospital. Follicular ovarian cells were co-cultured with SARS-CoV-2 and then analyzed by qPCR, immunofluorescence (IF), western blot (WB) and transmission electron microscopy (TEM). In addition, cell culture supernatant was used to infect VERO6 cells. We demonstrated the expression of cell host factors ACE2, TRPMSS2, BSG and CTSL, which are pivotal for the virus life cycle. Cultured GCs and CCs incubated with SARS-CoV-2 revealed productive SARS-CoV-2 infection at 24 h, 48 h and 72 h post-adsorption. Indeed, SARS-CoV-2 RNA, spike and nucleocapsid proteins were detected in GCs and CCs, and their cell culture supernatant successfully infected the standard VERO E6 cells. Finally, TEM showed full-size virions attached to the membrane and located inside the cytoplasm. This in vitro study reveals the susceptibility of human ovarian cells to SARS-CoV-2 infection, suggesting a potential detrimental effect of COVID-19 infection on female human fertility.
Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better ...characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.
Immune protection against pathogenic organisms has been shown to incur costs. Previous studies investigating the cost of immunity have mostly focused on the metabolic requirements of immune ...maintenance and activation. In addition to these metabolic costs, the immune system can induce damage to the host if the immune response is mis-targeted or over-expressed. Given its non-specific nature, an over-expressed inflammatory response is often associated with substantial damage for the host. Here, we investigated the cost of an over-expressed inflammatory response in the reproductive function of male mice.
We experimentally blocked the receptors of an anti-inflammatory cytokine (IL-10) in male mice exposed to a mild inflammatory challenge, with each treatment having an appropriate control group. The experiment was conducted on two age classes, young (3 month old) and old (15 month old) mice, to assess any age-related difference in the cost of a disrupted immune regulation. We found that the concomitant exposure to an inflammatory insult and the blockade of IL-10 induced a reduction in testis mass, compared to the three other groups. The frequency of abnormal sperm morphology was also higher in the group of mice exposed to the inflammatory challenge but did not depend on the blockade of the IL-10.
Our results provide evidence that immune regulation confers protection against the risk of inflammation-induced infertility during infection. They also suggest that disruption of the effectors involved in the regulation of the inflammatory response can have serious fitness consequences even under mild inflammatory insult and benign environmental conditions.
Despite the major target of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, being the respiratory system, clinical evidence suggests that the male ...reproductive system may represent another viral target organ. Revealing the effect of SARS-CoV-2 infection on testis and sperm is a priority for reproductive biology, as well as for reproductive medicine. Here, we confirmed that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on human testis and ejaculated sperm; moreover, we provide evidence for the expression of the co-receptors transmembrane protease/serine (TMPRSS2), Basigin (BSG), and Catepsin L (CTSL). Human sperm were readily infected, both in vivo and in vitro, by SARS-CoV-2, as demonstrated by confocal and electron microscopy. The demonstration that the seminiferous epithelium and sperm support SARS-CoV-2 viral replication suggests the possibility that the spermatogenetic process may be detrimentally affected by the virus, and at the same time, supports the need to implement safety measures and guidelines to ensure specific care in reproductive medicine.
PDF
