Role of transient receptor potential channels in mammalian oviduct and uterine epithelia

Calcium is an important secondary messenger and plays a major role in cell function, including proliferation, cell growth, secretion and death .It also plays a critical role in uterine smooth muscle contraction and embryo implantation. This thesis is concerned with calcium homeostasis in epithelial tissue lining the oviduct and uterus which are key players in early reproductive events, being involved in gamete transport, sperm capacitation and providing the micro-environment for the gametes and early embryo. Calcium transport across epithelial cells is either via tight junctions or calcium channels, specifically, members of the transient receptor potential (TRP) channel superfamily and the Na+/Ca2+ exchanger. TRP channels are an important class of calcium channels with more than 28 identified members and their potential involvement in calcium transport in uterus and oviduct epithelia has yet to be determined. The aim of this study was to discover which TRPC isoforms are expressed in epithelial cells lining the female reproductive tract in the bovine and human. Gene expression of TRPC channels changes was measured throughout the oestrous cycle in bovine oviduct and uterine epithelial cells using Real-Time PCR, while immunohistochemistry, immunocytochemistry and western blotting were used to discover the localization of TRPC channels in oviduct/uterine epithelium and changes in protein expression of TRPC isoforms induced by sex hormones. . to The physiological role of TRPC isoforms in regulating intracellular calcium concentration in bovine oviduct epithelial cells was determined using a calcium assay approach and finally. the potential clinical relevance of a possible role of TRP channels in female reproduction was investigated.# OF 7 members of TRPC family, TRPC1, 2, 3, 4 and 6 were expressed in bovine oviduct and uterine epithelia. In human endometrium, TRPC1, 6 and 7 genes were detected. Expression levels of all TRPC isoforms present in both bovine oviduct and uterine epithelia changed throughout the oestrous cycle. 17β-estradiol, FSH and LH individually and in combination up-regulated gene expression of TRPC isoforms in bovine oviduct epithelial cells. However, progesterone inhibited the upregulatory effect of 17β-estradiol, FSH and LH on TRPCs gene expression. TRPC1 and TRPC6 which are the common TRPC isoforms in bovine oviduct/uterine epithelium and human endometrium were localized on the apical, basal and lateral membranes of the epithelial tissue in bovine oviduct/uterus and human endometrium. TRPC isoforms were physiologically active in bovine oviduct epithelial cells (BOEC). SKF96365 which is a general TRP channel blocker inhibited the calcium influx into BOEC. Furthermore, Hyperforin which is a TRPC6 channel activator increased the intracellular calcium concentration in BOEC. TRPC1, 6 and 7 expression in endometrium of patients being treated for infertility by IVF illustrated that gene expression of TRTPC1 and 6 were up regulated in the endometrium of the IVF patients compared to controls. However, gene expression of TRPC7 in IVF patients was downregulated compared to that of the endometrium of the control group.Gene expression of TRPC6 and 7 in endometrium of women with Poly Cystic Ovarian Syndrome (PCOS) who have higher level of LH and normal FSH level, alongside the absence of the post-ovulatory increase in progesterone secretion, were up -regulated compared to that of the control group. However, the expression level of TRPC1 in endometrium of PCOS patients was not significantly different compared to the control group. Gene expression of TRPC isoforms in the epithelia lining the female reproductive tract is possibly regulated by sex hormones via nuclear factor-kappa B (NF-КB) signalling pathway. However, further investigation is required to determine the mechanisms underlying the endocrine regulation of TRPC channels

Expression and function of transient receptor potential channels in the female bovine reproductive tract

© 2016 Elsevier Inc. The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17β-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17β-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated to torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with TOR1A-AMC5 have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with fetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71% with higher mortality in males. Death occurred at a median age of 1.2 months (1 week - 9 years) due to respiratory failure, cardiac arrest, or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival

Role of transient receptor potential channels in mammalian oviduct and uterine epithelia

Calcium is an important secondary messenger and plays a major role in cell function, including proliferation, cell growth, secretion and death .It also plays a critical role in uterine smooth muscle contraction and embryo implantation. This thesis is concerned with calcium homeostasis in epithelial tissue lining the oviduct and uterus which are key players in early reproductive events, being involved in gamete transport, sperm capacitation and providing the micro-environment for the gametes and early embryo. Calcium transport across epithelial cells is either via tight junctions or calcium channels, specifically, members of the transient receptor potential (TRP) channel superfamily and the Na+/Ca2+ exchanger. TRP channels are an important class of calcium channels with more than 28 identified members and their potential involvement in calcium transport in uterus and oviduct epithelia has yet to be determined. The aim of this study was to discover which TRPC isoforms are expressed in epithelial cells lining the female reproductive tract in the bovine and human. Gene expression of TRPC channels changes was measured throughout the oestrous cycle in bovine oviduct and uterine epithelial cells using Real-Time PCR, while immunohistochemistry, immunocytochemistry and western blotting were used to discover the localization of TRPC channels in oviduct/uterine epithelium and changes in protein expression of TRPC isoforms induced by sex hormones. . to The physiological role of TRPC isoforms in regulating intracellular calcium concentration in bovine oviduct epithelial cells was determined using a calcium assay approach and finally. the potential clinical relevance of a possible role of TRP channels in female reproduction was investigated.# OF 7 members of TRPC family, TRPC1, 2, 3, 4 and 6 were expressed in bovine oviduct and uterine epithelia. In human endometrium, TRPC1, 6 and 7 genes were detected. Expression levels of all TRPC isoforms present in both bovine oviduct and uterine epithelia changed throughout the oestrous cycle. 17β-estradiol, FSH and LH individually and in combination up-regulated gene expression of TRPC isoforms in bovine oviduct epithelial cells. However, progesterone inhibited the upregulatory effect of 17β-estradiol, FSH and LH on TRPCs gene expression. TRPC1 and TRPC6 which are the common TRPC isoforms in bovine oviduct/uterine epithelium and human endometrium were localized on the apical, basal and lateral membranes of the epithelial tissue in bovine oviduct/uterus and human endometrium. TRPC isoforms were physiologically active in bovine oviduct epithelial cells (BOEC). SKF96365 which is a general TRP channel blocker inhibited the calcium influx into BOEC. Furthermore, Hyperforin which is a TRPC6 channel activator increased the intracellular calcium concentration in BOEC. TRPC1, 6 and 7 expression in endometrium of patients being treated for infertility by IVF illustrated that gene expression of TRTPC1 and 6 were up regulated in the endometrium of the IVF patients compared to controls. However, gene expression of TRPC7 in IVF patients was downregulated compared to that of the endometrium of the control group. Gene expression of TRPC6 and 7 in endometrium of women with Poly Cystic Ovarian Syndrome (PCOS) who have higher level of LH and normal FSH level, alongside the absence of the post-ovulatory increase in progesterone secretion, were up -regulated compared to that of the control group. However, the expression level of TRPC1 in endometrium of PCOS patients was not significantly different compared to the control group. Gene expression of TRPC isoforms in the epithelia lining the female reproductive tract is possibly regulated by sex hormones via nuclear factor-kappa B (NF-КB) signalling pathway. However, further investigation is required to determine the mechanisms underlying the endocrine regulation of TRPC channels

Role of transient receptor potential channels in mammalian oviduct and uterine epithelia

Calcium is an important secondary messenger and plays a major role in cell function, including proliferation, cell growth, secretion and death .It also plays a critical role in uterine smooth muscle contraction and embryo implantation. This thesis is concerned with calcium homeostasis in epithelial tissue lining the oviduct and uterus which are key players in early reproductive events, being involved in gamete transport, sperm capacitation and providing the micro-environment for the gametes and early embryo. Calcium transport across epithelial cells is either via tight junctions or calcium channels, specifically, members of the transient receptor potential (TRP) channel superfamily and the Na+/Ca2+ exchanger. TRP channels are an important class of calcium channels with more than 28 identified members and their potential involvement in calcium transport in uterus and oviduct epithelia has yet to be determined. The aim of this study was to discover which TRPC isoforms are expressed in epithelial cells lining the female reproductive tract in the bovine and human. Gene expression of TRPC channels changes was measured throughout the oestrous cycle in bovine oviduct and uterine epithelial cells using Real-Time PCR, while immunohistochemistry, immunocytochemistry and western blotting were used to discover the localization of TRPC channels in oviduct/uterine epithelium and changes in protein expression of TRPC isoforms induced by sex hormones. . to The physiological role of TRPC isoforms in regulating intracellular calcium concentration in bovine oviduct epithelial cells was determined using a calcium assay approach and finally. the potential clinical relevance of a possible role of TRP channels in female reproduction was investigated.# OF 7 members of TRPC family, TRPC1, 2, 3, 4 and 6 were expressed in bovine oviduct and uterine epithelia. In human endometrium, TRPC1, 6 and 7 genes were detected. Expression levels of all TRPC isoforms present in both bovine oviduct and uterine epithelia changed throughout the oestrous cycle. 17β-estradiol, FSH and LH individually and in combination up-regulated gene expression of TRPC isoforms in bovine oviduct epithelial cells. However, progesterone inhibited the upregulatory effect of 17β-estradiol, FSH and LH on TRPCs gene expression. TRPC1 and TRPC6 which are the common TRPC isoforms in bovine oviduct/uterine epithelium and human endometrium were localized on the apical, basal and lateral membranes of the epithelial tissue in bovine oviduct/uterus and human endometrium. TRPC isoforms were physiologically active in bovine oviduct epithelial cells (BOEC). SKF96365 which is a general TRP channel blocker inhibited the calcium influx into BOEC. Furthermore, Hyperforin which is a TRPC6 channel activator increased the intracellular calcium concentration in BOEC. TRPC1, 6 and 7 expression in endometrium of patients being treated for infertility by IVF illustrated that gene expression of TRTPC1 and 6 were up regulated in the endometrium of the IVF patients compared to controls. However, gene expression of TRPC7 in IVF patients was downregulated compared to that of the endometrium of the control group. Gene expression of TRPC6 and 7 in endometrium of women with Poly Cystic Ovarian Syndrome (PCOS) who have higher level of LH and normal FSH level, alongside the absence of the post-ovulatory increase in progesterone secretion, were up -regulated compared to that of the control group. However, the expression level of TRPC1 in endometrium of PCOS patients was not significantly different compared to the control group. Gene expression of TRPC isoforms in the epithelia lining the female reproductive tract is possibly regulated by sex hormones via nuclear factor-kappa B (NF-КB) signalling pathway. However, further investigation is required to determine the mechanisms underlying the endocrine regulation of TRPC channels.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Differing endometrial expression of calcium modulating transient receptor potential channels

Assisted reproductive techniques (ART) have increased the live birth success rate, but there is still a significant implantation failure rate. Epithelial cell calcium homeostasis is tightly regulated by mechanisms that include activation of the TRP channel superfamily of 9 families including TRPC (Canonical). TRPCs are located on the cell membrane and act as receptor-operated channels (ROC), whilst cytosolic localization of these channels indicates their role as store-operated channels (SOC): both ROCs and SOCs are key players in the regulation of intracellular calcium homeostasis. TRPC 1–4 and 6 expression in the bovine reproductive tract has been reported; these receptors exhibit hormone modulation and calcium dysregulation can lead to menstrual disturbances, suggesting that TRPC receptors may modulate calcium in the human endometrium and affect implantation and fertility.</p

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications

PPFIBP1 encodes for the liprin-β1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications. CC BY 4.0© 2022 The AuthorsCorrespondence: [email protected] (A.E.X.B.), [email protected] (K.P.)We thank all families that participated in this study. This project has received funding from the European Research Council under the European External Action Service Horizon 2020 Research and Innovation Program (grant agreement no. 714853) and was supported by the Medical Research Council through grant MC-A658-5TY30. H.T. was supported by the European External Action Service Seventh Framework Programme for research, technological development, and demonstration under grant agreement no. 608473.</p

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants.Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 +/- 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed.MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed.This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'.Biallelic variants in MED27 are associated with an ultra-rare neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. Maroofian et al. further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families.Peer reviewe