Genetic causes of inherited retinal diseases among Israeli Jews of Ethiopian ancestry.

Purpose: This study sought to describe the phenotype frequency and genetic basis of inherited retinal diseases (IRDs) among a nationwide cohort of Israeli Jewish patients of Ethiopian ancestry. Methods: Patients' data-including demographic, clinical, and genetic information-were obtained through members of the Israeli Inherited Retinal Disease Consortium (IIRDC). Genetic analysis was performed by either Sanger sequencing for founder mutations or next-generation sequencing (targeted next-generation sequencing or whole-exome sequencing). Results: Forty-two patients (58% female) from 36 families were included, and their ages ranged from one year to 82 years. Their most common phenotypes were Stargardt disease (36%) and nonsyndromic retinitis pigmentosa (33%), while their most common mode of inheritance was autosomal recessive inheritance. Genetic diagnoses were ascertained for 72% of genetically analyzed patients. The most frequent gene involved was ABCA4. Overall, 16 distinct IRD mutations were identified, nine of which are novel. One of them, ABCA4-c.6077delT, is likely a founder mutation among the studied population. Conclusions: This study is the first to describe IRDs' phenotypic and molecular characteristics in the Ethiopian Jewish community. Most of the identified variants are rare. Our findings can help caregivers with clinical and molecular diagnosis and, we hope, enable adequate therapy in the near future.

ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Azithromycin and Sildenafil May Have Protective Effects on Retinal Ganglion Cells via Different Pathways: Study in a Rodent Microbead Model.

Decreased blood flow to the optic nerve (ON) and neuroinflammation are suggested to play an important role in the pathophysiology of glaucoma. This study investigated the potential neuroprotective effect of azithromycin, an anti-inflammatory macrolide, and sildenafil, a selective phosphodiesterase-5 inhibitor, on retinal ganglion cell survival in a glaucoma model, which was induced by microbead injection into the right anterior chamber of 50 wild-type (WT) and 30 transgenic toll-like receptor 4 knockout (TLR4KO) mice. Treatment groups included intraperitoneal azithromycin 0.1 mL (1 mg/0.1 mL), intravitreal sildenafil 3 µL, or intraperitoneal sildenafil 0.1 mL (0.24 µg/3 µL). Left eyes served as controls. Microbead injection increased intraocular pressure (IOP), which peaked on day 7 in all groups and on day 14 in azithromycin-treated mice. Furthermore, the retinas and ON of microbead-injected eyes showed a trend of increased expression of inflammatory- and apoptosis-related genes, mainly in WT and to a lesser extent in TLR4KO mice. Azithromycin reduced the BAX/BCL2 ratio, TGFß, and TNFα levels in the ON and CD45 expression in WT retina. Sildenafil activated TNFα-mediated pathways. Both azithromycin and sildenafil exerted a neuroprotective effect in WT and TLR4KO mice with microbead-induced glaucoma, albeit via different pathways, without affecting IOP. The relatively low apoptotic effect observed in microbead-injected TLR4KO mice suggests a role of inflammation in glaucomatous damage.

Neuroprotective Effect of Azithromycin Following Induction of Optic Nerve Crush in Wild Type and Immunodeficient Mice.

This study evaluated the potential neuroprotective effect of azithromycin (AZ) intraperitoneal injections in male C57Bl/6 (wild type, WT) and female NOD scid gamma (NSG) mice subjected to optic nerve crush (ONC) as a model for optic neuropathy. Histologically, reduced apoptosis and improved retinal ganglion cell (RGC) preservation were noted in the AZ-treated mice as shown by TUNEL staining-in the WT mice more than in the NSG mice. The increased microglial activation following ONC was reduced with the AZ treatment. In the molecular analysis of WT and NSG mice, similar trends were detected regarding apoptosis, as well as stress-related and inflammatory markers examining BCL2-associated X (Bax), heme oxygenase 1 (Ho-1), interleukin 1 beta (Il1ß), superoxide dismutase 1 (Sod1), and nuclear factor-kappa B (Nfkb) levels. In the optic nerve, AZ increased the levels of expression of Sod1 and Nfkb only in the WT mice and decreased them in the NSG mice. In the retinas of the WT and NSG mice, the Bax and Ho-1 levels of expression decreased following the AZ treatment, while the Sod1 and Nfkb expression decreased only in the WT mice, and remained stable near the baseline in the NSG mice. Il1ß remained at the baseline in WT mice while it decreased towards the baseline in AZ-treated NSG mice. The neuroprotective effects demonstrated by the reduced RGC apoptosis in AZ-treated WT mice retinae, and in the optic nerves as stress-related and inflammatory gene expression increase. This did not occur in the immunodeficient NSG mice. AZ modulated the inflammatory reaction and microglial activation. The lack of an effect in NSG mice supports the assumption that AZ acts by immunomodulation, which is known to play a role in ONC damage. These findings have implications for the development and repurposing of drugs to preserve RGCs after acute optic neuropathies.

Genetic Alteration Analysis of IDH1, IDH2, CDKN2A, MYB and MYBL1 in Pediatric Low-Grade Gliomas.

Objective: To investigate pediatric low-grade gliomas for alterations in IDH1, IDH2, CDKN2A, MYB, and MYBL1. Materials and Methods: DNA and RNA were extracted from 62 pediatric gliomas. Molecular methods included PCR, RT-PCR, and RNA sequencing; Sanger sequencing was used for validation. Results: Analysis for hotspot genetic alterations in IDH1 R132 and IDH2 R172 (45 and 33 samples) was negative in all cases. CDKN2A deletions were detected in exons 1 and 2 in 1 (pleomorphic xanthoastrocytoma) sample of 9 samples analyzed. Of 10 samples analyzed for MYB translocation, 4 each were positive for translocations with exon 2 and exon 3 of PCDHGA1. Six samples showed MYBL rearrangement. The lack of IDH1/2 genetic alterations is in accordance with the literature in pediatric tumors. Alterations in MYB, MYBL were recently reported to characterize diffuse grade II, but not grade I, gliomas. Conclusion: We optimized methods for analyzing gene variations and correlated the findings to pathological grade. The high incidence of MYB and MYBL need further evaluation. We also compared DNA, RNA, and RNA sequencing results for fusion, translocation, and genetic alterations. More accurate identification of the underlying biology of pediatric gliomas has implications for the development of targeted treatment.

High Myopia and Strabismus Induced by a Deep Intronic Mutation in COL2A1.

Purpose: To characterize a genetic mutation causing Stickler syndrome in a previously undiagnosed family.Methods: Five generations of a single family suspected of having Stickler syndrome were evaluated clinically and genetically.Results: The demographic and clinical data yielded specific clinical phenotypes of Stickler syndrome in 13 family members; 7 had more than one clinical feature. Four family members underwent genetic analysis: the proband (index patient) and his mother, maternal grandfather, and healthy father. No relevant mutation was detected in the proband on whole exome analysis, but subsequent extension of the analysis to intronic areas yielded a deep intronic mutation, NM_001844.5:c.1527 + 135 G > A. Sanger sequencing was used to validate the results in the family members.Conclusions: Stickler syndrome has several subtypes with variable clinical features. Therefore, predicting the genetic locus of the disease based on clinical characteristics is challenging. We present a rarely described intronic mutation in COL2A1. Genetic testing may aid in the early diagnosis of Stickler syndrome, which is important for genetic counselling, proper clinical management, and improved prognosis.

Gene-Related Response of Basal Cell Carcinoma to Biologic Treatment with Vismodegib.

We aimed to characterise the response of locally advanced basal cell carcinoma (BCC) to systemic treatment with Vismodegib, a Hedgehog pathway inhibitor, by changes in the expression levels of Hedgehog pathway genes. Data were collected prospectively on 12 patients treated systemically for locally advanced BCC. Biopsy samples taken on admission and after treatment cessation were analysed pathologically and with the NanoString nCounter system to quantify the expression of 40 Hedgehog signaling pathway genes. Findings were compared before and after treatment, between complete and partial responders, and with localised BCC samples from 22 patients. Sixteen Hedgehog pathway genes changed significantly from before to after treatment. GAS1 was the only gene with a significantly different expression at baseline between complete responders (6 patients) and partial responders (4 patients) to Vismodegib (P = 0.014). GAS, GLIS2 and PRKACG1 showed different expression before treatment between the locally advanced and localised BCCs. The baseline expression level of GAS1 appears to be predictive of the response of locally advanced BCC to systemic Vismodegib treatment. A change in expression of many Hedgehog pathway genes, albeit expected by the known activity of Vismodegib, may nevertheless serve as an indicator of the response potential of the tumour.

Diffusion Reflection Measurements of Antibodies Conjugated to Gold Nanoparticles as a Method to Identify Cutaneous Squamous Cell Carcinoma Borders.

Diffusion reflectance spectroscopy measurements targeted with gold nanoparticles (GNPs) can identify residual cutaneous squamous cell carcinoma (SCC) in excision borders. Human SCC specimens were stained with hematoxylin and eosin to identify tumor borders, and reflected onto an unstained deparaffinized section. Diffusion reflection of three sites (normal and SCC) were measured before and after GNPs targeting. Hyperspectral imaging showed a mean of 2.5 sites with tumor per specimen and 1.2 tumor-free (p < 0.05, t-test). GNPs were detected in 25/30 tumor sites (sensitivity 83.3%, false-negative rate 16.6%) and 12/30 non-tumor sites (specificity 60%, false-positive rate 40%). This study verifies the use of nanotechnology in identifying SCC tumor margins. Diffusion reflection scanning has high sensitivity for detecting the residual tumor.

The co-occurrence of rare non-ocular phenotypes in patients with inherited retinal degenerations.

Purpose: To describe the coexistence of additional non-ocular genetic diseases in patients diagnosed with inherited retinal degenerations (IRDs). Methods: The study was based on a retrospective chart review of patients diagnosed with IRD and additional rare systemic diseases. The chart review included the ophthalmic and genetic aspects of each patient. The ophthalmic examination included best-corrected visual acuity, biomicroscopic examination, cycloplegic refraction, retinal imaging (fundus photos, optical coherence tomography, and fundus autofluorescence), and electroretinography. Genetic testing included homozygosity mapping, whole exome sequencing, and Sanger sequencing. Results: Fifteen index cases diagnosed with IRDs and one or more rare systemic diseases were identified. Six of the families were consanguineous. Of six patients with complete molecular diagnosis, four (66%) had pathogenic variants in two autosomal recessive (AR) disease genes, and of the total pathogenic variants identified, AR mutations were the most common (16/22, 72%). One patient was diagnosed with mutations in three different genes, underlying three distinct genetic conditions. Nine patients could have had an incorrect clinical diagnosis based on the clinical evaluation only (e.g., retinitis pigmentosa and hearing loss could have been diagnosed as Usher syndrome). Conclusions: The common working paradigm for the ophthalmologist is combining the different symptoms observed in a patient into one unifying diagnosis. However, IRD is a strikingly heterogeneous condition, and may coincide with other genetic (and non-genetic) rare conditions. Establishing a correct diagnosis is important for the patients and their family members, as it enables prediction of disease prognosis, aids in tailoring the correct follow-up and treatment, and allows patients to pursue prenatal counseling and reproductive planning.

Ocular Effects of Sildenafil in Naïve Mice and a Mouse Model of Optic Nerve Crush.

Purpose: To investigate the potential neuroprotective effect of sildenafil on the ocular circulation in mice with/without optic nerve crush (ONC). Methods: Male adult mice (n = 63) were treated with intravitreal (IVT) sildenafil 24 µg/3 µL, intraperitoneal (IP) sildenafil 24 µg/300 µL, or IP saline immediately before right ONC induction (ONC group). A second group (n = 123) received the same treatments without ONC induction (naïve group). Evaluations included fluorescein angiography (naïve group; day 0), molecular studies (days 1 and 3), and retinal and optic nerve histology (day 21). Results: Maximal retinal vessel dilatation and increased choroidal effusion were detected within 30 minutes of sildenafil injection. In the ONC group, moderate retinal ganglion cell (RGC) loss was noted at 21 days. However, molecular studies showed increased stress induced gene expression (IP superoxide dismutase [SOD]-1: 3.1-fold; heme oxygenase [HO]-1: 5.8-fold; IVT SOD-1: 1.47-fold), proapoptotic gene expression (IP BAX/B-cell lymphoma [BCL]-2 10.8-/2.3-fold), and glial gene expression (IP glial fibrillary acidic protein [GFAP]: 2.8- and myelin basic protein [MBP]: 2.5-fold). In the naïve group, IVT sildenafil was not associated with RGC loss or optic nerve stroke on histology, although in two samples, molecular parameters were compatible with stroke, showing increased gene expression of HO-1 (3.8-fold) and BCL-2 (2.5-fold). In the IP sildenafil subgroup, optic neuropathy was observed in 6/120 optic nerves, including 3 cyan fluorescence protein (CFP)-Thy-1 mice. Levels of antiapoptosis and anti-ischemia genes were decreased (<0.5-fold) except for three outliers. Conclusions: Sildenafil affects retinal and choroidal perfusion in mice. When injected immediately before ONC, molecular parameters showed a preconditioning neuroprotective effect while histologic studies did not. In the absence of ONC, it is associated with neuropathy, possibly dose-dependent.

Late diagnosis of Alstrom syndrome in a Yemenite-Jewish child.

BACKGROUND: We describe the ophthalmologic, clinical, and genetic findings in a patient of Yemenite-Jewish origin diagnosed with Alstrom syndrome due to a novel splice-site mutation 10 years after a clinical misdiagnosis of Leber congenital amaurosis. METHODS: Ophthalmological evaluations included visual acuity, cycloplegic refraction, slit-lamp, and optical coherent tomography. Genetic analyses included whole exome sequencing followed by bioinformatics analysis and segregation analysis. An in vitro splicing assay was used to evaluate the effect of the identified mutation on splicing. Taqman assay was used to determine the need for population screening for the identified mutation. RESULTS: Ophthalmologic findings at age 6 were impaired vision, nystagmus, and hyperopia. At age 16 years, the patient presented with obesity, hypothyroidism, and elevated transaminase levels in addition to reduced vision, wandering nystagmus, disc pallor, and degenerative retinal changes. Targeted genetic analysis of ALMS1 revealed a homozygous transversion, c.11544 + 3A>T, suggesting a novel splicing mutation, with elimination of the donor splice site and insertion of 73 nucleotides at the end of exon 16. These changes were validated by Sanger sequencing and co-segregation on family members. CONCLUSIONS: Ophthalmologists should be alert to the differential diagnosis of inherited retinal degeneration in young patients who present with impaired vision, especially if systemic symptoms are mild and there is no known family history. In the present case, targeted genetic analysis of a child with a syndromic cone-rod dystrophy yielded a novel splicing mutation in ALMS1 causing Alstrom syndrome.

Single-Nucleotide Polymorphisms in IL23R-IL12RB2 (rs1495965) Are Highly Prevalent in Patients with Behcet's Uveitis and Vary Between Populations.

Purpose: To test the frequency of single-nucleotide polymorphisms in the IL-10, IL23R-IL12RB2 genes in patients with Behcet's uveitis. Methods: Blood samples were collected from 89 Israeli and Turkish patients, and from healthy control subjects of different origins. Genomic DNA was extracted from peripheral blood leukocytes and genotyped. Results: The risk allele, A, in rs1800871, of IL-10 gene was highly prevalent in Behcet's uveitis and healthy control samples alike; highest among the Turkish groups. Prevalence of G allele, in rs1495965, in the IL23R-IL12RB2 gene was high in Behcet's uveitis patients, and among healthy Turkish and Israelis of Middle Eastern origin, while lower among the other Israeli control group (77.9%, 78.9%, 27.8%, respectively, P < 0.001). Conclusion: Our findings highlight the differences between populations and may account for the increased prevalence of the disease among Turkish and Israelis of Middle Eastern origin. Further studies are required to map other healthy and affected populations.

Comparison of Neuroprotective Effect of Bevacizumab and Sildenafil following Induction of Stroke in a Mouse Model.

To evaluate the effect of bevacizumab and sildenafil on stroke parameters in a mouse model, middle cerebral artery occlusion was induced in male C57Bl/6 mice using an intra-arterial filament method. The filament was removed after 60 minutes, and the mice were immediately given a single intraperitoneal injection of saline, bevacizumab, or sildenafil. An additional group of mice (n = 7) received bevacizumab 6 h after MCAO induction. The mice were euthanized 24 hours later and evaluated for infarct area and brain edema using triphenyltetrazolium chloride staining and ImageJ. In the saline-treated mice (n = 16), total stroke volume was 19.20 ± 6.38 mm(3), mean penumbra area was 4.5 ± 2.03 mm(3), and hemispheric asymmetry was 106.5%. Corresponding values in the bevacizumab group (n = 19) were 17.79 ± 5.80 mm(3), 7.3 ± 3.5 mm(3), and 108.6%; in the delayed (6 h) bevacizumab injected mice (n = 7) they were 9.80 ± 8.00 mm(3), 2.4 ± 2.0 mm(3), and 98.2%; and in the sildenafil group (n = 16) they were 18.42 ± 5.41 mm(3), 5.7 ± 2.02 mm(3), and 109.9%. The bevacizumab group had a significantly larger mean penumbra area when given immediately and smaller total stroke area in both groups than the saline- (p = 0.03) and sildenafil-treated (p = 0.003) groups. Only delayed bevacizumab group had reduced edema. Bevacizumab, injected immediately or delayed after injury, exerts a neuroprotective/salvage effect, whereas immediate treatment with sildenafil does not. Inflammation may play a role in the neuroprotective effect.

Relative neuroprotective effects hyperbaric oxygen treatment and TLR4 knockout in a mouse model of temporary middle cerebral artery occlusion.

PURPOSE: To examine the effects of hyperbaric oxygen (HBO) therapy and knockout of toll-like receptor 4 (TLR4) on the outcome of temporary middle cerebral artery occlusion (MCAO) in a mouse model. MATERIALS AND METHODS: MCAO was induced in anesthetized male C57Bl/6 mice (WT) and TLR4 knockout mice (TLR4(-/-)) using an intra-arterial filament method. After 30 or 90 min, the filament was removed, and the mice were given either no treatment (WT and TLR4(-/-) groups) or HBO (WT only). Mice were euthanized 24 h after MCAO, and the brain infarct area was examined using 2,3,5-triphenyltetrazolium chloride (TTC) staining. RESULTS: In the WT group, without treatment, lesion volume was 120 ± 13 mm(3) in the mice subjected to 30 min' MCAO and 173 ± 23 mm(3) in the mice subjected to 90 min' MCAO. Respective values with HBO treatment were 66.5 ± 36.7 mm(3) and 53.2 ± 17.2 mm(3). The difference was significant only for 90-minute MCAO (p < 0.01, nonparametric test). In the TLR4(-/-) group (all untreated), lesion volume was 95.9 ± 17.9 after 90 min of MCAO, which was significantly lower than in the untreated WT animals (p < 0.05, nonparametric test). CONCLUSIONS: A single treatment of HBO immediately after MCAO followed by 24 h' reperfusion significantly reduces edema and may improve perfusion. TLR4 knockout protects mice from MCAO damage, but to a lesser extent than HBO treatment.

Toll-like receptor-4 knockout mice are more resistant to optic nerve crush damage than wild-type mice.

BACKGROUND: This study aims to investigate the role of the inflammatory response following optic nerve crush (ONC) in knockout mice for the toll-like receptor-4 gene (TLR4-/-) compared to wild-type (WT) mice. METHODS: ONC was induced in TLR4-/- and C57BL6 WT mice. Histological sections of the retina and optic nerve were analysed on days 1, 3 or 21 after injury. Molecular analysis with real-time quantitative polymerase chain reaction was used to study the expression of CD45, tumour necrosis-alpha (TNF-α) and glial fibrillary acidic protein, as well as retinal ganglion cell (RGC) markers THY-1 and Brn3b. RESULTS: There was a 25.5% and 38% loss in the RGC layer of the ONC-injured eyes of the TLR4-/- and the WT mice, respectively (with 27% and 9% of the remaining cells positive for Brn3a, respectively). Mean levels of Thy-1 and Brn3b were higher in the TLR4-/- mice. CD45 and Iba1 staining revealed infiltration of inflammatory cells into the injured nerve and retina in both groups. Molecular analysis of the optic nerve on day 1 showed increased TNF-α expression and reduced CD45 and GFAP expression; on day 3, CD45 reverted to baseline but GFAP remained low; on day 21, all 3 markers were at baseline in the TLR4-/- group and decreased in the WT group. CONCLUSION: Inflammation plays a major role in the response to ONC injury. Reduced levels of inflammation are associated with improved RGC preservation. The increase in TNF-α and reduction in CD45 in both TLR4-/- and WT mice may indicate the presence of an alternative pathway for induction of RGC death.

Effect of intravitreal injection of bevacizumab on optic nerve head leakage and retinal ganglion cell survival in a mouse model of optic nerve crush.

PURPOSE: To evaluate the effect of bevacizumab, a VEGF inhibitor, on optic nerve edema and retinal ganglion cell (RGC) loss in a mouse model of optic nerve crush (ONC). METHODS: Two hundred C57BL/6 wild-type mice were anesthetized. Right ONC was induced in 150 mice, of which half (n = 75) received an intravitreal injection of bevacizumab immediately thereafter and half (n = 75) did not. The remaining 50 received only bevacizumab. The left eyes served as a control. Findings were analyzed by fluorescein angiography (days 0, 1, 3), histologic and immunohistochemical tests (days 1, 3, 4, 21), and quantitative real-time PCR. RESULTS: Angiography revealed a reduction in postinjury disc leakage following bevacizumab injection (days 1, 3), confirmed with IgG staining. On PCR, expression of HO-1 and SOD-1 mRNA increased following ONC and further increased with bevacizumab. VEGF gene expression decreased following bevacizumab injection without ONC, remained at baseline after ONC, and increased slightly after ONC+bevacizumab. Histologically, there was a 38% RGC loss 21 days after ONC alone, which dropped to 14% with bevacizumab treatment; it was close to 15% with bevacizumab alone. Mean (SEM) microvascular perfusion in the optic nerve 4 days after ONC was significantly higher in the bevacizumab-treated (85% ± 10%) than the vehicle-treated (33% ± 13%) animals. CONCLUSIONS: Bevacizumab treatment following ONC induction exerts a protective effect, manifested by reduced optic nerve head edema. The underlying mechanism probably involves a lesser interruption of axonal transport. Reduced expression of antioxidative and ischemic genes may contribute to RGC preservation.

Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction.

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca(2+) influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca(2+) influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10microM) could enhance EGFR phosphorylation, Ca(2+) influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 microM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.