cAMP -dependent protein tyrosine phosphorylation during bovine sperm capacitation

Mammalian sperm capacitation, defined as an obligatory maturational process leading to the development of the fertilization-competent state, is regulated by a series of poorly understood molecular events. This study examines signaling pathways associated with bovine sperm capacitation. Aims. (1) To determine the effects of modulators of sperm capacitation on protein tyrosine phosphorylation in ejaculated and epididymal bovine sperm. (2) To examine the role of glucose and cyclic nucleotide phosphodiesterases (PDE) in the regulation of capacitation-associated protein tyrosine phosphorylation. (3) To isolate and identify substrate proteins of tyrosine phosphorylation. Methods. Ejaculated and epididymal bovine sperm were incubated under conditions that support capacitation in vitro. Protein tyrosine phosphorylation was evaluated by immunoblotting and indirect immunofluorescence using antiphosphotyrosine antibodies. PDE activity was measured using a standard assay. Protein substrates of tyrosine phosphorylation were purified using two-dimensional gel electrophoresis and identified via mass spectroscopy and peptide sequencing. Results. Ejaculated and epididymal bovine sperm displayed a pattern of protein tyrosine phosphorylations that was regulated by a cAMP/protein kinase A (PKA) - dependent pathway. Enhancers (heparin, cAMP agonists) and an inhibitor (glucose) of bovine sperm capacitation enhanced or inhibited, respectively, protein tyrosine phosphorylation. Protein tyrosine phosphorylation was stimulated by a synthetic cholesterol acceptor, β-cyclodextrin, in the capacitation medium. PDE1 was responsible for a majority of the cAMP-PDE activity and regulated capacitation-associated protein tyrosine phosphorylation in bovine ejaculated sperm. A model was presented for the mechanism of glucose inhibition of bovine sperm capacitation. Protein tyrosine phosphorylation was localized to specific acrosomal and flagellar regions of bovine sperm incubated under capacitating conditions. The major proteins undergoing tyrosine phosphorylation were purified and identified as an A Kinase Anchoring Protein (AKAP82), the major protein of the fibrous sheath in the flagellum, ODF84, a structural protein from the outer dense fibers of the flagellum, and β-tubulin, most likely from the flagellar axoneme. Conclusions. These studies are the first to report a unique inter-relationship between tyrosine kinase/phosphatase and cAMP signaling pathways at the level of PKA in bovine sperm capacitation. The capacitation-dependent tyrosine phosphorylation of AKAP82, ODF84 and β-tubulin suggests that this signaling pathway might be involved in motility changes during capacitation

cAMP -dependent protein tyrosine phosphorylation during bovine sperm capacitation

Mammalian sperm capacitation, defined as an obligatory maturational process leading to the development of the fertilization-competent state, is regulated by a series of poorly understood molecular events. This study examines signaling pathways associated with bovine sperm capacitation. Aims. (1) To determine the effects of modulators of sperm capacitation on protein tyrosine phosphorylation in ejaculated and epididymal bovine sperm. (2) To examine the role of glucose and cyclic nucleotide phosphodiesterases (PDE) in the regulation of capacitation-associated protein tyrosine phosphorylation. (3) To isolate and identify substrate proteins of tyrosine phosphorylation. Methods. Ejaculated and epididymal bovine sperm were incubated under conditions that support capacitation in vitro. Protein tyrosine phosphorylation was evaluated by immunoblotting and indirect immunofluorescence using antiphosphotyrosine antibodies. PDE activity was measured using a standard assay. Protein substrates of tyrosine phosphorylation were purified using two-dimensional gel electrophoresis and identified via mass spectroscopy and peptide sequencing. Results. Ejaculated and epididymal bovine sperm displayed a pattern of protein tyrosine phosphorylations that was regulated by a cAMP/protein kinase A (PKA) - dependent pathway. Enhancers (heparin, cAMP agonists) and an inhibitor (glucose) of bovine sperm capacitation enhanced or inhibited, respectively, protein tyrosine phosphorylation. Protein tyrosine phosphorylation was stimulated by a synthetic cholesterol acceptor, β-cyclodextrin, in the capacitation medium. PDE1 was responsible for a majority of the cAMP-PDE activity and regulated capacitation-associated protein tyrosine phosphorylation in bovine ejaculated sperm. A model was presented for the mechanism of glucose inhibition of bovine sperm capacitation. Protein tyrosine phosphorylation was localized to specific acrosomal and flagellar regions of bovine sperm incubated under capacitating conditions. The major proteins undergoing tyrosine phosphorylation were purified and identified as an A Kinase Anchoring Protein (AKAP82), the major protein of the fibrous sheath in the flagellum, ODF84, a structural protein from the outer dense fibers of the flagellum, and β-tubulin, most likely from the flagellar axoneme. Conclusions. These studies are the first to report a unique inter-relationship between tyrosine kinase/phosphatase and cAMP signaling pathways at the level of PKA in bovine sperm capacitation. The capacitation-dependent tyrosine phosphorylation of AKAP82, ODF84 and β-tubulin suggests that this signaling pathway might be involved in motility changes during capacitation

Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

Abstract Background Laminitis is often associated with endocrinopathies that cause hyperinsulinemia and is also induced experimentally by hyperinsulinemia, suggesting that insulin initiates laminitis pathogenesis. Hyperinsulinemia is expected to activate pro-growth and anabolic signaling pathways. We hypothesize that chronic over-stimulation of these pathways in lamellar tissue results in endoplasmic reticulum stress, contributing to tissue pathology, as it does in human metabolic diseases. We tested this hypothesis by asking whether lamellar tissue from horses with naturally-occurring endocrinopathic laminitis showed expression of protein markers of endoplasmic reticulum stress. Results Three markers of endoplasmic reticulum stress, spliced XBP1, Grp78/BiP and Grp94, were upregulated 2.5–9.5 fold in lamellar tissues of moderately to severely laminitic front limbs (n = 12) compared to levels in controls (n = 6–7) measured by immunoblotting and densitometry. Comparing expression levels between laminitic front limbs and less affected hind limbs from the same horses (paired samples from 7 to 8 individual horses) demonstrated significantly higher expression for both spliced XBP1 and Grp78/BiP in the laminitic front limbs, and a similar trend for Grp94. Expression levels of the 3 markers were minimal in all samples of the control (n = 6–7) or hind limb groups (n = 7–8). Immunofluorescent localizations were used to identify cell types expressing high levels of Grp78/BiP, as an indicator of endoplasmic reticulum stress. Grp78/BiP expression was highly elevated in suprabasal epidermal keratinocytes and only observed in laminitic front limbs (10/12 laminitic samples, compared to 0/7 in sections from the hind limbs and 0/5 of controls). Conclusions These data demonstrate that the endoplasmic reticulum stress pathway is active in naturally occurring cases of laminitis and is most active within a subset of epidermal keratinocytes. These data provide the rationale for further study of endoplasmic reticulum stress in experimental models of laminitis and the links between laminitis and human diseases sharing activation of this stress pathway. Pharmacological options to manipulate the endoplasmic reticulum stress pathway under investigation for human disease could be applicable to laminitis treatment and prevention should this pathway prove to be a driver of disease progression

Interleukin-17A pathway target genes are upregulated in Equus caballus supporting limb laminitis.

Supporting Limb Laminitis (SLL) is a painful and crippling secondary complication of orthopedic injuries and infections in horses, often resulting in euthanasia. SLL causes structural alterations and inflammation of the interdigitating layers of specialized epidermal and dermal tissues, the lamellae, which suspend the equine distal phalanx from the hoof capsule. Activation of the interleukin-17A (IL-17A)-dependent inflammatory pathway is an epidermal stress response that contributes to physiologic cutaneous wound healing as well as pathological skin conditions. As a first test of the hypothesis that hoof lamellae of horses diagnosed with SLL also respond to stress by activating the IL-17A pathway, the expression of IL-17A, IL-17 receptor subunit A and 11 IL-17A effector genes was measured by RT-PCR or qPCR. Lamellar tissue was isolated from Thoroughbreds euthanized due to naturally occurring SLL and in age and breed matched non-laminitic controls. By RT-PCR, the IL-17 Receptor A subunit was expressed in both non-laminitic and laminitic tissues, while IL-17A was primarily detectable in laminitic tissues. IL-17A target gene expression was undetectable in non-laminitic samples with the exception of weak detection of DEFB4B, S100A9 and PTSG2. In contrast, all target genes examined, except CCL20, were expressed by some or all laminitic samples. By qPCR, severe acute (n = 7) SLL expressed ~15-100 fold higher levels of DEFB4B and S100A9 genes compared to non-laminitic controls (n = 8). DEFB4B was also upregulated in developmental/subclinical (n = 8) and moderate acute (n = 7) by ~ 5-fold, and in severe chronic (n = 5) by ~15-200 fold. In situ hybridization (DEFB4) and immunofluorescence (calprotectin, a dimer of S100A9/S100A8 proteins) demonstrated expression in keratinocytes, primarily in suprabasal cell layers, from SLL samples. These data demonstrate upregulation of a cohort of IL-17A target genes in SLL and support the hypothesis that similarities in the response to stresses and damage exist between equine and human epidermal tissues