Supporting Limb Laminitis.

Supporting limb laminitis (SLL) is a relatively frequent complication of painful limb conditions that alter normal weight-bearing patterns in horses. New evidence suggests that a lack of limb load cycling activity (normally associated with ambulation) interferes with normal perfusion of the lamellae in these cases, resulting in ischemia and dysfunction/death of cells critical to the mechanical function of the lamellae. Excessive weight-bearing load drives the progression to overt acute laminitis in the supporting limb. Monitoring and enhancement of limb load cycling activity are key strategies that may lead to successful prevention of SLL by ensuring adequate lamellar perfusion.

Genetics and Signaling Pathways of Laminitis.

Laminitis is a devastating disease with diverse etiologies and few, if any, effective treatments. Gene expression and hypothesis-generating genomic studies have provided a fresh look at the key molecular players at crucial timepoints in diverse experimental and naturally affected tissues. We summarize findings to date, and propose a unifying model of the laminitis disease process that includes several pathogenesis concepts shared with other diseases of epidermal and epithelial tissues. The value of these new pathways as potential therapeutic targets is exciting but will require careful future work to validate new methods and launch systematic clinical trials.

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

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.

Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof.

BACKGROUND: The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. HYPOTHESIS/OBJECTIVES: To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. METHODS: Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC transition to transit-amplifying (TA) cell] in epithelial tissues of the foot (haired skin, hoof coronet and hoof lamellae) and the eye (limbus and cornea). RESULTS: Expression of K14 was restricted to the basal layer of epidermal lamellae and to basal and adjacent suprabasal layers of the haired skin, coronet and corneal limbus. Coronary and lamellar epidermis was negative for both K3 and K10, which were expressed in the cornea/limbus epithelium and haired skin epidermis, respectively. Variable expression of p63 with relatively low to high levels of phosphorylation was detected in individual basal and suprabasal cells of all epithelial tissues examined. CONCLUSIONS: To the best of the author's knowledge, this is the first report of the characterization of tissue-specific keratin marker expression and the localization of putative epithelial progenitor cell populations, including ESCs (high p63 expression with low pp63 levels) and TA cells (high expression of both p63 and pp63), in the horse. These results will aid further investigation of epidermal and corneal epithelial biology and regenerative therapies in horses.

Wheat germ agglutinin as a counterstain for immunofluorescence studies of equine hoof lamellae.

Equine laminitis is a common, painful, debilitating condition of the hoof that is a leading cause of disability in horses, often necessitating euthanasia. The equine hoof represents an extreme evolutionary adaptation of an epidermal structure homologous to the human or murine nail units. Immunohistochemistry is frequently utilized in the study of the pathophysiology of laminitis. The complex, multilayered, extensively interdigitated epidermal-dermal lamellar interface renders precise interpretation of immunofluorescence localization difficult, especially when effective technique and reagents render non-reactive tissues completely dark. Fluorescent-conjugated wheat germ agglutinin (WGA) selectively labels dermal extracellular matrix fibres and epidermal cell membranes in tissue sections of horse hoof lamellae, is compatible with indirect immunofluorescence and augments interpretation of indirect immunofluorescence antigen localization. The current report details the use of WGA as a rapid, simple, economical counterstain for immunofluorescence studies of the equine hoof and may have application to other complex epidermal tissue structures.

Transcriptome diversity and differential expression in supporting limb laminitis.

Laminitis results in impaired tissue integrity and Inflammation of the epidermal and dermal lamellae connecting the hoof capsule to the underlying distal phalanx and causes loss-of-use, poor quality of life and euthanasia in horses. Historically, studies to better understand the etiology of laminitis by documenting changes in gene expression were hampered by the paucity of gene annotation specific to hoof tissues. Next-generation sequencing enables improvements to annotation by incorporating equine- and hoof-specific transcripts. Here we characterize the hoof lamellar tissue transcriptome of naturally occurring supporting limb laminitis (SLL) using archived lamellar tissue from Thoroughbred racehorses consisting of 13 SLL hospital cases and seven age-matched control horses. This was achieved using: 1) Applied transcriptome annotation by long-read sequencing to document transcript diversity and 2) short-read RNA sequencing to document changes in gene expression correlating to the developmental and acute stages of naturally occurring SLL. 1.99Gbp of long-read transcriptome sequencing deeply documented 5067 unique loci, while short read RNA-seq under very stringent quality filters described 66 differentially expressed loci. Functional analysis of these loci revealed alterations in cell replication and growth, stress response and leukocyte recruitment and activation pathways. Differential expression of the Ezrin and TIMP3 genes suggests they may have utility as biomarkers for laminitis disease, while NR1D1 and genes relevant to the inflammasome are promising targets for novel pharmacological treatments.

Witch Nails (Krt90whnl): A spontaneous mouse mutation affecting nail growth and development.

Numerous single gene mutations identified in humans and mice result in nail deformities with many similarities between the species. A spontaneous, autosomal, recessive mutation called witch nails (whnl) is described here where the distal nail matrix and nail bed undergo degenerative changes resulting in formation of an abnormal nail plate causing mice to develop long, curved nails. This mutation arose spontaneously in a colony of MRL/MpJ-Faslpr/J at The Jackson Laboratory. Homozygous mutant mice are recognizable by 8 weeks of age by their long, curved nails. The whnl mutation, mapped on Chromosome 15, is due to a 7-bp insertion identified in the 3' region of exon 9 in the Krt90 gene (formerly Riken cDNA 4732456N10Rik), and is predicted to result in a frameshift that changes serine 476 to arginine and subsequently introduces 36 novel amino acids into the protein before a premature stop codon (p. Ser476ArgfsTer36). By immunohistochemistry the normal KRT90 protein is expressed in the nail matrix and nail bed in control mice where lesions are located in mutant mice. Immunoreactivity toward equine KRT124, the ortholog of mouse KRT90, is restricted to the hoof lamellae (equine hoof wall and lamellae are homologous to the mouse nail plate and nail bed) and the mouse nail bed. Equine laminitis lesions are similar to those observed in this mutant mouse suggesting that the latter may be a useful model for hoof and nail diseases. This first spontaneous mouse mutation affecting the novel Krt90 gene provides new insight into the normal regulation of the molecular pathways of nail development.

Continuous digital hypothermia reduces expression of keratin 17 and 1L-17A inflammatory pathway mediators in equine laminitis induced by hyperinsulinemia.

The euglycemic hyperinsulinemic clamp model (EHC) of equine endocrinopathic laminitis induces rapid loss of lamellar tissue integrity, disrupts keratinocyte functions, and induces inflammation similar to natural disease. Continuous digital hypothermia (CDH) blocks tissue damage in this experimental model, allowing identification of specific genes or molecular pathways contributing to disease initiation or early progression. Archived lamellar tissues (8 horses, 48 h EHC treatment, including CDH-treated front limbs) were used to measure relative expression levels of genes encoding keratin 17 (KRT17), a stress-induced intermediate filament protein, and genes upregulated downstream of keratin 17 and/or interleukin 17A (IL-17A), as mediators of inflammation. Compared to front or hind limbs at ambient temperature, CDH resulted in significantly lower expression of KRT17, CCL2, CxCL8, PTGS2 (encoding COX2), IL6, TNFα, S100A8 and MMP1. By immunofluorescence, COX2 was robustly expressed in lamellar keratinocytes from ambient limbs, but not in CDH-treated limbs. Genes not significantly reduced by CDH were IL17A, DEFB4B, S100A9 and MMP9. Overall, 8 of 12 genes were expressed at lower levels in the CDH-treated limb. These 8 genes are expressed by wounded or stress-activated keratinocytes in human disease or mouse models, highlighting the role of keratinocytes in equine laminitis.

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.

Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation.

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus.

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Cryopreservation of boar semen and its future importance to the industry.

Whereas AI has arguably been the most important management tool leading to improved herd productivity, long-term storage of semen brings forth additional advantages to producers of agriculturally important animals and the AI industry. Semen cryopreservation greatly facilitates the distribution of agriculturally desirable genes, rapidly increasing herd productivity. Of particular importance to the pig industry, the use of frozen semen would help to control transmission of certain pathogens, thereby protecting the health status of the herd. Moreover, a reserve of cryopreserved semen would minimize the effects of a sudden outbreak of a contagious illness or a natural disaster. Successful cryopreservation of boar semen is necessary for international sales. Finally, effective gene banking depends on the availability of functional, cryopreserved germplasm. Despite these potential advantages of long-term semen storage, porcine sperm are notoriously sensitive to cold temperatures, and frozen-thawed semen is not routinely used by the industry. The objective of our laboratories is to develop protocols for efficient long-term storage of porcine semen using cryopreservation. We hypothesize that since the sperm plasma membrane is the primary site of cold-induced damage, reinforcing the membranes with molecules having particular properties, such as cholesterol, will improve the ability of boar sperm to withstand cold temperatures and cryopreservation protocols. Based on our data, such approaches should help alleviate the problems with sperm function after cooling, thereby resulting in better survival and motility characteristics, and reduced non-regulated capacitation and spontaneous acrosome reactions.

Case-control study of risk factors for pasture-and endocrinopathy-associated laminitis in North American horses.

OBJECTIVE To investigate risk factors for the development of pasture- and endocrinopathy-associated laminitis (PEAL) in horses and ponies in North America. DESIGN Case-control study. ANIMALS 199 horses with incident cases of PEAL and 351 horses from 2 control populations (healthy horses [n = 198] and horses with lameness not caused by laminitis [153]) that were evaluated in North America between January 2012 and December 2015 by veterinarian members of the American Association of Equine Practitioners. PROCEDURES North American members of the American Association of Equine Practitioners were contacted to participate in the study, and participating veterinarians provided historical data on incident cases of PEAL, each matched with a healthy control and a lameness control. Conditional logistic regression analysis was used to compare data on PEAL-affected horses with data on horses from each set of controls. RESULTS Horses with an obese body condition (ie, body condition score ≥ 7), generalized or regional adiposity (alone or in combination), preexisting endocrinopathy, or recent (within 30 days) glucocorticoid administration had increased odds of developing PEAL, compared with horses that did not have these findings. CONCLUSIONS AND CLINICAL RELEVANCE The present study identified several risk factors for PEAL that may assist not only in managing and preventing this form of laminitis, but also in guiding future research into its pathogenesis.

Current concepts of molecular events during bovine and porcine spermatozoa capacitation.

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.

Effects of 2-hydroxypropyl-beta-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation.

Porcine sperm are extremely sensitive to the damaging effects of cold shock. It has been shown that cholesterol-binding molecules, such as 2-hydroxypropyl-beta-cyclodextrin (HBCD), improve post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. The objective of this study was to determine the effects of HBCD and cholesterol 3-sulfate (ChS) on porcine sperm viability and capacitation following cold shock or incubation under conditions that support capacitation using a defined medium. We report here that porcine sperm incubated in medium containing both HBCD and ChS have significantly improved viability following cold shock (10 min at 10 degrees C) when compared to sperm incubated without HBCD or ChS, or with either component alone. Treatment with HBCD plus ChS also completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment or by incubation for 3 hr under conditions that support capacitation. Two assays of sperm capacitation, the rate of calcium ionophore-induced acrosome reactions and chlortetracycline (CTC) staining, were not significantly altered by HBCD and ChS following cold shock. However, 3-hr incubation with HBCD plus ChS or with 1 mM ChS alone decreased the percentage of sperm undergoing the induced acrosome reaction without significantly affecting viability when compared to the control. These results indicate that the manipulation of sperm plasma membrane cholesterol content affects porcine sperm viability and capacitation status and could therefore be useful to protect sperm from cold shock during cryopreservation by improving viability without promoting premature capacitation.

Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation.

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats.

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.