Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines

Anandamide Induces Sperm Release from Oviductal Epithelia through Nitric Oxide Pathway in Bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation

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\u27 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

Risk factors associated with health disorders in sport and leisure horses in the Netherlands

Horses are used for a wide variety of purposes from being used for recreational purposes to competing at an international level. With these different uses, horses have to adapt to numerous challenges and changes in their environment, which can be a challenge itself in continuously safeguarding their welfare. The objective of this study was to assess the prevalence of health disorders with clinical examination and identify possible risk factors of health disorders affecting horse welfare in professional husbandry systems in the Netherlands. With the use of fixed protocols for recording health aspects in horses, 150 horse farms voluntarily participating in the study were assessed by trained assessors. On each farm, 20 horses were clinically examined, in total almost 3,000 animals. This study recorded on the basis of the clinical examinations: the respiratory system (i.e., abnormal breathing [1%], coughing [1%], nasal discharge [1.9%]), body condition (i.e., 18.8% fat body condition and 6.4% poor body condition), locomotion (14.5% exhibited irregularity of locomotion and 4.8% were lame), back palpation (a light response [22.6%] and moderate to severe response [8.4%]), mouth (i.e., irregularities on mouth corners [3.4%] and bars [3.4%]), and ocular discharge (12%). Risk factor analysis, stepwise using mixed model regression, demonstrated several risk factors for health aspects. Horses used for instruction (riding lessons) were almost two times more at risk to develop moderate to severe back pain compared to horses used for recreation (odds ratios [OR] = 0.54) or for competition (OR = 0.61). Horses used for instruction (riding school lessons), breeding, or recreation all had a higher risk for irregular locomotion or lameness compared to competition horses (OR = 0.42, OR = 0.55, OR = 2.14, respectively). Horses used for recreation were more prone to have a higher BCS compared to horses used for breeding (OR = 3.07) and instruction (OR = 2.06). The prevalence of health problems and the identified risk factors are valid for the horses in the present study in which farms voluntarily participated. Furthermore, the results may provide the basis for horse welfare and health programs on farm and horse industry levels. With the development of a valid welfare monitoring system for the horse industry, the welfare of horses can be increased through improving awareness and stimulating changes in management