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A PCR-based DNA test for the genetic defect resulting in the deficiency of uridine monophosphate synthase (DUMPS) was developed for embryonic cells to identify the homozygous recessive genotype. This genotype is usually lethal at about day 40 of gestation. Oocytes from a cow, heterozygous for DUMPS, were fertilized in vitro with spermatozoa from a DUMPS-carrier bull and cultured in vitro until the morula or blastocyst stage. Embryos were matured in vitro, fertilized and cultured according to a method established in our laboratory. Heminested PCR was performed and the genotype of twelve embryos were unambiguously determined by the Aval digestion of the PCR products. Among these embryos two individuals homozygous for the defective DUMPS allele (R405→STOP were detected. From the remaining ten embryos, four were homozygous and six were heterozygous for the normal allele. The observed distribution is close to the expected ratio of 1:2:1. These results further support the monogenic recessive inheritance of the defect.
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To investigate the involvement of endogenous opioids in the regulation of gonadotrophin release in male horses, effects of the opioid antagonist naloxone (0.5 mg kg−1 i.v.) on plasma LH and testosterone concentrations and the possible influence of season and of gonadal steroids were investigated. To determine quantitative as well as qualitative changes in gonadotrophin release, LH concentrations were measured by radioimmunoassay and by an in vitro bioassay. Experiments were performed in May, August and December. In stallions, basal LH secretion in May and August was significantly higher than in December (May versus December: P< 0.01; August versus December: P< 0.05); plasma testosterone concentrations were highest in August (August versus May: P < 0.05, August versus December: P < 0.001). The basal bioactive LH concentration and the ratio of bioactive: immunoreactive LH in stallions were highest in May. Therefore, in addition to seasonal changes in quantitative LH secretion, the bioactivity of LH in the circulation also undergoes seasonal variations. Bioactive LH concentrations and the bioactive:immunoreactive ratio in geldings were higher than in stallions. Naloxone caused a significant increase in LH release in stallions in August and December (P< 0.001); no significant increase was found in May (P = 0.06). In geldings, naloxone did not induce any changes in LH secretion; in stallions, a highly significant correlation was observed between basal testosterone concentrations and the LH increment after injection of naloxone (P < 0.001). In August and December, the bioactive:immunoreactive ratio increased significantly (P< 0.05) after injection of naloxone in stallions, indicating a preferential release of LH molecules with high bioactivity. The bioactive:immunoreactive ratio did not change after naloxone injection in May. The naloxone-induced LH release was followed by a significant increase in plasma testosterone concentrations in stallions in August (P< 0.001) and December (P<0.05). In conclusion, endogenous opioid systems are involved in the regulation of LH and testosterone secretion in stallions. These mechanisms undergo seasonal changes: their activity is increased during winter and decreased during the breeding season. By affecting LH release, endogenous opioids, at least in part, regulate seasonal changes in reproductive activity in the stallion.
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Periglandular fibrosis and cystic dilation of uterine glands are associated with equine endometriosis. The presence of extracellular matrix proteins (collagen type I, III and IV, laminin and fibronectin) in healthy and endometriotic specimens was demonstrated by immunohistochemistry. The distribution of collagen I, but not collagen III, was dependent on the stage of the oestrous cycle. The arrangement of collagen I and collagen III in endometriotic specimens was similar to that in normal endometrium. In periglandular fibrosis, collagen IV, laminin and fibronectin deposition outside the basement membrane was observed. In these regions, stromal cells were characterized immunohistochemically as myofibroblasts because of their expression of a-smooth muscle actin, and occasionally tropomyosin and desmin. Periglandular differentiation of contractile cells could be interpreted as a reaction to support the extrusion of secretions in cystic dilated glands. Moreover, the changes of extracellular matrix proteins are characteristic for neoplastic lesions, although further development of endometriosis to benign or malignant tumours is not known in horses. Knowledge of the factors responsible for these fibroblastic modulations may be the key to explaining the pathogenesis of endometriosis.
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Effects of the opioid antagonist naloxone on concentrations of LH and FSH in plasma were measured in mares during different stages of the oestrous cycle. During the follicular phase of the cycle, naloxone (300 mg i.v.) had no discernible effects on basal concentrations of LH and FSH. A significant increase in plasma LH (P < 0.01) and FSH (P < 0.05) concentrations was observed after naloxone in mares during the luteal phase. This response was not different between suckled and non-suckled mares. The gonadotrophin-releasing hormone analogue buserelin (0.02 mg i.v.) caused a significant (P < 0.05) LH and FSH release irrespective of the stage of the oestrous cycle and a previous naloxone treatment. The results of this study indicate that endogenous opioid peptides are involved in the inhibition of LH and FSH release during the luteal phase of the oestrous cycle in mares and may partially mediate the suppressive influence of progesterone on gonadotrophin secretion. The opioid-mediated suppression of LH and FSH release does not seem to be affected by suckling.
