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Lexington, KY, United States

Klein C.,University of Calgary | Troedsson M.H.T.,Gluck Equine Research Center | Rutllant J.,Western University of Health Sciences
Anatomical Record | Year: 2013

The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, -3, -7, and -9). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, -5, -7, and -8). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, -2, -5, and -9) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells. © 2013 Wiley Periodicals, Inc. Source

Klein C.,Gluck Equine Research Center | Troedsson M.,Gluck Equine Research Center | Rutllant J.,Western University of Health Sciences
Reproduction in Domestic Animals | Year: 2013

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare. © 2012 Blackwell Verlag GmbH. Source

Klein C.,Gluck Equine Research Center | Troedsson M.H.T.,Gluck Equine Research Center
Reproduction in Domestic Animals | Year: 2013

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine expressed by a wide range of tissues, which has been implicated to be involved in reproduction. Relative abundance of MIF mRNA in conceptus and endometrial tissue was assessed using real-time RT-PCR. Western blot analysis and immunohistochemistry were used to detect MIF protein expression. MIF transcript abundance was lowest in conceptuses obtained 16days after ovulation, while the remaining stages of conceptus development that were analysed showed relatively constant expression levels. Migration inhibitory factor expression localized to trophectoderm cells, while capsular material was void of MIF immunoreactivity. Throughout the oestrous cycle, no clear statistically significant cycle-dependent expression pattern could be observed. During early pregnancy, the highest mRNA transcript levels were detected 16days after ovulation. Pregnancy status did not affect MIF mRNA expression. Using immunohistochemistry, MIF protein expression was primarily localized in luminal and glandular epithelial cells, while stromal cells displayed weaker immunoreactivity. Taken together, we suggest that MIF is part of the molecular repertoire that contributes to normal endometrial function. The detailed functional significance of MIF expression in equine endometrium and pre-implantation stages of conceptus development remains to be determined. © 2012 Blackwell Verlag GmbH. Source

Klein C.,Gluck Equine Research Center | Troedsson M.H.T.,Gluck Equine Research Center
Reproduction in Domestic Animals | Year: 2012

During the second and third week of pregnancy, the equine conceptus is covered by an acellular glycoprotein capsule. This capsule contains glycoproteins resembling those of the mucin family with sialic acid making up a high proportion of the carbohydrate. Coinciding with conceptus fixation, a marked decline in sialic acid content of the capsule occurs, which has been proposed to contribute to cessation of conceptus mobility. Herein, we describe the expression of neuraminidase 2 (NEU2) by pre-implantation stages of equine conceptus development. NEU2 transcript abundance was examined in conceptuses obtained 8, 10, 12, 14 and 16days after ovulation; highest levels were observed 16days after ovulation. Transcript abundance observed in endometrial tissue was on average 474-fold lower than in conceptus tissue. Protein expression was localized to trophoblast cells and capsular material. Functionality of NEU2 was shown using an Amplex Red reagent-based assay. NEU2, formerly known as sialidase 2, belongs to a family of enzymes that cleave sialic acid from polysaccharide chains. The expression of NEU2 described herein provides a mechanism by which the conceptus can regulate the sialic acid content of its own capsule. The timely desialylation coinciding with conceptus fixation has been suggested integral for establishment of normal pregnancy. © 2011 Blackwell Verlag GmbH. Source

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