Increasing expression of oxytocin and vasopressin receptors in the equine conceptus between Days 10 and 16 of pregnancy.

Oxytocin (OT) and arginine vasopressin (AVP) have been detected in the yolk sac of the pre-attachment equine conceptus. Therefore, we have assessed the presence of OT and AVP receptors in equine conceptuses between Days 10 and 16 of pregnancy by qualitative PCR, quantitative PCR and immunohistochemistry. Expression of OT receptor and of the AVP receptors V1aR and V2R could be verified after sequencing the RT-PCR products of the expected length. The size of conceptuses used for quantitative PCR significantly increased with day of pregnancy (P<0.01) as did their quantitative expression of OTR (P<0.01). Immunohistochemistry of OTR resulted in weak trophectodermal abundance on Day 10, increasing at Day 12. On Day 14, staining intensity increased in individual cells of the trophectoderm while it decreased in other cells; this trend became more apparent on Day 16. The endoderm of the trophoblast and surrounding subtrophoblastic compartments always showed moderate staining for OTR. On Day 10 immunoreactive V2R protein was localised in the trophectodermal apical membrane; on Day 12 it was also present in the basal membrane and weakly in the cytoplasm. On Day 14 only individual trophectodermal cells showed positive supranuclear cytoplasmic areas or V2R, whereas on Day 16 about one-third of the trophectodermal cells were stained entirely and intensely positive. These results suggest an involvement of OT and AVP action in the development and expansion of the early equine conceptus.

Expression of Enzymes Associated with Prostaglandin Synthesis in Equine Conceptuses.

In the horse, mobility of the conceptus is required for maternal recognition of pregnancy depending on secretion of prostaglandins by the conceptus. The aim of this study was to determine the expression and localization of key enzymes of the different pathways leading to synthesis of prostaglandin E2 and F2α in the equine conceptus during the mobility phase. Enzyme expression was analyzed via quantitative RT-PCR in total RNA samples of equine conceptuses collected on days 10 (n = 5), 12 (n = 12), 14 (n = 5) and 16 (n = 7) from healthy mares. Relative abundance of cyclooxygenase (COX)-2 mRNA was higher (p < 0.05) than of COX-1 irrespective of conceptus age and for phospholipase A2 on day 16 in comparison to all other days (p < 0.01). Abundance of mRNA of cytosolic and microsomal prostaglandin E synthase (PGES) and of carbonyl reductase (CBR) 1 was not influenced by conceptus age. Immunohistochemically, COX-1, COX-2, as well as cytosolic and microsomal PGES were present in both the ectodermal and endodermal layer of the yolk sac wall. CBR-1 was restricted to periembryonic disc area. The localisation of the key enzymes explains the mechanism of embryo mobility. In vitro incubation of primary trophoblast cell cultures with oxytocin had no effect on key enzyme synthesis.

Transmission electron microscopy (TEM) of equine conceptuses at 14 and 16 days of gestation.

The present study gives a detailed ultrastructural description of equine conceptuses at Day 14 (n = 2) and Day 16 (n = 3) after ovulation. Whereas on Day 14 only primitive structures were seen, on Day 16 neurulation and formation of mesodermal somites had taken place. The ectoderm of the embryo itself and the surrounding trophoblast ectodermal cells were characterised by specific cell surface differentiations. At the embryonic ectodermal cell surface (14 and 16 days) remarkable protruded and fused cytoplasmic projections were seen, typically associated with macropinocytotic events involved in macromolecule and fluid uptake. This finding adds an important point to the expansion mode of the hypotone equine conceptus, which is characterised by 'uphill' fluid uptake. Numerous microvilli and coated endocytotic pits at the apical trophoblast membrane emphasised its absorptive character. Endodermal cells were arranged loosely with only apically located cellular junctions leaving large intercellular compartments. At the border of the embryonic disc apoptotic cells were regularly observed indicating high remodelling activities in this area. Conspicuous blister-like structures between ectoderm and mesoderm were seen in the trilaminar part of Day-14 and -16 conceptuses. These were strictly circumscribed despite not being sealed by cellular junctions between germinal layers. It is possible that these blisters are involved in embryo positioning; however, further studies are needed to verify this.

The ABCA1 blocking agent probucol decreases capacitation in ejaculated dog spermatozoa.

BACKGROUND: The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay. RESULTS: In ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples. CONCLUSIONS: CTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus.

Expansion of the equine conceptus can be divided into blastocoel and yolk sac phases. The endodermal layer transforming the blastocoel into the yolk sac is completed around day 8 of pregnancy. From that time, the size of the spherical conceptus increases tremendously due mainly to the accumulation of fluid rather than cell multiplication. In this study, we have investigated the abundance and localisation of Na(+)/K(+)-ATPases and aquaporins (AQP) in the equine conceptus on days 8, 10, 12, 14 and 16 by multiplex reverse transcriptase PCR, Western blot and immunohistochemistry. During conceptus expansion, the ectoderm of the yolk sac exhibited basolateral abundance of alpha1ATPase, apical localisation of AQP5, and membrane and cytoplasmic expression of AQP3. With increasing conceptus size its cells showed an extensive enlargement of the apical membrane surface by microvilli. From day 14 onwards, the yolk sac endoderm forms arc-like structures with attaching sites to the ectodermal layer and shows intensive staining for alpha1ATPase, AQP5 and AQP3 in the membrane as well as in the cytoplasm. In the yolk sac ectoderm, the arrangement of these proteins is comparable with the collecting ducts of kidney with AQP2 being replaced by the closely related AQP5. The detection of phosphorylation sites for protein kinase A suggests a similar AQP5 traffic and regulation as known for AQP2 in the collecting ducts of the kidney. The arrangement of these proteins in equine embryos indicates at least partially the mechanism of conceptus expansion.

Uterine involution and endometrial function in postpartum pony mares.

OBJECTIVE: To determine endometrial regeneration in postpartum mares by analysis of histologic features, apoptosis and cell proliferation markers, lectin binding, cytokines, and progesterone and estrogen receptors in endometrial biopsy specimens. ANIMALS: 9 postpartum mares. PROCEDURES: Mares were examined on postpartum days 1, 9, and 16, and uterine biopsy specimens were obtained for histologic examination. Lectin binding was analyzed histochemically, and expressions of Ki-67 antigen (proliferation marker), lysozyme, and caspase 3 (apoptosis marker) were studied immunohistochemically. Gene expressions for cytokines (interleukin-1beta, -6 and -8 and tumor necrosis factor-alpha), cyclooxygenase 2, prostaglandin-E-synthase, and estrogen and progesterone receptors were determined by use of quantitative real-time PCR assay. RESULTS: On day 1, neutrophils predominated but by day 9 had largely been replaced by lymphocytes and macrophages. High numbers of cells with staining for caspase 3 were found on day 1, but numbers decreased by day 9. In contrast, the number of cells with staining for Kiel 67 antigen increased between days 1 and 9. Lectin binding to the endometrium changed over time. Relative mRNA expressions for cytokines and prostaglandin-E-synthase did not differ among days. Expressions of progesterone and estrogen receptors were minimal on day 1 and increased by day 9. CONCLUSIONS AND CLINICAL RELEVANCE: Early postpartum endometrial cells underwent apoptosis, but during the second week, postpartum proliferation of cells predominated. Lectin binding reflected changes in endometrial glycocalyx patterns. Increased expression of estrogen receptors allowed the endometrium to respond to estrogen during foal heat, and in subsequent diestrus, the endometrium was able to respond to progesterone.

Early pregnancy in the horse revisited - does exception prove the rule?

Early equine pregnancy shares many features with that of more intensively assessed domestic animals species, but there are also characteristic differences. Some of those are poorly understood. Descent of the equine conceptus into the uterine lumen occurs at day 5 to 6 after ovulation but is only possible when the embryo secretes prostaglandin E2. Although maintenance of equine pregnancy probably involves secretion of a conceptus derived anti-luteolytic factor, this agent has not been identified. Rapid growth, conceptus mobility and presence of an acellular capsule at the time of maternal recognition of pregnancy, i.e. between days 12 and 14, are prerequisites to avoid pregnancy loss. Progesterone together with 5α-pregnanes is secreted by the corpus luteum and induces the production of endometrial histotroph which is responsible for conceptus nutrition until placention. A stable contact between the outer trophoblast layer of the allantochorion and the luminal epithelium of the endometrium is not established before days 40 to 42 of pregnancy.