Effect of canine mesenchymal stromal cells loaded with paclitaxel on growth of canine glioma and human glioblastoma cell lines.

This study investigated whether canine mesenchymal stromal cells (cMSCs) are able to take up and release paclitaxel (PTX) in active form, and therefore whether they have potential as a tool for therapeutic delivery of this drug. cMSCs from bone marrow and adipose tissue were isolated, expanded and characterised phenotypically. cMSCs were loaded with PTX (cMSCs-PTX) and their capacity for release of PTX was determined by their effect on proliferation of cancer cells. cMSCs-PTX derived from bone marrow and adipose tissue were able to take up and then release active PTX. cMSCs-PTC inhibited proliferation of the canine glioma cell line J3T, and the human glioblastoma cell lines T98G and U87MG. The potential of canine cMSCs-PTX for treatment of canine gliomas should be investigated further.

Periovulatory time in the bitch: what's new to know?: Comparison between ovarian histology and clinical features.

The ability to recognize specific events happening in the ovaries during periovulatory time allows optimal management of canine reproduction. The objective of this study was to assess the efficacy of vaginal cytology and blood progesterone (P4) assay to identify accurately the changes occurring at the ovarian structures, mainly during the fertile period. Tertiary follicles, corpora hemorrhagica (CHs) and corpora lutea (CLs) from forty healthy bitches undergoing ovariohysterectomy were evaluated by histo-morphometry based on their aspect, number and size. The tertiary follicles distribution (small, medium and large) was statistically different (P<0.002) among all the stages of the reproductive cycle, except for small follicles (<2mm), which were always observed from proestrus to anestrus. Very large follicles (>4mm) were predominant (P=0.008) around ovulation when P4 mean level was 6.1±1.7ng/mL. The early postovulatory estrous period was characterized by CHs (P<0.002) and P4 level of 16.7±5.9ng/mL. The end of the fertile period - start of diestrus - coincided with the development of CLs (P=0.001) associated with a P4 mean level of 73.9±9.9ng/mL. The small (P<0.001) and medium (P<0.05) follicle diameters were positively correlated with the bitch size. The number of follicles larger than 4mm was significantly lower in bitches younger than 4 years (P<0.02). This study provides insight into some critical steps in the canine reproductive processes in the periovulatory phase and the end of the fertile period, essential to plan breeding programs.

Immunolocalization of aquaporin water channels in the domestic cat male genital tract.

Four different aquaporins (AQP1, 2, 5 and 9), integral membrane water channels that facilitate rapid passive movement of water, were immuno-localized in the excurrent ducts collected from sexually mature cats during orchiectomy. Aquaporins 1, 2 and 9, were immuno-localized at distinct levels, whereas AQP5 was undetectable all along the feline genital tract. No immunoreactivity was present at the level of the rete testis with any of the antibodies tested. In the efferent ducts, AQP1-immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells, and AQP9-immunoreactivity was shown at the periphery of both ciliated and non-ciliated cells. Aquaporins 2 was absent in the caput epididymidis, either in the efferent ducts or in the epididymal duct. Otherwise, AQP2 was increasingly localized at the adluminal surface of principal cells from the corpus to the cauda epididymidis and more weakly in the vas deferens epithelium. The supranuclear zone of the epididymal principal cells was AQP9-immunoreactive throughout the duct, with the exclusion of the vacuolated sub-region of the caput and with higher reaction intensity in the cauda region. AQP1 was present in the blood vessels all along the genital tract. AQP1 was expressed also in the smooth muscle layer of the vas deferens. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates and the dog, unique other carnivore species studied to date. The present information is possibly useful in regard to the regional morphology of the feline epididymis and correlated functions, which are still a matter of debate.

Expression of aquaporin water channels in canine fetal adnexa in respect to the regulation of amniotic fluid production and absorption.

Amniotic fluid (AF) is created by the flow of fluid from the fetal lung and bladder and reabsorbed in part by fetal swallowing and partly by the transfer across the amnion to the fetal circulation. Placental water flux is an important factor in determining AF volume and fetal hydration. In addition the fetal membranes might be involved in the regulation of fluid composition. To understand the mechanisms responsible for maintaining a correct balance of AF volume we evaluated the expression of aquaporins (AQPs) in canine fetal adnexa. AQPs are a family of integral membrane proteins permitting passive but physiologically rapid transcellular water movement. The presence of AQP1, 3, 5, 8 and -9 was immunohistochemically assessed in canine fetal adnexa, collected in early, middle and late-gestation during ovario-hysterectomies performed with fully informed owners' consent. Changes in AF volume and biochemical composition were also evaluated throughout pregnancy. Our results show distinct aquaporin expression patterns in maternal and extraembryonic tissues in relation to pregnancy period. AQP1 was localized in placental endothelia, allantochorion, amnion, allantois and yolk sac. AQP3 was present in the placental labyrinth, amnion, allantois and yolk sac. AQP8 was especially evident on the epithelia lining the glandular chambers, the amniotic and allantois sacs. AQP9, a channel highly permeable to water and urea, was observed in epithelia of amnion, allantois and yolk sac. In summary, AQP1, 3, 5, 8 and -9 have distinct expression patterns in canine fetal membranes and placenta in relation to pregnancy period, suggesting an involvement in mediating the AF changes during gestation.

Undernutrition during foetal to prepubertal life affects aquaporin 9 but not aquaporins 1 and 2 expression in the male genital tract of adult rats.

Expression of aquaporin water channels (AQPs) in the male excurrent ducts, is of major importance for local water movements. To study the influence of pre- and postnatal undernutrition on AQP-expression in the adult male genital tract, 4 pregnant female rats were fed ad libitum (control group) and 4 with 33.5% of gestational feed requirements (underfed group). Feeding restriction of underfed group pups continued up to weaning (25 days of age), then all pups were fed ad libitum until slaughtered at 100 days of age. Epididymides were sampled and processed for aquaporin immunohistochemistry. Expression of AQP1 was similar either in the control and underfed groups of rats, strongly evidenced at the apical and lateral plasma membrane of the efferent ducts non-ciliated cells, in the smooth muscle cells surrounding epididymal duct and in blood vessel endothelium throughout the epididymis. AQP2-immunoreactivity was present in the corpus and cauda regions, strongly expressed in the principal cells of both groups of rats. In contrast, AQP9 expression was modified by early life undernourishment, as it was weakly evidenced at the microvilli in the principal cells and strongly diminished or completely lacked in the clear cells of the cauda, in underfed group epididymides. Since it is known that clear cells are involved in luminal fluid acidification, this function might be altered in adult animals, which were underfed during early life.

An insight into testis and gubernaculum dynamics of INSL3-RXFP2 signalling during testicular descent in the dog.

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.

Absorptive activities of the efferent ducts evaluated by the immunolocalization of aquaporin water channels and lectin histochemistry in adult cats.

Ultrastructural and biochemical features of efferent ducts (EDs) are indicative of an intense absorptive activity towards the luminal fluid. This function was investigated by 1) the immunohistochemical localization of different aquaporins, integral membrane water channels that facilitate rapid passive movement of water, and 2) the histochemical localization of lectins, known to have specific affinity for glycoconjugate residues. AQP1 was mostly revealed at the apical surface and adluminal cytoplasm of non-ciliated cells and to a minor extent in their lateral plasma membrane, whereas it was absent in ciliated cells. Blood vessels showed AQP1-immunoreactivity, which was present in endothelial cells of venous vessels and capillaries and around the muscular sheath of arteries. AQP9 was expressed in the apical zone of ciliated and non-ciliated cells and in the lateral cell membrane. AQP2 and AQP5 were undetectable. Lectin histochemistry showed that non-ciliated cells contain glycans with terminal Neu5Acalpha2,3Galbeta1,3GalNAc, Neu5Acalpha2,3Galbeta1,4GlcNAc, Galbeta1,4GlcNAc, GalNAc (s-PNA, MAL II, RCA120, SBA reactivity) and with internal/terminal alphaMan (Con A affinity) at the luminal surface and the apical region. In addition, non-ciliated cells expressed oligosaccharides terminating with GalNAc and Neu5Acalpha2,6Gal/GalNAc (SNA reactivity) in the luminal surface and the apical zone, respectively. Ciliated cells revealed glycoconjugates only on cilia, which showed terminal Neu5Acalpha2,3Galbeta1,4GlcNAc (s-RCA120 staining) and GalNAc, as well as internal/terminal alphaMan and GlcNAc (s-WGA, GSA II staining). Data provide evidence for the involvement of different pathways in the bulk reabsorption of water and low molecular weight solutes by the non-ciliated cell of the cat EDs. AQP-mediated trans-cellular route can be hypothesized, together with fluid phase endocytosis mediated by the glycocalix and a well-developed endocytotic apparatus. Epithelial ciliated cells, whose main function is the movement of luminal content, might also participate in absorptive processes to a lesser extent.

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus.

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.