Recent investigations1 indicate that cat efferent ducts (EDs) play a role in reabsorption of the fluid and proteins leaving the testes, dependent also on androgen2. The males with Klinefelter syndrome (XXY) show a variable degree of androgen deficit responsible for testicular disfunction3. Since we demonstrated synthesis and secretion of glycoconjugates in normal cat EDs1, here we investigated the glycoprotein pattern of the EDs from a tricolor cat with Klinefelter syndrome (39,XXY), by means of the lectin histochemistry, utilizing a panel of 12 lectins in association with sialidase (s) treatment. Cilia of ciliated cells reacted with HPA, SBA, Con A, KOH-s-WGA, GSA II in normal cats and with MAL II, SNA, Con A and KOH-s-WGA in XXY cat. The luminal surface of non-ciliated cells bound MAL II, KOH-s-PNA, Con A in all samples, RCA120 and HPA only in normal subjects and PNA in XXY cat. The supra-nuclear cytoplasm of non-ciliated cells expressed SNA and Con A affinity in XXY cat and also MAL II, KOH-s-PNA, RCA120, SBA in normal cats. These results indicate that negative charges are mainly expressed on the cilia of XXY cat EDs, whereas a more complex glycoconjugate pattern, probably related to an more effective endocytotic apparatus, is expressed in the supra-nuclear cytoplasm of non-ciliated cells from normal EDs. References 1. Arrighi S et al. Histol. Histopathol. 2001, 25:433-44. 2. Jones RC. The epididymis: From molecule to clinical practice. Robaire and Hinton eds, Kluwer Academic/Plenum Publ. 2002,11-33. 3. Wikström AM and Dunkel L. Horm. Res. 2008, 69:317-26.

Fetal adnexa are a noncontroversial source of mesenchymal stem cells (MSCs) with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. Mesenchymal SCs have been characterized for both their stemness and their differentiation abilities. Recently, the scientific debate has focused on MSCs selection and on establishing predictable elements to discriminate the cells with most promising potential in regenerative medicine. In this study, we characterized and followed in vitro proliferation and differentiation potency of canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) isolated from fetuses at early (35-40 days) and late (45-55 days) gestational ages. We found that cells from both fetal gestational ages showed similar features. In all examined cell lines, the morphology of proliferating cells typically appeared fibroblast-like and the population doubling of cells, cultured up to passage 10, significantly increased with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age. In AM- and UCM-MSCs of both gestational phases, the expression of embryonic (Oct-4) and mesenchymal stemness (CD29, CD44) markers was observed. Hematopoietic and histocompatibility markers were never found in any sample. Cells of the two cell types at P3 showed multipotent abilities and differentiated to neurocytes and osteocytes, as demonstrated by specific stains and molecular analysis. These results indicated that MSCs retrieved from UCM and AM in early and late fetal phase of gestation could be used in regenerative medicine approaches in the dog.

The most commonly observed reproductive dysfunction in male fishes reared in captivity is reduction in sperm volume and quality. The Atlantic bluefin tuna Thunnus thynnus (Osteichthyes: Scombridae) is one of the few large pelagic and migratory marine fishes maintained in captivity with the purpose of establishing breeding populations to support an aquaculture industry. The objectives of the present study were to compare male germ cell proliferation and apoptosis between wild and captive individuals at two different phases of the spermatogenetic cycle, and to evaluate sperm motility characteristics of captive individuals. Histological observations were performed to analyze testicular activity, and germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase mediated d’UTP nick end labeling (TUNEL) method,respectively. Computer-assisted sperm analysis (CASA) was used to evaluate sperm motility. Results showed that germ cell proliferation was delayed and germ cell apoptosis increased in captive animals relative to wild individuals. Sperm motility of samples obtained from captive individuals was anomalous, both in terms of motility duration and swimming efficiency. Thus it appears that rearing in captivity impairs male reproductive function through, at least, changes in germ cell roliferation and apoptosis.

The study was carried out on 42 breeder couples (42 males and 42 females) of European brown hare (Lepus europaeus), divided into three groups fed three different experimental diets (14 couples/treatment). Two diets were supplemented with n-3 and n-6 polyunsaturated fatty acids (PUFAs; 2% of linseed oil and soybean oil, respectively) and were compared with a control diet supplemented with a monounsaturated fatty acids (2% of olive oil). During the experimental period (from 15 April to 30 September), the following parameters were recorded: days from the beginning of trial to the first parturition, parturition interval, number of parturitions, number of leverets born (alive and dead), dead during suckling, the total number of leverets weaned and feed intake per cage (of males, females and leverets until weaning). Feed intake was not influenced by treatments. In hares fed n-3 and n-6 diets, the days from the beginning of the trial to the first parturition and the parturition interval were similar and were lower compared with control group (63.1 v. 70.6 days, and 37.8 v. 40.9 days, respectively; P < 0.05). Hares from n-6 group had a higher (P < 0.05) number of parturitions per cage during the experimental period than the n-3 and control group that showed a similar value (3.00 v. 2.36, respectively). The total number of leverets born per cage and parturition in n-6 and n-3 groups increased with respect to those fed control diet (P < 0.05). The leverets' mortality rate at birth was higher in n-6 than in n-3 and control group (3.50 v. 2.17, respectively; P < 0.05). In control group, leverets' mortality rate during suckling was lower with respect to n-3 (P < 0.05) and n-6 (P < 0.05), showing the highest value for the latter (P < 0.05). In spite of this higher mortality, the number of leverets weaned per cage and parturition was higher (P < 0.05) in n-6 compared with n-3 group, being the latter higher than the control group (3.12, 2.79 and 2.43, respectively). Our results show that the dietary PUFAs, particularly n-6 supplementation, have a positive influence on the reproductive performances of the European brown hare.

ABSTRACT - Amniotic membrane (AM) and umbilical cord matrix (UCM) mesenchymal stem cells (MSCs) have been isolated and characterized in humans and large animal models. In order to distinguish which cells retain the best features for different purposes, the effects of gestational age on proliferation and differentiation potency of canine AM-MSCs and UCM-MSCs was analyzed. Samples were recovered after elective ovariohysterectomy from bitches in early (35 to 40 days) and late (45 to 55 days) fetal stage of pregnancy. The proliferation study and the molecular analysis of embryonic, mesenchymal and hematopoietic markers were performed. Cell neurogenic and osteogenic differentiation were followed. No differences were noticed when comparing data obtained from cells isolated at different gestational ages. Doubling times, cell viability and Oct-4, CD29 and CD44 stemness markers expression were similar in cell isolated from bitches in early or late pregnancy. In both gestational ages, morphological features of neuronal and osteogenic differentiation were observed which need to be confirmed by molecular analysis. In conclusion, our data indicate the possibility to isolate MSCs from canine fetuses at early and late gestational ages with the same proliferative and differentiative capabilities.

Opioid receptors (ORs) are G protein-coupled receptors. Other than antinociception, they have been recently shown to be involved in the crucial switch phase between cell proliferation and differentiation, from which the stem cell fate depend. We detected mu-OR subtype 1 (MOR-1) and kappa-OR subtype 1 (KOR-1) expression in canine umbilical cord matrix (UCM) mesenchymal stem cells (MSCs). MOR expression decreased with passage numbers, whereas KOR was expressed at constant levels throughout passages. Both ORs type were functionally active, since DAMGO and U69593, MOR- and KOR- selective agonists respectively, and CTAP and nor-BNI, MOR- and KOR- selective antagonists respectively, were significantly able to modulate cell proliferation. Both opioid agonists, when used at the concentration of 1μM, inhibited cell proliferation of canine UCM-MSCs. Inhibitory effect on cell proliferation was also observed after CTAP treatment, whereas no effect was noticed after nor-BNI treatment. By specific stain and morphology analysis, no differences were observed in the neurogenic differentiation potency of UCM-MSCs in both treatments and control conditions. Collectively our data suggest that, in canine UCM-MSC, opioids modulate cell proliferation, but further studies are needed to evaluate whether opioid modulation may play a role in directing these cells to neurogenic lineages.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canineMSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblastlike, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P<0.05).Amnion andUCM-MSCs expressed embryonic and MSC markers, such as Oct- 4 CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLADRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast-like, and the population doubling time (DT) significantly increased with passage number. For AF- and AM-MSCs, cell viability did not change with passages. In UCM-MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM-MSCs expressed embryonic and MSC markers, such as Oct-4, CD44, CD184, and CD29, whereas AF-MSCs expressed Oct-4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA-DRA1 and DLA-79) were expressed only in AF-MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs.

Stallion sperm from semen collected in Southern Italy during the breeding (June–July) and non-breeding (December–January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc, with Galb1,3GalNAc, a/bGalNAc and glycans with terminal/internal aMan and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Galb1,4GlcNAc (Ricinus communis120 affinity) (RCA120) and LFuca1,2Galb1,4GlcNAcb (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and postacrosomal region did not display glycans terminating with GalNAc, GlcNAc, and aL-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Aca2,3Galb1,4GlcNAc, Neu5Aca2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with aGalNAc, GlcNAc, and L-Fuca1,2- Galb1,4GlcNAcb (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and nonbreeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (0, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MILL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (1) small aggregates diffused throughout the cytoplasm; (11) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P<0.05; O vs. A: P<0.001), in EL (61%; O vs. EL P<0.01), or in MLL (0%; F vs. MLL: P<0.05; O vs. MLL: P<0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P<0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes. (C) 2009 Elsevier B.V. All rights reserved.

Introduction:While amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in humans and horses and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications. Methods:In our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion. Results:AECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic Marker SSEA-4,but not SSEA-3, as demonstrated by mmunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low mmunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity. Conclusions: In conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic Cell-based therapies in order to treat tissue lesions, especially with loss of substance

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5×10-4 to 25nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development.

The prevalence of feline haemoplasma infections in blood samples collected from cats in southern Italy between 2007 and 2011 was evaluated. A convenience-sample of 314 cats (136 healthy; 178 non-healthy;) was screened by polymerase chain reaction using several sets of primers. The overall prevalence of Haemoplasma infections was 18,0% in the samples collection examined in this study. The prevalence was 9,5% for 'Candidatus Mycoplasma haemominutum', 6,5% for Mycoplasma haemofelis and 2% for 'Candidatus Mycoplasma turicensis'. Interestingly, the prevalence was significantly higher in symptomatic (28%) rather than in healthy animals (6%). Also, free-ranging male individuals older than 2 years were more exposed to risks of infection by these pathogens. These findings indicate that haemoplasma infections are common in Italy.

Ochratoxin A (OTA) is one of the most frequently occurring mycotoxins in several food products and, under experimental conditions, it causes a wide spectrum of toxic effects, including teratogenicity, carcinogenicity, nephrotoxicity, and immunotoxicity. In laboratory animals, the exposure to OTA during pregnancy may interfere with embryonic development and may induce embryo lethality, growth delay and teratogenic effects. An alternative approach to in vitro predict the developmental risk of chemicals has been offered by mesenchymal stem cells (MSCs) isolated from fetal adnexa. The aim of this work is to study OTA effects at nanomolar concentrations, corresponding to levels detected in human placenta, on proliferation of canine umbilical cord matrix MSCs (UCM-MSCs). Cells were plated in 12-well dishes to perform doubling time (DT) analysis, cellular density and viability from passage (P) 1 to P7, by using Trypan blue dye exclusion test. OTA was added at concentrations ranging from 0.01ng/ml to 10ng/ml. Two experimental protocols of OTA treatment were performed: 1) continue exposure model; 2) alternate exposure model, in which cultures were performed for 3 days in standard medium followed by 4 days in presence of OTA. Control cultures received the same amount of OTA solvent (DMSO). After OTA exposure, cell proliferation was observed until P3, in contrast to control samples in which cells proliferated until P7. In both exposure models, DT values were higher than those found in control samples. In particular, lowest OTA concentrations (0.01–0.1ng/ml) significantly increased (P<0.0001) DT values compared with controls in both exposure models, with consequent inhibition of cell proliferation. No differences were observed between continue and alternate exposure models, leading to suppose that the cell damage early occurs and cannot be reversed independently of exposure model. At all passages, OTA treatment did not influence cell viability. In addition, changes in osteo- and neuron-like morphology were observed in treated cells at P3, in contrast with controls that maintained fibroblast-like morphology. The action mechanism of OTA on cell proliferation, at nanomolar concentrations, is not clear. It has been reported in renal epithelial cells that OTA may interact with cellular key targets, such as mitochondria, and interfere with Ca2+, pH and energy homeostasis, leading to the disturbance of cell signaling/proliferation and apoptosis. Moreover, OTA, in renal epithelial cells, may activate MAP-kinase, which cause cell dedifferentiation and transformation: this evidence could explain the morphological changes observed also in UCM-MSCs. In conclusion, OTA, at nanomolar concentrations, modulates physiological signals influencing cell growth, self-renewal and differentiation in UCM-MSCs as it may occur during embryo development.