Proliferation and Apoptosis of Cat (Felis catus) Male Germ Cells during Breeding and Non-Breeding Seasons.

The domestic cat (Felis catus) is a seasonal-breeding species whose reproductive period starts when the day length increases. Since the existing information on cat spermatogenesis is limited and somewhat contradictory, in the present study, germ cell proliferation and apoptosis in feral adult tomcats orchiectomized during reproductive (reproductive group, RG; February-July) and non-reproductive (non-reproductive group, NRG; November and December) seasons were compared. Cross-sections taken from the middle third of the left testis were chemically fixed and embedded in paraffin wax. Histological sections were processed for the immunohistochemical detection of proliferating germ cells (PCNA) and for the identification of apoptotic cells (TUNEL method). The percentage of PCNA-positive spermatogonia was higher in the RG than in the NRG. On the contrary, germ cell apoptosis was higher in the NRG than in the RG. Our results confirm that cat spermatogenesis is modulated on a seasonal basis and suggests that spermatogenesis control involves changes in germ cell proliferation and apoptosis according to a common paradigm of seasonally breeding species.

Respiratory and hemodynamic effects of 2 protocols of low-dose infusion of dexmedetomidine in dogs under isoflurane anesthesia.

The aim of this study was to evaluate the respiratory and hemodynamic effects of a low-dose dexmedetomidine infusion [1 µg/kg body weight (BW) per hour], with or without a loading dose (1 µg/kg BW), in dogs under isoflurane anesthesia. Thirty dogs were premedicated with methadone [0.3 mg/kg BW intramuscular (IM)], induced with propofol intravenous (IV) and maintained with isoflurane (1.3% to 1.4%) under mechanical ventilation. Animals were randomly assigned to 3 intravenous (IV) treatments (n = 10): 1 µg/kg BW dexmedetomidine, followed by 1 µg/kg BW per hour (group BI); or saline solution bolus, followed by either an infusion of 1 µg/kg BW per hour dexmedetomidine (group I) or saline solution (group C). The infusions were interrupted after 30 minutes. Respiratory system static compliance (Cstat) and respiratory system resistance (Rrs), partial pressure of oxygen/fractional inspired oxygen ratio (PaO2/FIO2), intrapulmonary shunt (Fshunt), and cardiac output (CO) were determined 5 minutes before the bolus (BASELINE), at the end of the bolus (BOLUS), and at 15 (T15), 30 (T30), and 45 minutes (T45) intervals. In group BI, Cstat and PaO2/FiO2 were higher at T15 and T30 than at BASELINE in the same group and than group C at the same times. In group I, the same parameters at T30 were higher than at BASELINE and than group C at the same time. In group BI, Rrs and Fshunt were lower than at BASELINE and than group C at the same time. In group I, the same parameters at T30 were lower than at BASELINE and those of group C at the same time. Cardiac output (CO) at T30 was higher in groups BI and I than in group C. The results of this study showed that low-dose dexmedetomidine infusion improves oxygenation and respiratory system mechanics and has a stabilizing hemodynamic effect in dogs anesthetized with isoflurane and mechanically ventilated.

L'objectif de la présente étude était d'évaluer les effets respiratoires et hémodynamiques de l'infusion d'une faible dose de dexmedetomidine [1 µg/kg de poids corporel (BW) par h], avec ou sans une dose de charge (1 µg/kg de BW), chez des chiens sous anesthésie à l'isoflurane. Trente chiens ont été pré-médicamentés avec de la méthadone [0,3 mg/kg BW intra-musculaire (IM)], induit avec du propofol intra-veineux (IV) et maintenu avec de l'isoflurane (1,3 % à 1,4 %) sous ventilation mécanique. Les animaux étaient attribués au hasard à trois traitements IV (n = 10) : 1 µg/kg BW dexmedetomidine, suivi de 1 µg/kg BW par heure (groupe BI); ou un bolus de solution saline, suivi par soit une infusion de 1 µg/kg BW par heure de dexmedetomidine (groupe I) ou une solution saline (groupe C). Les infusions étaient arrêtées après 30 min. La compliance statique du système respiratoire (Cstat) et la résistance du système respiratoire (Rrs), le ratio pression partielle d'oxygène/fraction d'oxygène dans l'inspiré (PaO2/FIO2), le shunt intrapulmonaire (Fshunt) et le débit cardiaque (CO) furent déterminés 5 min avant le bolus (BASELINE), à la fin du bolus (BOLUS), et à des intervalles de 15 (T15), 30 (T30) et 45 min (T45). Dans le groupe BI, Cstat et PaO2/FIO2 étaient plus élevés à T15 et T30 qu'à BASELINE dans le même groupe et similaire au groupe C aux mêmes temps. Dans le groupe I, les mêmes paramètres à T30 étaient plus élevés qu'à BASELINE et plus élevés que ceux du groupe C aux mêmes temps. Dans le groupe BI, Rrs et Fshunt étaient plus bas qu'à BASELINE et similaire au groupe C aux mêmes temps. Dans le groupe I, les mêmes paramètres à T30 étaient plus bas qu'à BASELINE et ceux du groupe C aux mêmes temps. Le CO à T30 était plus élevé dans les groupes BI et I que dans le groupe C. Les résultats de cette étude ont démontré qu'une infusion d'une faible dose de dexmedetomidine améliore l'oxygénation et la mécanique du système respiratoire et a un effet hémodynamique stabilisant chez les chiens anesthésiés avec de l'isoflurane et ventilé mécaniquement.(Traduit par Docteur Serge Messier).

Vitellogenin receptor and fatty acid profiles of individual lipid classes of oocytes from wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle.

The greater amberjack Seriola dumerili (Risso, 1810) is a large migratory pelagic fish occurring in tropical and temperate waters with a great potential for the world aquaculture industry. Previous studies showed that wild-caught female greater amberjack reared in sea cages and handled during the reproductive season, underwent extensive ovarian atresia. This atresia, however, was not related to an insufficient liver transcription or oocyte uptake of vitellogenin (Vtg). In the present study, the structure of two greater amberjack vitellogenin receptors, namely Vtgr (Lr8-) and Lrp13, was characterized. Moreover, vtgr and lrp13 gene expression and the fatty acid profiles of specific phospholipids and neutral lipids were compared in the ovaries of wild and captive-reared greater amberjack during different phases of the reproductive cycle (i.e. early gametogenesis, advanced gametogenesis and spawning). Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered into secondary growth (i.e. vitellogenesis). Rearing of wild-caught greater amberjack in captivity together with handling during the reproductive season was associated with a reduced vtgr and lrp13 transcription and with a diminished capacity of oocytes in the early phase of gametogenesis (primary oocyte growth) to enter into vitellogenesis. During early gametogenesis, remarkable differences in the fatty acid composition were observed between wild and captive-reared individuals: all phospholipids of captive fish displayed dramatic increases of saturates (16:0 and 18:0) and decreases of arachidonic acid (ARA) and docosahexaenoic acid (DHA). The present study confirms the susceptibility of greater amberjack reproductive function to handling stress and suggests that the consequent extensive atresia of vitellogenic follicles originated during the primary oocytes growth when the capacity of oocytes to synthesize vitellogenin receptors was reduced. The study also suggests that this reduced capacity was associated with an altered oocyte phospholipid fatty acid composition during early gametogenesis.

Cytotoxic and trypanocidal activities of cinchona alkaloid derivatives.

A series of 27 cinchona alkaloid derivatives (1f-w, 2a-e and 3a-d) were investigated for their cytotoxic and trypanocidal activities using seven different cancer cell lines (KB, HeLa, MCF-7, A-549, Hep-G2, U-87 and HL-60), two normal cell lines (HDF and CHO) and bloodstream forms of Trypanosoma brucei brucei, respectively. Four compounds (1u, 1w, 2e and 3d) were identified with promising cytotoxic activity with 50% growth inhibition (GI50 ) values below 10 µM. Two (2e and 3d) of the four compounds also exhibited potent anti-trypanosomal activity with GI50 values of 0.3-0.4 µM. All four active compounds represented derivatives modified at their C-9 hydroxy group. With respect to anti-proliferative activity and selectivity, 2e (epi-N-quinidyl-N'-bis(3,5-trifluoromethyl)phenylthiourea) proved to be the most promising derivative for both cancer cells and bloodstream forms of T. b. brucei. The cytotoxic activity of compounds 1u, 1w, 2e and 3d was attributed to their ability to induce apoptosis in cancer cells. The results demonstrate the potential of cinchona alkaloid derivatives as novel anti-cancer and anti-trypanosome drug candidates.

Ochratoxin A at low concentrations inhibits in vitro growth of canine umbilical cord matrix mesenchymal stem cells through oxidative chromatin and DNA damage.

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.

Characterization and in vitro differentiation potency of early-passage canine amnion- and umbilical cord-derived mesenchymal stem cells as related to gestational age.

Fetal adnexa are a non-controversial source of mesenchymal stem cells (MSCs) that have high plasticity, a high proliferation rate, and the ability to differentiate towards multiple lineages. MSC populations have been characterized for their stemness and differentiation capabilities; more recent work has focused on MSC selection and on establishing predictable elements to discriminate the cells with the most potential for regenerative medicine. In this study, we cytogenetically and molecularly characterized and followed the in vitro proliferation and differentiation potential of early-passage 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. Population doublings, passaged up to 10 times, increased significantly with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age at early passages. Passage-3 AM- and UCM-MSCs from both gestational phases also expressed embryonic (POU5F1) and mesenchymal (CD29, CD44) stemness markers, whereas hematopoietic and histocompatibility markers were never found in any sample. Passage-3 cell populations of each cell type were also multipotential as they could differentiate into neurocytes and osteocytes, based on cell morphology, specific stains, and molecular analysis. These results indicated that MSCs retrieved from the UCM and AM in the early and late fetal phases of gestation could be used for canine regenerative medicine.

Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH).

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.

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells.

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 immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, 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.

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells.

BACKGROUND: The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. METHODOLOGY/PRINCIPAL FINDINGS: A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. CONCLUSIONS/SIGNIFICANCE: In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage.

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 (O, n=2), early luteal (EL, n=7) and mid/late luteal phase (MLL, 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: (I) small aggregates diffused throughout the cytoplasm; (II) 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.