Michele Caira

Isolation, Proliferation, Cytogenetic, and Molecular Characterization and In Vitro Differentiation Potency of Canine Stem Cells From Foetal Adnexa: A Comparative Study of Amniotic Fluid, Amnion, and Umbilical Cord Matrix

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. Mol. Reprod. Dev. 78:361–373, 2011.

Oocyte mitochondrial bioenergy potential and oxidative stress: within-/between-subject, in vivo versus in vitro maturation, and age-related variations in a sheep model

OBJECTIVE To analyze within-/between-subject, in vivo versus in vitro maturation (IVM), and age-related variations of mitochondrial (mt) bioenergy potential and oxidative status of metaphase II (MII) oocytes recovered from hormonally stimulated sheep. DESIGN Prospective study. SETTING Academic basic research laboratory. SUBJECT(S) Ten adult ewes. INTERVENTION(S) Estrus synchronization, controlled ovarian hyperstimulation (COH), ovariohysterectomy; follicular and oviductal oocyte retrieval; IVM of follicular oocytes. MAIN OUTCOME MEASURE(S) Mean ± SD, within-subject (CV(w)) and between-subject (CV(b)) variation coefficients of mt activity, intracellular reactive oxygen species (ROS) levels, and mt/ROS colocalization in sheep oocytes from young and aged donors and matured in vivo (in vivo MIIs) or in vitro (IVM MIIs). RESULT(S) Within- and between-subject, in vivo versus IVM, and age-related variations of mt activity were observed in MII oocytes from hormonally stimulated donor sheep. ROS levels increased significantly in oocytes from aged donors. Mt-ROS colocalization was consistently higher in in vivo MIIs compared with IVM MIIs. Oviductal energy/antioxidant ability is influenced by COH. CONCLUSION(S) Oocyte energy/oxidative status is affected by within-/between-subject, in vivo versus IVM, and age-related variations. Mt/ROS colocalization is a reliable marker of in vivo MII oocytes.

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 ([Ca2+]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 [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca2+]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 [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]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 [Ca2+]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.

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. Mol. Reprod. Dev. 81: 539–551, 2014.

Expression of the μ opioid receptor and effects of the opioid antagonist naloxone on in vitro maturation of oocytes recovered from anoestrous bitches.

The mu-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus-oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 x 10(-6), 1 x 10(-8) and 1 x 10(-10) M. The rate of oocytes resuming meiosis after culture in presence of 1 x 10(-6) M Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as delta and kappa, and possible interactive effects of their antagonists on canine oocyte maturation.

Modifications of carbohydrate residues in the sheep oviductal ampulla after superovulation.

Epithelium of oviductal ampulla was studied in normal and in superovulated sheep using morphologic analysis and lectin glycohistochemistry. The lining epithelium consisted of two types of cells, ciliated and nonciliated cells. Unlike superovulated samples, the nonciliated cells from control ewes showed apical protrusions indicating an apocrine secretory activity. The ciliated cells showed lectin-binding sites mainly at the level of the cilia which bound all the used lectins except Peanut agglutinin, suggesting the lack of glycans terminating with Galβ1,3GalNAc. In superovulated specimens, the ciliated cells with high mannosylated glycans Concanavalin A (Con A) and GlcNAc and GalNac termini Griffonia simplicifolia agglutinin II (GSA II) and Dolicurus biflorus agglutinin (DBA) decreased. The luminal surface of nonciliated cells showed all investigated sugar residues in controls, whereas it was lacking in high mannosylated (Con A) and terminal GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 sequence (DBA) in superovulated ewes. Apical protrusions from control ampullae nonciliated cells showed glycans containing mannose, GlcNac, GalNAc, galactose, and α2,3-linked sialic acid (Con A, KOH-sialidase- Wheat germ agglutnin [WGA], GSA II, SBA, Griffonia simplicifolia agglutinin-isolectin B4 [GSA I-B4], Maackia amurensis agglutinin II [MAL II]). The supranuclear cytoplasm of nonciliated cells expressed terminal GlcNAc (GSA II) in all specimens, also O-linked glycans (mucin-type glycans) with GalNAc and sialic acid termini (Helix pomatia agglutinin [HPA] and MAL II) in control animals, and also N-linked glycans with fucose, galactose, lactosamine, and α2,3-linked sialic acid termini (Ulex europaeus agglutinin I [UEA I], GSA I-B4, Ricinus communis agglutinin120 [RCA120], and Sambucus nigra agglutinin [SNA] ) in superovulated ewes. These results report for the first time that the superovulation treatment affects the secretory activity and the glycan pattern of the epithelium lining the sheep oviductal ampulla.

Glycan profile of oviductal isthmus epithelium in normal and superovulated ewes

Glycans of oviductal isthmus are implicated in sperm-isthmus interaction, sperm storage, survival, and capacitation. Isthmus morphology and glycoprotein production are controlled by sex steroids, which could be responsible for alterations of some reproductive events in the superovulated ewes (SE). In this study, the oviductal isthmus epithelium was evaluated in normal and in SE using morphologic and lectin histochemical analysis. The epithelium of normal isthmi was significantly taller in folds than in crypts, whereas it significantly decreased in the folds of SE. Nonciliated cells (NCs) from normal, showed apical blebs revealing apocrine secretory activity, which was missing in SE. The quantitative analysis of lectin staining revealed higher Con A, DBA, and PNA reactivity but lower affinity to KOH-sialidase- (Ks)WGA, GSA II, LTA, UEA I, SBA, GSA I-B4, RCA120, KsPNA, MAL II, SNA in control isthmi compared with superovulated ones. The NCs apical blebs showed terminal fucose (Fuc), N-acetylgalactosamine (GalNAc), galactose (Gal), lactosamine, and O- and N-sialoglycans. In normal isthmi, the luminal surface of NCs and ciliated cells expressed Fuc, highly mannosilated N-glycans terminating with lactosamine as well as O-glycans ending with N-acetylglucosamine (GlcNAc) and GalNAc. Moreover, NCs microvilli contained Gal and α2-3-linked sialic acids. In SE, the luminal surface lacked Gal and GalNAcα1, 3(LFucα1,2)Galβ1,3/4GlcNAcβ1, whereas it was enriched with Fuc in the folds and with α2-3sialo-mucins both in crypts and in folds. The apical surface showed additional O- and N-linked sialoglycans in NCs and αGal in the cilia, which expressed α2-6-linked sialic acid only in the folds. The cytoplasm of control NCs showed highly mannosilated N-glycans throughout the epithelium and GlcNAc in the folds. After superovulation treatment, NCs expressed cytoplasmic terminal Fuc, βGalNAc, lactosamine, α2-3-, and α2-6-linked sialic acids in the folds. The cytoplasm of normal ciliated cells cells displayed a binding pattern similar to normal NCs except for the absence of higly mannosilated N-glycans in the folds, which appeared in superovulated samples. This study demonstrates glycan zone-specific distribution along the isthmus epithelium that is influenced by the superovulation treatment. Whether an alteration in the glycan distribution is implicated in the low-rate fertilization after natural mating of the superovulated sheep remains to be addressed.