Antonio Ciro Guaricci

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.

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs.

Expression and localization of the µ‐opioid receptor (MOR) in the equine cumulus–oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence

The µ‐opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT‐PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 × 10−8 M β‐endorphin in medium without additional hormones. Inhibitory effects of 10−3 M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10−8 M Naloxone. The exposure to 10−3 M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. β‐Endorphin 3 × 10−8 M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10−8 M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus–oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes. Mol. Reprod. Dev. 75: 1229–1246, 2008.

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.

Expression of the μ-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

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.

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

BackgroundOpioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.MethodsWe analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.ResultsWe showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.ConclusionsThe results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.

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.

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)

BackgroundInfertility 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.ResultsWe 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.ConclusionWe 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.

Changes in the expression of the μ-opioid receptor in the mare oviduct during oestrus and anoestrus

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mares physiological condition.