Localization of putative pituitary stem/progenitor cells in female dairy cattle

Research on sex-determining region Y-box 2 (SOX2)-positive pituitary stem/progenitor cells, as a source of hormone-producing cells, is progressing rapidly in rodents. However, the stem/progenitor cells supplying hormone-producing cells that are essential for growth, reproduction, and lactation in bovines have not yet been identified. In this study, we characterized SOX2-positive cells in the pituitary gland of dairy cattle (Holstein heifers) after sexual maturity. Immunofluorescence analysis revealed that the localization pattern of SOX2-positive cells in the dairy cattle pituitary gland was similar to that observed in the rodent pituitary gland; the marginal cell layer (MCL), dense cell clusters, and single cells scattered in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells were positive for the pituitary stem/progenitor cell niche markers E-cadherin and cytokeratin 8+18, which have been reported in rodents. In addition, in the MCL of the anterior lobe, there was a subpopulation of SOX2-positive cells positive for paired-related homeobox 1 and 2, whereas negative for S100β. Moreover, in the parenchyma of the anterior lobe, co-localization of SOX2 and pituitary hormones was infrequent. In summary, this study reveals the localization of putative pituitary stem/progenitor cells positive for SOX2 in dairy cattle. These results provide valuable information to support further investigation of cell supply in the dairy cattle pituitary gland.     

Characterization of Murine Pituitary-Derived Cell Lines Tpit/F1, Tpit/E and TtT/GF

The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.     

High level expression of Prop-1 gene in gonadotropic cell lines

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.     

Expression of the cannabinoid receptor type 1 in the pituitary of rabbits and its role in the control of LH secretion

The aim of this study was to elucidate the possible direct regulatory role of the endocannabinoids in the modulation of LH secretion in rabbits, a reflex ovulator species. The cannabinoid receptor type 1 (CB1) was characterized by RT-PCR techniques in the anterior pituitary of intact and ovariectomized does treated with GnRH and primed with estrogen and CB1 antagonist, rimonabant. Cannabinoid receptor type 1 immune reaction was evidenced by immunohistochemistry in the cytoplasm of approximately 10% of the pituitary cells with a density of 8.5 ± 1.9 (per 0.01 mm²), both periodic acid–Schiff positive (30%) and negative (70%). All CB1-immunoreactive cells were also immune reactive for estrogen receptor type 1. Ovariectomy, either alone or combined with estrogen priming, did not modify the relative abundances of pituitary CB1 mRNA, but decreased (P < 0.01) the expression of estrogen receptor type 1 mRNA. Treatment with CB1 antagonist (rimonabant) inhibited (P < 0.01) LH secretory capacity by the pituitary after GnRH injection, and estrogen priming had no effect. The present findings indicate that the endocannabinoid system is a potential candidate for the regulation of the hypothalamic-pituitary-ovarian axis in reflex ovulatory species.     

The effects of GnRH and adrenergic agents on PRL and beta-endorphin secretion by porcine pituitary cells in vitro

The direct effects of alpha- and beta-adrenergic agents on PRL and beta-endorphin (beta-END) secretion in vitro by porcine pituitary cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomised (OVX) one month before slaughter. Ovariectomised gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2) and (3) oestradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30-36 h (OVX+EB I) and 60-66 h (OVX+EB II) before slaughter, respectively; and (4) progesterone (P4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P4). Isolated anterior pituitary cells were submitted to 3.5 h incubation in the presence of GnRH, alpha- and beta-adrenergic agonists [phenylephrine (PHEN) and isoproterenol (ISOP), respectively], or alpha- and beta-adrenergic blockers [phentolamine (PHENT) and propranolol (PROP), respectively]. The culture media were assayed for PRL (exp. I) and beta-endorphin-like immunoreactivity (beta-END-LI) (experiment II). In experiment I, GnRH did not influence PRL release by pituitary cells in all experimental groups. Some of tested doses of adrenergic agonists, PHEN and ISOP, increased PRL release from pituitary cells of OVX gilts, but not from those of OVX+EB I animals. In the OVX+EB II group, PHEN alone, but ISOP with PROP, potentiated PRL secretion by the cells. In OVX+P4 animals, PHEN alone or in combination with PHENT and also ISOP alone or with PROP enhanced PRL output from the cells. In experiment II, addition of GnRH increased beta-END-LI release from pituitary cells only in the OVX+EB II group. PHEN and PHENT potentiated beta-END-LI secretion by pituitary cells in OVX+EB II and OVX+P4 groups, while ISOP and PROP increased beta-END-LI secretion by the cells of OVX and OVX+EB II animals. In turn, in the OVX+EB I group, effect of PHENT and PROP on PRL secretion by pituitary cells was inhibitory. In conclusion, our results suggest that adrenergic agents can modulate PRL and beta-END secretion by porcine pituitary cells in a manner dependent on the hormonal status of gilts.     

Effect of anaesthetic maintenance with isoflurane or propofol on ease of endoscopic duodenal intubation in dogs

ObjectiveTo compare the ease of endoscopic duodenal intubation (EDI) in dogs during maintenance of general anaesthesia with isoflurane or propofol infusion.Study designProspective, randomized, partially blinded clinical trial.AnimalsA total of 22 dogs undergoing upper gastrointestinal tract endoscopy to include EDI were recruited.MethodsDogs were randomly assigned isoflurane (ISO; n = 10) or propofol (PROP; n = 11) for maintenance of general anaesthesia. Following anaesthetic premedication with intramuscular medetomidine (0.005 mg kg^–1) and butorphanol (0.2 mg kg^–1), general anaesthesia was induced with propofol, to effect, maintained with 1.5% (vaporizer setting) isoflurane in 100% oxygen or 0.2 mg kg^–1 minute^–1 propofol. The dose of both agents was adjusted to maintain general anaesthesia adequate for the procedure. Degree of sedation 20 minutes post-anaesthetic premedication, propofol induction dose, anaesthetist and endoscopist training grade, animal’s response to endoscopy, presence of gastro-oesophageal and duodenal-gastric reflux, spontaneous opening of the lower oesophageal and pyloric sphincters, antral movement and time to achieve EDI were recorded. EDI was scored 1 (immediate entry with minimal manoeuvring) to 4 (no entry after 120 seconds) by the endoscopist, blinded to the agent in use. Data were tested for normality (Shapiro-Wilk test) and differences between groups analysed using independent t test, Mann-Whitney U test and Fisher’s exact test as appropriate.ResultsThere were no significant differences between groups for EDI score [median (interquartile range): 2 (3) ISO, 2 (3) PROP] or time to achieve EDI [mean ± standard deviation: 52.50 ± 107.00 seconds (ISO), 70.00 ± 196.00 seconds (PROP)]. Significantly more dogs responded to passage of the endoscope into the oesophagus in group PROP compared with group ISO (p = 0.01).Conclusions and clinical relevanceMaintenance of general anaesthesia with either isoflurane or propofol did not affect EDI score or time to achieve EDI.     

The Effect of 2-Hydroxypropyl-β-Cyclodextrin on Post-Thaw Parameters of Cryopreserved Jack and Stallion Semen

Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.     

Identification and characterisation of side population cells in the canine pituitary gland

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.     

Scanning 17 candidate genes for association with primary cataracts in the wire-haired Dachshund

Primary cataracts are breed-related eye diseases and are common in many dog breeds. In this study, 17 genes (BFSP2, EYA1, FOXE3, FTL, GCNT2, GJA3, GJA8, HSF4, MAF, MIP, PAX6, PITX3, SIX5, SORD, SOX1, SPARC, TRNT1) were evaluated as candidates for primary non-congenital cataracts (CAT) in the Dachshund using microsatellites adjacent to the candidate genes. Linkage and association with CAT was tested in 15 affected and six unaffected wire-haired Dachshunds. Non-parametric linkage analysis and association tests did not reveal significant linkage or association for the candidate gene flanking microsatellites tested. Thus, it is unlikely that the 17 investigated candidate genes harbour a causative mutation for CAT in these Dachshunds.     

The Endometrium of Cycling Cows Contains Populations of Putative Mesenchymal Progenitor Cells

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1–5) and late luteal phases (days 13–18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD‐117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin‐embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c‐KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.     

Expression of inflammatory-related factors in porcine anterior pituitary-derived cell line

Recent studies have shown that undifferentiated stem cells act as immunomodulators. To investigate the immunomodulatory function of the progenitor cells of the anterior pituitary gland, we attempted to establish a stem/progenitor cell line from the porcine anterior pituitary gland, and to detail its inflammatory cytokine expression. A cloned cell line from the porcine anterior pituitary gland was established and was designated as the porcine anterior pituitary-derived cell line (PAPC). PAPC expressed the mRNA of Nanog and Oct-4, and showed positive immunoreactivity for beta-catenin and Hes1 in its nucleus. PAPC grew stably by repeated passage and rapidly in the EGF and bFGF containing medium. RT-PCR showed that PAPC expressed mRNA of IL-1alpha, IL-6, IL-12, IL-15, IL-18 and TLR4. PAPC expressed S100alpha and IL-18 protein, which was localized in the marginal epithelial cells of Rathke's pouch. These results suggest that PAPC is a stem/progenitor cell and may regulate anterior pituitary cell function through an immuno-endocrine pathway.     

Culture of canine synoviocytes on porcine intestinal submucosa scaffolds as a strategy for meniscal tissue engineering for treatment of meniscal injury in dogs

Meniscal injury is a common cause of canine lameness. Tissue engineered bioscaffolds may be a treatment option for dogs suffering from meniscal damage. The aim of this study was to compare in vitro meniscal-like matrix formation and biomechanical properties of porcine intestinal submucosa sheets (SIS), used in canine meniscal regenerative medicine, to synoviocyte-seeded SIS bioscaffold (SSB), cultured with fetal bovine serum (SSBfbs) or chondrogenic growth factors (SSBgf). Synoviocytes from nine dogs were seeded on SIS and cultured for 30 days with 17.7% fetal bovine serum or recombinant chondrogenic growth factors (IGF-1, TGFβ1 and bFGF). The effect on fibrochondrogenesis was determined by comparing mRNA expression of collagen types Iα and IIα, aggrecan, and Sry-type homeobox protein-9 (SOX9) as well as protein expression of collagens I and II, glycosaminoglycan (GAG), and hydroxyproline.The effect of synoviocyte seeding and culture conditions on biochemical properties was determined by measuring peak load, tensile stiffness, resilience, and toughness of bioscaffolds. Pre-culture SIS contained 13.6% collagen and 2.9% double-stranded DNA. Chondrogenic growth factor treatment significantly increased SOX9, collagens I and IIα, aggrecan gene expression (P < 0.05), and histological deposition of fibrocartilage extracellular matrix (GAG and collagen II). Culture with synoviocytes increased SIS tensile peak load at failure, resilience, and toughness of bioscaffolds (P < 0.05). In conclusion, culturing SIS with synoviocytes prior to implantation might provide biomechanical benefits, and chondrogenic growth factor treatment of cultured synoviocytes improves in vitro axial meniscal matrix formation.