Presence and expression of apelin and apelin receptor in bitch placenta.

Apelin is a potent inotropic agent causing endothelium-mediated vasodilation and is involved in vessel formation by interacting with a specific receptor. Its cardiovascular profile suggests a role in the regulation of gestational hemodynamic changes. The expression of apelin and its receptor has been reported in some portions of the reproductive tract of different mammalian species. As far as we know, there are no reports describing the expression of apelin and apelin receptor in bitch's placenta. Therefore, the aim of this study was to investigate, for the first time, the presence and distribution of apelin and apelin receptor in bitch placenta by molecular biology and immunohistochemical techniques. Sixteen adult female half-breed bitches were used. The animals were divided into two groups based on the stage of pregnancy: group 1 (mid-gestation n = 8) and group 2 (end gestation n = 8). These bitches were subjected to ovariohysterectomy (group1) or non-conservative caesarean section (group 2). The immunohistochemical technique revealed the presence of positive immune reaction for apelin and apelin receptor in all the samples examined at 30 days and at the end of pregnancy. In particular, apelin and apelin receptor staining was evident in the cytoplasms of cytotrophoblasts and in epithelial cells of the maternal portion. Even if not included into the structure of the placenta, the uterine glands also exhibited a positive immune reaction for apelin and apelin receptor. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all the placenta samples examined. On the basis of our results it was also possible to hypothesize a potential role of apelin in the control of local placenta blood flow during pregnancy development in bitches.

The presence and distribution of cannabinoid type 1 and 2 receptors in the mandibular gland: The influence of different physical forms of diets on their expression in piglets.

We explored the expression and cell type distribution of cannabinoid receptors type 1 (CB1) and cannabinoid receptors type 2 (CB2) in the mandibular glands of pigs in relation to different physical forms of the diet. Thirty-two crossbred growing pigs (ages 5-6 weeks) were randomly allotted to four experimental groups (eight pigs/group) and fed four different physical types of the same diet for 4 weeks: finely ground pellet (FP), coarsely ground meal (CM), coarsely ground pellet (CP) and coarsely ground extruded (CE) with dMEAN of 0.46, 0.88, 0.84 and 0.66 mm respectively. At the end of the feeding trial, the pigs were euthanized and the mandibular gland was collected after dissection. By immunohistochemistry, positive signals for CB1 were found in the cytoplasm of duct epithelial cells of pigs fed CP, FP and CE diets and in the serous cells of mixed acini in pigs fed the coarser CM diet. Positive signals for CB2 were detected in duct epithelial cells and in neurons of ganglia close to major secretory ducts of all pigs. The differential expression and localization of these receptors in response to variable chewing activity due to the type of diet suggest that endocannabinoids may influence the functional activity of the mandibular gland by modifying qualitative and/or quantitative aspects of salivary secretion.

Nuclear Glycogen Inclusions in Canine Parietal Cells.

Nuclear glycogen inclusions occur infrequently in pathologic conditions but also in normal human and animal tissues. Their function or significance is unclear. To the best of the authors' knowledge, no reports of nuclear glycogen inclusions in canine parietal cells exist. After initial observations of nuclear inclusions/pseudoinclusions during routine histopathology, the authors retrospectively examined samples of gastric mucosa from dogs presenting with gastrointestinal signs for the presence of intranuclear inclusions/pseudoinclusions and determined their composition using histologic and electron-microscopic methods. In 24 of 108 cases (22%), the authors observed various numbers of intranuclear inclusions/pseudoinclusions within scattered parietal cells. Nuclei were characterized by marked karyomegaly and chromatin margination around a central optically empty or slightly eosinophilic area. The intranuclear inclusions/pseudoinclusions stained positive with periodic acid-Schiff (PAS) and were diastase sensitive, consistent with glycogen. Several PAS-positive/diastase-sensitive sections were further examined by transmission electron microscopy, also using periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining to identify polysaccharides. Ultrastructurally, the nuclear inclusions were composed of electron-dense particles that were not membrane bound, without evidence of nuclear membrane invaginations or cytoplasmic organelles in the nuclei, and positive staining with PA-TCH-SP, confirming a glycogen composition. No cytoplasmic glycogen deposits were observed, suggesting that the intranuclear glycogen inclusions were probably synthesized in loco. Nuclear glycogen inclusions were not associated with gastritis or colonization by Helicobacter-like organisms ( P > .05). Our findings suggest that nuclear glycogen inclusions in canine parietal cells could be an incidental finding. Nevertheless, since nuclear glycogen is present in several pathologic conditions, further investigations could be warranted to determine their true significance.

Gut complex carbohydrates and intestinal microflora in broiler chickens fed with oregano (Origanum vulgare L.) aqueous extract and vitamin E.

One hundred and seventy one-day-old female broiler chicks were randomly divided into three groups fed with different dietary treatments: basal control diet (C); C supplemented (2 g/kg) with an oregano aqueous extract (O); C supplemented (150 mg/kg) with vitamin E (E). Growth performance was evaluated at 21 (T1) and 42 days (T2). On the same days, morphological, histochemical and microbiological analyses were performed. The O group showed the highest (p < 0.01) body weight at T1, while no differences were observed at T2. Light microscopic observation and conventional histochemistry showed no differences with regard to the two sampling times, whereas significant differences emerged among the treatments. The O treatment generally enhanced goblet cell reactivity more than both the C and E treatments. Coliform count was lower in the ileum tract of the O group at both T1 and T2 (p < 0.05) and increased with age in all groups. Escherichia coli showed the lowest values in the caecum of the O group (p < 0.001) at both sampling times. Enterococci, lactobacilli and staphylococci populations showed no differences among the different experimental groups in the caecum. In the ileum, the O group did not exhibit the sharp decline (p < 0.001) in the lactic acid bacteria population observed in the other two experimental groups. In conclusion, oregano aqueous extract supplementation seemed to elicit the best response among treatments, enabling better growth performance, enhancing both the quantity and quality of glycoconjugates involved in indirect defence actions and significantly reducing both the coliform and E. coli counts.

Different physical forms of one diet fed to growing pigs induce morphological changes in mandubular glands and local leptin (Ob) production and receptor (ObR) expression.

The physical form of the diet plays an important role for morphological adaptations of organs in the gastrointestinal tract. It was hypothesized that different physical forms of one diet could exert extra-enteric effects, under local and systemic neuroendocrine regulation. Gross morphology, fresh mass and cytoarchitecture of mandibular glands (MG) were studied in growing pigs fed with one diet processed under four different physical forms. Four dietary treatments were offered for 4 weeks to 32 growing pigs (initial BW: 8.30 ± 0.83 kg) allotted into 4 experimental groups: FP, finely ground pellet (dMean, 0.46 mm); CM, coarsely ground meal (dMean, 0.88 mm); CP, coarsely ground pellet (dMean, 0.84); CE, coarsely ground extruded (dMean, 0.66). Conventional and immuonohistochemical techniques were used to immunolocalize, in particular, leptin (Ob) and its receptor (ObR). A significant effect was observed on the relative mass of the MG, depending on the diet (p < 0.03) and on the BW (p < 0.04), with no interactions (diet*BW). The immunohistochemical reactions for Ob and ObR showed a marked positivity in the MG from the group fed with the CM diet, displaying Ob-positive acinar cells and ObR-positive cells in the striated ducts, together with endocrine-like cells. The intensity of chromogenic reactions positively testing to ObR was used to evaluate the cytoarchitecture of the MG and its possible correlations. Pearson's correlation coefficient resulted to positively link (p < 0.0001) the ObR expression with the absolute mass of MG in the 61.1% of pigs. The physical form of the diet is related to extra-enteral effects, inducing changes in gross and microscopic morphology of the MG in the growing pig. The local production of Ob and the expression of the respective ObR in the striated duct cells shed a new light on the mitogenic activity of Ob in extra-enteral organs, like the MG, in relation to the physical form of the diet.

Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Ovarian hormones and fasting differentially regulate pituitary receptors for estrogen and gonadotropin-releasing hormone in rabbit female.

To investigate the mechanisms by which caloric restriction affects reproductive function in female rabbits, we measured, in animals intact or ovariectomized (OVX) estrogen-primed and fed ad libitum or fasted for 48 h, the adenohypophysial expression of estrogen receptor-alpha (ESR1) and gonadotropin releasing hormone receptor (GnRHR) and the dynamic secretion of LH following GnRH stimulation. Fasting increased the number of GnRHR-immunoreactive (-IR) cells in intact animals, whereas reduced the density of ESR1-IR cells in OVX rabbits. Estrogen priming decreased the number of ESR1-IR cells in fasted and OVX animals. Ovariectomy increased the number of ESR1-IR cells in fed rabbits, but caused an opposite effect in both fed and fasted animals treated with estrogen. Fasting down regulated the mRNA levels for ESR1 and GnRHR. Estrogen-priming reduced the abundance for ESR1 mRNA in both fed and fasted rabbits, and that for GnRHR in fasted rabbits. Ovariectomy halved ESR1 mRNA levels independently of treatment and feeding condition, whereas increased (P < 001) that for GnRHR in estrogen-primed rabbits. In all rabbits, an LH surge occurred 30 min after GnRH injection but the lowest levels were found in intact fasted rabbits and the highest in fasted, estrogen-primed animals. The LH profile was similar in intact and OVX rabbits and neither fasting nor estrogen priming modified it. In conclusion, fasting differentially modifies the ESR1 and GnRHR expression in the pituitary, depending on the presence of gonadal hormones, indicating complex interactions between metabolic signals and ovarian steroids.

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 mm(2)), 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.

Orexin system expression in the gastrointestinal tract of pigs.

The aim of the present study was to characterize the expression of both proteins and gene transcripts for orexins (OXA and OXB) and their cognate receptors (OX1R and OX2R) in the different gastrointestinal sections of pigs. Using immunohistochemistry, OXA and OXB were found to be co-expressed in the same endocrine cells localized in the basal third of the glands of the body portion of the stomach. Using double immunostaining technique, these orexin-immunoreactive (IR) cells co-stored ghrelin and gastrin. Apparently, OX1R was also expressed within the same cells, forming the tubular gastric gland which displayed positive immunostaining for orexins and the other peptides. Neurons of the enteric nervous system of the stomach were not immunolabeled. We did not find any definite OXA- or OXB-IR cells as well as any immunosignal for orexin receptors in sections of the duodenum, ileum, cecum and rectum. PPOX, OX1R, OX2R mRNA were similarly expressed in all the gastrointestinal tracts. Gastrin and ghrelin showed the highest levels of expression in the gastric mucosa, but their abundance decreased along the subsequent tracts. Thus, in pigs, orexins do not play any role in the local control of intestinal motility and secretion but may rather be involved as endocrine modulators for the regulation of feeding and metabolic homeostasis. However, the co-localization of ghrelin and gastrin with both orexins in the same endocrine cells of the gastric glands suggests that these gut peptides may collaborate in the regulation of gastric secretion, energy homeostasis, body weight and food intake.

Immunohistochemical distribution of leptin receptor in the major salivary glands of horses.

The presence of the leptin receptor (ObR) has already been highlighted in the human major salivary glands and it has been hypothesized that leptin may act by regulating the gland's growth. No data are reported on domestic animals so, considering the important role that these glands play, not only related to food ingestion and digestion, and the important functional role hypothesized to explain the presence of ObR in humans salivary glands, the aim of the present work was to investigate the presence and the distribution of the leptin receptor in horse parotid and mandibular glands, by immunohistochemical techniques. The presence of ObR was evidenced in parotid and mandibular glands, exclusively localized in duct epithelial cells; their positivity was localized in the cytoplasm and was most evident near its apical portion. Immuno-positivity not only affects the intralobular ducts (intercalated and striated) but also the interlobular ones. Our results indicate that horse major salivary glands, like those of humans, are likely targets of leptin actions, suggesting a functional role of leptin on these glands.

Immunohistochemical detection of the orexin system in the placenta of cats.

The aim of the present study was to investigate the presence and distribution of cells containing orexin A (OXA), and orexin type 1 and 2 receptors (OX1R and OX2R, respectively) in the feline placenta by means of immunohistochemical technique. OXA was identified in several decidual and syncytiotrophoblastic cells present in the lamellar portion of the placenta. In the same placental structures, few decidual and syncytiotrophoblastic cells showed the presence of OX1R-like immunoreactivity. Characteristically, immunopositivity for OX2R, but not for OX1R, was evidenced in the cells of the glandular layer. The orexinic system was not expressed in the uterine structures that were not engaged by the chorion. Our results provide the first evidence of the presence of a placental orexinic system in a mammalian species. Orexin A and both OX1R and OX2R are unequally distributed within the cat placenta. Local OXA production and the presence of specific receptors, differentially expressed in the placental structures of the cat, suggest that the orexinic system may participate in placental growth and development as well as in the regulation of its steroidogenic capacity via endocrine, paracrine and/or autocrine mechanisms.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.

Immunohistochemical localization of CB1 receptor in canine salivary glands.

CB1 is a member of the G-protein-linked receptor superfamily that is present in the central nervous system as well as in certain peripheral neuronal and non-neuronal tissues. Recently, the presence of CB1 was found in the ductal system of the major salivary glands of laboratory animals, but no data are available for domestic mammals. Thus, in the present study, we examined the presence and distribution of CB1 in the major salivary glands of dogs using immunohistochemical techniques. CB1 was found in the parotid and mandibular glands of adult dogs; positive immunoreaction was localized to the cells of the striated ducts, with a peculiar localization on or near the apical membrane. This particular localization may be explained by the characteristics of this receptor as membrane-associated. The acinar structures were completely negative for CB1. We conclude that CB1 is involved in the control of dog salivary secretion via endogenous substances, likely endocannabinoids. The localization of CB1 highlights that endocannabinoids promote qualitative and/or quantitative changes of the primary saliva in the ductal system.

Expression of luteal estrogen receptor, interleukin-1, and apoptosis-associated genes after PGF2alpha administration in rabbits at different stages of pseudopregnancy.

The dynamic expression for estrogen receptor subtype-1 (ESR1), interleukin-1beta (IL1B), and apoptosis-associated genes, as well as nitric oxide synthase activity, were examined in corpora lutea (CL) of rabbits after prostaglandin F(2alpha) (PGF(2alpha)) administration on either day 4 or day 9 of pseudopregnancy. By reverse transcriptase polymerase chain reaction, the steady-state level of ESR1 transcript was lower (P < 0.01) and that of anti-apoptotic B-cell CLL/lymphoma 2 (BCL2) -like 1 (BCL2L1) was greater in day 4 (P < 0.01) than in day 9 CL. Western blot analysis revealed that BCL2-associated X protein (BAX) abundance was greater in day 4 (P < 0.01) than in day 9 CL, whereas BCL2L1 protein was undetectable at both luteal stages. After PGF(2alpha), ESR1 transcript decreased (P < 0.01) in day 9 CL, whereas IL1B mRNA showed a transitory increase (P < 0.01) at both stages. The pro-apoptotic tumor protein p53 (TP53) gene had diminished (P < 0.01) on day 4 and on day 9 after a transitory increase (P < 0.01), whereas the BAX/BCL2L1 expression ratio increased (P < 0.01) in day 9 CL 24 h after treatment. Following PGF(2alpha), TP53 protein increased (P < 0.01) at both luteal stages, and BAX decreased (P < 0.01) in day 4 CL but increased (P < 0.01) 24 h later in day 9 CL; BCL2L1 became detectable 6 h later in day 4 CL. Nitric oxide synthase activity temporarily increased (P < 0.01) following PGF(2alpha). These findings suggest that PGF(2alpha) regulates luteolysis by ESR1 mRNA down-regulation and modulation of pro- and anti-apoptotic pathways in CL that have acquired a luteolytic capacity.

Immunohistochemical evidence of Orexin-A in the pancreatic beta cells of domestic animals.

A large body of information proves that Orexin-A is present in the pancreatic endocrine cells of humans and laboratory animals; more detailed studies identify Orexin-A-immunopositive cells as beta cells. Because no data have been reported on the pancreas of domestic animals, we investigated the presence and the distribution of cells containing Orexin-A in the pancreas of cattle, sheep and pigs by means of immunohistochemical techniques. Using a polyclonal antibody against Orexin-A, the immunopositive reaction was identified in the cytoplasm of many insular cells in the three species studied. Double immunohistochemical staining, using a polyclonal anti-insulin antibody, showed that Orexin-A is co-expressed with insulin. Our results, besides showing the presence of Orexin-A in the endocrine pancreas of domestic animals, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the functionality of the endocrine pancreas in domestic animals.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres. The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species.

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle. As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90+ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs.

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles.

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.

Intraluteal regulation of prostaglandin F2 alpha-induced prostaglandin biosynthesis in pseudopregnant rabbits.

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.