Dual origin, development, and fate of bovine pancreatic islets.

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo-acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of 'perilobular giant islets' are distinguishable from larger numbers of 'intralobular small islets'. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin-synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette-like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the 'interlobular small islets' persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T-cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo-acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.

Expression of KIT in the ovary, and the role of somatic precursor cells.

KIT is a type III receptor protein tyrosine kinase, and KITL its cognate ligand. KIT can mediate its effects via several intracellular signalling pathways, or by formation of a cell-cell anchor with its ligand. Through these mechanisms, KIT controls fundamental cellular processes, including migration, proliferation, differentiation and survival. These cellular processes are modulated by soluble KIT, a cleavage product of KIT, generated at the cell membrane. A cell-retained KIT cleavage fragment also arises from this cleavage event. This cleavage fragment must be distinguished from truncated KIT (trKIT), which originates through cryptic promoter usage. The expression of trKIT is highly restricted to postmeiotic germ cells in the testis. In contrast, KIT, together with its cleavage products, is present in somatic cells and germ cells in the gonads of both sexes. A functional KITL/KIT system is mandatory for normal population of the gonads by germ cells. Signalling via the KITL/KIT system promotes the growth, maturation, and survival of germ cells within the gonads, and prevents meiotic entry and progression. In addition to its importance in germ cell biology, the KITL/KIT system is crucial for gonadal stromal differentiation. During foetal life, KIT is expressed by testicular stromal precursor cells, which develop into Leydig cells. In the ovary, stromal cell KIT expression accompanies theca layer development around advanced follicles. After ovulation, KIT-immunopositive cells translocate from the theca layer to the luteal ganulosa where they contribute to a delicate cellular network that extends between the fully luteinised large luteal cells. In the outer regions of the developing corpus luteum, a highly conspicuous subpopulation of KIT/CD14-double-immunopositive cells can be observed. KIT/CD14-double-immunopositive cells are also seen in the haematopoietic-like colonies of long-term granulosa cultures established from late antral follicles. These cultures demonstrate expression of pluripotency marker genes such as octamer binding transcription factor-3/4 and sex determining region Y-box 2. The KIT/CD14-double-immunopositive cells can be purified and enriched by KIT-immunopositive magnetic cell sorting. Subsequent exposure of the KIT-expressing cells to the hanging drop culture method, combined with haematopoietic differentiation medium, provides the signals necessary for their differentiation into endothelial and steroidogenic cells. This suggests that monocyte-derived multipotent cells are involved in ovarian tissue remodelling. In summary, multicelluar KITL/KIT signalling organizes the stroma in the ovary and testis; monocyte-derived multipotent cells may be involved.

The CD34 surface antigen is restricted to glucagon-expressing cells in the early developing bovine pancreas.

Controversy remains regarding the origin of the pancreatic endocrine cells. It is generally accepted that the majority of insulin-secreting cells derive from the endodermal epithelium of the gastrointestinal tract. The aim of this study was to determine the contribution made by a particular cluster of differentiation (CD)-positive cells to the development of the bovine endocrine pancreas. In bovine embryos and foetuses with crown to rump lengths (CRL) ranging from 1 to 47 cm, cells staining positively for CD34 and/or CD133 were always more numerous in the left lobe and body of pancreas than in the right lobe. In the early stages of pancreatic development (CRL <5 cm), CD34 and/or CD133-reactive cells were concentrated within the epithelial cell cords that form the primitive pancreas. In later developmental stages (CRL >5 cm), individual or groups of CD34 and/or CD133-reactive cells were present in newly formed acini, which bulged out from the duct system that had arisen from the cords. Some of the positively stained cells accumulated in focal areas associated with hyperplastic intra-acinar cells. These "acino-insula-like complexes" appeared to enlarge with age and develop into intralobular Islets of Langerhans. Most of the described CD34 and/or CD133-reactive cells displayed co-localisation with glucagon. A negligible number of these cells showed co-localisation with insulin. Glucagon-stained cells were distinct from insulin-stained cells and were more abundant in embryonic and early foetal pancreata. Our data demonstrate that CD34 and/or CD133-reactive cells contribute to the pancreatic alpha cell population during early foetal development in cattle.

Progenitor cells harvested from bovine follicles become endothelial cells.

Hematopoietic-like colonies develop in post-confluent granulosa cell cultures derived from bovine antral follicles. Previously, we had shown that these colonies gave rise to macrophages. In the present study, we validated the presence of somatic KIT-positive (KIT(+)) progenitor cells in colony-containing granulosa cell cultures. The cultures expressed the progenitor cell markers Sox-2, Oct 3/4, KIT, and alkaline phosphatase in western blot analysis. The successful double immunofluorescence localization of KIT and CD14, CD45, CD133, or VEGF-R2 revealed a specific subpopulation of progenitor cells. Flow cytometry showed that cells doubly positive for KIT and CD14 or CD45 comprised less than 10% of the population. The KIT(+) cells were purified by magnetic selection and differentiated with the hanging drop technique using haematopoietic differentiation medium. Pure cultures of either granulosa cells or endothelial cells were obtained. The spindle-shaped and epithelioid phenotypes indicated endothelial cell heterogeneity of microvascular source. We conclude that progenitor cells are obtained from the follicle harvest, which differentiate into endothelial cells. The cells are relevant for findings to angiogenesis and luteinization of the corpus luteum.

Similar developmental patterns in immunolocalisation of stem cell factor and KIT in bovine meso- and metanephros.

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7-24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.

Characterization of bovine fetal Leydig cells by KIT expression.

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5-85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor alpha production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.

KIT variants in bovine ovarian cells and corpus luteum.

We report the presence of KIT variants in granulosa and thecal cells of the follicle and endothelial and steroidogenic cells of the corpus luteum. Transcripts of both full-length splice variants, KIT and KITA, were ubiquitously detected in all cell types, in contrast to transcripts for truncated KIT. RT-PCR with exon-intron-specific primers suggested that KIT transcripts retained intron sequences. We used domain-specific KIT antibodies to identify truncated KIT proteins in cell conditioned media and lysates. These proteins represented soluble KIT and a so far disregarded intracellular KIT fragment, and were ubiquitously present. In contrast, glycosylated variants of full-length KIT were predominantly detected in thecal and endothelial cells. All KIT variants were encountered again in COS-7 cells transfected with a vector containing KITA. Phorbol 12-myristate-13-acetate treatment induced levels of truncated KITs, and this effect was repressed by the metalloproteinase inhibitor TAPI-1. Our findings show that ectodomain cleavage of full-length KIT generates an intracellular KIT. Our experiments suggest that replenishing full-length KIT differs among various ovarian cell types.

KIT receptor-positive cells in the bovine corpus luteum are primarily theca-derived small luteal cells.

The tyrosine kinase KIT receptor, the protooncogene CD117, plays a key role in growth and maturation of oocytes and follicles. Relevant data are sparse for the corpus luteum (CL). We first confirmed the presence of KIT mRNA and KIT protein in bovine CL homogenates. We then localized KIT-positive (KIT+) cells in CL sections by immunohistochemistry. At the CL stage of early development, the former theca transforming into capsule/septa showed a strong band-like KIT+ immunoresponse. In addition, CD45+ leukocytes in septa included subpopulations of CD45+/KIT+ and CD14+/KIT+ leukocytes as validated by double immunofluorescence localization. At the early secretory stage, KIT+ cells appeared within the septa/capsule region and in the periphery of the CL parenchyma, there forming a complex network. This was separate from the capillary bed as determined by double staining for CD117 and FVIII-related endothelial cell antigen (FVIIIr). The KIT+ network coincided with cells positive for cytochrome P450 17alpha-hydroxylase, a thecal cell-specific enzyme. The late secretory stage was defined by an advanced manifestation of the KIT+ network in the CL periphery. At the stage of regression, the KIT+ network was absent. The CL of pregnancy expressed high levels of KIT mRNA and KIT protein uniformly throughout pregnancy. The KIT+ immunolocalization revealed small fibroblast-like cells, luteal cells with granules, and clusters of large luteal cells with staining of the cell membrane. We conclude that a majority of KIT+ cells in the bovine CL are primarily theca-derived small luteal cells, and that a minority represent KIT+ leukocytes, in some cases KIT+ monocytes.

Anti-bovine CD34 monoclonal antibody reveals polymorphisms within coding region of the CD34 gene.

OBJECTIVE: Monoclonal antibodies (mAbs) against CD34 are widely used for purification of CD34+ hematopoietic as well as nonhematopoietic stem/progenitor cells. We produced mAbs against bovine CD34 (boCD34) to facilitate the study of hematopoiesis in cattle. METHODS: MAbs were produced by immunizing BALB/c mice with BALB/3T3 cells transfected with boCD34 cDNA. Staining of bone marrow mononuclear cells (BMMNCs) from 10 newborn Holstein calves with the mAbs was examined by flow cytometry. The nucleotide sequence of the coding region for boCD34 in each calf was determined after amplification of the cDNA by reverse-transcription polymerase chain reaction (RT-PCR). BoCD34 fusion proteins, each representing one of the boCD34 alleles found to exist in the calves, were expressed in HeLa cells by DNA transfection, and the staining of these proteins with the mAbs was assessed. RESULTS: One mAb, N21, stained relatively high percentages of BMMNCs from 4 calves but failed to stain those from the other calves. RT-PCR analysis revealed single-nucleotide polymorphisms within the coding region, 3 of which led to amino-acid substitutions. A CD34 mutation experiment indicated that mAb N21 bound to a boCD34 allele with tryptophan at amino acid 167 but not to that with arginine. CONCLUSION: By using mAb N21 as an allelic cell marker, it would be feasible to detect and isolate boCD34+ cell species derived from N21+ donors in N21- recipients following allogeneic in utero transplantation; this would make cattle potentially useful as large animal models with a unique experimental advantage.

ephA9, a novel avian receptor tyrosine kinase gene.

Protein tyrosine kinase (TK) receptors are known to play crucial roles in various aspects of normal development and in tumorigenesis. Germ cells before their colonizing to the gonads during embryogenesis are called primordial germ cells (PGCs). To identify TK genes expressed in chicken PGCs, we purified these cells from the blood of 2.5-day-old embryos, extracted the polyA(+) RNA, and subjected it to reverse transcription-polymerase chain reaction (RT-PCR) amplification with TK gene-specific primers. As a result, we identified 13 receptor TK genes and 6 non-receptor TK genes. One of the receptor TK genes appeared to be novel, and thus the full-length DNA complementary to RNA (cDNA) sequence was retrieved by the rapid cloning of cDNA ends method. Sequence analysis of this cDNA indicated that it encoded a novel TK receptor of the Eph family. The putative amino-acid sequence of this TK was 63.0% identical to that of human EphA1; therefore, we designated our novel TK as EphA9. Northern blot hybridizations indicated that ephA9 RNA transcripts were present in the kidney, lung, testis, and thymus but not in the spleen, brain, or liver. Expression of a fusion protein containing the intracellular domain of EphA9 in bacterial cells showed that this domain had TK enzymatic activity. The EphA9 species produced in Cos-1 cells transfected with an expression vector were tyrosine-phosphorylated. These data suggest that EphA9 plays its biological role(s) in various organs of chickens as an active TK.

Production of a monoclonal antibody that recognizes bovine stem cell factor (SCF) and its use in the detection and quantitation of native soluble bovine SCF in fetal bovine serum.

Stem cell factor (SCF) is a pluritropic hematopoietic cytokine that acts predominantly on the proliferation and differentiation of hematopoietic progenitor cells. SCF has long been thought to be present in fetal bovine serum (FBS) as an endogenous factor that stimulates the growth of hematopoietic progenitor cells in FBS-supplemented cultures. To detect and quantitate bovine SCF in FBS, we produced a monoclonal antibody (mAb) by immunizing mice with recombinant soluble bovine SCF protein, which was expressed in insect cells by using a baculovirus system. Using the mAb, we purified native soluble bovine SCF from FBS by immunoaffinity chromatography. Western blot analysis revealed that the purified SCF protein had a molecular weight of 33 kDa. In addition, an enzyme-linked immunosorbent assay (ELISA) incorporating the mAb revealed that the levels of native soluble SCF in commercially available FBS were likely to be <100 pg/ml. These results suggest that the concentration of native soluble bovine SCF present in FBS may be insufficient to promote additive biologic effects on the growth of hematopoietic progenitor cells in FBS-supplemented cultures.