P. E. Høyer

Stem cell factor and c-Kit in human primordial germ cells and fetal ovaries.

The distribution of the tyrosine kinase receptor c-Kit and its ligand stem cell factor (SCF) was evaluated by immunohistochemistry in primordial germ cells (PGCs) and human embryonic gonads during weeks 5-8 of prenatal life, and fetal ovaries during weeks 9-36 of prenatal life. Distinct c-Kit and SCF staining was present in primordial germ cells in the wall of the hindgut and in the dorsal mesentery, particularly on level with the 10th thoracic columnar segment. Several PGCs were in close contact with c-Kit-negative but SCF-positive autonomic nerve fibers of the dorsal mesentery. Many fibroblasts and mesothelial cells of the dorsal mesentery were clearly stained for SCF, but not for c-Kit. Prominent c-Kit and SCF staining was present in germ cells of the embryonic gonadal anlage and in oogonia during further ovarian development. However, oocytes were either unstained or faintly stained for SCF. Oocytes not yet enclosed in follicles or present in primordial follicles were either unstained or exhibited faint cytoplasmic c-Kit staining, whereas oocytes of growing preantral follicles again showed distinct cell membrane staining which decreased during further follicular growth. Theca cells did not stain for c-Kit. Some pregranulosa cells and the first formed granulosa cells of primordial follicles were c-Kit stained. Granulosa cells of other follicles were not c-Kit stained. In the inner part of the cortex, SCF immunolabeling was detected in some pregranulosa cells surrounding cords containing germ cells and involved in formation of primordial follicles. Granulosa cells of primordial and growing follicles, including medium-sized antral follicles also revealed SCF staining. In conclusion, this first report on SCF in human PGCs and embryonic and fetal ovaries together with the c-Kit data lend substantial countenance to the notion that c-Kit and SCF play important roles during ascent of primordial germ cells towards the gonadal anlage, and during oogenesis and folliculogenesis in the human fetal ovary. We suggest that both autocrine and paracrine mechanisms are involved in the proposed anti-apoptotic effect of the c-Kit/SCF duet while PGCs are present in the dorsal mesentery. The SCF-positive autonomic nerve fibers of the dorsal mesentery, mesothelial cells and fibroblasts may nurse and perhaps guide PGCs during their ascent.

YKL-40 Protein Expression in the Early Developing Human Musculoskeletal System

YKL-40 is a growth factor for chondrocytes and fibroblasts. The aim was to evaluate YKL-40 expression in the musculoskeletal system during early human development. We studied sections from 15 human embryos [weeks 5.5-8; 7- to 31-mm crown-rump length (CRL)] and 68 fetuses (weeks 9-14; 33- to 105-mm CRL) for YKL-40 protein expression by immunohistochemistry. YKL-40 mRNA expression was evaluated in two human embryos (days 41 and 51). Initially YKL-40 is expressed in all germ layers: ecto-, meso-, and endoderm. YKL-40 mRNA and protein expression are found in tissues of the ecto-, meso-, and endoderm, and YKL-40 protein expression is present during development of cartilage, bone, joints, and muscles. At the cellular level, YKL-40 protein expression is high in tissues characterized by rapid proliferation, marked differentiation, and undergoing morphogenetic changes. Examples of rapid cell proliferation include the chondrogenic inner layer of perichondrium and the osteogenic inner layer of periosteum. Differences in YKL-40 expression during differentiation are found in the chondrogenic and osteogenic cell lineages. The initial shaping of cartilage and bone models and joints is concomitant with a strong outline of YKL-40-positive cells. This indicates that YKL-40 is associated with cell proliferation, differentiation, and tissue morphogenesis during development of the human musculoskeletal system.

Human primordial germ cells migrate along nerve fibers and Schwann cells from the dorsal hind gut mesentery to the gonadal ridge

The aim of this study was to investigate the spatiotemporal development of autonomic nerve fibers and primordial germ cells (PGCs) along their migratory route from the dorsal mesentery to the gonadal ridges in human embryos using immunohistochemical markers and electron microscopy. Autonomic nerve fibers in the dorsal mesentery, the pre-aortic and para-aortic plexuses and in the gonadal ridge were stained for beta III tubulin, neuron specific enolase and the glia fibrillary acidic protein. Electron microscopy demonstrated the presence of neurofilaments and neurotubules in these nerve fibers and their intimate contact with PGCs. PGCs expressed GAGE, MAGE-A4, OCT4 and c-Kit. Serial paraffin sections showed that most PGCs were located inside bundles of autonomic nerve fibers with the majority adjacent to the most peripheral fibers (close to Schwann cells). We also show that both nerve fibers and PGCs arrive at the gonadal ridge between 29 and 33 days pc. In conclusion, our data suggest that PGCs in human embryos preferentially migrate along autonomic nerve fibers from the dorsal mesentery to the developing gonad where they are delivered via a fine nerve plexus.

Simplified control experiments in the histochemical study of coenzyme-linked dehydrogenases

SummaryDifferent experiments concerning some of the most critical steps in the histochemical procedure for coenzyme-linked dehydrogenases (fixation procedure, cryoprotection, osmolar protection, substantivity of formazans, “nothing dehydrogenase” reaction, diffusion of enzyme, rediffusion of reduced coenzyme and/or reduced phenazine methosulphate) were carried out in order to improve or introduce simplified control methods by which the in situ localization of enzyme activity can be achieved without the need of expensive equipments.As a test-object glucose-6-phosphate dehydrogenase was used.Brief (5 min) prefixation of tissue (liver) at 0–4° C with 1% buffered (pH=7.2) methanolfree formaldehyde (from paraformaldehyde) gave excellent preservation of morphology during the procedures of freezing, cutting and incubation and caused no inhibition of G6PDH. With the named fixation no improvement was obtained by the simultaneous use of cryoprotection (DMSO) or osmolar protection (sucrose). Finally, the fixation caused an enhancement of Nitro BT penetration into the tissue as well as of formazan substantivity. On the other hand, the brief fixation did not abolish the diffusion of enzyme (proved by different methods) and of reduced coenzyme or reduced phenazine methosulphate.In a conventional aqueous incubation medium as well as in a gel incubation medium (PVA, grade Bo5/140) the rate of diffusion of reduced coenzyme and/or reduced phenazine methosulphate was investigated by using a special double-section incubation method. The concentrations of Nitro BT, NADP and PMS were balanced against each other and it was concluded that by using a gel medium containing 0.5 mg/ml Nitro BT, 0.1 mg/ml NADP and 0.003 mg/ml PMS, the in situ localization of G6PDH activity could be achieved at the cellular level with an incubation time not exceeding 10 min. With the incubation time mentioned, the “nothing dehydrogenase” reaction was out of the question. The sensibility of the double-section incubation method is discussed and provided that the dehydrogenase in question contains sulphydryl groups in the active enzyme centre, the method seems to exhibit a sufficient high level of sensitivity in the control of the diffusion of the different components operative in the histochemical dehydrogenase procedure.The recording of the incubation time needed for the appearance of the two formazans (red and blue) is recommended in order to follow the enzymatic reaction rate and the effect of different procedures (fixation, solvents added etc.) as well as the rates of “nothing dehydrogenase” reaction, diffusion of reduced coenzyme and/or reduced PMS.

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.

SummaryFor simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA.The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen analtion. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm.After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiffs reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum.We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.

Immunohistochemical markers for corneal stem cells in the early developing human eye.

The corneal epithelium is continuously being renewed. Differentiated epithelial cells originate from limbal stem cells (LSCs) located in the periphery of the cornea, the corneoscleral limbus. We have recently identified superoxide dismutase 2 (SOD2) and cytokeratin (CK) 15 as limbal basal cell markers and potential markers for LSCs and early transient amplifying cells in human adults. In this study, we describe the development of the ectodermally derived LSCs and the mesodermally derived niche cells from the time at which the cornea is defined (week 6) until the formation of the early limbal niche (week 14) in human embryos and fetuses. The expression of SOD2 and CK15 was investigated together with other recently identified limbal proteins. Previously suggested LSC and differentiation markers (PAX6, aquaporin-1 and nestin) were also investigated. Both SOD2 and CK15 were present in the corneal epithelium from week 6. However, in week 14 they were predominantly expressed in the limbal epithelium. Both proteins were expressed already from week 7 in a stromal triangular region from which the early mesodermal limbal niche most likely originates. PAX6 was expressed in both ectodermally and mesodermally derived parts of the limbal niche, underscoring the importance of PAX6 in niche formation.

Studies in succinate dehydrogenase histochemistry

SummaryThe activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of succinate dehydrogenase (SDH) as well as to the level of CoQ.It was concluded that the low activity in centrolobular areas of the liver lobules compared with the perilobular areas, exclusively is due to a naturally deprivation of CoQ.The level of SDH as well as of CoQ was very high in renal cortical tubules rich in mitochondria (proximal and distal convoluted tubules, the ascending thick limb of Henle). This was indicated by the facts that the initial reaction rate was high and no enhancement was obtained by the addition of CoQ10.In all experiments the activity of fresh frozen sections were compared with the activity of sections from briefly prefixed tissue. The influence of different fixatives, variation in Nitro BT concentration, cryoprotection (dimethyl sulfoxide, DMSO) and osmolar protection (sucrose) was investigated and discussed. Further, the substrate-carrying effect of DMSO was investigated and discussed.Brief (5 min) fixation at 0–4° C—especially with 1% buffered (pH=7.2) methanol-free formaldehyde (from paraformaldehyde) gave excellent preservation of morphology and caused no inhibition of SDH activity. Furthermore, the fixation caused an enhancement of Nitro BT penetration into the tissue and an enhancement of formazan substantivity.The incubation time needed for the appearance of both the red and blue formazan was recorded in order to follow the initial reaction rate. This procedure proved to be a sensitive indicator, when the influence of components added (CoQ10, DMSO, sucrose etc.) was studied.

Specificity in steroid histochemistry, with special reference to the use of steroid solvents. distribution of 11 β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse

SummarySeveral factors influencing the steroiddehydrogenase histochemistry were investigated: diffusion of enzyme; inactivation of enzyme; effects of the steroid solvents commonly used; the validity in localization of the enzyme activity; “nothing dehydrogenase” reaction. 1. The importance in controlling the diffusion of each enzyme system to be studied is emphasized. Provided that the presence of SH-groups in the active centre of the dehydrogenase can be proved, a control experiment using a double-section incubation method should be carried out. 2. A comparison between the use of unfixed and briefly prefixed sections is recommended in order to avoid a possible distortion of the tissue during the incubation. The influence of prefixation on diffusion of enzymes or reaction products as well as on inactivation of enzymes must be studied. 3. The steroid solvents—especially dimethyl formamide caused a morphological distortion, and an inactivation and/or extraction of reaction products (the red monoformazan) in fresh frozen sections, these solvents should therefore be handled with caution. A special mixture of dimethyl formamide and propylene glycol is recommended. 4. The steroid should be completely soluble in the incubation medium in order to secure zero order kinetics. 5. Avoidance of sulphydryl “nothing dehydrogenase” reaction, since the reaction predominantly manifests itself as a red formazan obscuring sites with low dehydrogenase activity. 6. The localization of the NAD(P)H oxidase systems must be controlled, in order to ensure that they should not be a limiting factor in the detection of the dehydrogenase activity. Secondly, this investigation may act as a control on diffusion of dehydrogenase and/or reduced coenzyme. 7. That the investigation of the incubation time needed for initial visual reaction allows a certain quantitative estimation of the concentration of enzyme localized at different sites in the same section. The investigation should also include the red formazan, since it has recently been proved to be an intermediary step in the enzymic reduction of Nitro BT, and as such may reflect sites with low enzyme concentration.Further, some of the functional aspects of the activity of 11β-hydroxysteroiddehydrogenaseNAD(P)H oxidase systems in the thymus were discussed, and lastly the localization of these systems in the kidney was revised.

Regional differences in expression of specific markers for human embryonic stem cells

Characterization of human embryonic stem cell (hESC) lines derived from the inner cell masses of blastocysts generally includes expression analysis of markers such as OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. Expression is usually detected by immunocytochemical staining of entire colonies of hESC, using one colony for each individual marker. Four newly established hESC lines showed the expected expression pattern and were capable of differentiating into the three germ layers in vitro. Neighbouring sections of entire colonies grown for 4, 11, 21 and 28 days respectively were stained with different markers to study the regional distribution and cellular co-expression. TRA-1-60 staining defined the hESC territory at all time points analysed. This territory comprised a characteristic OCT4 and NANOG staining often in overlapping subregions. Staining intensity of nuclei varied from strong OCT4 staining to weak or absent NANOG staining, and vice versa. SSEA4 staining was only observed in small clusters or single cells and not confined to the TRA territory. Co-expression of all markers was only detected in small areas. SSEA1 expression was found exclusively outside the TRA territory. In conclusion, pronounced regional differences in the expression of markers considered specific for undifferentiated hESC may suggest the existence of different cell populations.

Histochemistry of 3β-hydroxysteroid dehydrogenase in rat ovary

SummaryBy recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3β-HSD was investigated:1.Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced.2.Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% w/v for 5 min) thereby enhancing the Nothing dehydrogenase reaction.3.Preextraction of lipids with acetone for 20 min at −30° C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells).4.No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20° C.5.By using the double-section incubation method no diffusion of 3β-HSD or rediffusion of NADH or PMSH could be noticed within 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0 mg/ml).6.The utility of different inhibitors of alkaline phosphomonoesterase was tested and discussed.7.By inhibiting alkaline phosphomonoesterase with 0.1 mM ofl-p-bromotetramisole or 16 mM ofβ-glycerophosphate, 3β-HSD was shown to be exclusively NAD-linked.8.Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3β-HSD, but not of NADPH-tetrazolium reductase.9.3β-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited byn-ethyl maleimide.10.Whether alcohol dehydrogenases may use steroids as substrate is discussed. It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed.11.Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase.12.Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF).13.The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone.4.If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.