• Clinical and Experimental Reproductive Medicine
• /
• v.21 no.1
• /
• pp.121-130
• /
• 1994

Expression of gonadotropin releasing hormone(GnRH) has been described in the rat ovary. It remains, however, unkown whether GnRH is synthesized as a prohormone. Therefore, this study was performed to verify the expression of pro-GnRH by in situ hybridization and further to investigate the effect of gonadotropin on GnRH or GnRH mRNA in rat ovary by immunohistochemical and in situ hybridization techniques. Adult female Sprague-Dawely rats were used and the estrous cycle was synchronized by intraperitoneal injection of pregnant mare's serum gonadotropin(PMSG). Ovaries were fixed with 4% paraformaldehyde and embedded with G.C.T. compound and cut by cryostat. For immunohistochemistry, avidin-biotin peroxidase complex(ABS) method was employed and for in situ hybridization, $^{35}S$-end labeled oligonucleotide was used and followed by autoradiography. By in situ hybridization using GnRH oligomer and GAP(GnRH associated protein) oligomer, GnRH mRNA and GAP mRNA were co-localized in the fullicular cells, luteal cells, interstitial cells and theca cells. GnRH or GnRH mRNA signals in the ovary increased by human chorionic gonadotropin(hCG) injection. At the 3 and 6 hrs after hCG injection, the number of GnRH and GnRH mRNA containing cells increased rapidly and the density of GnRH and GnRH mRHA culminated at 9 hrs after heG injection. With the follicular development, the high expression of GnRH and GnRH mRNA was also observed within the follicles. After ovulation, the density of GnRH or GnRH mRNA decreased in the follicles but increased in the corpus lutea.

• Clinical and Experimental Reproductive Medicine
• /
• v.29 no.2
• /
• pp.139-147
• /
• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• Applied Microscopy
• /
• v.17 no.1
• /
• pp.47-64
• /
• 1987

Morphological difference of the renal glomerulus at different age groups have been studied in young (three month-old), adult (twelve month-old) and old (thirty month-old) Fisher strain 344 rats. Pieces of the tissues were taken from renal corticies prefixed with 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), following by post-fixation with 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3) and embedded within Araldite. The ultrathin sections contrasted with uranyl acetate and lead citrate were observed under a JEM 100CX electron microscope. The mean thickness of glomerular basal lamina and Bowman's capsule were determined by measuring the thinnest portion of basal lamina, and by taking the average of 50 readings from electron micrographs at different ages. The numerical changes of the slit pores were compared based upon the numbers over the length of 10um of glomerular basal lamina. The results were as follow: 1. The thickness of glomerular basal lamina is increased during aging; 140.4 nm in young rats, 270.0 nm in adult ones, and 437.8 nm in old ones. 2. The thickness of basal lamina of parietal cells of Bowman's capsule is 187.5 nm in young rats, 914.0 nm in adult ones, and 2850.0 nm in old ones. 3. The numbers of the slit pores of basal lamina are reduced during aging, 30.3 slit pores/$10{\mu}m$ in adult ones, and 24.2 slit pores/$10{\mu}m$ in old ones. 4. Accumulation of dense intracytoplasmic filamentous material in the parietal cells of Bowman's capsule is increased in the vicinity of the basal lamina during aging. The proximal tubule-like epithelial cell in Bowman's capsule is observed at one glomerulus in a young rat. 5. The endothelial cells are edematous and form balloon-like structure protruding into capillary lumen in young and old rats. 6. Cytoplasm of the podocyte shows a variety of alteration during aging, such as swelling of mitochondria and of endoplasmic reticulum, and increase of microtubules, microfilaments, lysosomes and lamellated myelin structures, etc. Accumulation of dense intracytoplasmic material in the foot processes is increased in the vicinity of the basal lamina during aging. The podocytic membrane-like structures are seen in young and o]d rats. 7. The mesangial matrices and mesangial cells are increased during aging, and slight swelling of endoplasmic reticulum and Golgi cisternae in young and old rats.

• Applied Microscopy
• /
• v.17 no.1
• /
• pp.83-97
• /
• 1987

In order to discriminate the enteroendocrine cell types in the mucosal epithelium of the normal duodenum of the Korean hedgehog (Erinaceus koreanus). The tissues were fixed in the mixture of 1% paraformaldehyde and 1% glutaraldehyde in phosphate buffer (pH 7.2), and postfixed in 2% osmium tetroxide (phosphate buffer, pH 7.2). They were embedded in Araldite, and the ultrathin sections were made by LKB-V ultratome following the inspection of semithin sections stained with toluidine blue-borax solutions. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed with JEM 100B electron microscope. At least six types of enteroendocrine cells distributed in the mucosal epithelium of the duodenum were identified according to their morphological characteristics mainly based on the size, shape, number and electron density of the secretory granules. Type I cells had moderately developed organelles. The secretory granules were pleomorphic ($370X510nm$), and the granule cores with high electron density were enveloped in limiting membrane and characterized by a narrow halo. Type II cells contained an indented nucleus and well-developed organelles. The secretory granules were round (350 nm) and classified in two kinds by electron density, moderate and high. Both granules were surrounded by limiting membrane and those with high electron density showed often a wide halo. Type III cells had an indented nucleus. The secretory granules with various electron density were round (220 nm) in shape. The granules with high electron density were enveloped in limiting membrane and characterized by a narrow halo, but those with low or moderate electron density had not been observed the limiting membrane. Type IV cells contained an indented nucleus and moderately developed organelles. The secretory granules were round (180 nm) in shape, and the granule cores with high electron density were enveloped in limiting membrane and showed often a wide halo. Type V cells had a large amount of rough endoplasmic reticulum. Secretory granules with low or moderate electron density were round (230 nm) in shape, and surrounded by limiting membrane and showed a narrow halo. Type VI cells contained an oval nucleus and well-developed organelles, especially Golgi complex. The secretory granules with high electron density were round (210 nm) in shape. The granules were enveloped in limiting membrane and showed often a wide halo.

• Korean Chemical Engineering Research
• /
• v.54 no.4
• /
• pp.574-581
• /
• 2016

In this study, CNT functionalized with pyrrolidine ring via 1,3-dipolar cycloaddition reaction with various amino acid and aldehyde was synthesized. Metallocene was subsequently immobilized on the functionalized CNT and CNT/polyethylene composite was prepared via in-situ ethylene polymerization. The polymerization activities of metallocene supported on CNT functionalized with glycine and benzaldehyde (Gly+BA-CNT) were similar to those of metallocene supported on CNT functionalized with N-benzyloxycarbonylglycine and paraformaldehyde (Z-Gly+PFA-CNT) although its Zr content was lower than that of Z-Gly+PFA-CNT. In the case of metallocene supported on Z-Gly+PFA-CNT, the even distribution of active sites hindered the diffusion of ethylene monomer and cocatalyst MAO due to steric hindrance during ethylene polymerization. Compared to polyethylene produced from homogeneous metallocene catalysts, CNT/PE composites had a higher initial degradation temperature ($T_{onset}$) and maximum mass loss temperature ($T_{max}$). It suggests that pyrrolidine functionalized CNT is uniformly dispersed and strongly interacted with the PE matrix, enhancing the thermal stability of PE.

• Applied Microscopy
• /
• v.24 no.2
• /
• pp.48-62
• /
• 1994

The acute irradiation effect on rat Purkinje cell was carried out. Anesthetized rats, weighing 200-250g each, were exposed their heads to the linear accelerator (ML-4MV) with the doses of 3,000 rads or 6,000 rads respectively. Irradiated rats were sacrificed by perfusion fixation under anesthesia, six hours, two days and six days following the irradiations. Rats were perfused with the fixative of 1% glutaraldehyde-1% paraformaldehyde solution (pH 7.4). Small pieces of cerebellar cortices were taken out. Tissue blocks were washed out, and were refixed in the 2% osmium tetroxide solution. After dehydration, tissues were embedded in the araldite mixture. Ultrathin sections stained with uranyl acetate-lead citrate solution, were examined with an electron microscope. The results observed were as follow; 1. Many dark Purkinje cells exhibited most severe cellular alterations on 6 hours. But after the 2 or 6 days, the cells exhibited only some alterations of cytoplasmic organelles. 2. Many granular and agranular endoplasmic reticula exhibited the fusion of cisterns. These reticular alterations were most severe on 6 hours following irradiation. But the alterations were hardly found on 6 days. 3. In the Golgi region, alterations including the adhesion of lamelliform cisterns, enlarged saccules, and increased number of vesicles, etc, were seen on 6 hours. But the Golgi complexes were almost recovered on 6 days. 4. Lysosomes were abundant on 6 hours or 2 days, but some residual bodies were found on 6 days. 5. Mitochondrial changes were also most severe at on hours, and they were recovered thereafter. From the results, it was concluded that the cerebellar Purkinje cells reacted to the high doses of irradiation by hyperactive protein synthesis, autolytic activities and energy metabolism. The reaction was most active in the early stage. It implies that motor-control function of Purkinje cells are severely disturbed in the early stage of irradiation.

• Applied Microscopy
• /
• v.40 no.4
• /
• pp.211-218
• /
• 2010

The mammalian ovary is innervated by sympathetic and sensory neurons which contribute to regulating several aspects of ovarian function, including blood flow, steroidogenesis and follicular development. The existence of a neural connection between central neurons and the ovary has been rarely reported, but the mechanism underlying integration of ovarian activity to broader neuroendocrine responses has not been reported. We have now used a viral transneuronal tracing technique combined with a conventional retrograde labeling procedure of CT-HRP to demonstrate that oxytocin-producing neurons of the hypothalamus are synaptically connected to the ovary. Since ovarian activity is suppressed but the activity of oxytocin neurons is increased during breast feeding. Our finding that the oxytocinergic neural connection is likely to provide a direct transsynaptic mechanism by which the central nervous system maintains the state of infertility that accompanies lactation in mammals.

• Applied Microscopy
• /
• v.27 no.4
• /
• pp.357-372
• /
• 1997

To study the age-related morphological differences of the retinal pigment cells and Bruch's membrane of mouse, retinae of one week-old, five weeks-old, eight weeks-old, six months-old, twelve months-old, eighteen months-old, twenty-four months-old and thirty months-old ICR mice were dissected out under anesthesia. Pieces of the tissue taken from the posterior region of the retina were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde (0.1 M Millonig's phosphate buffer, pH 7.3), and 1% osmium tetroxide (0.1 M Millonig's phosphate buffer, pH 7.3), and embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed under a JEM 100 CX-II electron microscope. Observed results were as follows: 1. Retinae of one week old mouse exhibit that some parts of the pigment cell provided with basal foldings, whereas other parts of the one contain without basal foldings. After (ive weeks-old, all retinal pigment cells have the basal infoldings. 2. In the one week-old, stage 1 and stage 2 melanosomes were observed in the retinal pigments cells, but after five weeks-old, most of the retinal pigment cells contain some matured stage melanosomes (stage III and stage IV). 3. The phagosomes in the retinal pigment cells were increased during aging. 4. After eighteen months-old, electron dense materials are observed within the basal infoldings. 5. After eighteen months-old, the thickness of the Bruch's membrane is prominently increased. The thickness of the basal laminae of the pigment cell and the choriocapillary endothelium is more prominently increased as compared with that of the other components of the Bruch's membrane. 6. The thickness of the basal lamina of the pigment cell is more prominently increased as compared with that of the choriocapillary endothelium on aging. From the above results, it was suggested that the pigment cell and Bruch's membrane matures structurally In five weeks, and the function of the pigment cell is prominently suppressed around eighteen months-old, and thereafter the functional suppression is continued on aging.

• Applied Microscopy
• /
• v.27 no.1
• /
• pp.13-30
• /
• 1997

The experiment was performed to study the morphological responses of the thymus of the mice, to antitumour agents (5-Fluorouracil or mitomycin C). Healthy adult mice weighing 25 gm each were divided into normal and experimental groups. 5-Fluorouracil (60 mg/kg) or Mitomycin-C $(400{\mu}g/kg)$ were injected subcutaneously to the animals every other day. Animals were sacrificed at 4 days and 7 days following the first injection. Pieces of the tissue taken from the thymus were prefixed with 2.5% paraformaldehyde-l.5% glutaraldehyde, followed by post-fixation with 1% osmium tetroxide. Ultrathin sections stained with uranyl acetate and lead citrate were observed with a JEM 100 CX-II electron microscope. The observed results were as follow: 1. Apoptoses of T-lymphocytes were observed more frequently in the thymus of the experimental groups than in those of a normal group. 2. In the experimental group, the plasma cells with distended cisternae of the granular endoplasmic reticulum and the eosinophile leukocytes were observed frequently. 3. In the experimental group, newly forming Hassall's corpurscles were observed frequently. 4. In the mitomycin-treated group, the epithelial reticular cells containing distended perinuclear cisternae, distended the granular endoplasmic reticula and pyknotic nuclei were observed in the cortico-medullary junctional area. 5. In the mitomycin-treated group, nuclear bodies with medium electron dense materials were often observed in the T lymphocyte. 6. In the 5-fluorouracil-treated groups, fused and dissolved tonofilament bundles and apoptotic bodies were observed in the some epithelial reticular cells in the medullary area. 7. In the 5-fluorouracil-treated groups, some elongated and bar-shaped lysosomes with electron lucent gap were often observed in the macrophages. 8. In the 5-fluorouracil-treated group, membrane complex of the smooth endoplasmic reticulum were ofen observed in the macrophage. From the above results, it was suggested that 5-fluorouracil or mitomycin could induce rapid involution of the thymus, and disturb maturation and differentiation of T lymphocytes, and, in turn, supress immunity.

• Applied Microscopy
• /
• v.27 no.4
• /
• pp.373-389
• /
• 1997

Morphological difference of the renal glomerulus at different age groups have been studies in one week-old, five weeks-old, eight weeks-old, six months-old, twelve months-old, eighteen months-old, twenty-four months-old, and thirty months-old ICR mice. Pieces of the tissue taken from the renal corticies were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde solution (0.1 Millonig's phosphate buffer pH 7.3), and 1% osmium tetroxide solution (0.1 M Millonig's phosphate buffer, pH 7.3), and were embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate solution, and were observed under a JEM 100CX-II electron microscope. The results were as follows: 1 In the one week-old mouse, thicknesses of the three layers of the glomeluar basal lamina (lamina densa, lamina rara interna and lamina rara externa) are similar, but in the five weeks-old mouse, thick lamina densa becomes a greater portion of the thickness of whole glomerular basal lamina. 2. No difference was noticed between thickness of the renal glomerular basal lamina of the five weeks-old mouse compare with that of the one week-old one, but basal lamina of the eight weeks-old one is thickened considerably and thicknesses were maintained through twelve months-old one. After eighteen months, the thickness of the glomerular basal lamina is increased remarkably. 3. After eighteen months, electron dense deposits within the basal lamina of the renal glomeruli are observed frequently. 4. Amount of the microfilaments in the mesangial cells and the mesangial matrices are increasing during aging. 5. The thicknesses of the basal laminae of the Bowman's capsule are increasing during aging. 6. After twenty four months, the proximal tubular cell-like parietal cells with well developed microvilli are observed frequently. From the above results, it was suggested that the renal glomerulus matures structurally in five weeks, and the function of the glomerulus is suppressed after eighteen months.