Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Depressed nNOS expression during spine transition in the developing hippocampus of FMR1 KO mice

Nitric oxide (NO), synthesized as needed by NO synthase (NOS), is involved in spinogenesis and synaptogenesis. Immature spine morphology is characteristic of fragile X syndrome (FXS). The objective of this research was to investigate and compare changes of postnatal neuronal NOS (nNOS) expression in the hippocampus of male fragile X mental retardation 1 gene knockout mice (FMR1 KO mice, the animal model of FXS) and male wild-type mice (WT) at postnatal day 7 (P7), P14, P21, and P28. nNOS mRNA levels were analyzed by real-time quantitative PCR (N = 4-7) and nNOS protein was estimated by Western blot (N = 3) and immunohistochemistry (N = 1). In the PCR assessment, primers 5’-GTGGCCATCGTGTCCTACCATAC-3’ and 5’-GTTTCGAGGCAGGTGGAAGCTA-3’ were used for the detection of nNOS and primers 5’-CCGTTTCTCCTGGCTCAGTTTA-3’ and 5’-CCCCAATACCACATCATCCAT-3’ were used for the detection of β-actin. Compared to the WT group, nNOS mRNA expression was significantly decreased in FMR1 KO mice at P21 (KO: 0.2857 ± 0.0150, WT: 0.5646 ± 0.0657; P < 0.05). Consistently, nNOS immunoreactivity also revealed reduced staining intensity at P21 in the FMR1 KO group. Western blot analysis validated the immunostaining results by demonstrating a significant reduction in nNOS protein levels in the FMR1 KO group compared to the WT group at P21 (KO: 0.3015 ± 0.0897, WT: 1.7542 ± 0.5455; P < 0.05). These results suggest that nNOS was involved in the postnatal development of the hippocampus in FXS and impaired NO production may retard spine maturation in FXS.

Rat Dlx5 is expressed in the subventricular zone and promotes neuronal differentiation

The molecular mechanisms and potential clinical applications of neural precursor cells have recently been the subject of intensive study. Dlx5, a homeobox transcription factor related to the distal-less gene in Drosophila, was shown to play an important role during forebrain development. The subventricular zone (SVZ) in the adult brain harbors the largest abundance of neural precursors. The anterior SVZ (SVZa) contains the most representative neural precursors in the SVZ. Further research is necessary to elucidate how Dlx5-related genes regulate the differentiation of SVZa neural precursors. Here, we employed immunohistochemistry and molecular biology techniques to study the expression of Dlx5 and related homeobox genes Er81 and Islet1 in neonatal rat brain and in in vitro cultured SVZa neural precursors. Our results show that Dlx5 and Er81 are also highly expressed in the SVZa, rostral migratory stream, and olfactory bulb. Islet1 is only expressed in the striatum. In cultured SVZa neural precursors, Dlx5 mRNA expression gradually decreased with subsequent cell passages and was completely lost by passage four. We also transfected a Dlx5 recombinant plasmid and found that Dlx5 overexpression promoted neuronal differentiation of in vitro cultured SVZa neural precursors. Taken together, our data suggest that Dlx5 plays an important role during neuronal differentiation.

Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of á and â transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. â2 integrin (CD18) associates with four different á (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that â2 integrin is also expressed by other types of cells. Since the gene for â2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that â2 integrin and the áL, áM, and áX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of â2 integrin or against its á subunit partners, showed that â2 integrin, as well as the áL, áM, and áX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of â2 integrin in these various locations in the embryonic heart. These results indicate that the â2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.

Distribution and ontogeny of gastrin- and serotonin-immunoreactive cells in the proventriculus of developing chick, Gallus gallus domestica

The ontogeny and distribution of gastrin- and serotonin-immunoreactive (IR) cell in the proventriculus of chicks (Gallus gallus domestica, n = 60) in different growth periods was examined immunohistochemically using antisera specific to gastrin and serotonin. Gastrin and serotonin-IR cells were detected in chick proventriculus. Gastrin-IR cells were first evident after 12 days of incubation in lamina epithelialis and compound glands, while serotonin-IR cells were observed only in compound glands at that same time. Gastrin-IR and serotonin-IR cells increased in frequency on incubation day 14 and 16, respectively. Towards the end of incubation, gastrin- and serotonin-IR cell numbers decreased. In adult chicken, both IR cells were present but not lower numbers. The observations demonstrate the presence of gastrin- and serotonin-IR cells in the proventriculus of developing chicks in temporally changing frequencies.

Expression of tyrosine kinase A in the cerebral cortex of postnatal developing rat

Tyrosine kinase A (TrkA)is an essential component of the high affinity nerve growth factor (NGF) receptor necessary to the mediate the biological effects of the neurotrophins, NGF. This study examined the distribution of TrkA-immunoreactivity (IR)cells in the postnatal rat cerebral cortex and the changes that occur in postnatal development as a result of the expression of this protein. TrkA-IR was detected at postnatal day (PD)3, PD6, PD9 and PD15. Base upon their somatodendritic morphology, the most commonly labeled cell type was the pyramidal neurons. At PD3 and PD6, layer I, II, III and V was immunopositive for TrkA, at PD9, not only at layer I, II, III, and V but also at layer VI. At PD15, the TrkA-positive cells were distributed in all layers. These TrkA-positive cells were not detected at PD0. In contrast, there was significant increase in the percentage of cells exhibiting TrkA-IR with development and the highest level was detected at PD15. These results suggest that the cerebral cortex expresses TrkA strongly during the postnatal period. Moreover, the postnatal development-related increase in the expression of TrkA-cells shows that NGF may have a trophic effect on these cerebral cortex neurons from the postnatal period.

Differential Expression of TGF-beta Isoforms During Differentiation of HaCaT Human Keratinocyte Cells: Implication for the Separate Role in Epidermal Differentiation

The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, beta2, beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell growth and differentiation induction of the human keratinocyte cell line, HaCaT by elevating the Ca2+ concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta1 mRNA were increased during calci-um-induced differentiation. In contrast, major differences were seen for TGF-beta 2 and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta 2 and TGF-3beta protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associ-ated with the more differentiated state, and TGF-beta 2 and TGF-beta 3 may be associ-ated the more proliferated state.

mRNA expression pattern of multiple members of connexin gene family in normal and abnormal fetal gonads in mouse.

Gap junctions are made of connexin (Cx) molecules and provide communications between adjacent cells through which small molecules (<1kDa) move between cytoplasms. The connexin gene family consists of at least 17 members. Gap junctions play important roles in ovarian and testicular function. Connexin 43 (Cx43) knockout mouse is a model for developmentally impaired fetal gonads. The aim of the current study was twofold: (a) to analyze mRNA expression pattern of multiple members of connexin gene family in normal fetal gonads; (b) to investigate any alteration of mRNA expression of connexins in developmentally impaired fetal gonads. The study was conducted in normal gonads obtained from 17.5 dpc wildtype (Cx43+/+) and heterozygote (Cx43+/-) fetuses and in developmentally impaired gonads obtained from 17.5 dpc knockout (Cx43-/-) fetuses. The mRNA expression pattern of connexins (Cx26, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, Cx43, Cx45, Cx46 and Cx50) was analyzed by RT-PCR. The mRNA transcripts for Cx32 and Cx50 were absent in fetal testis of all the genotypes, and the transcripts for Cx26, Cx30.3, Cx31.1, Cx32, Cx40, Cx46 and Cx50 were absent in fetal ovary of all the genotypes. The transcripts for Cx43 showed expression in Cx43+/+ and Cx43+/- gonads and were absent in Cx43-/- gonads, as expected. Additionally, the mRNA transcripts for 8 more connexins (Cx26, Cx30.3, Cx31, Cx31.1, Cx37, Cx40, Cx45 and Cx46) showed expression in Cx43+/+ and Cx43+/- fetal testes, but the transcripts for only 4 connexins (Cx26, Cx37, Cx40 and Cx45) showed expression in Cx43-/- fetal testis. In fetal ovary, the mRNA transcripts for 3 more connexins (Cx31, Cx37 and Cx45) were expressed in all the genotypes. In summary, from the mRNA expression analysis of 11 members of connexin, gene family in 17.5 dpc fetal gonads, besides the expression of Cx43, 8 additional connexins were expressed in normal fetal testis but only 4 connexins were expressed in developmentally impaired testis. The fetal ovary showed the expression of 3 additional connexins besides the expression of Cx43. In developmentally impaired fetal ovary, only Cx43 was not expressed as expected but other three connexins were expresed. The study may be useful in interpreting human testis defects in infertility cases.

Molecular biology of ageing.

The process of deterioration or ageing of functions that occurs in all organisms after the attainment of reproductive ability is the sum total of the decline in activity of various organs. The functions of different organs begin to deteriorate at different times of the life span and at different rates. It is believed that different genes are involved in the ageing of different organs. Studies on isoenzyme patterns of enzymes show that the genes responsible for coding of different subunits of the enzymes are sequentially expressed during the life span. Also, the decrease in the levels of enzymes seen after adulthood is reversible and can be raised to adult level by inducing their genes by steroid hormones. Another factor that contributes to the decrease in the levels of enzymes is increasing compaction of the chromatin that houses the genes as seen from digestion of chromatin by DNase I and MNase. This decreases the rate of transcription of genes. The expression of many genes declines after adulthood which is due to the decrease in trans-acting nuclear proteins that bind to specific cis-acting sequences in the promoter regions of genes. These proteins are inducible by steroid hormones. Hence the deterioration of functions that occurs after adulthood can be delayed, and the adulthood period can by prolonged by manipulation of the expression of genes.