Relationship between Sloan-Kettering virus expression and mammalian follicular development.

Sloan-Kettering virus gene, a product of a cellular proto-oncogene c-Ski is a unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. The aim of the present study was to locate Ski protein in rat ovaries in order to find insights into the possible involvement of Ski in follicular development. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RT-PCR and in situ hybridization revealed that c-Ski mRNA was expressed in the ovaries of the adult rat on the day of estrous and localized mainly in the granulose cells. Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in rats having atretic follicles with double staining. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles. Based on the present findings, Ski may play a role in the apoptosis of granulosa cells during follicular atresia.

Identification and promoter analysis of PERV LTR subtypes in NIH-miniature pig.

Porcine endogenous retroviruses (PERVs) are integrated into the genomes of all pigs. Since some PERVs can also infect human cells, they represent a potential risk for xenotransplantation involving pig cells or organs. The long terminal repeat (LTR) elements of PERVs show promoter activity that can affect human functional genes; therefore, we examined these elements in this study. We detected several expressed LTRs in the NIH-miniature pig liver, among which we identified 9 different subtypes. When these LTRs were compared, distinct structures that contained several insertion and deletion (INDEL) events and tandem repeats were identified in the U3 region. The transcriptional activity of the 9 LTR subtypes was analyzed in the PK15 porcine cell line and in the HepG2 and Hep3B human liver cell lines, and transcriptional regulation was found to be different in the 3 cell lines. The D LTR subtype was found to have stronger promoter activity than all other types in 4 different human cell lines (HepG2, Hep3B, U251, and 293). Using computational approaches, the D type was shown to contain 4 transcription factor-binding sites distinct from those in the U3 regions of the other subtypes. Therefore, deletion mutants were constructed and examined by a transient transfection luciferase assay. The results of this analysis indicated that the binding site for the Hand1:E47 transcription factor might play a positive role in the transcriptional regulation of PERV LTR subtype D in human liver cell lines.

Identification of ORF sequences and exercise-induced expression change in thoroughbred horse OXCT1 gene.

In the mitochondrial matrix, the OXCT1 gene catalyzes the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate in a reaction related to energy production from ketone bodies. Here, horse OXCT1 gene containing coenzyme A transferase domain was identified in the transcriptome analysis of cDNAs derived from skeletal muscles. Horse OXCT1 gene consisted of 1761 [corrected] nucleotide sequences with an open reading frame of 1560 nucleotides encoding a protein of 520 putative amino acid residues.The number of non-synonymous substitutions was lower than the number of synonymous substitutions in the OXCT1 genes of other species, indicating that purifying selection occurred in the OXCT1 genes during evolutionary radiation. Quantitative real-time RT-RCR analysis showed a dominant expression pattern of horse OXCT1 gene in the cerebrum, heart, and skeletal muscle. Different expression levels of horse OXCT1 transcripts between before- and after-exercise samples were also measured in the skeletal muscles of six horses. These data could be of great use for further investigation of the relationship between energy products and horse OXCT1 gene.

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-beta1 expression.

The objective of this study was to investigate hormonal and TGF-beta(1) characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n=12, 301+/-41.22 g) was significantly higher than that of AI-R (n=10, 204.8+/-24.89 g) (p<0.05). There were significantly lower E2 (p<0.05) or higher P4 (p<0.01) and TGF-beta(1) (p<0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-beta(1) protein level compared to that of AI-R (p<0.01). Interestingly, the TGF-beta(1) protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.

Age- and area-dependent distinct effects of ethanol on Bax and Bcl-2 expression in prenatal rat brain.

Cell proliferation and differentiation are critical processes in a developing fetal rat brain, during which programmed cell death (PCD) also plays an important role. One of the decisive factors for PCD is Bcl-2 family proteins, where Bax induces cell death, whereas Bcl-2 acts as an inhibitor of PCD. As maternal drinking is known to cause fetal alcohol syndrome (FAS) or malformation of the fetal brain during pregnancy, the objective of the present study was to investigate whether maternal ethanol exposure alters the PCD-related Bax and Bcl-2 protein expression during fetal brain development. Pregnant female rats were orally treated with 10% ethanol and the subsequent expressions of the Bax and Bcl-2 proteins examined in the fetal brain, including the forebrain, midbrain, and hindbrain, from gestational day (GD) 15.5 to GD 19.5, using Western blots, in situ hybridization, and immunohistochemistry. With regard to the ratio of Bcl-2 to Bax proteins (Bcl-2/Bax), the Bax protein was dominant in the forebrain and midbrain of the control GD 15.5 fetuses, except for the hindbrain, when compared with the respective ethanol-treated groups. Moreover, Bcl-2 became dominant in the midbrain of the control GD 17.5 fetuses when compared with the ethanoltreated group, representing an alternation of the natural PCD process by ethanol. Furthermore, a differential expression of the Bcl-2 and Bax proteins was found in the differentiating and migrating zones of the cortex, hippocampus, thalamus, and cerebellum. Thus, when taken together, the present results suggest that ethanol affects PCD in the cell differentiation and migration zones of the prenatal rat brain by modulating Bax and Bcl-2 expression in an age- and area-dependent manner. Therefore, this is the first evidence that ethanol may alter FAS-associated embryonic brain development through the alteration of Bax and Bcl-2 expression.

Biological activities of tethered equine chorionic gonadotropin (eCG) and its deglycosylated mutants.

Equine chorionic gonadotropin (eCG), which consists of highly glycosylated alpha- and beta-subunits, is a unique member of the gonadotropin family because it elicits response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. In this study, recombinant tethered-eCG as well as its deglycosylated mutants were produced to determine if alpha- and beta- subunits can be synthesized as a single polypeptide chain (tethered-eCG) and display biological activity. We found that tethered-eCG (T- betaalpha) had both LH- and FSH-like activities comparable to dimeric eCG. Luteinizing hormone-like activity of tethered-eCGs deglycosylated at Asn(56) (T-betaalpha56) was decreased. In contrast, LH-like activity of eCG without O-glycosylated carboxyl-terminal peptide (CTP) (T-betacalpha) was slightly decreased but still similar to T-betaalpha. Double mutation at Asn(56) and CTP (T-betacalpha56) caused marked decrease in the activity, indicating that both glycosylations at Asn(56) and CTP are involved in LH-like activity in the tethered form. Interestingly, FSH-like activity remained in all deglycosylated eCG mutants (T-betaalpha56, T-betacalpha and T-betacalpha56) as well as T-betaalpha. The biological roles of oligosaccharides at Asn(56) of eCG alpha-subunit and O-linked peptide of beta-subunit appear to be different in LH- and FSH-like activities in tethered-eCG.