The Journal of Reproduction and Development Supplement

The effect of glucose in various concentrations of 0%, 2%, 4%, 6%, 8%, and 10%, respectively, on sperm quality of Barbonymus gonionotus (Bleeker, 1850) 24 hours postcryopreservation has been studied. Milt was collected by hand-stripping method, and was diluted by combination of glucose, 10% of DMSO, and Kurokura solution. The ratio of milt and diluent was 1 : 3 according to Bozkurt (2005). Sample (milt + diluent) was then equlibrated at 4 oC for 45 minutes, and was freezed at -34 oC for 24 hours. Thawing was carried out at 30 oC for 30 seconds. The percentage of sperm motility, viability, and abnormality were 87.68%, 77.25%, and 24.25%, respectively. Based on ANAVA test there were significant effect (alpha = 0.05) of various concentration of glucose on post-thawed sperm viability and abnormality compared to control (0% of glucose). According to Tukey test, the various concentration of 4%, 6%, and 8% of glucose, respectively, were shown significant difference on post-thawed viability compared to control, while only the concentration of 6% glucose was significant difference on post-thawed abnormality. Six percent of glucose showed the highest post-thawed sperm motility (88.45%) and sperm viability (59.80%), and also showed the lowest post-thawed sperm abnormality (45%).

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[Introduction] Cryptorchidism (CO) is an anomaly which shows retained testis due to failure of descent to scrotum. CO frequently occurs in some small breeds of dogs. CO is considered to be inherited but the causative genes have not been determined in dogs. In human, association between CO and single nucleotide polymorphisms (SNP) in the 3' region of estrogen receptor alpha gene (ESR1) has been described recently. The aim of this study was to determine whether CO is associated with SNP in the 3' region of ESR1 in small breed dogs.
[Methods] Forty six CO and 86 normal small breed dogs, namely, Miniature Dachshunds (MD), Chihuahua (CHH) and Toy Poodle (TP), were used in this study. Thirteen DNA fragments located in the 70kb region at the 3' end of ESR1 were amplified by PCR and directly sequenced. Genotype for SNP was analyzed and genotype and haplotype frequencies compared among breeds and between CO and normal dogs.
[Results] Eight SNPs were identified in MD and CHH while 7 SNPs in TP. Frequency of dominant homozygous genotype at 6 SNPs was significantly different among three breeds. At 2 SNPs, a lowered tendency was observed for the frequency of heterozygous genotype in CO compared to normal MD. In another SNP, frequency of dominant homozygous genotype tended to be higher in CO than normal MD. No specific haplotype was found to be associated with CO.
[Conclusions] The genotype frequencies of SNPs in the 70 kb region at the 3' end of ESR1 are different among the three small breeds of dogs. There seems to be no clear association between the genotype of SNPs in the analyzed region of ESR1 and the occurrence of cryptorchidism in those dogs.

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[Introduction] Progesterone (P4) has been demonstrated to be an anti-apoptotic agent in bovine luteal steroidogenic cells (LSC). Since luteinizing hormone (LH) is one of the most potent stimulators of P4 production by the corpus luteum (CL), we hypothesized that LH suppresses apoptosis of LSC via P4. To confirm the above hypothesis, we examined the effect of LH on cell death and expression of apoptosis related proteins in LSC. [Materials and Methods] Cultured bovine LSC at the mid stage were treated for 24 h with LH (0.34 nM) in the presence or absence of tumor necrosis factor α (TNF; 2.9 nM) and interferon γ (IFNG; 2.5 nM) with or without P4 antagonist (onapristone, OP; 100 µM). Cell viability, and gene and protein expressions were measured by WST-1 assay, a real-time PCR and Western immunoblotting, respectively. [Results] LH reduced the levels of cell death induced by TNF and IFNG. LH attenuated mRNA expression of apoptosis related proteins, i.e. FAS, BAX and CASP3, as well as FAS and cleaved CASP3 protein expressions in TNF and IFNG treated cells. LH increased mRNA expression of BCL2 and BCL2:BAX mRNA ratio, whereas LH did not affect the BAX and BCL2 protein expression and BCL2:BAX protein ratio. Exposure to OP suppressed the anti-apoptotic effects of LH in the absence of TNF and IFNG. [Conclusion] The above results indicate that the anti-apoptotic effect of LH is mediated by increasing P4 production as well as by regulating the expression of apoptosis related proteins i.e. FAS, BAX, BCL2, CASP3 and cleaved CASP3. The overall results suggest that LH not only potentiates CL function but also protects LSC against apoptosis.

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Cellular FLICE-like inhibitory protein (cFLIP) is one of candidate regulator of apoptosis in germ cells. To elucidate the role of cFLIP, we examined the expression in ovarian germ cells in embryos of rodents. cFLIP mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Immunohistochemical staining was performed to demonstrate the localization of cFLIP protein. The ovarian germ cells were assigned as 3 stages base on development of each animal as follows: In mice, 13.5, 16.5 and 18.5 day post coitus (dpc) embryos were used. In hamsters, 11.5, 14.5 and 15.5 dpc embryos were examined. In rats, 15.5, 18.0 and 21.0 dpc embryos were removed and examined. The RT-PCR analyses for ovarian tissues showed that lower levels of cFLIPS and cFLIPL mRNAs were noted in 13.5 and 11.5 dpc in mice and hamsters, respectively, and no difference was noted among 15.5, 18.0 and 21.0 dpc in rats. Western blotting analyses showed that both cFLIPS and cFLIPL isoform were detected in ovarian tissues and lower levels in 13.5, 11.5 and 15.5 dpc embryos of mice, hamsters and rats were noted, but higher levels were demonstrated in other days examined in these rodents. Immunohistochemistry demonstrated positive staining of cFLIPS/L in ovarian germ cells but no difference in staining intensities in each day of these rodents. In the present study, we firstly show the changes in expression and localization of cFLIP in ovarian germ cells during embyonic development, indicating that cFLIP relates apoptotic cell death in ovarian grem cells and may acts as a survival factor during embryonic development.

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