In vitro development of marbled cat embryos derived from interspecies somatic cell nuclear transfer.

The objective of the study was to investigate interspecies Somatic Cell Nuclear Transfer (iSCNT) techniques in marbled cats (Pardofelis marmorata), using domestic cat and rabbit oocytes as the recipient cytoplasm. The recipient oocytes were obtained from ovariohysterectomized cats and superovulated rabbits. The donor cells were collected from a male marbled cat that had died in captivity. Experiment 1 was conducted to observe the development of cloned marbled cat embryos (marbled cat donor cells-domestic cat oocytes; MC-DC), derived from oocytes matured for 24, 36 and 42 h. The result showed that the developmental rates of MC-DC cloned embryos at the 4-8 cell and the morula stages derived from oocytes cultured for 24 h were significantly greater than those cultured for 36 and 42 h (p < 0.05). Experiment 2 was conducted to compare the fusion rate of MC-DC couplets, fused by inducing different fusion voltages, 2.1 or 2.4 kV/cm. The result showed that there was no difference in fusion efficiency between the 2.1 and 2.4 kV/cm fusion protocols. Experiment 3 was conducted to compare the developmental rate of MC-DC and domestic cat (DC-DC) cloned embryos. In vitro fertilized cat embryos served as a control. The development of MC-DC and DC-DC cloned embryos to the 4- to 8-cell, morula and blastocyst stages was not significantly different. However, the development rates at morula and blastocyst stages of control were significantly greater than those of cloned embryos (p < 0.05). Experiment 4 rabbit (RB) oocytes were used as a recipient cytoplasm for marbled cat and domestic cat cloned embryos (MC- RB and DC-RB). RB-RB cloned embryos served as a control. There were no differences in the developmental rates between MC-RB, DC-RB and RB-RB embryos. In conclusion, marbled cat fibroblast cells can be reprogrammed in domestic cat and rabbit oocytes, and by using iSCNT it might be possible to produce marbled cat offspring in the future.

Mouse blastocyst vitrification compared with the conventional slow-freezing method.

To compare mouse blastocyst survival after cryopreservation with vitrification and the slow-freezing method, one-hundred and forty-eight in vitro mouse blastocysts were randomly frozen by the two methods: vitrification and conventional slow-freezing. After being thawed, the blastocysts were assessed for survival and hatching rate. The survival rates of blastocysts cryopreserved by vitrification and slow-freezing were 68.33 and 65.52 per cent (p = 0.89), whereas hatching rates were 51.22 and 44.74 per cent, respectively (p = 0.64). Therefore, vitrification of blastocyst-stage-embryos may be a useful, economic method for freezing the excess blastocysts in some centers where blastocysts are routinely transferred.

Identification of essential subelements in the hHSD17B1 enhancer: difference in function of the enhancer and that of the hHSD17BP1 analog is due to -480C and -486G.

The function of the gene encoding human 17beta-hydroxysteroid dehydrogenase (17HSD) type 1, the hHSD17B1 gene, is regulated by a cell-specific enhancer at position -662 to -392. The adjacent hHSD17BP1 gene, whose function is not known, contains an analogous region in its 5'-flanking region. The identity between the hHSD17B1 enhancer and the hHSD17BP1 equivalent is as high as 98%, i.e. they differ by only five nucleotides. Results from reporter gene analyses showed that the hHSD17BP1 analog, a pseudoenhancer, has only 10% the activity of the hHSD17B1 enhancer. Furthermore, the results indicate that the reduced function of the pseudoenhancer is a consequence of the presence of G and A at positions -480 and -486, whereas the hHSD17B1 enhancer contains -480C and -486G. In addition, three protected areas were localized to regions -495/-485 (FP1), -544/-528 (FP2), and -589/-571 (FP3) in deoxyribonuclease I footprinting analysis of the hHSD17B1 enhancer. Replacement of the footprinted regions with a nonsense sequence demonstrated that the FP2 region is the most critical for enhancer activity. Mutations of FP2 or a short palindromic region within it led to almost complete abolishment of enhancer activity. We have identified several subelements that are essential for appropriate function of the hHSD17B1 enhancer. The results also show that the hHSD17B1 and hHSD17BP1 genes operate differently despite the high homology between them.

Fetal metabolism.

Normal reference ranges for apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, total triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol + chylomicron, plasma glucose, total protein, albumin and globulin were determined from 25 fetal plasma samples between 21-39 weeks' gestation. Pure fetal blood was obtained by cordocentesis under continuous ultrasound guidance. They were referred to us for advanced maternal age and a previous chromosomal aneuploidy baby. All these biochemical parameters excepts total protein and albumin showed no change with gestational age. These normal values of fetal metabolism will improve our knowledge of physiology and help to determine the specific values of a test in fetal pathology.

Estradiol and follicle-stimulating hormone levels in oophorectomized women using vaginal estrogen.

To assess the changing estradiol (E2) and follicle-stimulating hormone (FSH) level in oophorectomized women using vaginal estrogen. Serum estradiol and FSH were evaluated in 32 oophorectomized women using a daily dose of 2 g base of 1.25 mg vaginal conjugated equine estrogen (CEE) cream. The blood sample for hormone assay was collected 8-10 hours from the time of vaginal application. E2 and FSH levels were measured in the serum sample before and after commencing the study at 4, 8 and 12 weeks using the time-resolved fluoroimmunoassay method. Serum estradiol significantly increased from baseline value at 4, 8 and 12 weeks. (Mean +/- SD of E2 value at 0, 4, 8, 12 weeks: 9.97 +/- 12.13, 249.83 +/- 170.46, 299.38 +/- 190.65, 322.82 +/- 218.31 pmol/L, respectively, P < 0.05) On the other hand, serum FSH significantly decreased from baseline value at 4, 8 and 12 weeks. (Mean +/- SD of FSH value at 0, 4, 8, 12 weeks: 77.64 +/- 27.24, 40.33 +/- 21.64, 38.84 +/- 22.33, 30.90 +/- 24.32 IU/L, respectively, P < 0.05) In conclusion, a daily dose of 2 g vaginal CEE cream raised the serum estradiol level close to the normal level in the follicular phase of the normal menstrual cycle. However, even though FSH significantly decreased it did not reach the premenopausal level.