bluuebunny/crossref_metadata_2025 huggingface dataset:
307 TRANSGENIC OVINE EMBRYOS BY ARTIFICIAL INSEMINATION, IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION
Authors: Pereyra-Bonnet F. , Gibbons A. , Cueto M. , Fernández-Martín R. , Salamone D.
Published: December 2008

Nowadays, transgenesis in animals constitutes an important tool for pharmacological protein production and livestock improvement. In 1971 Brackett first described that heterologous DNA can be introduced into a mammalian oocyte using sperm cells as vectors. We evaluated the capacity of AI, IVF and ICSI to produce transgenic embryos, in ovine, using sperm that had been exposed to a pCX-EGFP plasmid in Long and Short incubation protocols. The pCX-EGFP plasmid contains an enhanced green fluorescent protein gene (egfp) under the chimeric cytomegalovirus-IE-chicken β-actin enhancer-promoter control. Sperm/pCX-EGFP incubation was carried out by Long Incubation (2 h at 17°C in 200 μL of SFM medium: 100 mL contains glucose 1.2 g, Na citrate 1.0 g, EDTA 0.4 g, Citric acid 0.3 g, Trizma 0.6 g) and Short Incubation (5 min at 0–5°C in 10–100 μL of extender medium: 100 mL contains Na Citrate 2.8 g and EDTA 4 mg). For AI, Merino sheep (n = 17) were superovulated and inseminated with fresh semen (200 millions sperm/sheep) from eight Merino rams. The embryos were recovered by flushing the uterine horns by standard procedures. In IVF and ICSI, slaughterhouse oocytes were fertilized with frozen/thawed sperm. IVF was carried out in Brackett-Oliphant medium with 5 mm of caffeine, 20 IU mL–1 of heparin with 20 million sperm mL–1 during 5 h in an atmosphere of 5% CO2 in air. In ICSI, the spermatozoon was immobilized by breaking its tail and injected into MII oocytes. Immediately the oocytes were activated by incubation in TALP-HEPES with 5 μm ionomycin for 4 min, cultured in TCM199 for 3 h and transferred to a droplet of 1.9 mm DMAP for 3 h. Maturation and cultivation conditions were determined by standard operating procedures. All embryos were exposed to blue light (488 nm) to determine the percentage of morulae/blastocysts showing green fluorescence. Results are shown in Table 1. Statistical analysis was done by Fisher test. AI and IVF were able to produce a high percentage of morula and blastocyst stage, but were unable to produce transgenic embryos. In contrast, regardless of the sperm/plasmid incubation protocol, high percentages of transgenic morulae and blastocysts were always obtained by ICSI and the highest rate was achieved with Short Incubation (P < 0.05). In order to demonstrate ICSI-Short incubation embryo viability, two-day-old non-selected fluorescence embryos (n = 45), were transferred into the oviducts of five surrogate mothers. Pregnancy was diagnosed at day 25 (2/5; 40%), and one normal female lamb was recently born (1/5; 20%). In conclusion, our results show that in ovine, ICSI seems to be the only method for producing transgenic embryos using sperm cells as vectors. In addition the offspring born confirm the viability of these embryos.

Table 1.Development and fluorescence expression of ovine embryos
308 DEVELOPMENT OF TRANSGENIC LIVESTOCK WITH REDUCED MYOSTATIN EXPRESSION USING RNA INTERFERENCE
Authors: Tessanne K. , Stroud T. , Long C. , Hannon G. , Sadeghieh S. , Hwang E. , Chen S. , Polejaeva I. , Westhusin M.
Published: December 2008

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and siRNAs have been extensively employed for manipulating gene expression in a wide range of species. However, the great majority of this work has involved in vitro studies with cells grown in culture. Our goal for this project is to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. In theory, livestock in which myostatin has been silenced should exhibit increased muscle growth and development. To that end, we designed shRNAs to target the bovine myostatin mRNA sequence. The shRNAs were cloned into a lentiviral vector that contains a cytomegalovirus promoter controlling green fluorescent protein and shRNA expression as well as neomycin resistance. Infective lentivirus was made in HEK293T cells through co-transfection of the lentiviral vector, a packaging plasmid, and a plasmid expressing the VSVG pseudotype. Bovine fetal fibroblasts were transduced, selected using Geneticin®, and nuclear transfer was utilized to produce cloned transgenic embryos. There were 186 fusion attempts resulting in 160 fused embryos (fusion rate = 86%). Of these, 54 reached the blastocyst stage (34%) and 10 embryos were transferred into 5 recipient females (2 embryos per recipient). At 40 days, ultrasound revealed 1 confirmed pregnancy. Current plans are to harvest this fetus at 90 days and analyze it for evidence of myostatin knockdown. The production of transgenic animals exhibiting myostatin knockdown through lentiviral-mediated RNAi will demonstrate the utility of RNAi in the study of gene function in large animal models without the need for homologous recombination techniques, which are currently inefficient in species other than mice.
309 PRODUCTION OF CATTLE BY NUCLEAR TRANSFER FROM CELLS IN WHICH A GENE IS DISRUPTED
Authors: Urakawa M. , Sendai Y. , Ideta A. , Hayama K. , Shinkai Y. , Sawada T. , Hoshi H. , Aoyagi Y.
Published: December 2008

Gene-targeted animals provide a powerful model to examine gene functionality. In this study, we examined the effect of gene targeting of donor cells for nuclear transfer (NT) on the pregnancy rate and on viability of the offspring after embryo transfer. Gene-targeted (tg; targeting of both alleles of the gene encoding bovine prion protein) or non-manipulated (control) bovine fetal fibroblasts were used for NT. A promoterless positive selection vector (pPrP5.2) containing an internal ribosome entry site-antibiotic resistance gene (neo) cassette and loxP sequences was used to disrupt the bovine prion protein gene. The cells (tg) in which homologous recombination was occurred were used for NT. The tg and control cells were cultured in DMEM with 10% FCS and were prepared in the early G1 phase to our previous report (Urakawa M et al. 2004 Theriogenology 62, 714–728). Each donor cell was inserted into an enucleated in vitro-matured (19 h) oocyte. Cell fusion (DC, 200 V mm–1, 10 μs) and activation (DC, 100 V mm–1 , 60 μs) were done in 0.3 m mannitol solution. The NT embryos were then activated with 5 μm Ca-ionophore and 10 μg of mL–1 cycloheximide and were cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were judged at 6 days after NT. The blastocysts were non-surgically transferred to recipient heifers. The recipients were monitored daily for heat behavior, examined by ultrasound at Day 30 and 60, and then observed monthly to confirm pregnancy. The offspring born in the tg group were confirmed by PCR to be transgenic. Statically significance was tested using a chi-square test or t-test. Developmental rate to the blastocyst stage, pregnancy rate at Day 30 and 60, and calving rate did not differ significantly between tg and the control group (Table 1). Gestation length (tg; 290.0 ± 2.2 days v. control; 290.5 ± 3.9 days) and birth weight (tg; 39.6 ± 8.0 kg v. control; 40.2 ± 4.1 kg) were not significantly different. These results indicate that gene targeting of donor cells used for NT does not significantly affect the development of embryos, pregnancy rate, or the viability of the offspring.

Table 1.Development of NT embryos with tg or control cells
310 MARKER-GENE EXCISION IN TRANSGENIC CLONED SOMATIC CELLS AND EFFECTS ON RECLONED EMBRYO DEVELOPMENT IN CATTLE
Authors: Wang S. H. , Sun X. Z. , Ding F. R. , Zhang K. , Zhao R. , Li S. , Li R. , Tang B. , Zhang L. , Li J. , Gao F. L. , Wang H. P. , Wang L. L. , Dai Y. P. , Li N.
Published: December 2008

Recently, it has become common to produce transgenic cattle by somatic cell nuclear transfer. As a major step, donor cells are transfected with expression vectors that contain not only the target gene but also marker genes such as neomycin resistant gene (neoR) used for subsequent cell screening. However, such markers in transgenic animals may be undesirable for further researches (Kuroiwa et al. 2004 Nature Genetics 36, 775–780). Therefore, the aim of this study was to excise the marker gene (neoR) in transgenic cattle by using transiently expressed CRE recombinase. Initially, the presence of loxP sites flanking neoR was confirmed in fibroblast cells derived from human lysozyme transgenic cloned cattle. By using cassettes from plasmids PBS185, pIRES-EGFP and pIREShyg3, IRES-EGFP element was inserted downstream of the CRE coding region, and the co-expression vector, which permits translation of GFP and CRE from one messenger RNA, was constructed. Then transgenic fibroblast cells were transfected by the CRE expression vector in circular format by Lipofectamine™ 2000 and screened by FACS after 48 h. GFP positive cells were selected to culture for 7 to 9 days, and removal of neoR was confirmed by PCR with 2000 to 3000 cells from each colony and the remaining cells used as donors for recloning. Then cleavage and blastocyst rate were evaluated; neoR excision was identified by single blastocyst PCR. Some blastocysts were harvested for differential staining and some used to conduct embryo transfer to evaluate in vitro and in vivo developmental ability. Data were analyzed using SAS (version 9.1) with one-way ANOVA. All experiments were replicated at least three times. We confirmed that loxP sites are correctly located at two sides of neoR. After transfection, neoR was removed from transgenic cloned fibroblast cells and cre was found to be expressed transiently. Additionally, neoR excision was confirmed by single blastocyst PCR. Furthermore, no significant difference was found on cleavage (P = 0.15) and blastocyst rate (P = 0.73) between cloned embryos when using neoR free donors and neoR included donors. The differential staining study showed similar numbers in terms of total cell number (P = 0.69) and the ratio of ICM to total cell number (P = 0.96). After embryo transfer, successful establishment of pregnancies were observed and pregnancy rate on Day 90 and Day 120 (Day 1 is the 1st day after embryo transfer) are similar with control (P > 0.05). In conclusion, we successfully excised resistant marker gene from transgenic cloned cattle by transient expression of CRE recombinase and we expect it will benefit bioreactor and animal transgenic research in the future.
312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME
Authors: Zhou Z. H. , Yan Z. J. , Zhang L. J.
Published: December 2008

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neor) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 105 cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neor and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.
314 EARLY PREGNANCY DIAGNOSIS USING A NOVEL TRANSRECTAL ULTRASONOGRAPHY PROTOCOL IN JAPANESE BLACK COWS
Authors: Gaja A. , Kubota C. , Kojima T.
Published: December 2008

The present study aims to establish a novel practical protocol for early pregnancy diagnosis in cows by using transrectal ultrasonography. The protocol is based on measurements of corpus luteum (CL) cross-sectional area (CL c-s area) change performed at 2 separate days before the coming estrus after AI. Fourteen cows were inseminated artificially, and transrectal ultrasonographical observation of the ovaries and blood collection for measurement of peripheral plasma progesterone (P4) concentration were carried out daily from Days 12 to 23 (Day 0 = the day of onset of estrus). Thereafter, cows were routinely diagnosed for pregnancy at Day 30 by transrectal ultrasonography. The largest CL c-s area was obtained at Day 14 in both pregnant and non-pregnant cows. Seven out of 8 non-pregnant cows showed significant CL c-s area regression between Days 14 and 20 (422 ± 112 v. 249 ± 63 mm2), whereas no regression was observed between Days 14 and 20 in pregnant cows (416 ± 65 v. 402 ± 78 mm2). The regression in the CL c-s area between pregnant and non-pregnant cows was significantly different during Day 18 (424 ± 65 v. 288 ± 88 mm2) to Day 23 (402 ± 71 v. 139 ± 64 mm2). P4 concentration was significantly low (less than 1 ng mL–1) at Day 20 in 3 out of 8 non-pregnant cows, whereas the pregnant cows showed significant increase of P4 between Days 14 and 20 (2.6 ± 0.2 v. 3.4 ± 0.5 ng mL–1). The pregnant cows showed significantly higher P4 concentration starting from Day 18 than non-pregnant cows. However, in non-pregnant cows, 4 cows returned to estrus on Day 20 or after, 3 cows showed no signs of estrus, and 1 cow came in estrus as early as Day 18 after AI. In conclusion, the results of the present study suggest that measuring the change in the CL c-s area at Days 14 and 20 makes it possible to detect the non-pregnant cows at Day 20 after AI. However, it was also indicated that measuring the change of P4 concentrations on the same days did not always successfully detect non-pregnant cows. The new protocol based on CL c-s area regression rate can detect almost certainly non-pregnant cows at Day 20 after AI. It is suggested that this method is advantageous in research and industrial breeding.
316 EFFECT OF ULTRASOUND TRANSDUCER FREQUENCY ON FOLLICLE IDENTIFICATION ACCURACY IN CATTLE
Authors: Tachibana C. , Kabeya S. , Sugulle A. H. , Koyama H. , Singh J. , Dochi O.
Published: December 2008

Ultrasoundsonography (US) is an essential tool for the study of reproductive physiology and is particularly useful in research on ovarian follicular dynamics in cattle. The resolution of the US images obtained, however, differs according to the frequency of the transducer used. The objective of this study was to investigate the effect of transducer frequency on the accuracy of follicle identification in cattle. A Honda HS-2000 sonograph equipped with 5.0, 7.5, and 10.0 MHz of B-mode linear rectal transducers was used in this study. A total of 22 ovaries with corpus luteum were collected from a local slaughterhouse. Each ovary was fixed on a paraffin block, immersed in de-gassed water, the transducer was placed at a distance of 1 cm from the surface, and sequential images from one end to other end of ovary were obtained at a distance of 1 mm. All follicles were aspirated by needle after imaging and were then injected with a pigment after counting by all frequencies. Subsequently, following freezing, 5-mm sections of ovaries were cut and the number of follicles in the sections was counted using a megascope. The follicles were classified into 4 groups according to their diameter: 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, and 11 mm. The number of follicles observed using the megascope was compared with those observed at the 3 US frequencies. The ovaries were classified according to corpus luteum (CL) by determining the condition of the CL. Very red and small-sized CL (stage 1: ovary ovulated within 3 days), elastic and bigger CL (stage 2: ovary ovulated at 4 to 14 days), and regressed CL with yellow color (stage 3: ovary ovulated at 15 to 20 days). The data were analyzed by ANOVA. The results revealed no difference (P > 0.05) in the number of follicles observed in any size category using the US transducers and the megascope. Further, there were no differences between the different transducer frequencies and megascopic observations in terms of the number of follicles ≥4 mm in diameter. The stage of estrus in the ovary also had no effect on the number of follicles observed regardless of follicular size or the US frequency used. In conclusion, these results indicated that follicles could be counted accurately in excised ovaries using 5.0-, 7.5-, and 10.0-MHz transducers.

Table 1.The average number of follicles in different frequencies and megascopic (n = 22)
33 REPRODUCTIVE CHARACTERISTICS OF CLONED DOGS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER
Authors: Hong S. G. , Park J. E. , Kang J. T. , Oh H. J. , Kim M. J. , Kim D. Y. , Kim M. K. , Jang G. , Lee B. C.
Published: December 2008

Several cloned dogs have been successfully produced by somatic cell nuclear transfer. However, there is no investigation of its reproductive characteristics including changes of hormone and ovarian follicles in cloned dogs. Thus, this study was to examine the onset of puberty, follicular dynamics, and reproductive hormone profiles in cloned beagle dogs. Two female cloned beagles, derived from fetal fibroblasts, were compared to 2 individual age- and weight-matched female beagles produced by natural breeding. All dogs were examined twice weekly from 6 months for the presence of swelling of the vulva and serosanguineous vaginal discharge, which were used as markers of the onset of proestrus. From the first day of proestrus, jugular venous blood samples were collected twice a day for hormonal analysis. Also, from the day when progesterone concentration was >1 ng mL–1 until 5 days after ovulation, blood sampling was carried out 3 times a day. Ultrasound examination of ovaries and uterus was performed daily from the onset of proestrous until 5th day post-ovulation. Cloned female dogs (332.5 ± 1.5 days) reached puberty later than controls (300 ± 8 days). The mean number of ovarian follicles was 4.5 ± 0.5 v. 6.5 ± 0.5 for clones and controls, respectively. The largest size of ovarian follicles in clones and controls detected by ultrasound were 0.6 ± 0.03 v. 0.69 ± 0.07 cm, respectively. It took 8 days from the initiation of vulvar bleeding to the LH peak in all beagles. Moreover, there were no differences in the profile of hormonal changes (LH, FSH, estradiol, and progesterone) in cloned and control beagles. These results demonstrate that cloned dogs have normal development of reproduction.

This study was financially supported by the Korean MEST, through KOSEF (grant # M10625030005-08N250300510) and the BK21 program for Veterinary Science.
45 DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM ADULT MARROW STROMAL CELLS AND FETAL MUSCLE CELLS
Authors: Murakami M. , Bai X. J. , Shi W. S. , Wang W. M. , Liu W. , Dong Y. J.
Published: December 2008

The use of less differentiated cells, such as marrow stromal cells (MSCs), as the nuclear donor may increase the efficiencies of somatic cell cloning in cattle. Healthy offspring was produced from bovine MSCs (Kato et al. 2004 Biol. Reprod. 70, 415–418); however, there is little information that directly compared the post-implantation survival among the clones originated from MSCs and other somatic cells. The objective of this study was to evaluate the developmental potential in vitro and in vivo of bovine NT embryos derived from adult MSCs and fetal muscle cells (FMCs). Primary cell populations of MSCs and FMCs were obtained from the femurs of 8- and 12-months-old Holstein cows (MSC1 and MSC2 groups, respectively) and a Holstein fetus at 8 months of gestation (FMC group), respectively. They were used as donor cells for the NT procedure (Murakami et al. 2005 Cloning Stem Cells 7, 77–81) at passages 1 to 3. Briefly, oocytes collected from cow ovaries were enucleated at 20 h post-in vitro maturation (IVM), and the donor cell was placed into the perivitelline space. The couplets were fused electrically, activated (10 μg mL–1 cycloheximide; 4 h), and cultured in CR1aa medium. Development in vitro of these embryos is summarized in Table 1. Data were analyzed by ANOVA. The fusion rates were higher in the MSC groups than in the FMC group. The rate of cleaved embryos was significantly lower (P < 0.05) in the MSC1 group than in the other groups. However, there were no significant differences among the groups in the rates of development into morulae/blastocysts on Day 6. A total of 8 and 3 fresh good quality Day 6 embryos in the MSC1 and FMC groups, respectively, were nonsurgically transferred to 6 naturally cycling Holstein females 6 days after estrus (3 recipients/group, 1–3 embryos/female). On Day 30 of gestation, none of the recipients were pregnant in the FMC group, while 2 recipients in the MSC1 group were diagnosed as pregnant via ultrasonography; they remained pregnant on Day 80 of gestation. In addition, a total of 4 Day 7 embryos cryopreserved in 1.8 m ethylene glycol plus 0.05 m trehalose were directly transferred to 4 synchronized recipients after thawing (1 embryo/female) in the MSC1 group. Of those, 2 females were pregnant on day 30 of gestation. These results indicate that the developmental potential in vitro of bovine NT embryos derived from adult MSCs was comparable to that of the embryos derived from fetal muscle cells, and that pregnancies were produced after transfer of the fresh and frozen–thawed NT embryos derived from the MSC, but the sample size was small. Further studies with more replicates are needed to evaluate viability in vivo of these cloned embryos for comparative purposes.

Table 1.Development in vitro of bovine NT embryos derived from different cell types
57 NUCLEAR TRANSFER WITH RABBIT EMBRYONIC STEM CELLS: SERUM-STARVATION IMPROVES THE QUALITY OF CLONED EMBRYOS
Authors: Zakhartchenko V. , Flisikovska F. , Hao R. , Li S. , Kind A. , Wolf E. , Schnieke A.
Published: December 2008

Rabbit cloning by NT with somatic cells is so far a rather inefficient process. However, this technology is urgently required to generate rabbits with a humanized immune system as a source of human polyclonal antibodies. Embryonic stem cells (ESCs) have a number of advantages over somatic cells as tools for cell-mediated transgenesis including long periods of proliferation in vitro, higher frequency of homologous recombination between exogenous and chromosomal DNA, and less requirements for reprogramming (Rideout et al. 2000 Nat. Genet. 24, 109–110). To improve rabbit cloning we have derived and characterized 19 putative rabbit ESC lines and tested cells from 6 lines as donors for NT. First, we assessed in vitro development of NT embryos. Blastocyst rates varied in the range of 6–68% depending on the particular cell line and passage number, but the quality of the resultant embryos was worse compared to NT embryos derived from adult fibroblasts [hatched blastocysts: 13/214 (6%) v. 36/86 (42%), respectively]. Transfer of NT embryos derived from the ESC line showing the highest development to blastocysts into recipients resulted only in implantations (70%, 7/10) but not in offspring. Assuming that poor quality of NT embryos derived from ESCs could be due to the incompatibility between cell cycles of donor and recipient cells we used serum starvation to make ESCs more suitable for nuclear transfer. Serum starvation of one of the ESC lines (0.5% FCS for 3 days) greatly improved the quality of cloned embryos compared to those derived from non-starved cells of the same ESC line as indicated by the high proportions of hatched [38/151 (25%) v. 4/153 (3%)] and attached [25/151 (17%) v. 0%] to the surface of a culture dish blastocysts. Moreover, some of these blastocysts grew in vitro for 14–25 days. Our study provides evidence that the quality of NT embryos derived from ESCs can be significantly improved using serum starvation of donor cells suggesting possible effect of this treatment on the cell cycle synchronization. We are currently testing whether serum starvation of ESCs would also improve post-implantation development of rabbit NT embryos.

This work is supported by Roche Diagnostic GmbH.
71 COMPARISON OF CRYOPROTECTANTS ON CRYOPRESERVATION OF VENDA COCK SPERMATOZOA
Authors: Mphaphathi M. L. , Raito M. B. , Makhafola M. B. , Luseba D. , Nedambale T. L.
Published: December 2008

Improving the cryopreservation technique for indigenous fowl semen may contribute to the development of cryogene banks in South Africa. The goal was to identify a cryoprotectant among dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (PND) that is compatible with survival after freezing of Venda cock spermatozoa. Six Venda cocks were used for semen collection. The abdominal massaging technique was applied for semen collection from cocks. Individual ejaculates were diluted with modified Kobidil+ (mK+) extender (extender A) at ratio of 1:2 (v/v) before freezing, and equilibrated for 2 h. Semen was diluted again at a ratio of 1:1 (v/v) with mK+ plus 8% DMSO, EG, and PND (extender B) and equilibrated for 2 h at 5°C. Semen were then transferred into 0.25-mL plastic straws and placed into a programmable freezer (Planer Kryosave). The temperature of the chamber was decreased in a stepwise manner, from 5°C at a rate of 1°C min–1 until it reached the target temperature of –20°C. Finally, the straws were exposed to liquid nitrogen (LN2) vapor and plunged into LN2 (–196°C). The semen straws were stored in an LN2 tank at –196°C. After 1 week, frozen semen straws were thawed at 5°C for evaluation of spermatozoa survivability and motility rate at 0, 30, 60, and 90 min, using contrast microscopy (20× magnification). Data were analyzed by ANOVA. Spermatozoa live and motility rates were greater before freezing (Table 1) in all groups. There was no significance difference between DMSO and EG with regard to live and motility rates. However, the lowest rates of live and motility spermatozoa were recorded in the PND group. In conclusion, this study demonstrated that the cryopreservation process reduces sperm quality and propanediol was not suitable for cryopreserving Venda cock spermatozoa.

Table 1.Comparison of three CPA on cryopreservation of Venda cock semen

This study was funded by the South African National Department of Agriculture, ARC, DST-PDP (RT19000), and National Research Foundation (NRF, Grant. no. RT21 and 24000).
85 DEOXYRIBONUCLEIC ACID METHYLATION IN PORCINE PARTHENOGENETIC PREIMPLANTATION EMBRYOS
Authors: Deshmukh R. S. , Svarcova O. , Li J. , Callesen H. , Vajta G. , Maddox-Hyttel P.
Published: December 2008

DNA methylation is one of the most important epigenetic mechanisms involved in gene silencing. Waves of DNA de- and re-methylation occur during mammalian preimplantation development. Whether the same happens in porcine parthenogenetic embryos has never been determined, and we set out to investigate this question. Porcine oocytes were aspirated from antral follicles, matured in vitro for 42 h, parthenogenetically activated and fixed in 4% paraformaldehyde at the 1-, 2-, 4-, and 8-cell stage as well as at the early and late blastocyst stage. The degree of DNA methylation was assessed by immunocytochemical staining (Anti-5MetC mouse monoclonal; Abcam, Copenhagen, Denmark) and DNA was counterstained with Hoechst 33258. Porcine fetal fibroblasts were used as standard. The fluorescent signals were detected using an epifluorescence microscope (Leica) and a Leica camera set at fixed exposure times. Signals were quantified using NIH ImageJ sofware. Total means of intensities (methylation and DNA) were calculated and exponential curves were obtained using Microsoft Excel-based statistics. DNA methylation and DNA content were highly correlated in porcine fetal fibroblasts demonstrating the effect of an immediate maintenance methylation taking place during the DNA S-phase of the cell cycle. A similar correlation was observed in the parthenogenetic embryos at all the developmental stages. The level of DNA methylation increased slightly from the early to the late 1-cell stage, and a pronounced increase in DNA methylation level was noted at the 2-cell stage. At the late 1-cell stage, the DNA methylation level of the two pronuclei was similar probably due to the maternal origin of both pronuclei. At the 4-cell stage, DNA methylation had decreased again but was higher compared with other developmental stages, except the 2-cell stage, and at the 8-cell stage, the level of DNA methylation reached a minimum. Subsequently, the level of DNA methylation increased slightly at the blastocyst stages. In conclusion, DNA methylation and DNA content were correlated in porcine fetal fibroblasts and parthenogenetic embryos. Furthermore, the levels of DNA methylation in parthenogenetic embryos exhibited an increase to the 2-cell stage followed by a decrease to the 8-cell stage and a final increase to the blastocyst stage. The initial increase in methylation to the 2-cell stage is different from what has been reported previously for in vivo derived porcine embryos.

We are thankful to H. Holm and J. Nielsen for excellent technical assistance. This project was supported by Marie Currie Action project MRTN-CT-2006-35468 (CLONET).
9 BIRTH OF CALVES AFTER ARTIFICIAL INSEMINATION WITH CRYOPRESERVED BOVINE EPIDIDYMAL SPERMATOZOA HARVESTED FROM POSTMORTEM BULLS
Authors: Guerrero C. A. , Gentry G. , Saenz J. , Bondioli K. R. , Godke R. A.
Published: December 2008

Recently, interest in the preservation of epididymal sperm as a potential source of valuable genes for genome resource banks has increased. The ability to recover and cryopreserve postmortem epididymal sperm will allow the use of valuable gametes from a breeding male that dies unexpectedly. The objective of this study was to produce pregnancies and live births from AI by using frozen–thawed bovine caudal epididymal sperm. Paired testes were obtained from mature, mixed-breed beef bulls (n = 3) from a local abattoir and transported to the laboratory (within 5 h postmortem) in a Styrofoam box at a temperature that ranged between 28 and 29°C. The mean ± SEM weights of the pair of testes and caudae epididymides were 345 ± 9g and 10 ± 2g, respectively. Epididymal sperm from each bull were harvested by multiple incisions from the caudae epididymides, pooled, and then rinsed with 4 mL of egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC). Harvested sperm samples were placed in a refrigerator at 4°C for 4 h before the addition of cryoprotectant. Samples were diluted slowly over a period of 30 min 1:1 in EYT-GC extender containing 14% glycerol to obtain a final concentration of 7% glycerol. Sperm concentration was adjusted to 35 million/straw and loaded into previously cooled 0.5-mL plastic straws. Straws were placed 2 cm above liquid nitrogen (LN2) vapors for 10 min and then plunged into LN2. Sperm were stored for 1 year before thawing. Luteal-phase crossbred beef cows were synchronized with a single 25-mg injection (i.m.) of prostaglandin, and those displaying standing estrus (n = 6) were each inseminated with 1 straw by a single technician from 1 bull 12 to 18 h after the onset of standing estrus. Straws were thawed for AI in a 37°C water bath for 40 s. Three of the 6 cows inseminated (50%) were diagnosed as pregnant by transrectal ultrasonography at 45 days post-AI. All 3 pregnant cows (100%) delivered healthy singleton calves (2 males and 1 female, with an average birth weight of 37 ± 2.5 kg), resulting in a mean gestation length of 286 ± 1.9 days (range: 282–288 days). We can conclude that epididymal sperm can be extracted by 5 h postmortem from bull testes, frozen, and subsequently used for the production of live offspring from AI. Further research is needed to improve this technology to optimize the utilization of valuable bovine male gametes.
91 INHIBITION OF TELOMERASE REVERSE TRANSCRIPTASE (TERT) INDUCES EMBRYO ARREST IN BOVINE PRE-IMPLANTATION EMBRYOS
Authors: Madan P. , King W. A. , Betts D. H.
Published: December 2008

Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. The objective of this study was to characterize the expression and function of the bovine telomerase reverse transcriptase (bTERT) subunit during bovine pre-implantation embryogenesis. Using RT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in pre-implantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and exhibited 94% sequence homology to the human hTERT cDNA sequence. In immature oocytes, the bTERT protein was localized within the germinal vesicle and after 18 to 20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctate staining, whereas others exhibited a more-uniform staining pattern within the cytoplasm. By the 8- to 16-cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, while in morulae and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 4′,6-diamidino-2-phenylindole (DAPI) staining. Treatment of zygotes with Telomerase Inhibitor III (telomere mimic that inhibits telomerase activity; Calbiochem, San Diego, CA) significantly induced embryos to permanently arrest in a senescence-like state at the 2- to 4-cell cleavage stage compared with nontreated controls. These arrested embryos showed abnormal nuclear phenotypes (elongated chromosomes, anaphase bridging) and phosphorylated histone γ-H2A.X foci suggesting a critical role(s) of TERT in chromosomal segregation and telomere integrity in early embryos. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the known telomere elongation event, whereas low levels of TERT at the 2- to 4-cell stage are associated with the previously documented state of permanent embryo arrest. Future studies would be directed toward understanding the relationship between TERT expression, telomere dysfunction, and permanent embryo arrest.

Operating grants from the Canadian Institutes of Health Research (CIHR) to D.H.B. and W.A.K. supported this research.
93 MICRORNA SEQUENCES OF BULL SPERMATOZOA
Authors: Robertson L. R. , Feugang J. M. , Rodriguez-Osorio N. , Kaya A. , Memili E.
Published: December 2008

MicroRNA sequences (miRNA) are small RNAs 19 to 22 nucleotides in length that play a crucial role during mammalian development and disease. They are found in plants and animals and regulate the expression of protein-coding genes. Mechanisms regulating gene expression during gamete and embryo development are critical for setting the stage in later development; however, very little is known about the roles of miRNAs in male gametes (spermatozoa). Therefore, the objective of this study was to profile miRNA populations in spermatozoa collected from high and low fertility bulls. Frozen–thawed straws containing spermatozoa from one low and one high fertility bulls were purified using a Percoll gradient (which removes somatic cells) followed by 3 washing steps in PBS. Total RNA were isolated from pelleted spermatozoa using Trizol (3 independent isolations for each bull, total of 6 samples). RNA samples were quantified (NanoDrop Spectrophotometer, Thermo Scientific, Wilmington, DE) and verified for potential somatic cells (Agilent BioAnalyzer, Agilent, Foster City, CA) and DNA (No-RT-PCR) contaminations. Samples devoid of any contaminations went through an enrichment process of miRNA, consisting of dephosphorylation (CIP) and labeling (pCp-Biotin) of the 3′-end of miRNA, followed by their purification (BioSpin6, Bio-Rad, Hercules, CA). The purified miRNAs were analyzed by a miRNA microarray containing approximately 30 000 probes from human, rat, mouse, and other organisms (Ambion/Affymetrix DiscovArray, Asuragen, Austin, TX). The results showed that roughly 48% of total miRNA probe sets (14 215) were successfully analyzed (P < 0.05); of which only 0.5% (7) were significantly differentially expressed between the 2 bulls (fold change >2 and t-test P < 0.001). The roles of these miRNAs are not yet identified because the annotations are unavailable. Nevertheless, our preliminary results suggest that bull spermatozoa are rich in miRNAs, which could play important roles during fertilization and embryo development. Future studies will be aimed at identifying potential molecular mechanisms by which these 7 miRNAs might regulate bovine embryonic development.

This study was funded by the Mississippi Agricultural and Forestry Experiment Station and Alta Genetics Inc.
Receptor noise limitations on chemotactic sensing
Authors: Rappel Wouter-Jan , Levine Herbert
Published: December 2008

Chemotactic eukaryotic cells are able to detect chemoattractant gradients that are both shallow and have a low background concentration. Under these conditions, the noise in the number of bound receptors can be significant and needs to be taken into account in determining the directional sensing process. Here, we quantify numerically the number of bound receptors on the membrane of a disk-shaped cell by using a numerical Monte Carlo tool. The obtained time traces of the receptor occupancy can be used as inputs for any directional sensing model. We investigate the response of the local excitation global inhibition model and a recently developed balanced inactivation model. We determine a measure for the motility of the cell for each model, based on the relevant output variable, as a function of experimental parameters, resulting in several experimentally testable predictions. Furthermore, we show that these two models behave in a qualitatively different fashion when the background concentration is varied. Thus, to properly characterize the sensitivity of cells to receptor occupancy, it is not sufficient to examine the input signal. Rather, one needs to take into account the response of the second messenger pathway.
The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing
Authors: Nguyen Hanh H. , Aronchik Ida , Brar Gloria A. , Nguyen David H. H. , Bjeldanes Leonard F. , Firestone Gary L.
Published: December 2008

Indole-3-carbinol (I3C), a naturally occurring component of
Brassica
vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G
1
cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive lower-molecular-mass 35-kDa form that is associated with cancer cell proliferation and poor clinical outcomes to the 50-kDa cyclin E form that typically is expressed in normal mammary tissue. An in vitro cyclin E processing assay, in combination with zymography, demonstrated that I3C, but not its natural dimer, 3,3′-diindolylmethane, disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct inhibition of human neutrophil elastase enzymatic activity. Analysis of elastase enzyme kinetics using either cyclin E or
N
-methoxysuccinyl-Ala-Ala-Pro-Val-
p
-nitroanalide as substrates demonstrated that I3C acts as a noncompetitive inhibitor of elastase activity with an inhibitory constant of ≈12 μM. Finally, siRNA ablation of neutrophil elastase protein production in MDA-MB-231 cells mimicked the I3C-disrupted processing of the 50-kDa cyclin E protein and the indole-induced cell-cycle arrest. Taken together, our results demonstrate that elastase is the first identified specific target protein for I3C and that the direct I3C inhibition of elastase enzymatic activity implicates the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.
Expanding metabolism for biosynthesis of nonnatural alcohols
Authors: Zhang Kechun , Sawaya Michael R. , Eisenberg David S. , Liao James C.
Published: December 2008

Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into
Escherichia coli
, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (
S
)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.
Cells of adult brain germinal zone have properties akin to hair cells and can be used to replace inner ear sensory cells after damage
Authors: Wei Dongguang , Levic Snezana , Nie Liping , Gao Wei-qiang , Petit Christine , Jones Edward G. , Yamoah Ebenezer N.
Published: December 2008

Auditory hair cell defect is a major cause of hearing impairment, often leading to spiral ganglia neuron (SGN) degeneration. The cell loss that follows is irreversible in mammals, because inner ear hair cells (HCs) have a limited capacity to regenerate. Here, we report that in the adult brain of both rodents and humans, the ependymal layer of the lateral ventricle contains cells with proliferative potential, which share morphological and functional characteristics with HCs. In addition, putative neural stem cells (NSCs) from the subventricular zone of the lateral ventricle can differentiate into functional SGNs. Also important, the NSCs can incorporate into the sensory epithelia, demonstrating their therapeutic potential. We assert that NSCs and edendymal cells can undergo an epigenetic functional switch to assume functional characteristics of HCs and SGNs. This study suggests that the functional plasticity of renewable cells and conditions that promote functional reprogramming can be used for cell therapy in the auditory setting.
Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ
Authors: Eltsov Mikhail , MacLellan Kirsty M. , Maeshima Kazuhiro , Frangakis Achilleas S. , Dubochet Jacques
Published: December 2008

Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.
Amino acids with an intermolecular proton bond as proton storage site in bacteriorhodopsin
Authors: Phatak Prasad , Ghosh Nilanjan , Yu Haibo , Cui Qiang , Elstner Marcus
Published: December 2008

The positions of protons are not available in most high-resolution structural data of biomolecules, thus the identity of proton storage sites in biomolecules that transport proton is generally difficult to determine unambiguously. Using combined quantum mechanical/molecular mechanical computations, we demonstrate that a pair of conserved glutamate residues (Glu 194/204) bonded by a delocalized proton is the proton release group that has been long sought in the proton pump, bacteriorhodopsin. This model is consistent with all available experimental structural and infrared data for both the wild-type bacteriorhodopsin and several mutants. In particular, the continuum infrared band in the 1,800- to 2,000-cm
−1
region is shown to arise due to the partially delocalized nature of the proton between the glutamates in the wild-type bacteriorhodopsin; alternations in the flexibility of the glutamates and electrostatic nature of nearby residues in various mutants modulate the degree of proton delocalization and therefore intensity of the continuum band. The strong hydrogen bond between Glu 194/204 also significantly shifts the carboxylate stretches of these residues well <1,700 cm
−1
, which explains why carboxylate spectral shift was not observed experimentally in the typical >1,700-cm
−1
region upon proton release. By contrast, simulations with the proton restrained on the nearby water cluster, as proposed by several recent studies [see, for example, Garezarek K, Gerwert K (2006) Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy.
Nature
439:109], led to significant structural deviations from available X-ray structures. This study establishes a biological function for strong, low-barrier hydrogen bonds.