Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes.

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum-fed donor cells. There were no significant differences (P > 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10,11, and 13 (7, 11.5, and 14%, respectively, P < 0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium.

BACKGROUND: Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. RESULTS: Analysis of the cell cycle stages revealed that 91.2 +/- 0.2% of confluent cells were at the G0/G1 phase and 54.1 +/- 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. CONCLUSIONS: Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

Comparison by restriction fragment differential display RT-PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serum.

A novel restriction fragment differential display (RFDD) RT-PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT-PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re-amplified. Re-amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re-amplified successfully as evidenced by size similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re-amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A-I, bovine mitochondrion DNA, human CGI-79 mRNA, human Ab1-interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT-PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes.

Acute acetaminophen toxicity in transgenic mice with elevated hepatic glutathione.

Previous studies demonstrated that elevation of hepatic glutathione (GSH) concentrations protect against acetaminophen (APAP) hepatotoxicity in mice. Employing transgenic mice overexpressing glutathione synthetase, this study was conducted to determine if sustained elevation of hepatic GSH concentrations could ameliorate or prevent APAP toxicity. International Cancer Research transgenic mouse males and matched (ie same strain, sex, and age) control nontransgenic mice were pretreated ip with GSH synthetase substrate gamma-glutamylcysteinyl ethyl ester (gamma-GCE) or with saline. After a 16-h fast, mice received a single dose of 500 mg APAP/kg bw in saline ip and were sacrificed 4 h later. Other mice similarly pretreated were killed without APAP challenge. The elevated GSH concentrations in transgenic mice livers did not lessen APAP hepatotoxicity. Instead higher degrees of hepatotoxicity and nephrotoxicity were observed in transgenic mice than in controls as indicated by higher serum alanine aminotransferase activity and more severe histopathological lesions in transgenic mice livers and kidneys. Pretreatment with gamma-GCE did not affect either initial or post-APAP treatment tissue GSH concentrations or observed degrees of toxicity. Detection of a higher level of serum APAP in transgenic mice and the histopathological lesions found in transgenic mice kidneys together with no observable nephrotoxicity in control mice indicated early kidney damage in transgenic mice. Our findings suggest that high levels of GSH-APAP conjugates resulting from increased GSH concentrations in the livers of transgenic mice caused rapid kidney damage. Compromised excretory ability may have caused retention of APAP, which, in effect, elicited higher hepatotoxicity than that observed in nontransgenic mice.

Developmental patterns of zygotes from transgenic female mice with elevated tissue glutathione.

Zygotes were collected from transgenic mice overexpressing glutathione synthetase to test the hypothesis that such zygotes are more capable of developing in suboptimal culture media than zygotes from non-transgenic (control) mice. The effects of injection of donor mice with gamma-glutamylcysteinyl ethyl ester (gamma-GCE) on embryonic development were also investigated. In addition, the effects of genetic background (i.e., transgenic vs. non-transgenic) and injection with gamma-GCE on developmental capacity in kSOM were studied after exposure of zygotes to diamide (0.01 mM, for 30 min). When cultured in modified Medium 16 significantly more pronuclear ova from transgenic females reached the morula (M) and early blastocyst (EB) stages than embryos derived from control mice. Genetic background significantly affected the proportions of embryos reaching 4- to 16-cell, M and EB stages during culture in modified Whitten's medium (WM); more zygotes collected from transgenic than from control mice developed. The injection of experimental mice with gamma-GCE significantly increased proportions of zygotes developing to M and EB stages in WM. Following exposure to diamide and subsequent culture in kSOM significantly more zygotes collected from transgenic mice reached the 4- to 16-cell stages than those from control females; a significant positive effect on developmental capacity was also seen after injection of donor mice with gamma-GCE. When cultured in suboptimal conditions, zygotes derived from transgenic mice overexpressing glutathione synthetase were more capable of developing than zygotes of non-transgenic control females. Zygotes from the transgenic mice also exhibited greater capacity to withstand toxic exposure to diamide. Present data suggest that commonly known strain differences in preimplantational development in vitro may reflect differences in the synthesis and/or metabolism of glutathione. J. Exp. Zool. 286:173-180, 2000.

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs.

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n = 6), day 1-2; II (n = 5), day 6-7; III (n = 5), day 11-12; and IV (n = 6), day 18-20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n = 5), day 35-40; II (n = 5), day 65-70; and III (n = 4), day 95-105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P < 0.01) in groups II and III (19.3 +/- 2.5 and 35.8 +/- 2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3 +/- 1.4 and 7.5 +/- 0.7 fmol/mg protein, respectively). Receptor affinity constants (Ka) were consistent (P > 0.05) throughout the estrous cycle [I, (5.1 +/- 1.5) x 10(9); II, (3.0 +/- 0.8) x 10(9); III, (3.2 +/- 0.9) x 10(9); IV, 5.5 +/- 0.7 x 10(9) 1m-1]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P < 0.05) in group II (85.4 +/- 18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8 +/- 2.3 and 26.7 +/- 6.6 fmol/mg protein, respectively. The Ka in group I was 10 times greater (P < 0.05) than Ka in groups II and III, (I, 3.1 +/- 0.9 x 10(10) lm-1; II, 3.4 +/- 0.3 x 10(9) lm-1; III, 3.3 +/- 1.1 x 10(9) lm-1). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.

Extragonadal gonadotropin receptors, their distribution and function.

Luteinizing hormone (LH) and a second pituitary gonadotropin, follicle-stimulating hormone (FSH), together control steroid secretion and gamete development in both males and females. LH and its agonist human chorionic gonadotropin (hCG), share a common receptor in gonadal cells. There is increasing evidence for the existence of extraovarian LH/hCG binding sites in mammalian females. High affinity, low capacity LH/hCG receptors have been detected in pigs, rabbits and rats. Gonadotropin receptors in the human uterus were also demonstrated using immunocytochemical techniques. The presence of LH/hCG receptors, both in the endometrium and myometrium, has been so far found in pigs and humans. Receptors in the endometrium are present in glandular and luminal epithelium and stromal cells. In the myometrium, they are present in circular and longitudinal myometrial smooth muscle and vascular smooth muscle. Recently, LH/hCG receptor gene expression was confirmed both in the endometrium and myometrium of the pig. The amount of receptors is higher in the luteal phase as compared to the follicular phase of the estrous cycle indicating possible regulation of these receptors by ovarian hormones. Treatment with estradiol benzoate increased the number of LH/hCG binding sites compared with ovariectomized gilts receiving corn oil. However, administration of progesterone caused an elevation of these receptors, when compared with estradiol. Combined administration of estradiol and progesterone increased receptor capacity similar to progesterone alone. It seems that there are species differences in the uterine binding response to estradiol since the positive response to estradiol by uterine receptors in the ovariectomized pig is quite different from that observed in rabbits and rats. In the endometrium, hCG and/or LH may regulate glandular and luminal epithelial cell function via cAMP modulation or by increasing the local synthesis of steroid hormones. Stimulation of LH receptors with hCG in estrogen primed ovariectomized gilts had a quiescent effect on myometrial contractility in vitro. We examined also the effect of hCG on electromyographic activities of the uterus in ovariectomized and estrogen treated pigs. The hCG treatment caused a significant reduction of total duration of electrical activity and mean burst duration. Based on our earlier and recent results we suggest that the role of LH/hCG receptors in the myometrium is the regulation of uterine contractility, though the second messenger system remains to be elucidated.

Effect of estradiol and progesterone on myometrial LH/hCG receptors in pigs.

High affinity luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors have been identified in porcine, rabbit and rat uteri and immunocytochemically demonstrated in the human uterus. We have now assessed the effect of estradiol and progesterone on the capacity and affinity of LH/hCG binding sites in crude membrane fractions of porcine myometria. Nineteen cross-bred gilts were ovariectomized at 6-7 months of age. Five weeks later, the experiment was conducted and gilts were given estradiol benzoate 2 mg (N = 5), progesterone 50 mg (N = 4) and 2 mg of estradiol benzoate plus 50 mg of progesterone in 2 ml of corn oil (N = 6), im for five consecutive days. Controls (N = 4) received 2 ml of the vehicle. Gilts were hysterectomized 24 h after the last injection. Blood samples for assays of LH, estradiol and progesterone were collected 1 h before hysterectomy. The numbers and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. The results indicate that treatment with estradiol benzoate increases (p less than 0.01) the number of LH/hCG binding sites compared with gilts receiving corn oil. Progesterone treatment caused elevation in the number of LH/hCG receptors (p less than 0.05), when compared with estradiol alone (2.9 +/- 0.3 vs 1.2 +/- 0.1 fmol/mg protein, respectively). Combined administration of estradiol and progesterone increased receptor capacity to 2.7 +/- 0.4. Steroid treatment did not alter the affinity (Ka) of [125I]hCG binding to receptors and it varied from 1.8 +/- 0.8 to 2.9 +/- 0.2 x 10(11) l/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenicity of bovine milk xanthine oxidase in guinea pigs.

Six female and four male albino guinea pigs were immunized with active purified xanthine oxidase of bovine milk mixed with equal volume of Freund's complete adjuvant. One male and one female were immunized with heat-inactivated xanthine oxidase mixed with equal volume of adjuvant. Two males and four females were controls and received a phosphate buffer mixed with equal volume of adjuvant. The mixtures were administered intradermally and subcutaneously at weekly intervals for 6 consecutive wk and blood samples collected weekly. The enzyme was antigenic by the coated tanned red blood cell method. After the third weekly immunization, precipitating antibodies were in the sera of animals that received the active enzyme. Hemagglutination titers increased during subsequent weeks and reached a maximum after the sixth weekly immunization. Antisera from animals immunized with heat-inactivated xanthine oxidase gave a positive response similar to that with animals immunized with the active enzyme. However, when the same antisera were tested with sheep red blood cells coated with heat-inactivated enzyme, no hemagglutination was observed. Ouchterlony double gel-diffusion tests showed that it may be possible to differentiate between antibodies elicited to active and heat-inactivated xanthine oxidase.