C.D. Nancarrow

Co‐culture, oviduct secretion and the function of oviduct‐specific glycoproteins.

Co‐cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co‐cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.

The effects of an estrus-associated oviductal glycoprotein on the in vitro fertilization and development of ovine oocytes matured in vitro

Abstract An estrus-associated glycoprotein (oEGP) secreted by the ampulla of the ovine oviduct for 3 to 6 d around the time of estrus is known to bind to the zona pellucida of newly ovulated oocytes, as well as embryos but does not appear to bind to spermatozoa. The objective of this paper was to establish whether or not the association of oEGP with the zona pellucida influences fertilization and subsequent embryo development. Four separate experiments were carried out to examine the specific effects of different concentrations (0, 2.5, 5.0, 10.0, 20.0% v/v) of an oEGP-enriched fraction of oviducial fluid (F1) in in vitro fertilization (IVF) medium. This medium was synthetic oviduct fluid (SOF) containing either no serum or supplemented with human serum (HS) or sheep serum. Fertilization did not occur in the absence of serum, while both cleavage rate and the developmental competence of embryos were significantly greater in SOF + sheep serum than in SOF + HS. The presence of F1 in the HS-containing medium significantly improved cleavage rates. The specificity of this effect was confirmed in a study which substituted F1 with a comparable concentration of BSA. In the IVF medium (SOF + sheep serum), the addition of F1 at a concentration of 10% significantly (P

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-aceB gene-ovine growth hormone (GH) gene (3′ GH sequence) construct was fused to the ovine MT-Ia promoter-aceA gene-ovine GH gene (3′ GH sequence). Therefore, in this single DNA sequence, bothaceA andaceB are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of theaceB-aceA gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressedEscherichia coli cells.

The effects of an ovine oviductal estrus-associated glycoprotein on early embryo development

Abstract Oviduct specific estrus-associated glycoproteins (EGP) secreted around the time of estrus are known to be present in several species. Many of these glycoproteins, including that of the sheep (oEGP), have been shown to bind to the zona pellucida and to become associated with individual blastomere membranes, indicating a role for these proteins in early embryo development. Given that the culture of ovine embryos can result in a number of developmental abnormalities as well as a reduction in viability, the present study examined the effects of oviducal fluid and gel filtration fractions of oviducal fluid on the in vitro development of ovine embryos. The first fraction of oviducal fluid (F1) contained predominantly oEGP, while the second (F2) contained albumin and was included in the study as a control. The culture medium was synthetic oviduct fluid (SOF) supplemented with 20% human serum (HS). The study consisted of 2 experiments; the first examined the effects of oviducal fluid, F1 and F2 in combination with 0, 10 and 20% HS; while the second, which aimed to investigate the effect of F1 during the first 72 h of culture, examined the influence of oviducal fluid and the fractions in the presence of either a low concentration of serum or no serum for the first 72 h. The presence of Fl significantly decreased the proportion of zygotes undergoing first cleavage, increased the mean number of nuclei per blastocyst and increased the mean time taken for blastocyst formation. Unfractionated oviducal fluid appeared to be detrimental to embryo development while F2 had no influence. The use of a low concentration of serum during the first 72 h of culturewas not beneficial to development. Removing F1 from the culture medium after the initial 72 h of culture did not reverse the effects of this protein, implying that it is required only during the first 2 to 3 d of development. It is postulated that oEGP is involved in a selection mechanism at the zygote stage and may improve the viability of in vitro cultured embryos.

The commercial and agricultural applications of animal transgenesis.

The commercial potential for transgenesis techniques is substantial, particularly in the fields of animal and plant agriculture. This results from productivity being a function of genetic potential and interaction with the environment, but environmental factors being only partially subject to influence by the farmer. Thus, concentrating on genotype improvement becomes an important goal if substantial cumulative gains in productivity are to be made. Historically, the genetic potential associated with important animal production traits, such as wool growth, milk yield, and body growth, has been improved by selective breeding, whereby phenotypically superior animals are used as parental stock for following generations. The high quality of the domestic animals in use in farming today compared with those of earlier centuries is witness to the success of the approach, but nevertheless, the method has significant limitations that have frustrated animal breeders for many years. The complex genetic interactions that combine to produce a particular animal phenotype result in slow genetic gain, averaging at best about 1-3% per year. In addition, separating a desired production trait from one or more undesirable traits is often very difficult. However, the most important of these limitations is the inability to transfer genetic information between species, because of the biological barrier that prevents interspecific breeding.

Production of Transgenic Sheep

The production of transgenic sheep has proven difficult compared to the mouse and lower animals. The work load is far greater and the rates of success far less by most criteria. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nancarrow, Chapter 5) and support for the continuation of the work is assured. Unfortunately, the low rate of transgenesis for sheep, at about 1% of injected, transferred embryos, means that investigation of the regulation of expression of the transgenes, their phenotypic effects, and optimization of the fusion gene constructs, all of utmost importance to the agricultural industry, can seldom be addressed. We know now that the mouse may not be a good model for the sheep, an example being the ovine metallothioneinovine growth hormone fusion gene, GH9, for which expression and phenotypic effects were quite different for sheep and mice. In sheep, pronuclear microinjection of several hundred copies of the foreign gene into embryos is the only published method used to regularly produce transgenics and it will be the standard by which future methods for incorporation of the transgene are judged.

Immunization against 5α-androstane-3α, 17β-diol affects ovarian folliculogenesis in Merino ewes

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.

Ovarian response to PMSG and GnRH in ewes immunised against oestradiol-17 beta

Forty-six adult merino ewes were immunised against oestradiol-17 beta-6 carbomethyloxime:human serum albumin and 48 comparable ewes were used as controls in an experiment to study the effects of gonadotrophin releasing hormone (GnRH) on ovulatory responses after treatment with pregnant mares serum gonadotrophin (PMSG). All the ewes were treated with progestogen sponges for 14 days and received 1500 iu PMSG on the 12th day. Twenty-four control and 24 immunised ewes received 25 micrograms GnRH 21.5 hours and 23 hours after the sponges were withdrawn. Plasma samples were collected between 17 and 50 hours after the sponges were withdrawn and assayed for luteinising hormone (LH). Immunisation reduced the proportion of ewes which ovulated and their rate of ovulation. Injection of GnRH increased the proportion of immunised ewes ovulating (P less than 0.0005) and their rate of ovulation (P less than 0.0001). More unovulated follicles were observed in immunised ewes regardless of GnRH treatment (P less than 0.0001). The rate of recovery of eggs was reduced after immunisation. Treatment with GnRH produced a surge of LH of equal magnitude in the control and immunised ewes although not as many immunised ewes ovulated.