Showing papers in "Reproduction, Fertility and Development in 1994"
TL;DR: The glucocorticoids, cortisol and corticosterone, have a unique function in the fetus in inducing a wide range of enzymes before birth that have little or no function during fetal life but on which survival after birth is dependent.
Abstract: The glucocorticoids, cortisol and corticosterone, have a unique function in the fetus in inducing a wide range of enzymes before birth that have little or no function during fetal life but on which survival after birth is dependent. The loss of the placenta at birth deprives the fetus of a source of oxygen, glucose and heat (among many other things) for which alternatives must be available immediately if survival is to be assured. In anticipation of these needs several organs undergo maturational changes in late pregnancy aimed at meeting these requirements. The lungs mature structurally and functionally, becoming distensible and capable of coping with high surface tension when air enters the alveoli with the first breath. In the liver, glycogen accumulates and gluconeogenesis is initiated to meet the demands for glucose until feeding begins. There is an increase in the production of tri-iodothyronine and catecholamines in preparation for the sharp increase in metabolic rate and thermogenesis associated with breathing and the cold environment. All these dramatic maturational events are regulated by cortisol as are numerous others in most organ systems that contribute to neonatal well-being but on which survival is less dependent. Pharmacological manipulation of these systems before birth has made a substantial contribution to improving human health.
446Â citations
TL;DR: The lipid peroxides thereby generated exhibit powerful negative correlations with the movement characteristics of the spermatozoa and their capacity for sperm-oocyte fusion should have important implications for the development of rational techniques for the diagnosis and treatment of male infertility.
Abstract: There is a growing body of evidence to indicate that a significant factor in the aetiology of male infertility involves a loss of sperm function as a consequence of oxidative stress. This stress originates from the excessive generation of reactive oxygen species by the spermatozoa and results in the peroxidation of unsaturated fatty acids in the sperm plasma membrane. It is possible that reactive oxygen species originating from infiltrating leucocytes could also stress the spermatozoa although the protective properties of seminal plasma would render this unlikely in vivo. Whatever the source of the reactive oxygen species, the lipid peroxides thereby generated exhibit powerful negative correlations with the movement characteristics of the spermatozoa and their capacity for sperm-oocyte fusion. These findings should have important implications for the development of rational techniques for the diagnosis and treatment of male infertility.
282Â citations
TL;DR: Porcine (Sus scrofa) embryonic stem (ES) cell lines from preimplantation blastocysts and their ability to develop into normal chimaeras are summarized, indicating a pluripotent cell.
Abstract: The establishment of embryonic cell lines from swine should be useful for studies of cell differentiation, developmental gene regulation and the production of transgenics. This paper summarizes the establishment of porcine (Sus scrofa) embryonic stem (ES) cell lines from preimplantation blastocysts and their ability to develop into normal chimaeras. ES cells can spontaneously differentiate into cystic embryoid bodies with ectodermal, endodermal, and mesodermal cell types. Further, culture of ES cells to confluence or induction of differentiation with retinoic acid or dimethylsulfoxide results in morphological differentiation into fibroblasts, adipocytes, and epithelial, neuronal, and muscle cells. These ES cells have a normal diploid complement of 38 chromosomes. Scanning electron microscopy of the ES cells reveals a rounded or polygonal, epithelial-like cell with numerous microvilli. The differentiation of these embryonic cell lines into several cell types indicates a pluripotent cell. Furthermore, chimaeric swine have been successfully produced using such ES cells.
200Â citations
TL;DR: Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma, and no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial.
Abstract: During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)
146Â citations
TL;DR: Although there is a paucity of information on the post-translational modification and extracellular processing of these molecules which result in matrix formation, a number of properties have been elucidated allowing better correlation between the structure and function of different ZP proteins among species.
Abstract: Many studies of the molecular and biochemical aspects of mammalian fertilization have focused on the interaction of the spermatozoa with the zona pellucida (ZP). The zona pellucida, a unique extracellular matrix surrounding the mammalian oocyte, is formed during ovarian follicular development. Following ovulation of the mature ovum, the spermatozoa must bind to and penetrate this matrix before the fertilization process is completed and the male and female genetic information combine. Although numerous models for this interaction have been proposed, the complete process has yet to be elucidated. The precise mechanisms by which these interactions occur also vary markedly among different mammalian species, making it more difficult to establish a unified model. To a great extent, the study of the molecules involved in these interactions have been limited because small numbers of female gametes are available for these studies. The recent development of techniques to isolate large numbers of zonae pellucidae as well as advances in immunological and molecular biology techniques have permitted the detailed characterization of ZP proteins. Although there is a paucity of information on the post-translational modification and extracellular processing of these molecules which result in matrix formation, a number of properties have been elucidated allowing better correlation between the structure and function of different ZP proteins among species. This review reflects these studies in relation to protein nomenclature and the molecular complexity of ZP antigens.
142Â citations
TL;DR: Changes of progesterone receptor (PR) protein and cellular localization in the endometrium were evaluated during the oestrous cycle and early pregnancy of the gilt to indicate down-regulation of epithelial PR might be one factor involved in the timing of luteolysis during the OES.
Abstract: Changes of progesterone receptor (PR) protein and cellular localization in the endometrium were evaluated during the oestrous cycle and early pregnancy of the gilt. During the oestrous cycle, the concentration of total PR protein within the endometrium was highest on Days 0-5 and decreased on Day 10. The endometrial concentration of PR reached a nadir on Day 12 and this level was maintained throughout the remainder of the oestrous cycle (Day 18). In pregnant gilts, the concentration of endometrial PR protein from Day 10 to Day 18 was similar to that in cyclic gilts. Western blot analysis with antiserum specific for the A and B isoforms of PR indicated that porcine endometrium expresses both isoforms of PR. Immunostaining was detectable for both the A and B isoforms of PR from Day 0 to Day 12 of the oestrous cycle. However, no staining was observed on Day 15 and Day 18 of the oestrous cycle or pregnancy Immunocytochemical localization of PR in the endometrium of cyclic gilts and pregnant gilts indicated that there was intense staining for PR in surface epithelium and glandular epithelium during oestrus (Day 0) and on Day 5. However, the staining was less intense on Day 7 and Day 10 of the oestrous cycle and no epithelial staining was observed after Day 12. PRs were present in the stroma and myometrium throughout the oestrous cycle and early pregnancy. The presence of conceptuses during pregnancy did not affect the loss of PR from the uterine epithelium after Day 10 of gestation. Down-regulation of epithelial PR might be one factor involved in the timing of luteolysis during the oestrous cycle as well as conceptus growth and placentation during early pregnancy.
137Â citations
TL;DR: Key questions about the efficacy of fertility control and the means for delivering antigens expressed in recombinant viral vectors are discussed and the legal and social concerns that relate to its possible future use are raised.
Abstract: The potential value of immunosterilization as a means to control species of wildlife that are widespread, numerous and undesirable is assessed. Key questions about the efficacy of fertility control and the means for delivering antigens expressed in recombinant viral vectors are discussed and the legal and social concerns that relate to its possible future use are raised.
137Â citations
TL;DR: Strategies for overcoming Chromosomal position effects in embryonic stem cells are described with particular reference to their application in transgenic animals.
Abstract: Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.
118Â citations
TL;DR: Pharmacological stimulation of sperm motility may increase yields but, for in vitro fertilization (IVF), such spermatozoa must be used to inseminate oocytes as soon as possible after exposure to the stimulant, although after its removal.
Abstract: Because seminal plasma contains factors that inhibit the fertilizing ability of spermatozoa, it is essential that spermatozoa be separated from it quickly and efficiently. Although the success of a sperm preparation method is often assessed by the yield of motile spermatozoa, the choice of a method also depends on its technical complexity, the materials and apparatus required and time costs. Any exposure of spermatozoa during preparation to factors that may cause iatrogenic sperm dysfunction must obviously be avoided. Consequently, methods involving centrifugal washing prior to the selection of motile spermatozoa should be avoided. Direct swim-up from semen is the simplest way to obtain highly motile sperm populations and can be a very rapid procedure with normal semen samples. Two-layer discontinuous Percoll gradients give excellent yields when the lower layer contains 81% (v/v) Percoll. However, for severely asthenozoospermic cases the results can be disappointing and a Nycodenz gradient may be better, although the 'mini-Percoll' technique might be useful if special care is taken to protect the spermatozoa from damage induced by free radicals. In such cases the migration-sedimentation approach can also be successful. Abnormal samples, especially those with increased viscosity, may benefit from prior dilution with culture medium, or even chymotrypsin-induced liquefaction, before density gradient centrifugation. Finally, pharmacological stimulation of sperm motility may increase yields but, for in vitro fertilization (IVF), such spermatozoa must be used to inseminate oocytes as soon as possible after exposure to the stimulant, although after its removal.
113Â citations
TL;DR: All cryptorchidism models, despite their different primary cause, have in common an abnormality of the GNF and/or CGRP which is consistent with the hypothesis that normal testicular descent in the rodent may be mediated by the GFN.
Abstract: This paper briefly reviews the literature on testicular descent and presents new observations from the authors' laboratory which suggest new ways of looking at old problems. There is now good evidence that testicular descent occurs in two morphologically and hormonally distinct phases. Relative 'transabdominal migration' of the testis compared with the ovary occurs at 10-15 weeks of gestation in the human and 'inguinoscrotal' migration occurs at 26-35 weeks of gestation. We have proposed previously that the first phase is controlled by Mullerian inhibiting substance although this remains controversial. The second phase is androgen dependent and is possibly mediated indirectly through the release from the genitofemoral nerve (GFN) of the neuropeptide calcitonin gene-related peptide (CGRP). Recently we have used three different rodent models of undescended testis to determine the involvement of the GNF and/or CGRP. The testicular feminization mouse with complete androgen resistance and the rat exposed prenatally to the antiandrogen flutamide have a deficiency of CGRP in the GFN. In contrast, the mutant trans-scrotal rat which has normal androgen levels has an excess of CGRP in the GFN. All cryptorchidism models, despite their different primary cause, have in common an abnormality of the GNF and/or CGRP which is consistent with the hypothesis that normal testicular descent in the rodent may be mediated by the GFN.
96Â citations
TL;DR: The results indicate differential prostaglandin responses by the two major endometrial cell types (epithelium and stroma) to regulatory agents such as bIFN tau and oxytocin in cattle is supportive of an antiluteolytic effect of bIFn tau.
Abstract: The effects of bovine interferon tau (IFN tau) and oxytocin on secretion of the prostaglandins PGF2 alpha and PGE2 by epithelial and stromal cells in the endometrium were assessed in two experiments. Endometrial tissues were collected from cyclic cows at Day 15 after oestrus for subsequent isolation of epithelial cells (4 cows) and stromal cells (4 cows). In both experiments, confluent cells were treated with 0, 2, 10 or 50 ng mL-1 natural bovine IFN tau (nbIFN tau) or 0, 0.4, 2, 10, 50 and 250 ng mL-1 recombinant bIFN tau (rbIFN tau). Culture medium was sampled at 24 h. Oxytocin (2.0 x 10(-7) M) or placebo was then added to wells and the medium was sampled 30 and 90 min later. Epithelial cells secreted more PGF2 alpha than stromal cells whereas stromal cells predominantly secreted PGE2. Oxytocin stimulated secretion of PGF2 alpha and PGE2 (P < 0.01) from epithelial cells, but both basal secretion and oxytocin-induced secretion of PGF2 alpha and PGE2 decreased with increasing dose of either nbIFN tau or rbIFN tau (P < 0.01). At comparable doses, rbIFN tau inhibited PGF2 alpha and PGE2 secretion more strongly than did nbIFN tau (either in the absence or the presence of oxytocin). The minimal effective dose of rbIFN tau was 0.4 ng mL-1 and 50% inhibition was obtained with 1 ng mL-1 (0.043 nM). Neither nbIFN tau nor rbIFN tau nor oxytocin altered PGF2 alpha or PGE2 secretion by stromal cells. The results indicate differential prostaglandin responses by the two major endometrial cell types (epithelium and stroma) to regulatory agents such as bIFN tau and oxytocin in cattle. Suppression of prostaglandin secretion by bIFN tau in epithelial cells of endometrial tissue is supportive of an antiluteolytic effect of bIFN tau.
TL;DR: The development of totipotent bovine embryonic cell cultures has great value in cattle breeding and provides a mechanism for making large numbers of clonal offspring by nuclear transfer, an efficient gene transfer system through the use of selectable markers to select transgenic cells and an mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination.
Abstract: The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.
TL;DR: Future studies with cultured ES cells of mouse and other species should provide insights into the factors regulating the differentiation of intermediate stem cells and terminal cells for the various embryonic lineages, contributing profoundly to the understanding of mammalian embryogenesis as well as providing cells for therapeutic applications.
Abstract: Embryonic stem (ES) cells were first cultured from mouse embryos little more than a decade ago, yet they are now widely used in transgenic studies which are revolutionizing mammalian genetics. Although drawing less attention, in vitro studies of mouse ES cells have also contributed widely to the understanding of mechanisms of embryonic cell differentiation and proliferation. This review focusses on the application of ES cells as in vitro models for cellular and molecular events in the early mammalian embryo. Future studies with cultured ES cells of mouse and other species should provide insights into the factors regulating the differentiation of intermediate stem cells and terminal cells for the various embryonic lineages, thus contributing profoundly to the understanding of mammalian embryogenesis as well as providing cells for therapeutic applications.
TL;DR: Overall, there are now good reasons to be optimistic that transgenesis will eventually be available to all livestock breeders with the proviso that there are no further unanticipated phenomena to threaten the predictability of outcome of ES cell-derived pregnancies and further limit the potential usefulness of this futuristic technology to the livestock industry.
Abstract: The creation of transgenic livestock is a complex multistep procedure the successful execution of which demands a high level of skill and application. Useful animals have been generated by transfer of genes to zygotes by microinjection, but further extension to livestock breeding is severely limited by the present low efficiency and lack of precision in gene transfer procedures. There are major developments in alternative approaches to gene transfer and those based on embryonic stem (ES) cell lines show particular promise as a broadly adaptable means of allowing precise manipulation of specific genes within the animal genome. Rapid progress is being made in adapting ES cell technology to livestock species but as yet no one has demonstrated the totipotency of the putative cell lines so far generated. The demonstration of the feasibility of the chimaeric route for reinstating an ES cell genome into the germ line of the pig is a major advance. For other livestock breeds, particularly those with long generation times and bearing single young where the chimaeric route is much less useful, there are encouraging developments in nucleus transfer (cloning) technology which could provide practical solutions. Overall, there are now good reasons to be optimistic that transgenesis will eventually be available to all livestock breeders with the proviso that there are no further unanticipated phenomena such as the effect of tissue culture on imprinting, to be discovered to threaten the predictability of outcome of ES cell-derived pregnancies and further limit the potential usefulness of this futuristic technology to the livestock industry.
TL;DR: The method involves the use of embryonic stem cells, gene targeting and the FLP recombinase system to allow single copy insertion of transgenes into a defined site in animal genomes.
Abstract: Methods to improve the production of transgenic animals are being developed Conventional transgenesis, involving microinjection of DNA into fertilized eggs, has a number of limitations These result from the inability to control both the site of transgene insertion and the number of gene copies inserted The approach described seeks to overcome these problems and to allow single copy insertion of transgenes into a defined site in animal genomes The method involves the use of embryonic stem cells, gene targeting and the FLP recombinase system
TL;DR: This review summarizes current understanding of the production of the zona pellucida during folliculogenesis, the structure of the conserved proteins and genes in the zzon pellUCida, and the progress made in the development of immunocontraceptive strategies that focus on this oocyte-specific structure.
Abstract: Although reversible interference of sperm-egg interactions with pharmacological agents has not yet been achieved, animal models have provided increasing evidence that immunological reagents directed against mammalian gametes can effectively inhibit fertilization. One potential target of immunocontraception is the zona pellucida, an extracellular matrix that surrounds the growing oocyte and ovulated egg. Recent advances in our knowledge of the biosynthesis and molecular biology of the zona pellucida have provided much information useful in the rational design of immunocontraceptive vaccines. There remain, however, major obstacles to using immunological reagents to prevent fertilization, including potential toxic side effects, the lack of adequate delivery systems and the possibility of incomplete reversibility. This review summarizes current understanding of the production of the zona pellucida during folliculogenesis, the structure of the conserved proteins and genes in the zona pellucida, and the progress made in the development of immunocontraceptive strategies that focus on this oocyte-specific structure.
TL;DR: It is concluded that activin has a role in the development and maintenance of healthy oestrogenic follicles, preventing premature luteinization, whereas follistatin opposes these effects of activin and promotes lute inization or atresia.
Abstract: The role of the gonadotrophins follicle-stimulating hormone (FSH) and luteinizing hormone and the putative local regulators, activin and follistatin, in the control of folliculogenesis is reviewed. An account of early work on the development and application of assays for FSH and inhibin is given, together with a summary of the data on the ovarian responsiveness to gonadotrophin and follicular atresia. Models for studying local regulation of granulosa cells in vitro are described and the data from these experiments reviewed. It is concluded that activin has a role in the development and maintenance of healthy oestrogenic follicles, preventing premature luteinization, whereas follistatin opposes these effects of activin and promotes luteinization or atresia.
TL;DR: Bovine primordial germ cell-derived cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase and preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.
Abstract: The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The mouse-specific murine cytomegalovirus (MCMV) is a large DNA virus which establishes persistent, non-lethal infection, it is a suitable vector for the insertion of foreign genes and has a number of properties that should assist the recombinant virus to persist in wild mouse populations and induce an immunocontraceptive effect.
Abstract: Wild populations of house mice (Mus domesticus) regularly undergo population eruptions in the cereal growing regions of S.E. Australia, causing significant damage to crops. Rodenticides are costly and are of limited usefulness, and the need for a biological control agent is widely recognized. The options for the use of an infectious agent to control mouse populations are considered. The three main types are: infectious agents which establish lethal infection; those which directly interfere with fertility; and recombinant virus vectors encoding fertility-associated proteins such as zona pellucida or sperm antigens in order to induce immunocontraceptive responses in infected mice. Ectromelia, a murine pox virus, has the potential for reducing mouse populations by lethal infection but it is not present in wild mice in Australia. The disadvantages of using ectromelia are that it would pose a significant threat to colonies of laboratory mice, there appears to be substantial innate resistance in Australian wild mice and it may not be entirely mouse-specific, thus placing native rodents at risk. A number of factors influencing the selection of a virus as a vector for immunocontraception are discussed. The mouse-specific murine cytomegalovirus (MCMV) fits most of these criteria. Infection with MCMV is already widespread in Australia with 80-90% of Mus domesticus tested being seropositive. It is a large DNA virus which establishes persistent, non-lethal infection, it is a suitable vector for the insertion of foreign genes and has a number of properties, including the capacity for superinfection, that should assist the recombinant virus to persist in wild mouse populations and induce an immunocontraceptive effect.
TL;DR: It is indicated that bicarbonate is essential for pig fertilization in vitro, but that caffeine exerts a synergistic stimulatory effect.
Abstract: Fertilization of pig oocytes was performed in vitro in modified Tyrode's media in which either HEPES or bicarbonate/CO2, or both, were included as buffer systems; caffeine (2 mM) was also included in some of the media because it is a reported stimulant of fertilization. The composition of the bicarbonate-containing media was designed so as to maintain the same pH and osmolality as bicarbonate-free media. The inclusion of bicarbonate during gamete co-incubation in caffeine-containing medium led to high levels of fertilization (66% of 238 mature oocytes were fertilized). However, essentially no fertilization occurred if bicarbonate was replaced with HEPES (0.7% of 146 oocytes were fertilized; significantly different, P < 0.001). Inclusion of HEPES in bicarbonate-containing medium during gamete co-incubation did not affect fertilization, showing that HEPES did not exert an inhibitory effect. Omission of bicarbonate during sperm preincubation also did not affect fertilization. If caffeine was included in bicarbonate-containing medium, 73% of 311 oocytes were fertilized whereas if caffeine was omitted only 14% of 326 oocytes were fertilized (significantly different, P < 0.001). In the absence of bicarbonate, when fertilization was very low, caffeine had no stimulatory effect. The results indicate that bicarbonate is essential for pig fertilization in vitro, but that caffeine exerts a synergistic stimulatory effect.
TL;DR: The development of an immunocontraceptive vaccine to control fox populations in Australia would confer considerable advantages in controlling the long-term impact of this predator on native and endangered species.
Abstract: The development of an immunocontraceptive vaccine to control fox populations in Australia would confer considerable advantages in controlling the long-term impact of this predator on native and endangered species. Studies are currently under way to identify sperm antigens that might be used in such a vaccine, and some of these studies are described. It is proposed that such a vaccine would be delivered orally in a bait, thereby stimulating a mucosal immune response to the foreign antigen(s). Such a vaccine requires a detailed understanding of reproductive-tract mucosal immunity in foxes, and selection of the most effective form of antigen delivery. Those under consideration include viral or bacterial vectors and microencapsulated antigens.
TL;DR: Recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses.
Abstract: The realization that induction of immune responses at mucosal surfaces may prevent colonization, invasion or dissemination of pathogenic microorganisms has spurred intensive efforts to develop vaccines which elicit effective mucosal immunity. In this paper, recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses. Oral administration of cholera toxin alone or with a co-administered protein vaccine tetanus toxoid induces a strong T helper type 2 (TH2) cell response in both Peyer's patches and spleen. Both serum IgG and secretory IgA antibodies specific for cholera toxin or for the co-administered protein tetanus toxoid were induced. When administered parentally, however, no mucosal antibody responses were evident and a mixed TH1- and TH2-type CD4+ T cell response was noted in the spleen. Various vectors are being employed in an effort not only to induce mucosal immune responses but also to direct the response to a TH1-type response, thought to promote strong cell-mediated immune responses, or to a TH2-type response for maximum B cell antibody responses. The ability to manipulate the TH cell responses may provide a more rational approach for the design of vaccines. Although lymphoid tissues of the female reproductive tract differ from that of the gut, many of the strategies and evolving principles may be directly applicable to the development of vaccines designed to prevent sexually transmitted diseases.
TL;DR: Although yet to be developed, bovine embryonic stem cells would alleviate many of the problems of transgenic cattle production and permit a wider range of genetic manipulations.
Abstract: The production of transgenic cattle presents a number of unique challenges not encountered in other species. First, the survival of microinjected zygotes is low; only 15% in vivo-derived develop into morulae and blastocysts and, of these, only about 18% yield live calves. Second, transgene integration frequency is relatively low, around 3%. Thus, more than 1000 zygotes must be injected to produce a single transgenic calf. Obtaining sufficient zygotes from donor cattle to sustain a transgenic cattle programme is logistically and financially prohibitive, since the average superovulated donor yields only about four microinjectable zygotes per collection attempt. In vitro oocyte maturation and fertilization techniques may be used to alleviate this problem, although initially the developmental potential of in vitro-derived microinjected zygotes is lower than their in vivo-produced counterparts (8% v. 15%, respectively, yield morulae and blastocysts). Since only 3-5% of calves born from microinjected zygotes produced in either fashion yield transgenics, at least 20-30 pregnancies must be carried to term for every transgenic calf born. These conditions require that large herds of donor and recipient cattle be maintained. Recipient requirements could be reduced if transgene integration frequency could be increased, but improvements in the near future are unlikely since the mechanism of integration after pronuclear microinjection is poorly understood. Alternatively, embryos could be screened for integrated transgenes before transfer; however, efforts in this area have been complicated by high frequencies of false positive results. Although yet to be developed, bovine embryonic stem cells would alleviate many of these problems and permit a wider range of genetic manipulations.
TL;DR: Although it is only a decade since the use of transgenic livestock for the production of pharmaceutical proteins in milk was first suggested, great progress has been made, and there is every reason to expect that many proteins will be produced at high concentrations in the milk of livestock species.
Abstract: Although it is only a decade since the use of transgenic livestock for the production of pharmaceutical proteins in milk was first suggested, great progress has been made. There is every reason to expect that, as methods for the transfer of genomic clones are applied with efficient regulatory elements, many proteins will be produced at high concentrations in the milk of livestock species. In most cases, these proteins will be biologically active, however, it remains to be shown whether they will be identical in structure and function to the natural product.
TL;DR: A review on current knowledge of sperm and embryo transport in the female reproductive tract of marsupials and the unique features of gamete structure-function and female genital tract morphology are described and compared with data available on eutherian mammals.
Abstract: A review on current knowledge of sperm and embryo transport in the female reproductive tract of marsupials. Some of the unique features of gamete structure-function and female genital tract morphology will be described and compared with data available on eutherian mammals.
TL;DR: The expression of the genes for murine interleukin-5 (IL-5) or IL-6 in recombinant vaccinia virus vectors markedly increased IgA reactivity to co-expressed heterologous antigen in the lungs of mice inoculated intranasally with the viruses, suggesting that their expression in mucosal vaccine vectors may stimulate local immune responses.
Abstract: The expression of the genes for murine interleukin-5 (IL-5) or IL-6 in recombinant vaccinia virus vectors markedly increased IgA reactivity to co-expressed heterologous antigen in the lungs of mice inoculated intranasally with the viruses. These elevated local IgA responses reached a peak four times higher than those elicited by control viruses 14 days after infection and these peak levels were maintained for at least four weeks. Elevated IgA responses, reaching a peak 3-4 weeks after immunization, were also observed in the lungs of mice inoculated with IL-6 expressed by another vector, fowlpox virus. The results indicate that these factors enhance the development of mucosal IgA reactivity in vivo and suggest that their expression in mucosal vaccine vectors may stimulate local immune responses. The approach described in this study may be useful in stimulating mucosal immunity to a wide range of vector-encoded antigens, not only for vaccination against disease but also for immunocontraception by the co-expression of antigens involved in reproduction.
TL;DR: This review surveys these specializations of spermatozoa from this branch of therian evolution and examines current knowledge on their respective functions and the forces which shaped their evolution.
Abstract: Marsupial sperm structure has been the focus of many comparative studies in the last 30 years. Although the basic organization of the marsupial spermatozoon is similar to that of eutherian mammals, spermatozoa from this branch of therian evolution have developed a specific suite of characters which clearly distinguish them from the Eutheria. This review surveys these specializations and examines current knowledge on their respective functions and the forces which shaped their evolution. Nuclear shaping and stability, the asymmetric positioning of the acrosome, and the unusual neck articulation are discussed. Although recent observations have provided evidence of a marsupial equatorial segment and posterior ring, the marsupial equivalent of the eutherian postacrosomal sheath has not been identified. The unusual neck structure of marsupial spermatozoa and the mobile articulation of the connecting piece are discussed in relation to nuclear rotation and the events associated with this process. Increasing flagellar length in some species is associated with extremes in flagellar organization and its effect on sperm motility is discussed.
TL;DR: An efficient sheep transgenesis programme is established with an overall transgenic rate of 2.1% from 516 lambs born, and one transgenic sheep (bearing the small proline-rich protein promoter constructs) has produced cysteine in the rumen, although the amount was low at 3 months of age and not detectable at 6 months.
Abstract: Merino wool is the result of generations of selection, yet improvements in wool quality and performance are still being sought. Through gene manipulation, sheep transgenesis offers possibilities of understanding the relationship between wool keratin protein composition and fibre structure and properties and of introducing novel changes to fibre properties and growth rates. We have established an efficient sheep transgenesis programme with an overall transgenic rate of 2.1% of zygotes injected. However, by incorporating in vitro culture and assessment of injected zygotes, this equates to a transgenic rate of 13% from 516 lambs born. With the first keratin gene construct, a wool keratin type II intermediate filament gene, four live F0 transgenic sheep have been produced and all express the transgene. In one of them, the highest expressor, phenotypic and ultrastructural changes were evident in the fleece. To improve wool growth rate by increasing the supply of cysteine to the follicle, transgenic sheep are being produced carrying the two genes necessary for endogenous cysteine synthesis. Three promoters have been tested driving the cysteine synthesis genes: two general promoters, the Rous sarcoma virus long terminal repeat and mouse phosphoglycerate kinase promoter, and a rumen-specific promoter from the sheep small proline-rich protein gene. To date, one transgenic sheep (bearing the small proline-rich protein promoter constructs) has produced cysteine in the rumen, although the amount was low at 3 months of age and not detectable at 6 months.
TL;DR: In the future, livestock will play an increasingly more important role in biomedical research through the application of genetic engineering methodologies and will allow for increased sophistication of experimental design and wider use of genetically modified livestock.
Abstract: Genetic models of human disease can lead to new insights concerning disease aetiology or suggest novel therapeutic interventions. Livestock species, especially pigs, cows, sheep and horses, are often good animal models of human disease. However, genetic models in livestock species have included only the study of spontaneous mutations. Production of transgenic livestock is now possible but owing to a low efficiency of production, it is very expensive and its application limited. Anticipated application of improved technologies such as embryonic stem cells and homologous recombination will allow for increased sophistication of experimental design and wider use of genetically modified livestock. In all cases, the appropriate species for a genetic model of human disease should be the species which best models the physiology of the organ or system under consideration. In the future, livestock will play an increasingly more important role in biomedical research through the application of genetic engineering methodologies.
TL;DR: This study demonstrates that the peptide pin-block method of Chiron Mimotopes, Australia, can lead to the identification of a B-cell epitope with strong immunocontraceptive potential.
Abstract: The selection of immunological targets present on gametes is an important first step in the successful production of animal or human vaccines for immunocontraception. One strategy with regard to sperm antigens is to select antigens with physiological roles in gamete interaction, obtain the mRNA sequence for such an antigen, and then determine which region or domain of the molecule is available to the immune system when spermatozoa are presented to the female reproductive system. To illustrate this strategy, we have used the rabbit sperm antigen Sp17 (RSA-3), which has been cloned and sequenced. The peptide pin-block method of Chiron Mimotopes, Australia, has been used to analyse the 146 amino acids of Sp17 with various homologous and heterologous antisera. This study demonstrates that such an analysis can lead to the identification of a B-cell epitope with strong immunocontraceptive potential.