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D. A. Clark, B. A. Croy, J. Rossant and G. Chaouat

Summary. Putatively immuno-incompetent Mus musculus females exhibited failure to support pregnancy of Mus caroli embryos. These results for M. musculus females (i.e. treated by cyclosporine A, of the nu/nu genotype, and as an interspecies chimaera) can be explained in immunological terms. Mus musculus females possessed pre-sensitized cytotoxic T cells against Mus caroli antigens. Nu/nu mice possessed activated NK cells and macrophages, and selectively discriminated against Mus caroli embryos early in pregnancy unlike normal +/+ females; the requirement for T cells to activate non-specific cytotoxic effector mechanisms was bypassed in nu/nu mice. Mus caroli are not inbred, and interspecies chimaeras which are tolerant of the antigens on the Mus musculus donor strain were not tolerant of cells from unrelated Mus caroli. Interspecies chimaeras also behaved as if they were pre-sensitized to Mus caroli. Our results show that Mus caroli embryos recruit fewer active suppressor cells even when gestating in Mus caroli decidua as compared to Mus musculus embryos in Mus musculus decidua and that the ability of Mus caroli placental cells to directly inhibit cytotoxic effector cell killing was inherently less than the inhibitory activity of placental cells from Mus musculus. Mus caroli embryos therefore appear to be less well defended against maternal immune attack even when gestating in a uterus possessing compatible Mus caroli decidual tissue.

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B. A. Croy, J. Rossant and D. A. Clark

Summary. The role of the immune system in promoting the midterm death of Mus caroli embryos transferred to the Mus musculus uterus was studied in vivo by transferring M. caroli blastocysts to recipients with altered immune status. Transfers of embryos to chimaeric mothers (Mus musculusMus caroli), which were expected to be tolerant of species antigens, resulted in survival of M. musculus embryos but death of M. caroli embryos. The preferential survival of M. musculus embryos was explained by showing that M. musculus embryos can survive in the M. caroli uterus. Transfers to T celldeficient mice of genotype nu/nu and to NK cell-deficient mice of genotype bg/bg as well as treatment of normal transfer recipients with Cyclosporin A or anti-Ia antiserum failed to prolong survival. However, immunization of recipients with M. caroli lymphocytes promoted more rapid and uniform failure of the interspecies pregnancy. Cytotoxic cells were detected in the resorbing embryos on Day 10·5 in immune pregnancies and on Day 12·5 in non-immune pregnancies and these cells were promiscuous in their pattern of lysis, showing equal reactivity against M. caroli, transfer recipient and 3rd party target cells. These experiments show that failure of M. caroli embryos in the M. musculus uterus is complex, but probably does not involve responses by classical cytotoxic T lymphocyte or natural killer cell pathways. Participation of the immune system in the resorption process, however, is confirmed and is associated with generation of promiscuous cytolytic cells.

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J. R. McNeilly, N. P. Evans, T. A. Bramley, P. Brown, A. J. Clark and R. Webb

Selection of the luteinizing hormone (LH) response to exogenous gonadotrophin-releasing hormone (GnRH) in sheep has resulted in the establishment of two lines (High and Low) with a fivefold difference in pituitary sensitivity to GnRH. The effect of selection on gonadotrophin gene expression in the presence or absence of an exogenous gonadotrophin-releasing hormone (GnRH) challenge in twenty-week-old ram lambs from both lines was examined. Before treatment with either GnRH or saline, LH and follicle-stimulating hormone (FSH) concentrations were significantly higher in the High line than in the Low line animals (LH and FSH: P < 0.01). One hour after either GnRH or saline, all animals were slaughtered. In the absence of a GnRH challenge, there were significantly higher concentrations of all three gonadotrophin subunit mRNAs in the High line compared with the Low line, corresponding to the higher basal concentrations of LH and FSH. When comparing treatments between the lines, following a GnRH challenge, LHβ subunit mRNA was significantly (P < 0.001) higher in both lines than before the GnRH, whereas there was no significant change in either α or FSHβ subunit mRNA. These results indicate that the differences in basal gonadotrophin secretion are related to differences in gonadotrophin subunit mRNAs with the High line animals having an inherently greater amount of all three gonadotrophin subunit mRNAs. Selection has not altered the differential amounts of gonadotrophin subunit mRNAs, since there is an overall increase in all three gonadotrophin subunits. GnRH appears to preferentially control LHβ mRNA in both High and Low line animals.

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S. Harris, M. McClenaghan, J. P. Simons, S. Ali and A. J. Clark

AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH25 9PS, UK

Keywords: Mammary gland; β-lactoglobulin; transgenic; gene expression

Introduction

The mammary gland is the specialized secretory organ that provides essential nourishment to mammalian young in the form of milk. Milk is primarily composed of water, fats, lactose and proteins; the major protein components are the various caseins and the whey proteins α-lactalbumin, β-lactoglobulin (ruminants) and whey acidic protein (rodents).

Mammary development and milk protein gene expression are regulated by a number of peptide and steroid hormones, as well as cell-cell and cell-substratum interactions within the gland (Topper & Freeman, 1980; Levine & Stockdale, 1985; Li et al., 1987). During gestation the secretory capacity of the mammary gland increases due to cellular proliferation and differentiation; concomitantly milk protein gene expression is initiated in preparation for sustained milk production after parturition. In late lactation milk production

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M. X. ZARROW, S. A. ESTES, V. H. DENENBERG and J. H. CLARK

Summary.

Facilitation of pmsg- or hcg-induced ovulation occurs in the immature female mouse exposed to the presence of, but not in contact with, an adult male. The effect is not seen if an immature or castrated adult male is used. Removal of the olfactory bulbs in the female also prevents facilitation due to the presence of the male. It is suggested that a number of different effects seen in the female due to a pheromone released by the male may be mediated through a single environmental stimulus.

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D. E. Clark, D. J. Tisdall, A. E. Fidler and K. P. McNatty

The c-kit protein is a transmembrane tyrosine kinase receptor that binds the growth factor stem cell factor. Mutant alleles of the genes coding for both the receptor (c-kit) and its ligand (stem cell factor) affect gametogenesis, development of melanoblasts and some aspects of haematopoiesis. The aim of this study was to examine expression of the c-kit gene during folliculogenesis in fetal sheep ovaries using in situ hybridization. A 422 bp cDNA encoding the extracellular domain of the c-kit protein was amplified from sheep ovarian RNA using reverse-transcription–polymerase chain reaction (RT–PCR), cloned and sequenced. Riboprobes transcribed from the ovine cDNA encoding c-kit were used to detect the presence of mRNA encoding c-kit within the ovaries of fetal sheep on days 90, 100, 120 and 135 of gestation (term = 147 days). In day 90 and 100 fetal ovaries, mRNA encoding c-kit was not detected in association with oogonia during the period of meiosis (to prophase I) but was present in some of the isolated oocytes. In ovaries from day 90 to day 135, mRNA encoding c-kit was detected in the oocytes at every stage of follicular growth – primordial through to antral follicles. This pattern of localization is consistent with that demonstrated in mice.

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A R Clark, Y M Stokes, M Lane and J G Thompson

Immature oocytes benefit from nutrient modification of the follicular environment by the surrounding cumulus mass. However, the oxygen concentration that the oocyte may be exposed to could be lower than the antral follicular concentration due to the metabolism of surrounding cumulus cells. Using metabolic data previously determined, we have developed a mathematical model of O2 diffusion across the bovine and murine cumulus–oocyte complex. From this we have determined that across a physiological range of external pO2, less than 0.25% and 0.5% O2 is removed by cumulus cells within the bovine and murine cumulus–oocyte complex respectively. Our model differs from others as it: incorporates a term that allows for nonlinear variation of the oxygen consumption rate with oxygen concentration; considers two regions (oocyte and cumulus) sharing a common boundary, both of which consume oxygen at different non linear rates. Cumulus cells therefore remove little O2, thus sparing this essential gas for the oocyte, which is dependent on ATP generation via oxidative phosphorylation.

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J. B. K. CLARK, E. F. GRAHAM, B. A. LEWIS and F. SMITH

Summary.

An investigation into the distribution of freely occurring polyhydric alcohols in normal bovine semen has revealed the presence of three previously unreported constituents, namely d-mannitol (17 mg/100 ml), erythritol (6·9 mg/100 ml) and glycerol (circa 2 mg/100 ml). These three polyols, together with inositol and sorbitol were characterized by the preparation of suitable derivatives. Where applicable, the physical constants of the crystalline materials and of their derivatives were determined.