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Biomedical subjects

R F Seamark

Publications and source records attributed to R F Seamark.

At least 19 recordsLinked to original sources
PubMed1992

Cytokines in rodent reproduction and the cytokine-endocrine interaction.

Insights derived from recent studies employing rodent models demonstrate that the synthesis of pluripotent cytokines is an important function of resident cells in the female reproductive tract. Through steroid hormone regulated secretion of these mediators, resident cells appear to coordinate the recruitment and action of leukocytes that are centrally implicated in the dramatic remodelling processes characteristic of reproductive events.

Animals↗
PubMed1992

Granulocyte-macrophage colony stimulating factor (GM-CSF): one of a family of epithelial cell-derived cytokines in the preimplantation uterus.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of a number of lympho-haemapoietic cytokines, including CSF-1, interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) now known to be synthesized by epithelial cells in the murine uterus. GM-CSF synthesis is regulated primarily by the ovarian steroid hormone oestrogen, but is also subject to modulation by factors including a seminal component of seminal vesicle origin which stimulates a 20-fold increase in luminal fluid content at mating, and bacterial lipopolysaccharide (LPS) and the T-lymphocyte and natural killer (NK) cell product interferon-gamma (IFN gamma). In the non-pregnant mouse GM-CSF synthesis peaks at oestrus. Synthesis is maintained at comparable or moderately higher levels during the preimplantation period of pregnancy and in the non-decidualized endometrium during mid gestation. An embryotrophic activity is suggested by studies in vitro that indicate that GM-CSF stimulates attachment and outgrowth of blastocysts. It is postulated that GM-CSF is of major importance to the physiology of pregnancy through its role as a component of a local cytokine circuit acting to recruit and regulate function of endometrial leukocytes, and by its action as interlocutor and important effector arm in embryo-maternal interactions during gestation.

Animals↗
PubMed1992

Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnant and nonpregnant mice.

Cytokine secretion by endometrial cells from estrous and mated mice was measured using specific bioassays. The granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) contents of uterine intraluminal fluid were elevated greater than 20-fold and 250-fold respectively following mating, and both cytokines were synthesized in abundance in vitro by uterine cells harvested at estrus and on Day 1 of pregnancy. Synthesis was not impaired in genetically lymphocyte-deficient nude, SCID, or beige mice. To determine the cellular origin of the cytokines, a panning technique employing monoclonal antibodies against a range of leukocyte and other lineage markers was used to isolate uterine cell subsets in vitro. These experiments identified glandular and/or luminal epithelial cells as the major source of GM-CSF and IL-6 in estrous and pregnant uteri. Stromal fibroblasts also synthesized IL-6, as did macrophages in mated mice. Epithelial cells harvested from midgestation uteri secreted GM-CSF and IL-6 in quantities similar to those of cells from estrous and mated mice. Bioactivities of both cytokines derived from epithelial cells were neutralized by specific antibodies, and size-exclusion chromatography of conditioned media from uterine cells revealed peaks of GM-CSF and IL-6 bioactivity with M(r) 23,000 and 23,000-26,000, respectively. Bioassay of luminal fluids and culture supernatants were negative for the cytokines interleukin-1, interleukin-2, interleukin-3, and tumor necrosis factor-alpha. These studies identify murine uterine epithelium as a potent source of the cytokines GM-CSF and IL-6, which we postulate have potentially important functions in pregnancy through actions on target cells in both the uterus and the conceptus.

Animals↗
PubMed1992

Plasma melatonin profiles of Romney Marsh sheep in natural photoperiod and in acutely extended darkness.

Plasma melatonin was measured at the summer and winter solstices and the autumn and spring equinoxes in Romney Marsh sheep held under natural conditions in South Australia (35 degrees S). The amount of melatonin detected was generally related to the extent of natural darkness, though the melatonin onset was particularly delayed after dusk in winter compared with other seasons. The duration of detectable melatonin was shorter in summer than at any other season. After each initial 24 h sampling, the sheep were resampled for a further 24 h in acutely extended darkness to mark the phase and duration of suprachiasmatic nuclei activity which is believed to be the source of the melatonin signal. The onset of high plasma melatonin was earlier than the time of natural sunset in spring and summer, but not different from the time of natural sunset in autumn and winter. The offset of high plasma melatonin was later than the time of natural sunrise at all times of year and particularly so in summer. Under the extended dark conditions, the duration of detectable melatonin was longer than that under natural photoperiod at all seasons of the year and the duration of melatonin was again shorter in summer than winter. If melatonin measurements under the conditions of extended darkness do reflect the phase and duration of suprachiasmatic nuclei function then the natural light of the photoperiod can, particularly during long photoperiod conditions, mask the expression of the pacemaker. The findings may have implications for the timing of the breeding season in Romney Marsh sheep.

Animals↗
PubMed1991

Distribution of extraneuronal uptake1 in reproductive tissues: studies on cells in culture.

Cultures of stromal cells from pregnant mouse uterus, and an FL cell line derived from human amnion, displayed significant capacities to O-methylate noradrenaline. O-methylation was inhibited in the stromal cells by uptake1-inhibitors, and in the FL cell line by uptake2 inhibitors. These findings are discussed in terms of the distribution and possible functional importance of catecholamine metabolising systems in the female reproductive system.

Amnion↗
PubMed1991

The use of transgenic animals to study the role of growth factors in endocrinology.

Transgenesis is identified as being of special interest in the study of growth factors where their multicellular origins and complex interactions make them particularly difficult to characterize using classical experimental approaches developed to investigate hormones originating in specialized cells in discrete glands. Through allowing molecular 'tinkering' in intact animals, transgenesis enables specific growth factors to be 'ablated or replaced' from specific tissues and organs and target cell response and impact of modulatory factors such as binding proteins to be explored in the intact animal. To the endocrinologist, the potential applications of such technology are legend. This chapter provides a brief overview of the technique and provides linkages to the rapidly developing body of literature in establishing transgenesis in growth factor research.

Animals↗
PubMed1991

Exogenous progesterone and embryo survival in Booroola-cross ewes.

To investigate if exogenous progesterone improves embryo survival, 209 multiparous Booroola Merino x South Australian Merino ewes, heterozygous for the F gene (F+) were allocated to seven treatment groups and inseminated at a synchronized oestrus. Six groups received progesterone from controlled internal drug release G dispensers on the following days after ovulation: 4-7, 4-11, 4-14, 7-11, 7-14 and 11-14. Concentration of peripheral progesterone increased (P less than 0.05) in most supplemented groups, but there were no significant differences in pregnancy rates between treatments. However, the number of fetuses per pregnancy was increased for progesterone treatments starting on Day 4 (Days 4-7, 4-11 and 4-14 combined v. control; P less than 0.05) and for all supplemented treatments compared with the control (P less than 0.05).

Animals↗
PubMed1991

Lymphokines, including interleukin-2, alter gonadotropin-stimulated progesterone production and proliferation of human granulosa-luteal cells in vitro.

The effects of human interleukin-1 (IL-1) and IL-2 on human granulosa-luteal cell progesterone production were examined with or without hCG stimulation in vitro. Human granulosa-luteal cells were recovered from follicular fluid obtained from women undergoing in vitro fertilization procedures and cultured for up to 7 days before supernatant progesterone level measurement. Lymphokine-rich conditioned medium was prepared from mitogen-stimulated human peripheral blood leukocytes (HPL-CM). The influence of HPL-CM on both granulosa-luteal cell progesterone production and cell growth was inhibitory. In contrast, supernatants of the IL-2-producing cell line MLA-144 (MLA-CM) stimulated both basal progesterone secretion and cell proliferation. Human recombinant IL-2 (from 0.1-100 IU) alone did not change progesterone levels, compared to control values, after 24 h of cell culture. However, 1, 10, and 100 IU IL-2 significantly inhibited progesterone secretion from cells stimulated by 5 IU hCG (P less than 0.01). The enhanced progesterone levels stimulated by forskolin were also significantly inhibited by 10 IU IL-2 (P = 0.01). This effect was not mediated through decreased cAMP, since the forskolin-enhanced cAMP level was not influenced by IL-2, IL-1, with or without hCG, did not show any effect on progesterone production during either 24 or 48 h of cell culture. It is concluded that 1) human recombinant IL-2 significantly inhibits progesterone production stimulated by hCG in human granulosa-luteal cells; 2) IL-2 also had a marked inhibitory effect on forskolin-induced progesterone release, but did not influence the increased cAMP level stimulated by forskolin; 3) the inhibitory influence of IL-2 on progesterone synthesis may be down-stream in the signal transduction pathway from cAMP activation; and 4) HPL-CM and MLA-CM produced inhibitory and stimulatory effects, respectively, on both basal and hCG-stimulated progesterone levels as well as on granulosa-luteal cell proliferation. These activities cannot be completely attributed to IL-2, and other mediators of leukocyte origin may, therefore, exist.

Cell Division↗
PubMed1990

Feeding lupin grain for six days prior to a cloprostenol-induced luteolysis can increase ovulation rate in sheep irrespective of when in the oestrous cycle supplementation commences.

Three groups of 40 parous Merino ewes were supplemented with 500 g of lupin grain/ewe/day for 7 days starting on either Day 3, 7 or 11 of the oestrous cycle and induced to ovulate by injecting cloprostenol on the sixth day of feeding. Supplementation with lupin grain significantly increased ovulation rate in all groups compared with corresponding controls by increasing the proportion of ewes with twin ovulations. Increases in ovulation rate did not depend on the stage of the cycle at which supplementation began or when during the luteal phase luteolysis was induced. It is concluded that the ovulatory response to lupin grain is initiated near the time of luteolysis.

Animal Feed↗
PubMed1990

Granulocyte macrophage colony stimulating factor (GM-CSF) in the murine reproductive tract: stimulation by seminal factors.

The activity of GM-CSF during early pregnancy in the murine uterine lumen in vivo and in media conditioned by uterine cells in vitro has been assessed. GM-CSF was detected in uterine luminal fluid recovered by lavage on the morning after syngeneic mating (median level 5.7 CFUc U/uterus) and following mating with vasectomized (5.1 U/uterus) or allogeneic males (4.4 U/uterus), with significantly lesser (P less than 0.05) amounts recovered from the uteri of superovulated, mated mice. By contrast, GM-CSF was only detectable (greater than 0.5 U/uterus) in the luminal fluid of three of 22 unmated oestrous mice examined. No activity was detected in secretions from male accessory glands including seminal vesicle, epididymis, prostate and coagulating gland (less than 0.5 U/gland). GM-CSF was found at higher levels in supernatants from cell monolayers prepared by tryptic digest of the uteri of Day 1 mated mice than those from unmated oestrous mice (P less than 0.05). Little GM-CSF was detected in supernatants from ovariectomized mice. An alpha-GM-CSF polyvalent antibody neutralized the FD5/12 bioassay response confirming the identity of the lymphokine. The interleukins IL-2 and IL-3 were not detected in uterine luminal fluid nor in media conditioned by cell monolayers. We postulate that elevated uterine GM-CSF activity after mating is elicited by a non-sperm associated, non-MHC component of the ejaculate and synthesized by a hormone-responsive endometrial cell population. This cytokine may have an embryotrophic role or contribute to priming of the uterus for implantation.

Animals↗
PubMed1990

In vitro embryo culture in the production of identical merino lambs by nuclear transplantation.

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.

Animals↗
PubMed1990

In vitro assessment of the viability of sheep zygotes after pronuclear microinjection.

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Temporal changes in the pattern of melatonin secretion in sheep held in constant darkness.

The pattern of melatonin secretion was investigated in six 2 yr-old Suffolk ewes transferred from a long-day lighting regime (14L:10D) to continuous darkness. During the 9 d in continuous darkness, five of the ewes maintained a characteristic diurnal rhythm in melatonin secretion. However, there was a systematic change in the duration of each episode of melatonin secretion with the result that it extended from 10 h under the entraining lighting regime to about 19 h by the fourth cycle and then contracted to about 14 h by the eighth cycle under continuous darkness. The pattern of individual ewes varied, but overall the time of offset showed a consistent delay each successive day of about 1 h, indicative of control by a pacemaker with a period of greater than 24 h. By contrast, the time of onset tended to occur earlier each day during the first 4 d of continuous darkness, then changed so that after 8 d, it was delayed in all animals compared to the time of onset on d 4. If, as previously postulated, onset is controlled by a separate pacemaker, then this change is accounted for by interaction with the dominant offset control pacemaker; however, control of both onset and offset through a single pacemaker cannot be excluded. The study confirms that the patterns of melatonin secretion developed in sheep held in continuous darkness may be used to gain insight into the activity of the light-sensitive pacemaker centre(s) controlling the offset and onset of pineal function. Further experimentation is required to differentiate whether control is being exerted by one or two or more independent but interacting pacemaker centres.

Activity Cycles↗
PubMed1990

Mechanisms controlling the offset of melatonin secretion in the ewe.

Melatonin secretion was investigated in ewes maintained in continuous darkness for 6 days and following an acute delay of lights off as part of a study of factors controlling the melatonin rhythm. In continuous darkness, the interval between successive offsets in circulating melatonin was always greater than 24 hr, indicating that the decline in melatonin secretion in sheep is controlled by endogenous mechanisms having a period longer than 24 hr. In ewes placed under extended darkness with no delay in the time of lights off, the decline in circulating melatonin was delayed by 2.5 +/- 0.2 hr. Animals that had dusk delayed by 4 hr continued to secrete melatonin for at least 4 hr after subjective lights on. Under these conditions, it is proposed that the offset of melatonin secretion is influenced by the timing of lights off and the onset of melatonin secretion. The brain centers controlling melatonin secretion in sheep appear to operate in a manner similar to several species, and thus the sheep may prove to be a useful experimental animal for further studies of circadian mechanisms.

Animals↗
PubMed1989

The status of the corpus luteum during pregnancy in Miniopterus schreibersii (Chiroptera: Vespertilionidae) with emphasis on its role in developmental delay.

Developmental delay is correlated with torpor in the temperate zone bent-winged bat, Miniopterus schreibersii (latitude 37 degrees S) as a period of pre-implantation delay (delayed implantation) followed by a short post-implantation delay (delayed development). During this time, the number of steroidogenic organelles in luteal cytoplasm is greatly reduced compared with normal embryogenesis, and granular endoplasmic reticulum is prominent. Nidation, which occurs while the animals are hibernating, is not accompanied by marked changes in luteal ultrastructure, although the number of lipid droplets decreases somewhat. Progesterone rises slightly but not significantly; however, a pre-nidation decrease in high 17 beta-estradiol levels may play a role in implantation. Following implantation, the conceptus remains delayed at the blastocyst stage for several weeks. During this time the bats remain torpid and the only change in luteal cell ultrastructure is an increase in smooth endoplasmic reticulum as differentiation begins toward the trilaminar stage. At the end of developmental delay hypertrophy of the luteal cell begins and mitochondria and lipid droplets increase, markedly. By this time arousal from hibernation has occurred, placentation takes place and normal development is underway. At placentation, smooth endoplasmic reticulum reaches its maximum in luteal cytoplasm; estrogen and progesterone levels peak about 6 weeks later. For the remaining 2 months of gestation, signs of luteolysis appear. These observations suggest that the corpus luteum of developmental delay, though sub-optimally functional, is prolonged in its luteinization by the arrival of winter when the bats enter torpor. The capacity for maximal steroidogenesis is acquired at the end of winter, some weeks after implantation, when arousal occurs and normal development ensues.

Analysis of Variance↗
PubMed1989

Addition of steroids to embryo-uterine monolayer co-culture enhances embryo survival and implantation in vitro.

Co-culture with a mixed cell monolayer established from trypsinized uterine tissue increased the numbers of embryos developing in a minimum essential medium beyond the hatching blastocyst stage in the 5-day period of culture to 56.0% (343/613) compared with 30.2% (93/308) for embryos cultured in medium alone. The inclusion of progesterone (3.2 x 10(-6), 3.2 x 10(-5), or 3.2 x 10(-4) M) or oestradiol (3.7 x 10(-5) M) to the co-cultures increased the mean percentage of embryos developing to the advanced stages to 72.7-78.4%. The addition of progesterone (3.2 x 10(-6) M) together with oestradiol (3.7 x 10(-5) M) resulted in additional improvement to a mean of 86.5% (787/910, P less than 0.001) but the combination was without significant effect on embryos cultured in media alone. Blastocyst viability was not impaired by co-culture as assessed in embryo survival following surgical transfer to pseudopregnant recipients. This study confirms the feasibility of establishing co-culture systems to facilitate investigation of pre-implantation events in vitro and highlights a role of steroid hormones in enhancing the capacity of uterine cells to support pre-implantation-stage embryos.

Animals↗
PubMed1989

Transgenesis: the challenge for the reproductive biologist.

The last decade has witnessed dramatic developments in molecular biology to a point where it is now possible for genetic engineers to consider programmes aimed at isolating and cloning specific genes from any genomic source for introduction into the germ line of other organisms. The impact of this technology on livestock breeding is potentially revolutionary.

Animals↗