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D. C. Skinner and J. E. Robinson

The presence of melatonin-binding sites in the ovine pars tuberalis is well established, but data on melatonin binding in the pars distalis are inconsistent. The distribution of melatonin-binding sites in the ovine hypophysis was investigated using in vitro auto-radiography and the high-affinity, high specific-activity ligand 2-[125 I]iodomelatonin. The histology of sections was visualized with Heidenhain's azan stain and sections were immunoreacted against ovine LH (β-subunit) using standard immunocytochemical techniques. Melatonin binding in the hypophysis was restricted to the pars tuberalis and the zona tuberalis. The zona tuberalis is histologically similar to the pars tuberalis and appears to be a ventral extension of this region, although the shape and size of the zona tuberalis are extremely variable between individuals. Like the anteroventral pars tuberalis, there is a high concentration of immunoreactive gonadotrophs in the zona tuberalis. The density of immunoreactive gonadotrophs alone is sufficient to discriminate between the zona tuberalis and the pars distalis. Our data suggest that the zona tuberalis and the pars tuberalis are part of the same endocrine tissue and that melatonin-binding sites are not present in the pars distalis proper.

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C. H. CHEN, D. C. KLEIN and J. C. ROBINSON

The present report is the first in a projected series of investigations on biogenic amine-synthesizing and -degrading enzymes in trophoblast. The work is being undertaken to gain information relating to two questions. First, do the placental enzymes which degrade biogenic amines and related compounds provide an important biochemical barrier which protects the fetus against undesirable stimulation by physiological and pharmacological compounds in the maternal circulation? Secondly, do critical changes in biogenic amine metabolism in the placenta precede or accompany parturition, and might they be involved in the actual mechanism underlying the initiation of parturition?

Catechol-O-methyltransferase (COMT, E.C.2.1.1.6) is an enzyme which inactivates certain catecholamines and O-methylates 2-hydroxyoestradiol-17 β (Ball, Knuppen, Haupt & Breuer, 1971); it has been purified 66-fold from human placenta (Gugler, Knuppen & Breuer, 1970).

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C. H. Chen, D. C. Klein and J. C. Robinson

Summary.

The activity of monoamine oxidase (MAO), an enzyme which metabolizes catecholamines and indoleamines, was determined in rat placenta at various stages of gestation, in human term placenta, and in choriocarcinoma grown in culture. From Day 15 to Day 20 of gestation the specific activity (units/mg protein) of MAO in rat placenta increased at least 3-fold; from Day 20 to the time of parturition, it decreased about 50 %. The specific activity of MAO in human placenta at term was about 8 times higher than that of rat placenta at term. No MAO activity was found in choriocarcinoma grown in culture.

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S. M. Rhind, J. J. Robinson, C. Fraser and I. McHattie

Summary. Finnish Landrace × Dorset Horn ewes of two flocks comprising 47 and 51 animals were mated to Suffolk rams at a synchronized oestrus on 9 December (Flock D) and 8 April (Flock A) after treatment with 500 i.u. PMSG. The ewes were slaughtered between 50 and 145 days of pregnancy. The mean ± s.e.m. ovulation rates were 4·5 ± 0·24 (Flock D) and 5·0 ± 0·32 (Flock A) for ewes which conceived to the induced oestrus and the corresponding litter sizes were 2·8 ± 0·15 and 2·8 ± 0·18. Mean concentrations of progesterone in the peripheral plasma during pregnancy were higher for Flock D than for Flock A ewes both in early (P < 0·001) and late (P < 0·05) pregnancy. An increase in ovulation rate, up to 4 or 5, was associated with an increase in mean litter size. There was evidence that higher ovulation rates were counterproductive.

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M. PHILLIPPO, G. H. SWAPP, J. J. ROBINSON and J. C. GILL

Summary.

The genital tract was examined by laparoscopy in anaesthetized sheep at various intervals after mating. A positive diagnosis could be made as early as the 17th day after mating. A correct diagnosis of pregnancy was made in twenty-eight out of thirty-one ewes and the diagnosis of non-pregnancy was correct in twenty-one out of twenty-three ewes. The number of lambs carried was correctly estimated from the number of corpora lutea in twenty out of twenty-four ewes. The results of the method are comparable to those reported by other methods of pregnancy diagnosis in the ewe.

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J. Keating, C. E. Grundy, P. S. Fivey, M. Elliott and J. Robinson

Defective sperm function has been identified as one of the most common causes of human infertility. The aim of this investigation was to identify whether the presence of retained cytoplasm on the human sperm midpiece is associated with defective sperm function. Statistical analysis of data demonstrated a strong negative correlation between the presence of residual cytoplasm on the midpiece of spermatozoa in the inseminate and fertilization rate during IVF. Significant negative correlations were also identified between the percentage of spermatozoa in the ejaculate bearing cytoplasmic residues and (i) spermatozoa having membrane integrity and (ii) sperm concentration. A highly significant positive correlation was also revealed between the percentage of spermatozoa in the ejaculate with membrane integrity and the percentage of motile spermatozoa. These correlations suggest that retained cytoplasm is a cause of subfertility. Measurements of the percentage of spermatozoa bearing residual cytoplasm in the IVF inseminate could provide the basis for a simple predictive test before IVF.

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C. Bielert, J. A. Czaja, S. Eisele, G. Scheffler, J. A. Robinson and R. W. Goy

Summary.

Behavioural and hormonal data were collected throughout pregnancy in captive rhesus monkeys. Heterosexual pairs observed in daily time-limited mating tests showed two distinct periods of increased sexual interaction, as measured by the incidence of ejaculation. One period was coincident with the preovulatory oestradiol peak, while the other occurred between the 6th and 10th weeks of pregnancy. The positive relationship found during the menstrual cycle between the ratio of circulating oestradiol: progesterone and degree of sexual activity continued for the first 8 weeks of pregnancy. However, sexual activity ceased in the second half of pregnancy despite a continued high oestradiol: progesterone ratio.

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R. D. Shanks, R. G. Popp, G. C. McCoy, D. R. Nelson and J. L. Robinson

Summary. Holstein–Friesian cattle heterozygous for the deficiency of uridine monophosphate (UMP) synthase have half-normal activity of UMP synthase. The homozygous recessive genotype would result in little or no activity, has not been observed among live animals and apparently leads to embryonic mortality at ∼Day 40 of gestation. Activity of UMP synthase averaged 2·74 ± 0·61 units/mg protein for 19 obligatory normal embryos (from normal × normal matings). Activity for 18 embryos from heterozygote × heterozygote matings yielded three non-overlapping groups as follows: (i) five presumed normals with > two-thirds normal activity, (ii) ten apparent heterozygotes with one-third to two-thirds normal activity and (iii) three putative homozygous recessive embryos with < one-third normal activity. The distribution among these groups was consistent with the 1:2:1 ratio expected for autosomal inheritance. Conception of embryos homozygous recessive for this disorder was demonstrated.

Keywords: embryonic mortality; inherited disorder; uridine monophosphate synthase; embryo; cow

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Y. Zhao, L. M. Williams, L. T. Hannah, A. W. Ross, W. A. C. McKelvey and J. J. Robinson

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0–3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0–3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0–3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.

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K. D. Sinclair, T. G. McEvoy, E. K. Maxfield, C. A. Maltin, L. E. Young, I. Wilmut, P. J. Broadbent and J. J. Robinson

The effects of in vitro culture systems for sheep zygotes on subsequent fetal growth and development to day 61 and day 125 of gestation were studied. Zygotes recovered from superovulated Scottish Blackface ewes approximately 36 h after intrauterine insemination using semen from a single Suffolk sire were cultured for 5 days in (a) a granulosa cell co-culture system (co-culture); (b) synthetic oviductal fluid medium without serum (SOF−); and (c) synthetic oviductal fluid medium supplemented with human serum (SOF+). Control embryos were recovered from superovulated donor ewes at day 6 after oestrus. Embryos were transferred at day 6 to synchronous Scottish Blackface recipient ewes. In total, 146 gravid uteri were recovered, comprising 97 at day 61 (20 co-culture, 27 SOF−, 25 SOF+ and 25 control) and 49 at day 125 (13 co-culture, 8 SOF−, 6 SOF+ and 22 control) of gestation. Fetuses derived from co-cultured embryos were 14% heavier (P < 0.01) by day 61 of gestation than those derived from control embryos. Growth coefficients derived from the linear allometric equation loge y = loge a + b loge x (where y = organ mass; x = fetal mass) were significantly greater (P < 0.05) for liver, heart, kidneys and plantaris muscle in fetuses derived from co-cultured embryos, and for liver in fetuses derived from SOF+ embryos than those for control fetuses. Fetuses derived from co-cultured embryos were 34% heavier (P < 0.001) and fetuses derived from SOF+ embryos were 18% heavier (P < 0.01) by day 125 of gestation than those derived from control embryos. Growth coefficients for liver and heart for fetuses derived from co-culture and SOF+ embryos were also significantly greater (P < 0.05) at this stage of gestation than those for control group fetuses. In contrast, allometric coefficients for these organs in fetuses derived from embryos cultured in SOF without serum supplementation were not different from those for controls. Excessive volumes of amniotic fluid (polyhydramnios) were observed in 23% of conceptuses derived from co-cultured embryos. In vitro embryo culture can significantly influence fetal growth and this study provides quantitative evidence of major shifts in the patterns of organ and tissue development.