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M Shemesh

Binding sites for LH/hCG are found in the uterus of several species, including humans. In cattle and pigs, the LH receptor, its mRNA and LH receptor protein are present in the uterus throughout the oestrous cycle, and maximum expression occurs at the luteal phase. GnRH receptor is also expressed maximally in the human endometrium at the luteal phase. LH activates both the adenylate cyclase and phospholipase C pathways and increases the concentrations of cyclooxygenase and its products. Activation of LH receptors in the endometrium is associated with PGF production. In contrast, bovine uterine vein LH receptor mRNA and LH receptor concentrations are greatest during pro-oestrus-oestrus and LH increases the production of both PGE and PGF. FSH receptor and its mRNA are present in the bovine cervix and the concentrations are greatest at the time of the FSH peak value in the blood, indicating a physiological role for FSH in the relaxation and opening of the cervix. The presence of gonadotrophin and releasing factor receptors with a dynamic pattern in the endometrium, myometrium, oviduct and cervix of different species provides evidence that gonadotrophins and GnRH play a substantial role as molecular autocrine-paracrine regulators of the oestrous cycle and implantation.

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N. AYALON and M. SHEMESH

A preovulatory rise in peripheral plasma progesterone concentration has been reported in women, monkeys, rats, mice, hamsters, guinea-pigs and rabbits (Kazama & Hansel, 1970). In the cow, a preovulatory rise in plasma progesterone levels was not found when blood samples were collected once daily (Shemesh, Lindner & Ayalon, 1971) every 2 to 3 hr for 10 to 12 hr, starting with the onset of oestrus (Henricks, Dickey & Niswender, 1970), or every 6 hr from the onset of oestrus until ovulation (Kazama & Hansel, 1970). The latter authors noted the possibility that a transient rise and fall in plasma progesterone concentration may have been missed due to infrequency of sampling during the pro-oestrous period. The present investigation was undertaken to examine this possibility.

The blood samples examined were aliquots from the same collections of jugular

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L. Shore and M. Shemesh

Summary. Ovaries were taken from 12 freemartins of crown-rump length 4·2–27·5 cm (47–130 days of gestation), 16 singleton fetuses of 5–8 cm (55–75 days) and 18 singleton fetuses of 10–20 cm (78–108 days). Ovaries were incubated for 24 h in tissue culture Medium 199 supplemented with 5% calf serum. The freemartin ovaries produced 0·16–2·1 ng testosterone/ovary/24 h compared with < 50 pg for normal singleton ovaries. Oestrogen was undetectable in the ovaries (< 50 pg) of freemartin fetuses of crown-rump length 4·2–9·4 cm (47–77 days), while 16 normal ovaries of the same stage produced 1375 ± 300 pg/ovary/24 h. The presence of abnormally high amounts of testosterone and the absence of normally occurring oestrogen in the freemartin ovary indicate that the pattern of steroidogenesis in the freemartin is altered as early as 47 days of gestation.

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M. SHEMESH, H. R. LINDNER and N. AYALON

Summary.

The non-steroidal phyto-oestrogens, coumestrol and genistein, were able to compete with oestradiol-17β (Oe2) for binding sites on a macromolecular component of the uterine cytosol from 6-day-pregnant rabbits. Binding affinity for these compounds was related to their reported oestrogenic potency: approximately one part by weight of Oe2, seventy of coumestrol and 175 of genistein produced equivalent inhibition of the uptake in vitro of [3H]Oe2 by this uterine receptor. Biochanin-A, formononetin, 12-O-methylcoumestrol, sativol and medicagol did not significantly inhibit Oe2 binding, suggesting that free hydroxyl groups in both position 7 and 12 (= 4') of coumestans and isoflavones are essential for effective interaction with the oestrogen receptor. The 7- and 12-methoxy-coumestans and isoflavones tested appear to be pro-oestrogens, able to bind to the uterine receptor only after O-demethylation in vivo.

A rapid competitive protein-binding radioassay for phyto-oestrogens in the blood that makes use of the uterine cytosol receptor is described. Its useful range is 0·5 to 40 ng for coumestrol and 2·5 to 200 ng for genistein. Prior chromatographic separation is required to discriminate between plant-derived and endogenous steroidal oestrogens. Coumestrol was present in the blood of goats (2·0 to 3·9 ng/ml) after feeding alfalfa hay at a rate supplying 12 mg coumestrol/24 hr/animal.

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M. SHEMESH, N. AYALON and H. R. LINDNER

Progesterone levels in jugular venous blood are being measured by us in cycling (unmated) and inseminated (pregnant or non-pregnant) cows as part of a comparative study of cows with normal and difficult breeding histories. Hawk, Wiltbank, Kidder & Casida (1955) reported a high incidence of embryonic death in cows during the period 16 to 25 days after insemination. The 3rd week of gestation may indeed be a critical phase in the regulation of luteal function, since towards the end of this week regression of the corpus luteum normally occurs in the absence of a conceptus. This note records that in a herd of normal Friesian cattle with a cycle length of 21·2 days±1·5 s.d. the presence of a conceptus in the uterus first exerted a significant

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M. Shemesh, N. Ayalon and T. Mazor

Summary. Plasma and milk progesterone concentrations in pregnant sheep (18–22 days after mating) were similar, about 3·7 ng/ml whereas values in non-pregnant sheep were <1 ng/ml. Lambing results indicated identical accuracy for both methods (82 and 84% in 2 flocks). The accuracy was 92–100% for ewes diagnosed non-pregnant in the breeding season, but for ewes tested in the non-breeding season the diagnosis of non-pregnancy according to milk progesterone levels was only 50% accurate.

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M. SHEMESH, H. R. LINDNER and N. AYALON

Summary.

A rapid radiometric assay based on thin-layer-chromatography and the competition by progesterone for binding sites on the corticosterone-binding globulin of human plasma was used to determine changes in the level of progesterone in peripheral bovine plasma during the oestrous cycle. The blood level (ng/ml, mean ± S.E.) of the hormone was minimal (0·6± 0·15 to 0·8± 0·20) from Day −2 to +2 of the cycle (Day 0 = day of oestrus), rose steeply between Day 3 and Day 7, and either continued to rise very slowly or maintained a plateau fluctuating about a mean of 5·4± 0·16 for 8 to 10 days, before declining; the most abrupt fall in plasma progesterone concentration occurred on Day −4 or −3. The results agreed well with control data obtained by sequential paper- and gas-chromatography (r = 0·93).

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Y. Shalev, M. Shemesh, T. Levinshal, S. Marcus and H. Breitbart

Ejaculated bovine spermatozoa were examined for their capacity to synthesize prostaglandins E2 and F (PGE2, PGF). It was found that in the absence of exogenous substrate, arachidonic acid, basal PGF production was less than that of PGF2. However, addition of 61 μmol arachidonic acid l−1 resulted in at least a twofold increase in PGF2 and PGF above control values (1.3 ng and 0.3 ng per 108 spermatozoa, respectively). Addition of calcium and the calcium ionophore A23187 to the incubation medium did not cause a significant increase in the production of either PG. The presence of indomethacin (100–200 μg ml−1) caused a 50–70% inhibition of the production of both PGs. Activity of cyclooxygenase was determined by western blot analysis, using a specific polyclonal antiserum, and by fluorescence immunohistochemistry using a monoclonal antibody. The western blot displayed a clear signal for the presence of cyclooxygenase in ejaculated and epididymal spermatozoa. The immunohistochemical studies showed that the enzyme is localized in the apical region of the head, the post-acrosomal region and the mid-piece of the tail. Since the synthesis of PGs in the absence of exogenous arachidonic acid is low, the effect of melittin, a known phospholipase A2 activator, on PG production was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE2 and a sixfold increase in PGF. Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A2 by protein kinase C is an obligatory step in PG synthesis by bovine spermatozoa. To determine the physiological role of PG synthesis by bovine spermatozoa the effect of PGE2 and PGF on Ca2+ uptake was examined. PGE2, but not PGF, increased the Ca2+ uptake linearly during the first 10 min of incubation. These data provide evidence that bovine spermatozoa can synthesize prostaglandins in the presence of arachidonic acid or melittin. Furthermore, it was demonstrated that the spermatozoa contain the key enzyme for prostaglandin synthesis, cyclooxygenase.

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M. Shemesh, F. Milaguir, N. Ayalon and W. Hansel

Summary. Bovine blastocysts were collected at Days 13, 15 and 16 and placed in TCM-199 supplemented with 5% fetal calf serum; some blastocysts were immediately frozen while the others were cultured for 48 h and then frozen. Samples (tissue + medium, 5–12/group) were thawed, homogenized and analysed by radioimmunoassays. Measurable amounts of progesterone were found in all blastocysts but values were higher (P < 0·01) after culture. Testosterone was not found in the cultured or uncultured blastocysts at Day 13, but was detectable on Days 15 and 16 and in greater amounts (P < 0·05) in the cultured blastocysts. PGF and PGE-2 were increased (P < 0·05) in the cultured blastocysts on all 3 days. Oestradiol was measurable in some but not all blastocysts. It is suggested that PG synthetase and enzymes capable of synthesizing progesterone, testosterone and, possibly, oestradiol are present in these early bovine blastocysts.