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  • Author: J. E. A. McIntosh x
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As an extension of studies on the chemical composition of rabbit blastocysts (Lutwak-Mann, 1966, 1971), and blastocyst-uterine relationships (Lutwak-Mann, Boursnell & Bennett, 1960), we have measured the content of calcium (Ca) and the uptake of 45Ca in free-lying (5- and 6-day-old) and attached (7-, 8- and 9-day-old) blastocysts, and endometrium, entire uterine wall, early placental tissue, blood plasma, peritoneal fluid and uterine fluid. Oestrous uterine fluid (Lutwak-Mann, 1962) was used because rabbit progestational secretion was too scarce for analysis.

Calcium content was determined by atomic absorption spectrophotometry using lanthanum chloride to eliminate interference from phosphates. Samples (50 to 150 mg) were incinerated with fuming nitric acid and hydrogen peroxide; the mean standard deviation of duplicates was 5 %. Some of the preliminary values for blastocysts were obtained by the method

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The process involved in the disappearance of PMSG from the blood of sheep, following a single intravenous injection, has been separated into two exponential components. Values (mean±S.E.) calculated from experiments on five animals were: metabolic clearance rate (37·8±1·6 ml hr−1); rate constant of disposal (0·0315±0·0016 hr−1); half-time of disposal (21·2±1·1 hr). The stage of the oestrous cycle, ovariectomy and the dose of PMSG used had no apparent effect on these values.

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Large intact follicles and granulosa cells were obtained from sheep ovaries at various stages of the oestrous cycle and were cultured in vitro for 7 days. The daily output of steroid hormones into the culture medium was determined using a procedure capable of measuring a wide range of steroids including immunoreactive oestrogen, testosterone, androstenedione, progesterone, 20α-hydroxy-pregn-4-en-3-one, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-pregn-4-en-3-one, pregnenolone, 17α-hydroxypregnenolone and certain hydrogenated metabolites.

Intact follicles explanted from sheep on Day 4 of the cycle produced oestrogen (40 ng/mg follicular tissue per 24 hr) during the first 3 days in culture followed by large amounts of testosterone (100 ng/mg per 24 hr) and finally 17α-hydroxylated progestin. Follicles explanted at midcycle produced constant amounts of oestrogen (30 ng/mg per 24 hr) throughout the culture period; no other steroids were produced in significant quantities. The highest levels of oestrogen (75 ng/mg per 24 hr) were produced by follicles which had been explanted from sheep on Day 14 of the cycle. Oestrogen production declined rapidly in follicles explanted just before oestrus (late Day 15) and was replaced by the production of testosterone and 17α-hydroxylated progestin. The production of oestrogen and testosterone was very low in follicles explanted at oestrus; these follicles produced a transient peak of 17α-hydroxypregnenolone (30 ng/mg per 24 hr) followed by large amounts of progesterone (300 ng/mg per 24 hr) and 20α-hydroxy-pregn-4-en-3-one (250 ng/mg per 24 hr).

Monolayer cultures of granulosa cells produced only progesterone, 20α-hydroxy-pregn-4-en-3-one and pregnenolone, thus implicating the cells of the theca interna as the principal source of oestrogen, androgen and 17α-hydroxylated progestin.

Our findings indicate that the biosynthesis of oestrogen in the cells of the theca interna involve a sequence of steps including pregnenolone → 17α-hydroxypregnenolone→17α-hydroxyprogesterone→ testosterone or androstenedione→ oestrogen. During the transformation of follicles from oestrogen to progesterone secretors, steroid synthetic capacity is transferred from the theca interna to the membrana granulosa. The accumulation first of testosterone and then of 17α-hydroxypregnenolone suggests that the aromatase and then the desmolase systems limit steroid production in the theca interna during the period of transformation.

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T. J. Weiss, R. F. Seamark, J. E. A. McIntosh and R. M. Moor


This study was undertaken (i) to establish a relationship between cyclic AMP (cAMP) production and the degree of LH and FSH stimulation; (ii) to determine the effects of various gonadotrophins on follicular formation of cAMP; and (iii) to identify the precise intrafollicular site of cAMP formation.

The formation of cAMP increased rapidly in follicles exposed to LH. Maximum concentrations were reached after 90 min and were maintained for 180 min. Extracellular release of cAMP increased steadily throughout the 180-min experimental period. Tissue levels of cAMP increased proportionally and significantly when LH concentrations in the medium were increased from 0 to 200 mi.u. ml-1.

Tissue levels of cAMP were significantly increased by HCG, prostaglandin E-2 and noradrenaline, but not by prolactin, prostaglandin F-2α, serotonin or melatonin.

Cyclic AMP formation occurred predominantly in the thecal compartment; the membrana granulosa contributed less than 3 % of the total amount of cAMP formed after gonadotrophic stimulation. A significant amount of cAMP from the thecal cells was released into the extracellular compartment and appeared to pass into the granulosa cells.