Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells.

The phytoestrogens genistein, daidzein and the daidzein metabolite equol have been shown previously to possess oestrogen agonist activity. However, following consumption of soya diets, they are found in the body not only as aglycones but also as metabolites conjugated at their 4'- and 7-hydroxyl groups with sulphate. This paper describes the effects of monosulphation on the oestrogen agonist properties of these three phytoestrogens in MCF-7 human breast cancer cells in terms of their relative ability to compete with [(3)H]oestradiol for binding to oestrogen receptor (ER), to induce a stably transfected oestrogen-responsive reporter gene (ERE-CAT) and to stimulate cell growth. In no case did sulphation abolish activity. The 4'-sulphation of genistein reduced oestrogen agonist activity to a small extent in whole-cell assays but increased the relative binding affinity to ER. The 7-sulphation of genistein, and also of equol, reduced oestrogen agonist activity substantially in all assays. By contrast, the position of monosulphation of daidzein acted in an opposing manner on oestrogen agonist activity. Sulphation at the 4'-position of daidzein resulted in a modest reduction in oestrogen agonist activity but sulphation of daidzein at the 7-position resulted in an increase in oestrogen agonist activity. Molecular modelling and docking studies suggested that the inverse effects of sulphation could be explained by the binding of daidzein into the ligand-binding domain of the ER in the opposite orientation compared with genistein and equol. This is the first report of sulphation enhancing activity of an isoflavone and inverse effects of sulphation between individual phytoestrogens.

Oestrogenic activity of p-hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben in human breast cancer cell lines.

This paper addresses the question of whether p-hydroxybenzoic acid, the common metabolite of parabens, possesses oestrogenic activity in human breast cancer cell lines. The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in consumer products to which the human population is exposed and have been shown previously to possess oestrogenic activity and to be present in human breast tumour tissue, which is an oestrogen-responsive tissue. Recent work has shown p-hydroxybenzoic acid to give an oestrogenic response in the rodent uterotrophic assay. We report here that p-hydroxybenzoic acid possesses oestrogenic activity in a panel of assays in human breast cancer cell lines. p-Hydroxybenzoic acid was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor of MCF7 human breast cancer cells by 54% at 5 x 10(6)-fold molar excess and by 99% at 10(7)-fold molar excess. It was able to increase the expression of a stably integrated oestrogen responsive reporter gene (ERE-CAT) at a concentration of 5 x 10(-4) M in MCF7 cells after 24 h and 7 days, which could be inhibited by the anti-oestrogen ICI 182 780 (Faslodex, fulvestrant). Proliferation of two human breast cancer cell lines (MCF7, ZR-75-1) could be increased by 10(-5) M p-hydroxybenzoic acid. Following on from previous studies showing a decrease in oestrogenic activity of parabens with shortening of the linear alkyl chain length, this study has compared the oestrogenic activity of p-hydroxybenzoic acid where the alkyl grouping is no longer present with methylparaben, which has the shortest alkyl group. Intrinsic oestrogenic activity of p-hydroxybenzoic acid was similar to that of methylparaben in terms of relative binding to the oestrogen receptor but its oestrogenic activity on gene expression and cell proliferation was lower than that of methylparaben. It can be concluded that removal of the ester group from parabens does not abrogate its oestrogenic activity and that p-hydroxybenzoic acid can give oestrogenic responses in human breast cancer cells.

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells.

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.

Concentrations of parabens in human breast tumours.

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng x g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng x g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues.

Oestrogenic activity of benzylparaben.

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10,000-fold molar excess, by 57% at 100 000-fold molar excess and by 100% at 1,000,000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive MDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping.

Oestrogenic activity of isobutylparaben in vitro and in vivo.

The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in foods, pharmaceuticals and cosmetics to which the human population is exposed. Recent studies have reported that methylparaben, ethylparaben, n-propylparaben and n-butylparaben all possess oestrogenic activity in several in vitro assays and in animal models in vivo. This study reports on the oestrogenic activity of isobutylparaben in a panel of assays in vitro and in vivo. Isobutylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor alpha of MCF7 human breast cancer cells by 81% at 100 000-fold molar excess. Using a clonal line of MCF7 cells containing a stably transfected oestrogen-responsive ERE-CAT reporter gene, CAT gene expression could be increased by isobutylparaben such that the magnitude of the response was the same at 10(-5) M isobutylparaben as with 10(-8) M 17beta-oestradiol. Isobutylparaben could also increase expression of the endogenous oestrogen-responsive pS2 gene in MCF7 cells and maximal expression at 10(-5) M isobutylparaben could be inhibited with the anti-oestrogen ICI 182 780. The proliferation of two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1 could be increased with isobutylparaben such that at concentrations of 10(-5) M the proliferation response was of the same magnitude as with 10(-8) M 17beta-oestradiol. Evidence for oestrogen receptor mediation of proliferation effects was provided by the inability of isobutylparaben to influence the growth of oestrogen-unresponsive MDA-MB-231 human breast cancer cells and by the ability of the anti-oestrogen ICI 182 780 to inhibit the isobutylparaben effects on MCF7 cell growth. The proliferation response to 10(-10) M 17beta-oestradiol was not antagonized with isobutylparaben at any concentration from 10(-9) M to 10(-4) M in either MCF7 or ZR-75-1 cells. Finally, subcutaneous administration of isobutylparaben was able to increase the uterine weight in the immature mouse after three daily doses of 1.2 or 12.0 mg per mouse. Previous work using linear-alkyl-chain parabens has shown that oestrogenic activity increases with alkyl chain length from methylparaben to n-butylparaben. The results here show that branching of the alkyl chain to isobutylparaben increases oestrogenic activity beyond that of the equivalent length linear alkyl chain in n-butylparaben.

Oestrogenic activity of parabens in MCF7 human breast cancer cells.

Parabens (4-hydroxybenzoic acid esters) have been recently reported to have oestrogenic activity in yeast cells and animal models. Since the human population is exposed to parabens through their widespread use as preservatives in foods, pharmaceuticals and cosmetics, we have investigated here whether oestrogenic activity of these compounds can also be detected in oestrogen-sensitive human cells. We report on the oestrogenic effects of four parabens (methylparaben, ethylparaben, n-propylparaben, n-butylparaben) in oestrogen-dependent MCF7 human breast cancer cells. Competitive inhibition of [3H]oestradiol binding to MCF7 cell oestrogen receptors could be detected at 1,000,000-fold molar excess of n-butylparaben (86%), n-propylparaben (77%), ethyl-paraben (54%) and methylparaben (21%). At concentrations of 10(-6)M and above, parabens were are able to increase expression of both transfected (ERE-CAT reporter gene) and endogenous (pS2) oestrogen-regulated genes in these cells. They could also increase proliferation of the cells in monolayer culture, which could be inhibited by the antiestrogen ICI 182,780, indicating that the effects were mediated through the oestrogen receptor. However, no antagonist activity of parabens could be detected on regulation of cell proliferation by 17 beta-oestradiol at 10(-10)M. Molecular modelling has indicated the mode by which paraben molecules can bind into the ligand binding pocket of the crystal structure of the ligand binding domain (LBD) of the oestrogen receptor alpha (ERalpha) in place of 17beta-oestradiol; it has furthermore shown that two paraben molecules can bind simultaneously in a mode in which their phenolic hydroxyl groups bind similarly to those of the meso-hexoestrol molecule. Future work will need to address the extent to which parabens can accumulate in hormonally sensitive tissues and also the extent to which their weak oestrogenic activity can add to the more general environmental oestrogen problem.

Relationships of peri-partum, plasma concentrations of progesterone, oestrogens and 13,14-dihydro-15-ketoprostaglandin F2alpha in heifers and of anatomical measurements of dam and calf with difficulty of calving in early-bred Hereford x Friesian heifers.

Plasma concentrations of progesterone, oestradiol-17beta, oestrone, oestrone sulphate and PGFM have been measured daily during the first peri-partum period of 45 Hereford x Friesian heifers bred at 11 months of age. Anatomical measurements of dam and calf were also recorded. Twelve of the calvings were scored easy, 33 difficult. Each of five models (fitted by linear logistic regression) relating difficulty of calving to the hormonal and anatomical measurements, predicts with at least 94% accuracy the calving score (easy or difficult) among the calvings. The models predict that increases of progesterone concentration on the day before calving, of oestrone sulphate concentration on the day after calving and of heifer heart girth decrease the odds of difficult calving, whereas increases of heifer body length and of calf head circumference increase the odds of difficult calving.

Possible roles for prolactin, thyroxine and insulin in the growth of heifers before and during early pregnancy.

Two groups of British Friesian heifers with cyclic ovarian function and the potential for further growth were individually fed a restricted diet at two levels over a 12-week period. Eight heifers receiving the higher level of feed (SP group) and five receiving the lower level (LP group) became pregnant by artificial insemination at week 6. Mean (and SEM) body weight changes in the SP and LP groups were respectively 0.24 (+/- 0.024) kg day-1 and -0.19 (+/- 0.039) kg day-1. Over the period from weeks 3-12, mean concentrations of plasma prolactin, thyroxine and insulin in the five LP heifers were 39%, 67% and 74%, respectively, of those in the eight SP heifers. The results suggest that prolactin, thyroxine and insulin have roles in the growth of heifers before and during early pregnancy.

Effects of luteolytic doses of prostaglandin F2 alpha and cloprostenol on concentrations of progesterone, luteinizing hormone, follicle-stimulating hormone, glucose, insulin, growth hormone, thyroxine, prolactin and cortisol in jugular plasma of lactating dairy cows.

Three groups of five lactating dairy cows in the mid-luteal phases of oestrous cycles were given an injection (at time 0 h) of the naturally-occurring prostaglandin F2 alpha (PGF2 alpha) or cloprostenol (a synthetic analogue of PGE2 alpha) at doses recommended for inducing luteolysis, or injection vehicle. Concentrations of glucose and hormones in jugular plasma were measured from 26 h before to 12 h after the injections and the significance (P < 0.05) of the effects of the prostaglandins on these concentrations was determined. Both prostaglandins induced falls in progesterone concentration and rises in luteinizing hormone concentration; neither influenced follicle-stimulating hormone. PGF2 alpha increased glucose concentration; neither prostaglandin influenced insulin concentration. Both prostaglandins increased growth hormone concentration and resulted in declining thyroxine concentration. PGF2 alpha increased prolactin and cortisol concentration. There were, however, no significant differences between the effects of the two prostaglandins on any hormone (or glucose) concentration.

Oestradiol-17 beta in the milk of cows from 6 days before to 14 days after their insemination.

In artificially inseminated cows (AI on day 1) peak concentrations of oestradiol-17 beta in defatted milk occurred at median times of day 0 during the pre-ovulatory period, days -5 to 2, and day 6 during the post-ovulatory period, days 2-15. Median peak concentrations during these periods were approximately 5 pg/ml and 3 pg/ml respectively. There were no significant differences in the timing or magnitude of oestradiol-17 beta concentrations between cows that became pregnant to the AI and those that entered normal length oestrus cycles immediately after AI.

Progesterone concentration in defatted milk of dairy cows in early pregnancy.

Progesterone concentrations have been measured in defatted milk of British Friesian cows of four herds during the oestrus cycles (other than short cycles) immediately before artificial insemination (AI) at oestrus and immediately after AI (in non-pregnant cows), and during early pregnancy. Differences in mean progesterone concentrations between herds were significant (P less than 0.05) on all days within the day 10-18 period after AI, both in pregnant and in non-pregnant, inseminated cows but were not significant between pregnant and non-pregnant cows within herds until day 17 or 18. It is concluded that up to this time (that of luteolysis in non-pregnant cows) undefined factors, variable among herds, can have a much greater influence on the rate of progesterone secretion by corpora lutea and consequent progesterone concentration in plasma and milk than does the presence of conceptuses. Maximum mean progesterone concentration reached during early pregnancy in two herds did not differ significantly; it was reached in the 11-15-day period in one herd but not until 46-50 days in the second. Mean progesterone concentration declined after day 90.

The value of progesterone, oestradiol benzoate and cloprostenol in controlling the timing of oestrus and ovulation in dairy cows and allowing successful fixed-time insemination.

The relative merits of three hormone treatments of dairy cows: (1) intravaginally administered progesterone and oestradiol benzoate; (2) intravaginally administered progesterone and injected cloprostenol; and (3) injected cloprostenol; begun 35-75 days after calving and designed to synchronize oestrus and ovulation and allow successful artificial insemination (AI) at fixed times, have been assessed utilizing information from progesterone concentrations in milk. From this it was concluded that 89% of the cows had ovulated one to three times between calving and the beginning of treatment. Treatment (2) was more effective than (1) in synchronizing ovulation. This was due to the fact that when treatments began early in the ovulation cycle, the requirement for a rapidly effective luteolytic agent was provided by cloprostenol but not by oestradiol benzoate. Treatment (2) was also more effective than (3) in synchronizing ovulation. This is interpreted as meaning that progesterone treatment for 12 days had a beneficial effect in restoring normal cyclic ovarian function in the cows after calving. Whilst cloprostenol administered alone did not have this beneficial effect, there is no evidence that it had a detrimental effect. Based on all cows in treatment groups, the proportion that became pregnant to the fixed-time AI was significantly greater after treatment (2) than after (1), but when based on numbers of cows with synchronized ovulation, there were no significant differences among treatments in the proportions becoming pregnant. The progesterone/cloprostenol treatment had a disadvantage in that when begun during the 11-22 day period of the ovulation cycle, so resulting in a long, total period of suppression of ovulation (mean, 32.1 days), fertility to the fixed-time AI was poor despite effective synchronization of ovulation. Ovulation cycles immediately following the failed, fixed-time AI were normal, both in length and in maximum, luteal-phase progesterone concentration and indicated normal corpus luteum function. Thus the infertility could be ascribed neither to poor timing of AI nor to gross degeneration of follicles prior to their synchronized ovulation following the prolonged suppression of ovulation. The 12-day progesterone treatments when given to anovulatory cows gave, within 5.5 h of their beginning, a concentration of progesterone in milk that was not significantly different from the maximum reached. This concentration declined during the 12 days of the treatment but remained above pretreatment level until 5.5 h after treatment withdrawal; the maximum reached was about half that in normal ovulation cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Post-partum, ovarian function in dairy cows as revealed by concentrations of oestradiol-17 beta and progesterone in defatted milk.

Concentrations of oestradiol-17 beta and progesterone in defatted milk have been used to study the post-partum restoration of ovarian function in 52 autumn-calved dairy cattle. In 32 cows the first preovulatory peaks in oestradiol-17 beta concentration (indicative of imminent ovulation) were less than or equal to 15 days and in 49 cows less than or equal to 49 days post partum. Delay to first ovulation was mainly due, not to failure of ovarian secretion of oestradiol-17 beta at preovulatory level, but to failure of oestradiol-17 beta at this level to exert its normal preovulatory function. Longer intervals to first ovulation were associated with longer intervals from calving to the clearance of placental oestradiol-17 beta, high peak milk yields and high body weight losses, suggesting that in high-yielding dairy cows these factors (themselves inter-related) may be associated with others, which are the immediate cause of the inhibition of the normal, preovulatory function of oestradiol-17 beta. Forty-eight per cent of cows had short, first ovulation cycles. Ovarian function, comprising oestradiol-17 beta secretion at preovulatory level, normal preovulatory function of oestradiol-17 beta and normal corpus luteum progesterone secretion were almost fully restored in this herd by the 49th day post partum.

Concentrations of oestradiol-17 beta in plasma and milk and progesterone in plasma during the oestrus cycle and in early pregnancy in goats.

Pre-ovulatory peaks in oestradiol-17 beta concentrations were observed on days 1 or 2 and post-ovulatory peaks between days 4 and 7, both in jugular venous plasma and defatted milk, day 1 being the day of the onset of oestrus in the goats. Mean values of the magnitudes of these concentration peaks and of their timing (relative to oestrus) during the oestrus cycle did not differ significantly (P greater than 0.05) from those when the goats were mated and became pregnant. Pre-ovulatory oestradiol-17 beta peaks were invariably greater than the corresponding post-ovulatory peaks, as were peak concentrations in plasma relative to those in defatted milk collected on the same day. Mean intervals between the pre- and post-ovulatory peaks in oestradiol-17 beta concentrations were respectively 4.2 days for plasma and 4.0 days for defatted milk. Concentrations of oestradiol-17 beta in jugular venous plasma and defatted milk were strongly correlated: rank correlation coefficients for the three goats studied were 0.871, 0.668 and 0.739. It is suggested that in goats, as in cattle, ovarian follicular oestradiol-17 beta secretion approaching pre-ovulatory level is restored by 4 days after oestrus and its rapid decline after this time may be due to the inhibitory influence of the rapidly rising plasma progesterone concentration.

Seasonal changes in the testes and accessory reproductive organs and seasonal and circadian changes in plasma testosterone concentrations in the male grey squirrel (Sciurus carolinensis).

A 27-month study of cycles of regression and recrudescence of testis function in adult, grey squirrels in a natural environment in southern England has shown concomitant variation in mean testes weights, mean plasma testosterone concentrations, and mean weights of accessory sex organs, this variation being closely associated with months of the year. Testis regression occurred in the period June to August, the exact timing differing among individuals. In 1979-1980 the testes of all squirrels then remained regressed for 7 months, whereas in the autumn of 1980 testes were regressed in most squirrels for 4 months. There was also evidence of testis regression in some individuals in March 1979 and March 1981. Males born and housed in a small woodland enclosure in 1979-1980 and well fed with grain did not experience the long period of regressed testes. Plasma testosterone concentrations measured hourly over 24 hr in squirrels with large, active testes varied from 0.4 to 20 nmol/l, both within and between individuals, the higher concentrations being observed between midday and midnight. The range in the age of males at puberty, based on fusion of the epiphyses of the wrist, was 1.0 to 1.25 year. Juvenile males housed in a woodland enclosure together with adult males and females remained prepubertal up to 2 years of age.

Testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3, 17-dione in the plasma of male and female grey squirrels (Sciurus carolinensis).

Extracts of squirrel plasma have been chromatographed on partition columns (using a hydrophilic stationary phase) at atmospheric pressure (Celite support) and a reversed phase system at high pressure (HPLC). Both methods effectively separated testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one (DHT) and 4-androstene-3,17-dione; they gave elution patterns that differed considerably. Chromatographic mobility of the three androgens on the two systems was identical with that of fractions of squirrel plasma extracts that gave responses measured by appropriate androgen radioimmunoassays; good evidence for the occurrence of these androgens in squirrel plasma is thus provided. Plasma testosterone levels were 300 pmol/l in juvenile males, 800-7000 pmol/l in sexually-active males but undetectable (less than 50 pmol/l) in sexually-regressed males. Plasma DHT levels were also high in sexually-active males, but undetectable in other males except for one regressed individual. Plasma androstenedione was higher in juvenile males than in adult males, in which it was similar whether or not they were sexually regressed. Plasma testosterone and DHT, unlike androstenedione, were totally dependent on the presence of the testes. In females testosterone and DHT were undetectable in plasma but androstenedione levels were high, especially at oestrus. Androstenedione was dependent on the presence of the ovaries.

Fertility of dairy cattle following oestrus and ovulation controlled with cloprostenol, oestradiol benzoate and progesterone or progesterone and cloprostenol.

There have been several approaches to the control of the timing of the oestrous cycle and ovulation in dairy cattle in the last three decades. The first phase involved the use of progestins which were administered in various forms for prolonged periods. Although the timing of oestrus was controlled in most animals after withdrawal of the treatment, this control was not very precise and pregnancy rates from insemination at the first oestrus after treatment were reported to be below normal. Attempts were then made to combine short-term progestin treatments with oestrogens as luteolytic agents to gain better control of the timing of oestrus and ovulation. These studies resulted in some cases in better synchronization of oestrus and improved pregnancy rates. The discovery that prostaglandin F2 alpha (PGF2 alpha) and its synthetic analogue, cloprostenol were potent luetolytic agents in the cow led in the past decade to the use of these agents for oestrus and ovulation control in cattle. Prostaglandins for this purpose are ineffective in anovulatory cows, in cows with deficient luteal function and in the first 5 days of the oestrus cycle when a new corpus luteum is being formed. This limitation in their use has encouraged investigations into the combined use of short-term progestin treatment with prostaglandins to give more effective control of the timing of oestrus and ovulation and to avoid the adverse effects on fertility of long-term progestin treatment. Short-term progestin treatment combined with prostaglandins should mean that fewer cows would have ovulation suppressed for long periods and fertility of treated cows should be improved. A comparison of three procedures of ovulation control and fertility results shows the short-term progestin treatment combined with prostaglandin to be the most effective.

Oestrogens in milk.

The steroid oestrogens oestradiol-17 beta, oestrone and oestradiol-17 alpha have all been adequately identified in various body fluids and tissues of cattle. There is also good evidence for the presence of oestrone sulphate. Oestriol (or similar triols) may also be present. The oestrogens found in the systemic plasma of cattle are present in milk in similar concentrations; whether their passage into milk involves metabolism by the mammary gland is uncertain. Oestrone sulphate, at least at relatively high levels of secretion, is believed to be found only in pregnant cattle and measurement of its concentration in milk is in use as a practical test for pregnancy. A close correlation has been found between the concentrations of oestradiol-17 beta in systemic plasma and milk of non-pregnant cows and levels of this oestrogen in milk may, together with those of progesterone, now be used in studies of post-partum ovarian function.