Immunisation of ewes with polyandroalbumin to improve fecundity.
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Androstenedione↗
Biomedical subjects
R J Scaramuzzi
Publications and source records attributed to R J Scaramuzzi.
The induction of oestrus and ovulation in seasonally anovular ewes by exposure to rams.
In anoestrus ewes of many breeds, the introduction of rams will evoke an increase in the tonic secretion of LH, pre-ovulatory surges of LH and FSH, and ovulation, provided the ewes have been preconditioned by a period of complete isolation from the rams. The "ram effect" is apparently mediated by a pheromone which seems to inhibit or reverse the effects of photoperiod on the secretion of LH pulses. In the absence of progesterone "priming" the ewes rarely show oestrus behaviour around the time of the first ovulation. In some of the ewes, the corpus luteum formed at the first ovulation is apparently normal, and the second ovulation and first oestrus is observed about one oestrus cycle later. In the remainder of the ewes, the first corpus luteum regresses prematurely, a second ovulation is seen about 6 days after the first, and the third ovulation and first oestrus are seen after a further 17 days. Progesterone treatment prior to the introduction of rams prevents the occurrence of short cycles, apparently by delaying the LH surge. The combination of the ram effect and progesterone treatments can provide useful synchrony of oestrus for artificial insemination, and also some gains in fecundity. There is considerable potential for using the effect in intensive breeding programmes.
Animals↗
Effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus.
The effects of progesterone on the responses of Merino ewes to the introduction of rams during anoestrus were investigated in two experiments. In the first experiment, the introduction of rams induced an increase in the levels of LH in entire ewes. The mean levels increased from 0.68 +/- 0.04 ng/ml (mean +/- s.e.m.) to 4.49 +/- 1.32 ng/ml within 20 min in ewes not treated with progesterone (n = 10). In ewes bearing progesterone implants that provided a peripheral concentration of about 1.5 ng progesterone per millilitre plasma, the LH response to the introduction of rams was not prevented, but was reduced in size so that the concentration was 1.38 +/- 0.15 ng/ml after 20 min (n = 5). Progesterone treatment begun either 2 days before or 6 h after the introduction of rams and maintained for 4 days prevented ovulation. In the second experiment ovariectomized ewes were used to investigate further the mechanism by which the ram evoked increases in tonic LH secretion. In ovariectomized ewes treated with oestradiol implants, the introduction of rams increased the frequency of the LH pulses and the basal level of LH. In the absence of oestradiol there was no significant change in pulse frequency but a small increase in basal levels. Progesterone again did not prevent but reduced the responses in ewes treated with oestradiol. It is suggested that following the withdrawal of progesterone treatment, the secretion of LH pulses in response to the ram effect would be dampened. This effect could be a component of the reported long delay between the introduction of rams and the preovulatory surge of LH in ewes treated with progesterone. Continued progesterone treatment prevented ovulation, probably by blocking positive feedback by oestradiol.
Animals↗
Modification of sheep pineal beta-adrenoceptors by some gonadal steroids but not by melatonin.
The effects of active immunization against a range of gonadal steroids on pineal beta-adrenoceptors were studied in cycling Merino ewes. In selected cases, the effects of exogenous steroids were also investigated. Immunization against progesterone had no detectable effect on pineal beta-adrenoceptor density or ligand binding affinity. Immunization against estrone or 17 beta-estradiol significantly increased binding affinity and decreased beta-receptor density. Testosterone immunization in ewes caused similar effects but to a lesser degree. In contrast, immunization against androstenedione resulted in a decreased affinity and an increase in receptor density in ewes. In subsequent experiments, some parameters of pineal function in intact cycling and ovariectomized ewes were compared; a significant decrease in beta-receptor density and an increase in binding affinity were noted in the ovariectomized animals. Androstenedione-releasing implants decreased beta-receptor density and increased beta-receptor affinity in pineals from intact ewes, but these implants had no effect on pineal parameters in ovariectomized ewes. Immunization of ewes against 17 beta-estradiol had no significant effects on basal or isoprenaline-stimulated N-acetyltransferase or adenyl cyclase activities. Immunization of ewes against melatonin, or in vitro incubation of pineal glands with the hormone had no effects on the pineal parameters studied. It is concluded that estrogens affect the pinealocyte beta-receptors in ewes, while specific androgens may act indirectly on these receptors to modify their interaction with estrogens.
Androstenedione↗
Effect of the introduction of rams during the anoestrous season on the pulsatile secretion of LH in ovariectomized ewes.
Introduction of rams to ovariectomized ewes treated with oestradiol implants (N = 10) increased the frequency of LH pulses from 4 X 8 to 10 X 6 pulses per 12 h. This effect was reflected by increases in mean levels of LH and the basal levels upon which the pulses were superimposed. In ewes that had not been treated with oestradiol (N = 5), there was no significant increase in pulse frequency but mean and basal levels of LH increased slightly after the introduction of rams. In a second experiment, similar effects of the introduction of rams were seen in ovariectomized ewes treated with oestradiol or oestradiol + androstenedione (N = 16), but no significant effects of the rams were observed in untreated ewes (N = 8) or ewes treated only with androstenedione (N = 7). No preovulatory surges of LH were observed in the 30-h period after the introduction of rams. It was concluded that the ram stimulus probably evokes the increase in pulse frequency by inhibiting the negative feedback action of oestradiol, and that the surge normally observed in entire ewes is dependent on the ovarian response to these pulses. However, the observation of responses in some ewes not treated with oestradiol also raises the possibility that the ram stimulus can act directly on the hypothalamic neurones that control the secretion of LH, and that this effect is enhanced in the presence of oestrogen.
Anestrus↗
Factors regulating ovulation rate in the ewe.
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Androgens↗
Effects of oestradiol, progesterone and androstenedione on the pulsatile secretion of luteinizing hormone in ovariectomized ewes during spring and autumn.
The effects of oestradiol-17 beta, androstenedione, progesterone and time of the year on the pulsatile secretion of LH were tested in ovariectomized Merino ewes (n = 32). The steroids were administered by small subcutaneous implants, and the LH pulses were observed in samples taken at intervals of 15 min for 12 h in spring 1979, autumn 1980 and spring 1980, seasons corresponding to successive periods of anoestrus, breeding season and anoestrus. During spring, oestradiol alone was able to reduce the frequency of the LH pulses, while progesterone, either alone or in combination with oestradiol, had little effect. During autumn, on the other hand, neither oestradiol nor progesterone could significantly reduce the frequency of the pulses when administered independently, whereas the combined treatment was very effective. Androstenedione had no significant effect on pulse frequency at either time of the year, either alone or in any combination with oestradiol and progesterone. The basal levels of LH, over which the pulses are superimposed, were reduced by oestradiol alone in both seasons. Progesterone alone had no consistent effects, but interacted significantly with oestradiol and this combined treatment maintained low basal levels most effectively at all times. Androstenedione had no significant effect. The amplitude of the pulses increased throughout the course of the experiment. Within seasons, the amplitudes were significantly higher in the presence of oestradiol and progesterone, but were not significantly affected by androstenedione. It was concluded that certain of the ovarian steroids exert negative feedback on the tonic secretion of LH primarily by reducing the frequency of the pulses, and that the changes in LH secretion attributable to season and phases of the oestrous cycle can be accounted for entirely by the responses of the hypothalamus to oestradiol and progesterone. The role of the androstenedione secreted by the ovary in the ewe remains obscure.
Androstenedione↗
Effects of steroids on beta-adrenergic binding sites in sheep pineal glands.
As an initial step in investigations of putative differences between central nervous system light-sensitive mechanisms in seasonally shedding and non-shedding breeds of sheep, some beta-adrenoceptor characteristics of Merino sheep pineal glands were determined, using [3H]dihydroalprenolol as the labelled ligand. Overall, a dissociation constant of 17.2 +/- 2.6 nmoles/l and a daytime beta-receptor density of 1.6 +/- 0.3 pmoles/mg were determined at 37 degrees. The binding sites exhibited stereospecificity, saturability and apparent homogeneity. 17 beta-Estradiol and progesterone implants that provided hormone concentrations in the physiological range had no significant effect on pineal beta-receptors in male sheep castrated shortly after birth. Dexamethasone injections, on the other hand, in doses sufficient to loosen the attachment of wool fibres to the skin, resulted in decreased pineal beta-receptor density and increased receptor affinity for dihydroalprenolol. This effect was apparently not mediated by altered plasma catecholamine concentrations, since the glucocorticoid treatment did not affect jugular venous noradrenaline, adrenaline or dopamine levels. The possible involvement of glucocorticoids in the regulation of wool growth could thus have a central neuronal component, medicated via action on pineal beta-adrenoceptors in sheep; however, the existence of the putative gonadal steroid feedback on beta-adrenoceptor-mediated pineal function remains to be demonstrated in this species.
Animals↗
Induction of ovulation in seasonally anovular ewes by the introduction of rams: effects of progesterone and active immunization against androstenedione.
The effects of progesterone priming and active immunization against androstenedione on ovulation, oestrus ad premature luteal regression were observed in seasonally anovular Merino ewes which had been induced to ovulate by the introduction of rams. Ovulation was induced in 27 out of 36 ewes, and the response was not affected by either immunization or progesterone priming. The ovulation rate (mean number of ovulations per ewe ovulating) was higher in ewes immune to androstenedione, but there was no significant effect of this treatment on oestrus or luteal maintenance. Priming with progesterone prior to the introduction of rams prevented premature regression of the corpora lutea but had no effect on the other measures of reproductive function. If active immunization were to be applied on a commercial scale, it would not interfere with the practice of mating ewes during the anoestrous season. The increase in ovulation rate in these ewes, with no increase in the proportion of ewes ovulating, indicates that the mechanism for the control of ovulation is separate from that controlling ovulation rate. The mechanism by which progesterone pretreatment prevents premature luteal regression awaits further investigation.
Androstenedione↗
Ovarian function in the ewe after active immunization against testosterone.
The secretion rates of testosterone measured during the last half of the oestrous cycle in 2 Merino crossbred ewes with utero-ovarian autotransplants were 1.95 +/- 0.34 ng/min and 2.48 +/- 0.63 ng/min. The secretion rate of testosterone was not correlated with the secretion rate of oestradiol or androstenedione in either ewe. Welsh Mountain ewes were actively immunized against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin (testosterone-3-BSA) or BSA alone (controls). Immunization against testosterone-3-BSA (5 ewes) resulted in anovulation (2 ewes) and the disruption of oestrous cycles and irregular ovulations (2 ewes). The ovaries showed morphological and endocrinological evidence of over-stimulation. Numerous large non-atretic follicles were present and in those ewes still ovulating the numbers of corpora lutea (ovulation rate) were increased. The concentrations of androstenedione and of oestradiol in the ovarian venous plasma were also markedly increased when compared to those in control ewes. The plasma binding capacity for steroids and the jugular venous concentrations of steroids were higher in immunized ewes. The binding capacity of follicular fluid for testosterone was similar to that of jugular venous plasma from the same ewe. These results show that immunization against testosterone-3-BSA leads to a disruption of ovarian cycles and ovarian over-stimulation.
Androstenedione↗
Ovarian morphology and the concentration of steroids during the oestrus cycle of sheep actively immunized against androstenedione.
Ewes were immunized against androstenedione-11 alpha-hemisuccinyl-bovine serum albumin (androstenedione-11-BSA) or BSA alone (controls). The ovaries were examined macroscopically and ovarian venous blood was collected at laparotomy by in-situ cannulation of the ovarian vein on Day 10 of the oestrous cycle, or by needle puncture of the ovarian vein 36 h after prostaglandin treatment. Follicular fluid was also collected from follicles greater than 5 mm diameter in some ewes. Ovaries and adrenal glands were collected and examined histologically. Oestrone, oestradiol-17 beta, androstenedione, testosterone and progesterone were measured by radioimmunoassays. Immunization against androstenedione-11-BSA was followed by an increase in the number of surface follicles greater than 3 mm in diameter and in ovulation rate. These changes were accompanied by an increased production of androgens (especially androstenedione) and of oestrogens during the preovulatory period. There were no observed changes in the adrenal cortex or pituitary glands, but the ovaries were heavier in immunized ewes and luteinized follicles were present in the ovaries of 1 of 4 immunized ewes. These results show that immunization against androstenedione-11-BSA leads to increased ovulation rate and suggest two possible mechanisms: (1) interference with the negative feedback effects of oestradiol-17 beta and/or (2) a reduction in the incidence of follicular atresia.
Androstenedione↗
Ovarian morphology and the concentration of steroids, and of gonadotrophins during the breeding season in ewes actively immunized against oestradiol-17 beta or oestrone.
Ewes were actively immunized against oestrone-6-(O-carboxymethyl)-oxime-bovine serum albumin, 17 beta-oestradiol-6-(O-carboxymethyl)oxime-bovine serum albumin or bovine serum albumin (controls). All 4 control ewes, 1 of 5 oestradiol-immunized ewes and 1 of 5 oestrone-immunized ewes had regular oestrous cycles. The other animals displayed oestrus irregularly or remained anoestrous. The plasma concentrations of LH and, to a lesser degree, FSH were increased relative to those in control ewes on Days 11-12 after oestrus or a similar total period after progestagen treatment in ewes not showing oestrus. The ovaries were examined and jugular venous blood, ovarian venous blood and follicular fluid were collected at laparotomy on Days 9-10 of the oestrous cycle. The ovaries of immunized ewes were heavier than those of control ewes. There were no CL in 5 of the immunized ewes but in the other 5 there were more CL than in the control ewes. Ovaries from 4 of 5 oestrone-immunized ewes contained luteinized follicles, while ovaries from 4 of 5 oestradiol-immunized ewes contained very large follicles with a degenerated granulosa and a hyperplastic theca interna. Both types of follicles produced progesterone, detectable in ovarian venous plasma and production of other steroids, particularly androstenedione, was also increased. The steroid-binding capacity of plasma was increased in the immunized ewes. The binding capacity of follicular fluid for oestradiol-17 beta and oestrone was similar to that of jugular venous plasma from the same ewes. These results suggest that immunization against oestrogens disrupts reproductive function by interfering with the feedback mechanisms controlling gonadotrophin secretion.
Animals↗
Growth of Graafian follicles in cows following luteolysis induced by the prostaglandin F2 alpha analogue, cloprostenol.
The ovaries of six multiparous cows 6-8 years old were collected 49-71 h after injection of a synthetic prostaglandin F analogue (cloprostenol) and serially sectioned. All follicles greater than 0.4 mm in diameter were counted and measured. The number of normal follicles ranged from 46 to 360 per cow. Atresia was not seen in follicles of less than 1.7 mm diameter. The number of early atretic follicles ranged from 4 to 20 per cow. The volume and mitotic index of the granulosa tissue was calculated for all follicles. Follicles were then grouped into classes according to granulosa volume, each class having twice the volume of granulosa of the preceding class. In all cows the rate of growth of follicles was slow up to 0.5 mm diameter (class 4). Above this size growth accelerated to a maximum in follicles of 1.2-1.7 mm diameter (class 8) and declined thereafter to a minimum in follicles 8-10 mm in diameter (class 13). Follicles beyond class 13 were not present in any of the ovaries. It was estimated that 22.1 days were required for a follicle to grow from 0.4 mm to 10 mm in diameter (classes 4 through to 13). The present data suggest that the pattern of synchronization of oestrus in cattle treated with cloprostenol may be related to the size of the largest non-atretic follicle present at the time of treatment because of the time required for such a follicle to complete its development.
Animals↗
Antibodies to androgens, and ovulation in the ewe.
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Androgens↗
Detection of early pregnancy in sheep by the rosette inhibition test.
The rossette inhibition test, an established test for determining the immunosuppressive potential of antilymphocyte serum, has been applied to the serum of sheep after mating. The rosette inhibition titre was much higher (12--26) in 7 sheep which were fertilized and remained pregnant for up to 21 days than in 5 sterile ewes mated with intact rams (8--10). The difference was apparent by 24 h after mating. One ewe had high titres for 6 days after mating but these then dropped and she returned to oestrus; early embryonic loss was suspected. Another ewe which returned to oestrus had consistently low titres. The results indicate that the rosette inhibition test can be used to detect fertilization, early embryonic death and continued pregnancy in sheep.
Animals↗
Detection of induced death of embryos in sheep by the rosette inhibition test.
The rosette inhibition test has been used to monitor the decrease of an 'early pregnancy factor' in 2 groups of pregnant sheep (19-21 days) in which embryos were removed surgically or by induction of luteolysis with cloprostenol. The rosette inhibition titres of sera taken from sheep of each group declined from high (16-18) to low (8-10) levels within 48 h of treatment. Surgical removal of embryos caused little change in serum progesterone concentration whereas cloprostenol prompted a rapid decrease over the same period. Death of the embryo can therefore be detected by the rosette inhibition test within 48 h of occurrence, but not necessarily by the measurement of progesterone in blood within this period.
Animals↗
Plasma concentrations of luteinizing hormone, follicle-stimulating hormone and progesterone during the breeding season in ewes immunized against androstenedione or testosterone.
Levels of plasma LH, FSH and progesterone during the breeding season were measured by radioimmunoassay in control ewes and ewes actively immunized against androstenedione-11 alpha-hemisuccinyl--bovine serum albumin or testosterone-3(O-carboxymethyl)oxime--bovine serum albumin. Immunization against androstenedione resulted in normal oestrous cycles with raised plasma LH and progesterone levels and a reduction in the concentration of FSH during the luteal phase. It is tentatively suggested that androstenedione, or its metabolites, could modify the oestrogenic control of LH secretion and facilitate the release of FSH in the ewe. Immunization against testosterone prevented oestrus and resulted in markedly increased levels of LH without alteration of the FSH concentration. Since evidence of increased binding of oestradiol-17 beta was found in the ewes immunized against testosterone, these results cannot be attributed solely to a reduction in the biologically active faction of testosterone.
Androstenedione↗
Plasma concentrations of luteinizing hormone and follicle-stimulating hormone during anoestrus in ewes actively immunized against oestradiol-17 beta, oestrone or testosterone.
The concentrations of LH and FSH in jugular venous plasma were measured during anoestrus in control ewes and ewes actively immunized against 17 beta-oestradiol-6-(O-carboxymethyl)oxime--bovine serum albumin, oestrone-6-(O-carboxymethyl)oxime--bovine serum albumin or testosterone-3-(O-carboxymethyl)oxime--bovine serum albumin. During a 12 h sampling period, the basal level of LH and the frequency of LH pulses were significantly increased in all the immunized animals. The plasma FSH concentration was increased in two out of five oestradiol-immunized ewes and two out of three oestrone-immunized animals. The plasma FSH concentration in the testosterone-immunized animals was similar to the control values. Significant titres of antibodies to oestradiol-17 beta were found in all the immunized animals. It is suggested that binding of oestradiol-17 beta in the circulation by antibodies reduced the negative feedback action normally eserted by this steroid on LH secretion and, to a lesser extent, on FSH escretion. However, since the oestradiol antibody titres in the testosterone-immunized and oestrone-immunized ewes were considerably lower than those found in the oestradiol-immunized animals, it is possible that testosterone and oestrone, perhaps by acting as prehormones, could influence the negative feedback control by oestradiol-17 beta of gonadotrophin secretion in the anoestrous ewe.
Anestrus↗