ABSTRACT

Anti-progesterone immunization leads to reversible infertility in mice; this can be achieved by passive immunization with a monoclonal antibody to progesterone (DB3), or by active immunization with either a progesterone–protein (bovine serum albumin; BSA) conjugate or anti-idiotype directed against DB3. Recovery of fertility in treated females varied from 39·5 to 75·5 median days after passive or active (progesterone–BSA) immunization respectively. Litter size after the first pregnancy also differed from 8·6 ±0·8 to 5·0 ±0·6 (mean ± s.e.m.) per mother after passive or active immunization respectively. When litter size was standardized to a maximum of four pups per litter, aberrant maternal responses were observed in the first 5 days after delivery in 40–70% of the nursing mothers. These responses took the forms of cannibalism and failure to retrieve or to nurse pups and resulted in a high incidence of pup rejection (up to 40%), compared with no rejection in control mothers. When mothers were allowed to keep entire litters, an even higher incidence of pup rejection occurred (51% compared with 8% in controls). There was an apparent relation between the degree of negative maternal behaviour and the progesterone antibody concentration in the circulation during the infertile period. Whereas aberrant behaviour occurred mainly within the first 5 days of lactation, it was significantly reduced thereafter. Aberrant behaviour of the mother towards pups may be a consequence of the presence of residual progesterone antibodies in the circulation which affects the process of progesterone withdrawal at parturition that is essential for the establishment of normal maternal responses to the neonate.

Journal of Endocrinology (1992) 134, 257–267

ABSTRACT

The anti-fertility effect of passive immunization against progesterone is influenced by genotype in mice. In order to quantify this finding we determined the effective dose that blocks pregnancy in 50% of treated mice (ED₅₀) for two different anti-progesterone monoclonal antibodies (DB3 and 11/32) in four different strains of mice (BALB/cJ, CBA/Ca, Tuck's no. 1 and F₁C). Efficacy was greatest in the two inbred strains (BALB/cJ and CBA/Ca) with ED₅₀ values of 0·46–1·4 nmol. The F₁C hybrid mice were more resistant to antibody treatment (ED₅₀ 2·2–3·0 nmol), while the outbred Tuck's no. 1 strain required much higher doses (ED₅₀ 7·0–8·2 nmol). There were no intrastrain differences between the two monoclonal antibodies.

We have examined the possible role of the H-2 haplotype on antibody efficacy in different strains and crosses. The antibody was highly effective in blocking implantation in three congenic BALB strains of different H-2 haplotype, in another inbred strain CBA/Ca, and in reciprocal BALB/cJ×CBA/Ca crosses. The F₁ hybrid crosses were somewhat resistant, but the C57BL/10Sn and B10.BR congenic strains were most resistant to treatment. The results show that the pregnancy-blocking effect of the anti-progesterone antibody was not influenced by the H-2 haplotype, but rather by background genes.

Journal of Endocrinology (1990) 125, 257–262

Abstract

In situ oestrogen synthesis makes an important contribution to the high oestrogen concentration found in breast tumours. Cytokines, including interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), have been shown to regulate aromatase activity in fibroblasts derived from normal and malignant breast tissues. In the present study, the ability of other cytokines in the IL-6 superfamily (IL-11 and oncostatin M) to stimulate aromatase activity has been confirmed. Formation of oestrone via the oestrone sulphatase pathway may be the major route of tumour oestrogen synthesis and in the present study TNFα was found to stimulate sulphatase activity in a dose-dependent manner. Human serum albumin was also found to be a potent stimulator of oestrone sulphatase activity. Its stimulatory effect was blocked by basic fibroblast growth factor, but not by several other growth factors tested. Insight into the regulation of oestrogen synthesis in breast tumours should enable the development of novel compounds to inhibit oestrogen synthesis in women with breast cancer.

Journal of Endocrinology (1996) 150, S65–S71

Abstract

Passive transfer of a monoclonal antibody against progesterone produces a high incidence of maternal rejection in mice after recovery from antibody-induced infertility. To investigate the mechanisms involved in this reduction of maternal care, we have examined whether the effect is due to long-term exposure to antibody. Antibody was administered i.p. either on day 2 or day 17 of pregnancy. When a low dose (1·0 nmol) was given on day 2, pregnancy proceeded normally but 44·8% pups delivered at term were rejected compared with 12·7% in the control group. When a higher dose (4·5 nmol) of antibody was given on day 17, pregnancy continued normally to term and the rejection rate was 48·8% (control: 11·1%). When the same amount of antibody was injected after delivery (day 1 of lactation), no detrimental effect was found on subsequent maternal care to the young, the rejection rate being comparable between antibody-treated and control groups (5·3% vs 4·6%).

To determine if the presence of antibody interfered with lactation or suckling, a bolus injection of 10 μCi [³H]H₂O was given to mice treated at day 17 with antibody or saline. The levels of radioactivity present in both mothers and pups and the first 5-day pup growth curves showed identical patterns, indicating that milk availability and the suckling process were not affected. Crossfostering studies revealed that antibody-treated mothers rejected 25·5% of fostered pups compared with 8·5% found in the control females when antibody was administered on day 17 of pregnancy and the entire litters were crossfostered between the two groups immediately after delivery. Detailed analyses using a retrieval test further demonstrated that the reduction in maternal care was most pronounced during the first 3 days after delivery.

These results demonstrate that antibody-treated mothers show a higher frequency of pup rejection which is (i) not restricted to their own litter; (ii) not due to lack of milk; and (iii) not a result of defects in the pups. They are consistent with the hypothesis that anti-progesterone antibody interferes with the priming mechanism(s) necessary for the onset of maternal behaviour during the 3-day period prior to delivery, leading to impaired maternal care after parturition.

Journal of Endocrinology (1995) 145, 363–369

Abstract

The rapid onset of normal maternal behaviour after parturition in mice, consisting of cleaning, warming, feeding and protection of offspring, is primed by oestrogen, progesterone and oxytocin. Previous studies showed that passive transfer of monoclonal antibodies against progesterone significantly increases the incidence of maternal rejection of pups. To test the hypothesis that aberrant maternal behaviour is due to partial progesterone withdrawal leading to hormonal imbalance during late pregnancy, maternal rejection was assessed following treatment with a progesterone receptor antagonist. Mifepristone (RU486) was given subcutaneously on either day 2 (100 μg) or day 17 (50 μg) of pregnancy, or on the first day of lactation (100 μg). Maternal behaviour was monitored twice daily for the first 6 days of lactation and pup rejection recorded for a further 15 days. Maternal rejection was significantly greater after mifepristone administration on either day 2 or day 17 (28·6% and 38·3%) compared with controls (11·1% and 5·2% respectively). Rejection was negligible in both treated and control groups if mifepristone was given after parturition. When mothers were treated at day 17, the length of the latent period before pups were retrieved and returned to the nest was markedly increased in mifepristone-treated mothers (46·3 s) compared with controls (4·4 s) though the effect was transient. The results indicate that mifepristone interferes with the hormonal priming mechanism(s) necessary for the onset of normal maternal behaviour by a receptor-mediated effect. The similarity of the present results and those obtained with anti-progesterone antibodies implies that receptor antagonism or antibody scavenging of progesterone influence a common central nervous mechanism that is essential for the normal priming process.

Journal of Endocrinology (1995) 145, 371–377

Leptin, administered either into the ventricles of the brain or systemically, has been shown to normalize blood glucose concentrations in streptozotocin (STZ)-induced diabetic rats. We hypothesized that an intact sympathetic nervous system is necessary for centrally administered leptin to normalize or attenuate high blood glucose concentrations in STZ-induced diabetic rats. Young male Wistar rats (∼50 g) were treated every other day with either s.c. guanethidine (100 mg/kg) or vehicle for 2 weeks. Rats were then implanted with an intracerebroventricular cannula directed to the lateral ventricle and made diabetic with an i.v. injection of STZ (50 mg/kg). Half of the animals in each group were given daily injections of leptin (10 μl), while the remaining animals received vehicle injections. Blood glucose concentrations were measured daily and tissue norepinephrine content was determined by high performance liquid chromatography at the end of the study. Guanethidine pretreatment did not block the ability of centrally administered leptin to decrease blood glucose concentrations in diabetic rats. This suggests that the sympathetic nervous system does not mediate the leptin-induced attenuation of high blood glucose concentrations observed in diabetic rats.

Abstract

Anti-progesterone treatment using specific anti-progesterone antibodies or a progesterone receptor (PR) antagonist during first pregnancy impairs postpartum maternal behaviour in mice. This effect is demonstrable only if the treatment is given during pregnancy but not immediately after parturition. The purpose of the present studies was to investigate if maternal behaviour is also impaired by anti-progesterone treatment in subsequent pregnancies.

Studies with a monoclonal antibody to progesterone (DB3; 4·5 nmol/mouse) showed that injection of females on day 17 of second pregnancy did not cause maternal rejection but the latency of pup retrieval was prolonged especially during the first 3 days of lactation. This phenomenon was not observed in animals that had previous experience of full length lactation. Experiments were carried out with mifepristone (RU486; 10 μg/mouse) injected at day 17 of first, second or third pregnancies. Pup rejection (22·5% vs 12·3%) and prolongation of the retrieval latency (62·3 ± 13·3 vs 19·7 ± 6·5 s; P<0·02) were observed following the first pregnancy. No abnormal behavioural effects were found in mothers treated in second or third pregnancy who had prior full length lactation experience. Control females subjected to only one pup retrieval test after first delivery rejected their pups if treated in their second pregnancy (27.3% vs 4·4%; P<0·001) and displayed a marginal prolongation of the retrieval latency period (20·9 ± 7·0 vs 7·4 ± 2·6 s). Anti-progesterone treatment had no negative influence when administered during third pregnancy.

To determine whether treatment with RU486 (50 μg/mouse, day 17) during first pregnancy has any residual effects, maternal response was monitored after completion of second pregnancy where no treatment was given. Females who exhibited both maternal rejection and prolonged retrieval latency following first pregnancy did not demonstrate any carryover effects during second lactation, indicating that there is no long-term consequence of RU486 treatment. These results suggest that: (i) anti-progesterone treatment of pregnant mice prevents maternal recognition and disrupts postpartum behaviour in females who had no, or very limited, nursing experience; (ii) there is a progesterone-dependent imprinting mechanism during the first pregnancy that is disrupted by anti-progesterone antibody or PR antagonist; and (iii) this imprinting mechanism and first lactation are important components of the consolidation of neural pathways that are associated with the establishment of normal maternal behaviour.

Journal of Endocrinology (1995) 147, 331–337

ABSTRACT

Synthetic ovine corticotrophin-releasing factor (oCRF) was infused continuously into the jugular veins of six ovine fetuses for 5–11 days. Two fetuses receiving 0·1 and 1·0 μg oCRF/h from gestational days 134 and 135 respectively, lambed prematurely on days 141 and 140 respectively. Three out of four fetuses receiving oCRF at 2·4 μg/h, from 125 days of gestation, delivered spontaneously at 131, 131 and 136 days, whilst one died in utero at 132 days. Two fetuses receiving vehicle only or oCRF intra-amniotically, were born at 148 and 145 days respectively, whilst six fetuses chronically cannulated but not infused were born at 149·8 ±2·1 (s.d.) days. In ewes lambing at term, maternal plasma progesterone concentrations were 41·4±11·4 (s.e.m.; n = 5), 28·8±7·8 (n = 6), 17·1 ±4·8 (n = 5) and 7·9± 1·1 (n = 4) nmol/l on 3, 2, 1 and 0 days respectively before the lambs were born. No such decrease in maternal plasma progesterone concentrations was seen in the oCRF-infused fetuses. Fetal plasma concentrations of immunoreactive ACTH were maintained above normal in oCRF-infused fetuses, but some desensitization to bolus oCRF injections occurred in these fetuses. Four of the five fetuses born prematurely were sufficiently mature to survive, being able to stand, breathe and suckle. It is concluded that continuous oCRF infusions into immature fetuses can accelerate maturation of a number of organs and systems culminating in the premature delivery of viable lambs.

J. Endocr. (1986) 111, 469–475

ABSTRACT

Anti-progesterone monoclonal antibody prevents the establishment of pregnancy in BALB/c mice by the prevention of implantation when injected i.p. 32 h after mating. To determine the specificity of this effect, mice were injected with immune and non-immune purified mouse immunoglobulins. The results show that anti-implantation efficacy was due to high-affinity antibody which bound progesterone since two further mouse immunoglobulin (Ig) G₁ preparations, mouse IgA and mouse IgM which failed to bind the steroid, had no effect on pregnancy rates. From a panel of anti-progesterone monoclonal antibodies, six with a high affinity (affinity constant, 0·24–0·80 litres/nmol) and specificity for progesterone were selected for additional studies. Anti-implantation efficacy for five antibodies was similar, with a 50% effective dose within the range of 0·8–2·0 nmol. Antibody reached high concentrations in plasma within 12 h after i.p. injection, and declined with a half-life of about 80 h. Purified F(ab′)₂ fragments of antibody also bound progesterone, but were less effective than the native molecule in blocking pregnancy. The results show that implantation in the mouse can be blocked by a high-affinity antibody that binds progesterone and which is removed from the blood at a slow rate.

J. Endocr. (1988) 118, 69–80

ABSTRACT

The interleukin-6 soluble receptor (IL-6sR) may regulate the ability of IL-6 to stimulate oestrogen synthesis in breast cancer cells and breast tumours. Significant aromatase activity was detectable in IL-6 stimulated fibroblasts derived from subcutaneous adipose tissue, but the combination of IL-6sR plus IL-6 resulted in a marked 21-fold stimulation of aromatase activity. To examine the control of IL-6sR release, the effects of oestradiol, 4-hydroxytamoxifen (4-OHT), dexamethasone, TPA, TNFα or IL-6 on this process was examined using MCF-7 breast cancer cells. Oestradiol, TNFα and dexamethasone all markedly increased IL-6sR release. While 4-OHT had a small stimulatory effect on IL-6sR release, it blocked the ability of oestradiol to increase IL-6sR release. Significant concentrations of IL-6sR were also detected in conditioned medium collected from lymphocytes and macrophages and in cytosols prepared from normal and malignant breast tissues. These results indicate that IL-6sR may have an important role in potentiating the effect of IL-6 on oestrogen synthesis in breast cancer cells. The abilities of oestradiol or tamoxifen to potentiate or inhibit the IL-6 stimulation of oestrogen synthesis in breast cancer cells may result from their effects on IL-6sR release.