The effect of bovine somatotropin and diet on somatotropin binding sites in hepatic tissue of lactating dairy cows.

In the lactating cow, galactopoiesis is stimulated by treatment with recombinant bovine somatotropin (bST) and by an improved plane of nutrition. The present study determined the interaction between these variables and examined whether a positive galactopoietic effect was accompanied by a change in hepatic binding sites for bST. Lactating dairy cows received one of three diets with increasing nutrient density; diet 1, 150 g/kg of dry matter (DM) of crude protein (CP) and 10.5 MJ/kg of DM of metabolizable energy; diet 2, 170 g/kg of DM of CP and 11.3 MJ/kg of DM of metabolizable energy; and diet 3, 190 g/kg of DM of CP and 12.1 MJ/kg of DM of metabolizable energy. At 90 d after calving, half of the cows in each dietary group were treated with bST every 14 d for the rest of the lactation. Both nutrient density and administration of bST increased milk yield significantly in mid and late lactation; there was no significant treatment by diet interaction. Treatment with bST significantly increased plasma concentrations of insulin-like growth factor (IGF)-I compared with IGF-I concentrations in controls in both mid and late lactation. Comparisons within diet revealed that concentrations of IGF-I were significantly higher in cows fed diet 3 than in cows fed diets 1 and 2 at both stages of lactation. Increases in plasma insulin were confined to cows in late lactation, and no changes were observed for nonesterified fatty acids. Liver biopsies showed that concentrations of hepatic binding sites for bST were not affected significantly by bST treatment but were increased in midlactation for cows fed diet 3. Concentration of hepatic binding sites per unit weight of tissue were greater for cows in midlactation than for cows in late lactation. In summary, exogenous bST treatment and increased nutrient density were associated with elevated plasma IGF-I concentrations and increased milk yield; however, only nutrient density in midlactation increased the number of hepatic binding sites for bST. Exogenous bST treatment had relatively little effect on the concentration of hepatic bST receptors compared with nutrient density.

In-vivo gene transfer to the uterine endometrium.

Therapeutic interventions in reproductive biology have relied largely on steroids and antisteroids which act to regulate gene expression in target tissues. Whilst their use has transformed women's lives, few conceptual advances have been made in contraceptive technology, no means identified to improve human implantation and no new strategies developed for the treatment of benign gynaecology. A novel alternative is direct gene transfer to the organ of interest. As a first step to achieving this goal in the uterus, we used reporter gene constructs to transfect mouse endometrium in vivo and human endometrial epithelial cells in vitro. We injected DNA-liposome complexes into the uterine lumen of mice on day 2 of pseudopregnancy and detected reporter gene activity 2 days later. The liposomes used were a 3:1 (w/w) mixture of 2,3-dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N-N-dimethyl-1-propanaminium trifluoroacetate and dioleoylphosphatidyl ethanolamine. Freshly isolated human endometrial epithelial cells were successfully transfected in vitro with similar DNA-liposome complexes. These data suggest that endometrial gene transfer may be effective in humans. This may lead to the development of new therapeutic agents, including contraceptives, for the improvement of women's health.

Functional specialization in the ruminant placenta: evidence for two populations of fetal binucleate cells of different selective synthetic capacity.

Trophoblast binucleate cells (BNC) in the ruminant placenta demonstrate a characteristic development, mature structure and migratory capacity whether situated in cotyledonary or intercotyledonary regions of the placenta. However, previous immunocytochemical studies demonstrated clear differences in gene expression in granule contents of BNC according to their anatomical location with some proteins being expressed in all BNC (e.g. ovine placental lactogen) whereas others were unique to a particular origin (e.g. SBU3 antigen in cotyledonary BNC only). We have used enriched preparations of binucleate cells and showed differences in steroid metabolic capacity in vitro which is more related to their species origin (sheep or goat) than to their anatomical location. The predominant product from [3H]pregnenolone is progesterone (sheep) and 5 beta-pregnane-3 alpha, 20 alpha-diol (goat) and the amount formed (corrected for the number of BNC) is similar irrespective of whether BNC were derived from the cotyledonary or intercotyledonary regions. These studies indicate specific forms of regional functional specialization of BNC and emphasize their multifunctional role in the ruminant placenta.

Progesterone and oestrogen receptors in the decidualized mouse uterus and effects of different types of anti-progesterone treatment.

Pseudopregnant mice were treated systemically with monoclonal anti-progesterone antibody (DB3) (model 1), or progesterone receptor antagonists RU486 or ZK98,299 (ZK299) (model 2) on day 3 post coitum. On day 4, sesame oil was administered intraluminally into one uterine horn to induce decidualization. On day 7, the average mass of the oil-injected horn was 335.2 +/- 52.4 mg, eight times greater than that of the non-injected horn (40.8 +/- 5.3 mg; P < 0.001). After treatment with DB3, RU486 or ZK299, the masses of the injected horns did not differ significantly from those of non-injected horns. In the control group, concentrations of progesterone receptors (ligand-binding assay) increased twofold in the decidualized (52.2 +/- 7.4 fmol mg-1) compared with the non-injected horn (26.0 +/- 7.6 fmol mg-1; P < 0.05), whereas oestrogen receptor content (ligand-exchange assay) decreased by 53% (104.9 +/- 18.2 versus 224.3 +/- 18.1 fmol mg-1; P < 0.001). In model 1, antibody-treated animals showed a tenfold increase in the concentration of progesterone receptors (261.7 +/- 81.1 fmol mg-1; P < 0.001), but there was no differential distribution of progesterone or oestrogen receptors in the oil-injected versus non-injected uterine horns. In model 2, uterine progesterone and oestrogen receptors again showed no differential response between injected and non-injected horns regardless of the route of administration (systemic or intraluminal). Concentrations of progesterone receptors in RU486-treated (35.8 +/- 9.4 fmol mg-1) and ZK299-treated (32.0 +/- 10.2 fmol mg-1) mice were comparable to those in non-injected horns (35.3 +/- 6.3 and 34.2 +/- 5.1 fmol mg-1, respectively) and were not significantly different from the control group (26.0 +/- 7.6 fmol mg-1). The results show that oil-induced decidualization is accompanied by increased concentrations of progesterone receptors and decreased concentrations of oestrogen receptors. When decidualization is blocked by anti-progesterone treatment (antibody against progesterone or progesterone receptor antagonist), there are differing effects on receptor responses with an increase in progesterone receptors and decrease in oestrogen receptors after passive immunization, and no change in progesterone receptors and a reduction in oestrogen receptors after anti-progestins. The anti-decidualization effect in the two models was therefore achieved via dissimilar uterine receptor responses.

Anti-progesterone effects on maternal recognition and behaviour imprinted during first pregnancy in mice.

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 micrograms/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.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific interactions of progestins and anti-progestins with progesterone antibodies, plasma binding proteins and the human recombinant receptor.

This structure-activity study compares the affinity of a series of progestins, progesterone metabolites and anti-progestins for a panel of monoclonal antibodies to progesterone, coypu (Myocastor coypus) or guinea pig plasma progesterone-binding proteins (PPBPs) and the human recombinant progesterone receptor A form (PR-A). The compounds tested were progesterone, Promegestone (R5020), Mifepristone (RU486), ZK98,734, Onapristone (ZK98,299), 11 alpha-hydroxyprogesterone, 11 alpha-progesterone hemisuccinate, androsterone, etiocholanolone, 5 alpha- and 5 beta-pregnane-3,20-diones, and 20 alpha- and 20 beta-hydroxyprogesterones. The Ki values for these ligands were determined by competitive binding assays using radiolabelled progesterone as the binding site ligand. For anti-progesterone antibodies (e.g. DB3 and 11/32), only progesterone (3.6-8.8 nM), the 11 alpha-derivatives (1.0-5.5 nM) used to prepare the immunogen and the two 5-pregnanediones (20.9-45.1 nM) were bound with high affinity. For PR-A, high affinity binding was found with receptor agonists (Ki = 1.1-6.2 nM), both 5- and 20-reduced metabolites, and antagonists (0.6-28.0 nM), but not with the 11 alpha-derivatives (950 nM-1.0 microM). In contrast, the PPBPs displayed high affinity interactions with progesterone (3.5-4.2 nM) and both 5 alpha- and 20 alpha-reduced metabolites (2.4-3.4 nM). Binding with the beta-isomers and R5020 was less pronounced (22-170 nM) and there was no evidence of high affinity binding with PR antagonists (> 1.0 microM). Analogs with the 17-keto group did not bind to any of the binders studied. Thus, commonalities among the three types of protein binders were their comparable binding affinities for progesterone (3.5-8.8 nM) and 5-pregnanedione isomers (2.4-330 nM), and a lack of binding for two C17-keto steroids (androsterone and etiocholanolone). The results imply that the tertiary features of the binding domain of these three types of proteins are sufficiently different to result in unique binding structures.

The local differential effect of prostacyclin, prostaglandin E2 and prostaglandin F2 alpha on mammary blood flow of lactating goats.

Mammary blood flow (MBF) and milk yield are closely related in dairy ruminants, but little is known about the regulation of MBF in vivo. The local effects on MBF of injections or continuous infusions into the mammary artery of prostaglandins (PG) or indomethacin (an inhibitor of prostaglandins) respectively, were investigated in surgically prepared conscious goats. Prostacyclin (PGI2) was found to be a potent stimulator of MBF which increased linearly over the dose range 50-1000 ng. PGE2 was almost as potent as PGI2 at low doses, but tachyphylaxis occurred at doses at and above 100 ng. The response to repeated injections of PGE2 quickly declined depending on the dose. PGF2 alpha had no effect on MBF. During infusion of indomethacin into the mammary artery MBF was reduced markedly, showing that endogenous mammary prostaglandins are involved in the regulation of vasodilatation. The results indicate that PGI2 (and to a lesser extent PGE2) has an important role in the local regulation of vascular tone in the mammary gland.

Anti-progesterone antibody administration and the impairment of postpartum maternal care in mice.

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 microCi [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.(ABSTRACT TRUNCATED AT 250 WORDS)

Aberrant maternal behaviour in mice treated with a progesterone receptor antagonist during pregnancy.

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 micrograms) or day 17 (50 micrograms) of pregnancy, or on the first day of lactation (100 micrograms). 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.

Oestrogen synthesis by the peri-implantation conceptus of the one-humped camel (Camelus dromedarius).

The ability of the embryonic tissues of the dromedary camel (Camelus dromedarius) to synthesize oestrogens in vitro was studied in 15 conceptuses recovered by non-surgical uterine lavage between 10 and 33 days after ovulation. Definitive evidence for strong aromatase activity with synthesis of considerable quantities of oestrogens was obtained at all stages when conceptus tissues were incubated with [3H]androstenedione. A high proportion of the oestrogens was in the form of oestradiol, which contrasts to the higher ratio of oestrone:oestradiol in the oestrogens synthesized by embryonic tissues of horses and pigs. Biopsies of endometrial tissue recovered from pregnant and nonpregnant camels showed great ability to conjugate both oestradiol and oestrone when incubated with tritium-labelled forms of these two hormones.

Unilateral control of ovarian oxytocin release and the facilitatory effects of insulin-like growth factor-I in sheep.

Prostaglandin F2 alpha (PGF2 alpha)-induced release of ovarian oxytocin was investigated to determine whether the effect in vivo was local. [3H]PGF2 alpha infused downstream into a single ovarian lymphatic was transferred into the adjacent ovarian vasculature (estimated transfer 1.1 and 1.7%, two experiments). When unlabelled PGF2 alpha was infused in a similar manner (76 pmol min-1), there was a prompt eightfold increase in ovarian oxytocin release from the adjacent ovary containing a corpus luteum, but no effect on the opposite corpus luteum, showing that the effect was local. Instillation of 2% lignocaine into the ovarian vascular pedicle did not affect PGF2 alpha-induced oxytocin release, supporting the idea that neural mechanisms are not involved. Repeated doses of PGF2 alpha given close-arterially produced a successive reduction in oxytocin release. This effect was prevented by a prior infusion of insulin-like growth factor-I (IGF-I), which itself gave a small, but significant, increase in oxytocin release. The results show that PGF2 alpha in ovarian lymphatics acts locally and directly to stimulate ovarian oxytocin secretion, that repeated exposure of the corpus luteum to pulses of PGF2 alpha can result in tachyphylaxis, and that this latter effect can be ameliorated by IGF-I infused in vivo.

Paracrine and autocrine functions of the placenta: a key to the success of viviparity?

The evolution of specific nuclear transcriptional regulators has endowed tissues of the reproductive system with responsiveness to small hydrophobic compounds such as steroids. Steroids are widely distributed in Nature and their distribution in prokaryotes and eukaryotes has given rise to the concept that their hormonal role came about by target organ specialization and not by the evolution of steroids themselves. Specific nuclear receptors for progesterone in the uterus are prominent during the establishment and maintenance of pregnancy. Anti-progesterone antagonists which interfere with receptor-mediated DNA activation abrogate pregnancy and thus emphasize the functional importance of the pathways by which the effects of progesterone as an extracellular signal are transduced. Comparative studies show that progesterone itself can be ovarian or placental in origin. This seems to reflect the evolution of different mechanisms of endocrine function rather than any obvious selective advantage being associated with the source of hormone secretion. For this reason, the question of whether the endocrine function of the placenta is obligatory for the adoption of viviparity in mammals is far from certain, and should be considered as an evolutionary option rather than a sine qua non. Of growing importance is the idea that the interaction between trophoblast and endometrial cells controls the degree of invasiveness at implantation and immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular dynamics of growth in sheep and goat synepitheliochorial placentomes: an autoradiographic study.

This paper demonstrates that in sheep and goats the two definitive fetomaternal interface layers are developmentally related. The fetal trophectoderm consists of binucleate and uninucleate cells. The apical microvilli of the trophectoderm interdigitate with a layer consisting of syncytial plaques of limited area bounding the maternal connective tissue. Our previous histological ultrastructural and immunocytochemical work has indicated that throughout pregnancy the fetal binucleate cells migrate to and fuse with the uterine epithelium or its derivatives to form these syncytial plaques which constitute a persistent fetomaternal tissue unique to ruminants. This quantitative autoradiographic study of thymidine incorporation into sheep and goat placentas confirms the central role of the binucleate cell in placental growth, demonstrates that throughout pregnancy all binucleate cells migrate and indicates that most of the nuclei of the syncytial plaques, which appear to have a limited lifespan, derive from binucleate cell fusion.

Abnormal maternal behaviour in mice previously immunized against progesterone.

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.

Proto-oncogene erbA expression and increased abundance of progesterone receptors in the mouse uterus after passive immunisation against progesterone before implantation.

Passive immunisation with a monoclonal anti-progesterone antibody (DB3) prevents pregnancy in the mouse, and antibody is localised in the endometrium before the onset of implantation. BALB/c female mice were injected intraperitoneally with 9 nmol of DB3 (a dose known to cause 100% infertility) 32 h post coitum, and the uterus was removed at various times after injection. Using a monoclonal anti-progesterone receptor antibody (PR6), expression of progesterone receptors was found to be abundant in uterine tissue of DB3-treated mice; this was associated with substantial progesterone receptor mRNA levels and with maximum localisation of DB3 antibody as detected by anti-idiotype antibody. Control animals treated with an equal amount of the mouse myeloma protein P3 showed very low levels of progesterone receptor in the uterus. DB3 treatment also affected uterine expression of the proto-oncogene erbA product (which shows primary sequence homology with the progesterone receptor) as revealed by specific antiserum to the ERBA protein and by in situ hybridisation with a cDNA probe to v-erbA. Time-course studies indicated that the erbA gene was expressed at a high level before progesterone receptor expression increased, that its expression was dependent on the presence of the embryo and that erbA expression persisted longer in DB3-treated females. The observations suggest that anti-progesterone immunisation has a direct effect within the uterus, involving persistence of proto-oncogene erbA expression (which itself may represent an early maternal response to pregnancy) and increased progesterone receptor levels resulting from an unopposed oestrogen effect derived from local ligand withdrawal.

Biotinylated anti-progesterone monoclonal antibodies specifically target the uterine epithelium and block implantation in the mouse.

Post-coital administration of a mouse monoclonal antibody (mAb) against progesterone (DB3; IgG1) prevents pregnancy in several species. Our previous studies in mice have shown that passively transferred DB3 specifically targets the uterus before the expected time of implantation, probably through progesterone-binding sites. DB3 and two other anti-progesterone mAbs, i.e. 11/34 (IgG1) and 11/64 (IgM), were biotinylated and 9 nmol of each (a dose known to reduce pregnancy rate by greater than 80% with unconjugated mAbs) was injected into BALB/c female mice 32 h post coitum (p.c.). The biotin-mAb complexes were highly effective in blocking pregnancy (83-100%) compared with control animals that had received the biotinylated MOPC21 myeloma protein P3 (IgG1). Using the streptavidin-FITC (fluorescein isothiocyanate) reporter complex or second-stage anti-biotin-FITC antibodies, biotinylated conjugates of DB3, 11/34 and 11/64 were specifically localized on the uterine luminal epithelium at 68 h p.c. (i.e. 36 h after i.p. injection). Neither P3-biotin-treated pregnant mice nor biotinylated anti-progesterone mAb-treated pseudopregnant females showed a positive reaction. In addition, the localization of biotin-conjugated anti-progesterone mAbs could be blocked by absorption of the antibodies with free progesterone or progesterone conjugates prior to injection. These results show that localization to the uterine epithelium occurs with different anti-progesterone mAbs, and that this phenomenon is probably associated with progesterone-binding sites on the luminal epithelium.

Vaccination against pregnancy.

PIP: Considerable research on immunological control of pregnancy has occurred within the last several decades. It indicates that effective contraceptive and interceptive vaccines against pregnancy are possible. It also allows for further research on basic reproductive processes. Studies of the basic reproductive process have revealed potential target antigens. Immunization against structural antigens associated with the sperm surface, zona pellucida, and cumulus oophorus may be a way to prevent fertilization, thereby circumventing the problems associated with abortifacients. Clinical trials are now testing the active immunization technique against human chorionic gonadotropin. If the application of the new immunogens strengthen the reproducibility of the immune response, the active immunization technique may successfully terminate pregnancy. Even though progesterone antagonists, e.g., RU-486, interrupt pregnancy, safe and effective postcoital and anti-implantation vaccines, which do not cause menstrual irregularities, are still needed. Development of these types of vaccine is not going to result in an acceptable vaccine in the near future. Research of basic reproductive biology should continue, because it may lead to the development of a type of fertility control which is more appealing than the longterm hormonal contraceptives now used.^ieng