[Effect of jiantai liquid on expression of estrogen and progesterone receptors in uterus of mice with embryo implantation dysfunction induced by mifepristone].

OBJECTIVE: To explore the molecular mechanism of jiantai liquid (JTL) in improving endometrial receptivity of mice with embryo implantation dysfunction (EID). METHODS: Mice model of EID induced by mifepristone were intervened with JTL (Twig of Chinese Taxillus, Red Sage root, Chinese Angelica, Milkvetch root, Chuanxiong rhizome), and sacrificed on day 8 of pregnancy. The endometrial estrogen receptor (ER) and progesterone receptor (PR) protein and their gene expressions were assessed by Western blot and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Levels of ER and PR protein and their gene expressions in the JTL treated group were significantly higher than those in the model group respectively (all P < 0.05), and showed insignificant difference from those in the normal control group (all P > 0.05). CONCLUSION: JTL could promote the development of endometrium and improve the embryo implantation by way of regulating the levels of ER and PR protein and gene expression in mice with EID.

Tokishakuyakusan directly attenuates PACAP's luteolytic action on luteal function in the rat ovary.

We investigated the potential direct effects of Tokishakuyakusan (TS) on progestin [progesterone and 20alpha-hydroxyprogesterone (20alpha-OH-P)] and cyclic adenosine-3',5'-monophosphate (cAMP) production in cultured rat luteal cells. In addition, we examined whether TS regulates the inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP), a newly found peptide, on luteinizing hormone (LH)-stimulated progesterone production. TS significantly stimulated progesterone, but not 20alpha-OH-P, production and cAMP accumulation through 24 hours of culture. PACAP-38 significantly elevated progesterone, 20alpha-OH-P and cAMP levels at all concentrations studied. On the other hand, PACAP-38 inhibited the production of progesterone and the accumulation of cAMP enhanced by LH, while the ratio of progesterone to 20alpha-OH-P was significantly decreased by PACAP-38 + LH. Concomitant treatment with TS and PACAP-38 + LH increased the ratio of progesterone to 20alpha-OH-P more than with PACAP-38 + LH. The present data have demonstrated that TS stimulates progesterone production in rat luteal cells, reconfirming our previous evidence that TS stimulates luteal steroidogenesis. The data further suggest that TS tends to attenuate PACAP's inhibition of LH-stimulated progesterone production, suggesting a luteotrophic effect within the corpus luteum.

Maternal recognition of pregnancy.

Enhanced secretion of PGF2 alpha from endometrial explants in vitro in response to oxytocin is associated with augmented activities of phospholipase A2, phospholipase C and prostaglandin endoperoxide H synthase (PGS). In early pregnancy, maintenance of the corpus luteum is associated with an absence of pulsatile PGF2 alpha secretion; an increase in endometrial inhibitors of phospholipase A2 and PGS contribute to the antiluteolytic alterations of PGF2 alpha secretion. Linoleic acid is a competitive inhibitor of arachidonic acid metabolism by PGS, and microsomal concentrations of free linoleic acid are increased in the endometrium of pregnant cattle. The trophoblast produces large quantities of interferon tau (IFN-tau). Inhibition of increases in endometrial oestradiol receptor mRNA and protein are associated with intrauterine administration of recombinant (r) ovine (o) IFN-tau in sheep. Intrauterine injections of ovine (b) IFN-tau in cattle (days 14-17) altered endometrial function so that secretion of PGF2 alpha from cultured endometrial epithelial cells was reduced. Antiluteolytic effects were not expressed in 20% of cows receiving IFN-tau or rbIFN-alpha I1 indicating that an inadequate endometrial responsiveness may contribute to embryo mortality. IFN-tau may activate a signal transduction system similar to that induced by other type I IFNs; activation of an intracellular tyrosine kinase ultimately leads to activation of an IFN-stimulated response element to induce gene transcription. Biological responses associated with pregnancy and IFN-tau treatment are integrated into a multifactorial antiluteolytic model. Strategies to enhance embryo survival could include supplementation with rIFN-tau and alterations in endometrial responsiveness to this cytokine through dietary manipulation of lipid metabolism.

Differential expression of distinct mRNAs for ovine trophoblast protein-1 and related sheep type I interferons.

An antiluteolytic substance secreted by the ovine conceptus and primarily responsible for maternal recognition of pregnancy is ovine trophoblast protein-1 (oTP-1), a new type I interferon (IFN). The objectives of this research were 1) to investigate whether multiple, distinct genes encode oTP-1 and other type I IFNs in the ovine genome and 2) to examine expression of oTP-1 and other IFN mRNAs during conceptus development. Genes for type I IFNs were isolated from a subgenomic library constructed from Day 25 (Day 0 = estrus) ovine conceptus high-molecular-weight DNA. Six clones were isolated and nucleotide-sequenced from -1000 to +900 (bases relative to cap site). Comparisons of inferred amino acid sequences demonstrated that four clones were distinct oTP-1 genes and that two clones, defined as o9 and o12, were related type 1 IFNs (deduced aa homology of o9 and o12 to oTP-1 was 71% and 54%, respectively). The presence of mRNAs encoded by oTP-1 and type I IFN genes was examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from Day 13-45 concepti. Total cellular RNA obtained from Day 75 placenta and adult lymphocytes was also analyzed by RT-PCR, coupled with Southern blot hybridization of the PCR reaction products with specific DNA probes. PCR products were sequenced in order to confirm primer specificity, and mRNAs corresponding to two of the four oTP-1 genes and to both related IFN clones (o9 and o12) were identified. Furthermore, quantitation of the PCR products revealed that of the two oTP-1 genes examined, one was highly expressed on Days 13-20 and transcripts were weakly detectable on Days 30 and 45. In contrast, the other oTP-1 gene examined was weakly expressed on Days 13-20 only. Densitometric analysis of hybridization signals revealed that IFN o9 mRNA was detected in Day 75 placenta but only weakly detected in conceptus (Days 13-45) and adult lymphocytes. IFN o12 mRNA was abundant in lymphocytes relative to the other tissues examined. Collectively, these results demonstrate the existence of distinct oTP-1 and related type I IFN genes. The data suggest that these genes display differential, tissue-specific expression and developmental regulation during pregnancy.

Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1: purification and antiviral activity.

Ovine trophoblast protein-1 (oTP-1) is a unique, Type I, trophoblast interferon (IFN) that possesses potent antiviral activity and is thought to be primarily responsible for maternal recognition of pregnancy in sheep. To provide sufficient amounts of protein for detailed studies, a synthetic gene for oTP-1 was designed and assembled in Escherichia coli, subcloned into a yeast expression plasmid, and used to overproduce recombinant oTP-1 in Saccharomyces cerevisiae. Recombinant oTP-1 was purified from soluble yeast extract using sequential ion-exchange and molecular sieve chromatography. Recombinant oTP-1 purified in this fashion exhibited potent antiviral activity (0.6 x 10(8) U/mg) similar to native oTP-1. This expression system will enable production of large quantities of soluble, biologically active, and correctly processed recombinant oTP-1. Furthermore, the synthetic gene construct facilitates introduction of mutations for ongoing structure/function studies of this unique, Type I, trophoblast IFN.

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiproliferative activity of a pregnancy recognition hormone, ovine trophoblast protein-1.

Ovine trophoblast protein-1 (oTP-1) is the alpha-interferon (IFN alpha) variant, secreted by conceptuses and referred to as type I trophoblast interferon, that is responsible for maternal recognition of pregnancy in sheep. We have previously shown that oTP-1 is as potent an antiviral agent as any known IFN. IFNs also possess anticellular activity and are, in fact, used in cancer therapy and have been found to be effective in the treatment of cancer such as myelogenous and hairy cell leukemias. A significant problem with the currently used IFNs is the undesirable side effect of toxicity at high concentrations. In this study, we examined the anticellular activity and toxicity of oTP-1. It inhibited proliferation but did not exhibit toxicity at high concentrations, unlike known IFN alpha S. In an anticellular assay using colony formation of both the human amnionic line, WISH, and the bovine epithelial line, MDBK, oTP-1 inhibited both colony size and number. oTP-1 was as effective as human and bovine IFN alpha s on human and bovine cells, respectively; thus, it displays potent cross-species activity. Its activity was dose dependent, and inhibition of proliferation could be observed at concentrations as low as 1 unit/ml. Concentrations as high as 50,000 units/ml stopped proliferation, while viability was not impaired. Cell cycle analysis revealed an increased proportion of cells in S phase and a corresponding decreased proportion of cells in G2/M after 48 h of oTP-1 treatment. Therefore, oTP-1 appears to inhibit progress of cells through S phase. oTP-1 antiproliferative effects can be observed as early as 12 h after after the initiation of culture and are maintained through 6 days. Thus, oTP-1 exhibits potent anticellular activity without toxicity across species and may have therapeutic potential as an antitumor agent without the toxic effects generally associated with IFNs.

Quantitative immunogold ultracryomicrotome studies of the distribution of periimplantation proteins in the sheep.

Several mammalian uterine and conceptus proteins are produced at specific stages of implantation. Ovine trophoblast protein-1 (OTP-1) is only synthesised in vitro by conceptus tissue from between 13 and 21 days of pregnancy (dpc). This immunogold ultracryosection study shows that, during this period, OTP-1 immunoreactivity is only found in the Golgi body of the trophectodermal cells. A second protein, of 14 kD molecular weight (14 K protein), has a more varied distribution being found in membrane-bounded crystals in uterine epithelium and trophectodermal cells, and distributed throughout the cytosol and nucleoplasm of the uterine epithelium. There are only trace amounts of the 14 K protein in the fetomaternal syncytium which replaces the uterine epithelium during implantation, and no crystals are found in the trophectoderm after cotyledonary villus formation is initiated at 24-25 dpc. The crystals containing 14 K protein persist throughout pregnancy in the intercotyledonary areas. The narrow time window of OTP-1 occurrence reinforces the suggestion that this represents an important developmental signal, whereas the distribution of the 14 K protein indicates a more general nutritive function.

Induction of trophoblastic interferon expression in ovine blastocysts after treatment with double-stranded RNA.

Ovine trophoblast protein-1 (oTP-1) is an interferon (IFN) related to the IFN-omega. The objectives of this research were: (i) to attempt to induce oTP-1 mRNA in day-11 ovine conceptuses with polyinosinic-polycytidylic acid (poly(I).poly(C], and (ii) to determine if IFN-omega mRNA is also produced on day 11 of gestation. In experiment I, conceptuses were cultured in presence of 100 micrograms/ml poly(I).poly(C) (n = 5) or medium alone (control, n = 3) for up to 8 h. In situ hybridization was used to assess effects of treatment on mRNA concentrations for oTP-1 and actin (positive hybridization control). Poly(I).poly(C) increased oTP-1 mRNA concentrations approximately 2.5-fold (p less than 0.01), but had no effect on actin mRNA. In experiment II, the presence of mRNA for oTP-1 and ovine IFN-omega was determined by using reverse transcription-polymerase chain reaction (RT-PCR) analysis of conceptus total RNA coupled with Southern blot hybridization of the PCR reaction products with specific cDNA probes. oTP-1 mRNA was detectable in all poly(I).poly(C)-treated (n = 7) and control (n = 6) conceptuses, whereas IFN-omega mRNA was detected in only three of seven poly(I).poly(C)-treated conceptuses and not in any controls. Together these results demonstrate that expression of oTP-1 mRNA can be enhanced by treatment with poly(I).poly(C) and that oTP-1 is the primary but not the only type I-IFN inducible in conceptuses on day 11 of gestation.

Regulation of bovine endometrial secretion of prostaglandins and synthesis of 2',5'-oligoadenylate synthetase by interferon-alpha molecules.

Experiments were performed to (1) verify the inhibitory effect of bovine trophoblast protein-1 (bTP-1) on uterine prostaglandin synthesis, (2) evaluate whether other interferon-alpha (IFN-alpha) molecules also inhibit prostaglandin secretion, and (3) test whether the enzyme 2',5'-oligoadenylate synthetase (2-5A synthetase) can be induced in endometrium by interferon-alpha. In experiment 1, all interferon molecules (bTP-1, oTP-1, bIFN-alpha and hIFN-alpha) equally inhibited secretion of PGF and PGE2 from endometrial explant cultures obtained at day 17 of the estrous cycle. In experiment 2, endometrial explants obtained from day 17 of the cycle were cultured with and without bovine serum albumin (BSA; 50 micrograms/ml) and bIFN-alpha (0, 0.84, 4.2, and 42 nM). Addition of BSA to the culture medium greatly enhanced the accumulation of PGF into the medium. The bIFN-alpha inhibited accumulation of PGF and PGE2 in both the presence or absence of BSA by 12 h. All three concentrations of bIFN-alpha were equally effective in inhibiting prostaglandin accumulation. Additionally, all concentrations of bIFN-alpha increased the amounts of 2-5A synthetase in endometrium. In conclusion, these results confirm the inhibitory effect of bTP-1 on PGF release from endometrium and demonstrate that bTP-1 can also inhibit PGE2 secretion. Furthermore, other interferon-alpha molecules, including bIFN-alpha, hIFN-alpha, and oTP-1, also reduced PGF and PGE2 secretion in culture. It is likely, therefore, that conceptus and other interferon-alpha molecules exert similar effects on endometrium in vitro and that the antiluteolytic effects of bIFN-alpha in vivo are mediated in part by changes in endometrial prostaglandin synthesis.

A role for interferons in early pregnancy.

In order to survive, the developing conceptus must interrupt the normal ovarian cycle of the mother and extend the production of progesterone by the corpus luteum. An unusual Type 1 interferon (IFN), related structurally to the IFN-alpha molecule and produced in massive amounts for only a few days by the first epithelium (trophectoderm) of the preimplantation conceptus, has been implicated as the antiluteolytic agent in sheep and cattle. IFN-alpha therapy during this critical period can also improve pregnancy success in sheep. It remains unclear, however, whether the trophoblast IFN have specialized biological properties or whether they are unique merely in the timing, magnitude and site of their expression.

The polypeptides and genes for ovine and bovine trophoblast protein-1.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) have been strongly implicated as antiluteolytic agents and responsible for maternal recognition of pregnancy in sheep and cattle, respectively. Both are interferons (IFN) belonging to the IFN-alpha family, but their length (172 residues versus 166 for most IFN-alpha) places them in an unusual subclass (the IFN-alpha II). The various isoforms of oTP-1 and bTP-1 produced by trophoblast tissue appear to arise in part from translation of multiple mRNAs which are themselves the products of distinct genes. These genes, like those for other IFN-alpha, are without introns. However, the genes for oTP-1 and bTP-1 form a distinct subgroup within the IFN-alpha II on the basis of their overall primary sequences and the high conservation of the 3'-untranslated ends of their transcription units. The bTP-1 genes also differ from the bovine IFN-alpha II in the organization of the promoter regions upstream from the transcription start site. Nevertheless, computer-aided analysis of the primary polypeptide sequences of oTP-1 and bTP-1 indicates that the molecules are likely to have approximately the same shapes and dimensions as all other IFN-alpha molecules. It remains to be determined whether they have unique biological properties which distinguish them from other IFN-alpha molecules.

Physiological mechanisms of pregnancy recognition in ruminants.

Maternal recognition of pregnancy in sheep, cattle and goats involves physiological mechanisms that result in protection of corpora lutea from luteolysis by modification or inhibition of uterine production of luteolytic pulses of prostaglandin (PG) F-2 alpha. Ovine, bovine and caprine luteal cells release oxytocin in a pulsatile manner during late dioestrus. Oxytocin then binds to its endometrial receptors and initiates luteolytic pulses of PGF-2 alpha. Ovine, bovine and caprine trophoblast protein-1 (oTP-1, bTP-1 and cTP-1) are secreted by the trophectoderm of conceptuses between Days 10 and 21-24 of pregnancy. These antiluteolytic proteins (oTP-1 and bTP-1) are primarily responsible for inhibiting uterine production of luteolytic amounts of PGF-2 alpha. During early pregnancy, the numbers of endometrial receptors for oxytocin are significantly lower in ewes and cows, and stimulatory effects of exogenous oxytocin on uterine production of PGF-2 alpha are correspondingly reduced or absent for ewes, cows and goats. Exogenous oestrogens can, through a uterine-dependent mechanism, stimulate synthesis of endometrial receptors for oxytocin and uterine production of PGF-2 alpha; an effect which is significantly attenuated during early pregnancy. These results suggest that oTP-1, bTP-1 and possibly cTP-1 exert their antiluteolytic effect(s) by: (1) inhibiting effects of oestrogen and/or progesterone necessary for synthesis of endometrial receptors for oxytocin; (2) inhibiting endometrial synthesis and/or recycling of oxytocin receptors directly; or (3) inducing the endometrium to synthesize an inhibitor of an enzyme(s) necessary for synthesis of PGF-2 alpha.

Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes.

In experiment (Exp) 1, 12 cyclic ewes had catheters placed into each uterine horn on Day 7 (estrus = Day 0). On Days 11-15, 6 ewes received twice-daily intrauterine infusions of 1.5 mg serum protein (SP) into each uterine horn and 6 ewes received infusions of 1.08 mg SP + 0.42 mg ovine conceptus secretory proteins (oCSP) containing 25 micrograms ovine trophoblast protein-one (oTP-1) as determined by radioimmunoassay (25-35% bioactive by antiviral assay). SP-infused and oCSP-infused ewes had similar plasma 13,14-dihydro-15-keto prostaglandin F2 alpha (PGF2 alpha) profiles in response to oxytocin on Day 11, but SP ewes became more responsive (p less than 0.01) to oxytocin on Days 13 and 15 than oCSP ewes. SP ewes also had greater incorporation of [3H]inositol into inositol trisphosphate (IP3) (+3449%, p less than 0.01) and total inositol phosphate (IP) (+760%, p less than 0.08), in response to oxytocin, than did oCSP ewes (+553 and +168% for IP3 and total IP, respectively) in endometrium collected at ovariectomy/hysterectomy on Day 16. Mean CL weights on Day 16 and mean concentrations of progesterone in plasma collected at 12-h intervals on Days 6-16 were not different for SP and oCSP ewes, but concentrations of progesterone were lower (p less than 0.05) in SP ewes on Days 15-16 than for oCSP ewes. These results indicate that oTP-1 may prevent luteolysis by inhibiting development of endometrial responsiveness to oxytocin and, therefore, reduce oxytocin-induced synthesis of IP3 and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Trophoblastic factors and the maternal recognition of pregnancy in sheep and cattle.

The establishment of pregnancy in domestic ruminants depends upon the continued secretion of progesterone by the corpora lutea. In non-pregnant cycles the corpora lutea regress between days 12-15 after oestrus in the sheep; this process must be blocked to ensure continued exposure of the uterus to progesterone. This review discusses the evidence that embryonic products are involved in the maintenance of corpus luteum function, the identification of factors which may be responsible for this maintenance and the probable mechanism of action. The discussion centres on the recent identification of a trophoblast interferon which is thought to be the major trophoblastic factor preventing luteolysis in sheep and cattle.

Interferons at the placental interface.

The antiluteolytic factors secreted by sheep and cattle conceptuses are closely related structurally to alpha-interferons (IFN-alpha s). They are known as ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1), respectively. The mRNAs for oTP-1 and bTP-1 are transcribed from multiple genes and are the major translatable messages of Day 13-17 sheep conceptuses and Day 15-20 cattle conceptuses. The proteins belong to the 172-amino acid IFN-alpha II (or IFN-omega) subfamily and have the typical antiviral and antiproliferative properties of the 166-residue IFN-alpha Is. These embryonic interferons also bind to the IFN-alpha receptor, which is present in uterine endometrium in high concentrations, and can influence the production of prostaglandin F-2 alpha and the pattern of protein secretion in that tissue. Through use of in-situ hybridization procedures on tissue sections and Northern and dot blot analyses of extracted conceptus RNA, ovine oTP-1 mRNA has been shown to increase markedly around Day 13 and to decrease after about Day 15 of pregnancy. The mRNA is confined entirely to cells of the trophectoderm. Significant induction of mRNA that hybridizes to an oTP-1 cDNA occurs in response to exposure to polyI:polyC in Day 11 sheep blastocysts which normally have low levels of oTP-1 expression. However, the basis for induction in the normal progression of embryonic development remains unclear. The fact that preimplantation conceptuses of other species, e.g. pig, release substances with antiviral activity suggests that IFNs may have an important role in pregnancy that extends beyond the domestic ruminants.

Cellular localization of an embryonic interferon, ovine trophoblastin and its mRNA in sheep embryos during early pregnancy.

The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.

Partial reversal of behavioral action of the agonist D-Trp-6-LH-RH by naloxone in mice.

The effects of the superactive agonist analog D-Trp-6-LH-RH were investigated in several neuropharmacological tests: inhibition of picrotoxin-induced seizures, open-field behavior, hot-plate and tail-flick tests, assessment of catalepsy and apomorphine-induced cage-climbing. In most tests, D-Trp-6-LH-RH was administered subcutaneously (sc.) at the dose of 100 micrograms/kg. The opiate involvement in the peptide action was checked by using naloxone HCl (NX) in a dose of 1 mg/kg intraperitoneally (ip.), with the exception of the analgesic tests where the dose was 0.5 mg/kg. The analog significantly suppressed the open-field parameters of ambulation, rearing and grooming; except for grooming, these actions were fully antagonized by NX. Similarly, NX pretreatment restored to the control levels the latencies of seizure parameters increased by D-Trp-6-LH-RH. The hot-plate latencies did not change after pretreatment with NX but the opiate antagonist was fully able to antagonize the analgesic effect of the peptide in the tail-flick test. The cataleptogenic effect and the inhibition of apomorphine-induced cage-climbing demonstrated after D-Trp-LH-RH were not antagonized by NX.