Progesterone's effects to reduce anxiety behavior of aged mice do not require actions via intracellular progestin receptors.

RATIONALE: Aging is associated with reduced secretion of, and down-regulation of receptors for, progesterone (P); yet, P's effects when administered to younger and older animals have not been systematically investigated. Some of P's antianxiety effects may be due to its conversion to 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) and its subsequent actions as a positive modulator at GABAA receptor complexes (GBRs). OBJECTIVES: We investigated whether P administration can decrease anxiety behavior of progestin receptor (PR) knockout (PRKO) or wild-type control mice. METHODS: P (10 mg/kg) or vehicle (propylene glycol) were administered subcutaneously to intact, female or male wild-type or PRKO mice that were either 9-12 or 18-24 months of age. Behavior in tasks that assess spontaneous activity (activity monitor and roto-rod), free exploration of a novel environment (open field, elevated plus maze, and elevated zero maze), and conflict behavior (mirror chamber, dark-light transition, and punished drinking) were examined 1 h after injection. RESULTS: P significantly decreased anxiety behavior of both PRKO and wild-type mice. P did not alter motor behavior but increased central entries in the open field, time in the open quadrants of the elevated zero maze, time in the mirrored chamber, time in the light compartment of the dark-light transition, and punished drinking in young and old mice. P-administered mice had higher levels of hippocampal 3alpha,5alpha-THP and GABA-stimulated chloride flux than did vehicle-administered PRKO or wild-type mice. CONCLUSIONS: The effects of P to decrease anxiety behavior of younger and older mice do not require classic PRs and may involve actions of 3alpha,5alpha-THP at GBRs.

Testosterone reduces pentylenetetrazole-induced ictal activity of wildtype mice but not those deficient in type I 5alpha-reductase.

Testosterone's (T) anti-seizure effects may be mediated in part by actions of its 5alpha-reduced metabolites. To test this hypothesis, T was administered to knockout mice deficient in the 5alpha-reductase type I enzyme and wildtype controls and their ictal activity following pentylenetetrazole (PTZ; 85 mg/kg i.p.) was compared to mice administered vehicle. T to wildtype mice increased latencies to forelimb clonus, tonic clonic seizures, hindlimb extension, and death compared to that seen with vehicle administration. Moreover, incidence of tonic clonic seizures and hindlimb extension were reduced in wildtype mice administered T compared to vehicle-administered mice. T administration to wildtype mice reduced ictal activity compared to T to knockout mice, which were not different than vehicle-administered control mice. T to wildtype mice increased the latencies and decreased the incidence of forelimb clonus compared to T to knockout mice, which were not different from vehicle-administered mice. These data are consistent with T having anti-convulsant effects and that 5alpha-reduced metabolites may mitigate some of T's anti-seizure effects.

Basic fibroblast growth factor-2 and interleukin-1 beta regulate S100 beta expression in cultured astrocytes.

Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo. S100 beta is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 beta achieve their effects by altering S100 beta gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 beta messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 beta suppressed S100 beta messenger RNA. We measured levels of S100 beta nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 beta primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 beta protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 beta influence the expression of the S100 beta gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.

Aging of the female reproductive system: a window into brain aging.

The menopause marks the permanent end of fertility in women. It was once thought that the exhaustion of ovarian follicles was the single, most important explanation for the transition to the menopause. Over the past decade, this perception has gradually changed with the realization that there are multiple pacemakers of reproductive senescence. We will present evidence that lends credence to the hypothesis that the central nervous system is a critical pacemaker of reproductive aging and that changes at this level contribute to the timing of the menopause. Studies demonstrate that an increasing de-synchronization of the temporal order of neuroendocrine signals may contribute to the accelerated rate of follicular loss that occurs during middle age. We suggest that the dampening and destabilization of the precisely orchestrated ultradian, circadian, and infradian neural signals lead to miscommunication between the brain and the pituitary-ovarian axis. This constellation of hypothalamic-pituitary-ovarian events leads to the inexorable decline of regular cyclicity and heralds menopausal transition.

Developmentally regulated expression of interleukin-1 beta by peri-implantation conceptuses in swine.

Interleukin-1 beta (IL-1 beta) is one of the critical inflammatory cytokines involved in many physiological processes. This study investigated the temporal expression of IL-1 beta in pig conceptuses. A human IL-1 beta cDNA probe and a anti-human IL-1 beta antibody were used to examine IL-1 beta gene and protein expression in pig conceptuses on days 11, 12, 13, 14, 15, 20, 30, 45, 60, 75, 90, 105 and 112 of pregnancy using Northern, Slot and Western blot analyses. A human cell line (A375.S2) was used to determine IL-1 activity in pig conceptuses. High levels of IL-1 beta mRNA were detected in days 11, 12 and 13 conceptuses. IL-1 beta protein was also detected in conceptuses on days 11, 12, 13, and 14, but not in conceptuses recovered after day 15 of pregnancy. IL-1 beta biological activity was demonstrated in days 11 and 12 conceptus homogenates, but not in homogenates of days 112 allantochorion. Low levels of IL-1 beta mRNA were detected by Northern blot analysis in Day 15 conceptuses, endometrium and myometrium only when poly(A+) RNA was used. The production of IL-1 beta by peri-implantation pig conceptuses was temporally associated with maternal recognition of pregnancy. The results suggest that conceptus IL-1 beta may be important for conceptus development and establishment of pregnancy.

In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges.

In mammals, the suprachiasmatic nuclei (SCN) regulate the timing of LH surges. Recent evidence suggests that vasoactive intestinal peptide (VIP), an abundantly expressed neuropeptide of the SCN, communicates time of day information from the SCN to GnRH neurons. VIP levels in the SCN decrease with age and may be responsible for alterations in LH surges that become apparent in middle-aged rats. We wished to determine whether suppression of VIP synthesis, through antisense oligonucleotides (oligos) directed at the SCN, results in 1) selective suppression of VIP levels in the SCN and 2) aging-like changes in the secretion of LH and PRL. To test the specificity of antisense oligo treatment, rats were ovariectomized and treated with estradiol. Antisense or control random oligos were infused into the peri-SCN region through stereotaxically placed bilateral cannulas. Beginning at lights off, rats were maintained in constant dim red illumination throughout the remainder of the experiment. They were killed at specific times, brains were microdissected, and VIP concentrations in the SCN, paraventricular nuclei, and cortex were assayed. As a control for the specificity of antisense VIP treatment, we monitored the levels of arginine vasopressin in the SCN. To test the effects of antisense treatment on the pattern of plasma LH and PRL secretion, blood samples were collected from atrial catheters from 1200-2000 h, and plasma samples were assayed for LH and PRL. The results indicate that the effects of antisense treatment were discrete, as they suppressed VIP concentrations in the SCN, but had no effect on VIP concentrations in the paraventricular nuclei or cortex or on arginine vasopressin concentrations in the SCN. Peak LH levels during the surge were delayed and attenuated in antisense-treated animals compared to random oligo-treated control rats in a manner strikingly similar to that observed previously in middle-aged rats. Likewise, PRL, which was unaffected in middle-aged rats, was also unaffected by targeted suppression of VIP. In summary, our findings clearly demonstrate that antisense VIP oligos suppress VIP levels in the SCN and do not affect peptide concentrations in other regions of the brain or other neuropeptides in the SCN. Further, we show that suppression of a single neuropeptide in the SCN can mimic the effects of age on the estradiol-induced surges of LH and PRL. These data support a central role for suprachiasmatic VIP in the regulation of the LH surge and suggest that age-related perturbations in the integrity of this axis may account for alterations in the pattern of LH secretion observed during middle age.

Acute effects of antisense antagonism of a single peptide neurotransmitter in the circadian clock.

The circadian clock that resides in the suprachiasmatic nucleus (SCN) of the hypothalamus is the major neural pacemaker driving most 24-h rhythms in mammals. Several neurotransmitter peptides are synthesized within this nucleus and communicate rhythmically with other cells in the SCN as well as with cells in other regions of the brain. At the present time, little is known about their role in regulating outputs of the clock. We demonstrate that antisense oligodeoxynucleotides corresponding to the NH2-terminus and the translation start site of vasoactive intestinal peptide (VIP) mRNA infused into the suprachiasmatic region of rats temporarily abolishes the circadian rhythm of corticosterone secretion without influencing stress-related corticosterone secretion in the same animals. Levels of VIP peptide are suppressed 30% on the second day after antisense treatment. These results indicate that a single neuropeptide transmitter in the circadian clock may serve a distinct role in the control of specific circadian rhythms.

Purification and characterization of a uterine retinol-binding protein in the bitch.

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

Colony-stimulating factor-1 in conceptus and uterine tissues in pigs.

Colony-stimulating factor-1 (CSF-1), a growth factor for cells of monocyte/macrophage lineages, is produced by uterine and placental tissues in humans and mice and may stimulate placental growth and development. The present study characterized CSF-1 mRNA and protein expressed by porcine uterine, conceptus, allantochorion, and fetal tissues at various stages of pregnancy. A human CSF-1 cDNA and an anti-human CSF-1 monoclonal antibody were utilized to examine CSF-1 mRNA and protein. Northern blot analyses detected mRNA transcripts of 3.6-5.1 kb in pig tissues. A 4.0-kb transcript was common to all tissues examined. Endometrial CSF-1 mRNA increased (p < 0.05) as pregnancy proceeded, with highest levels at term, and was temporally associated with concentrations of estrogen (E) in plasma. Placental expression of the CSF-1 gene increased (p < 0.05) throughout gestation with the major increase occurring between Days 20 and 30, after which time high levels of mRNA were maintained to term. The changes in placental CSF-1 mRNA were temporally associated with periods of rapid placental and fetal growth. High levels of CSF-1 mRNA were also detected in skeletal muscle, kidney, and intestine of fetuses. Immunoreactive CSF-1 was detected in all of the tissues examined. Partial cloning of the porcine CSF-1 gene indicated greater than 98% identity with the human CSF-1 gene. CSF-1 gene expression in endometrium was not affected by exogenous E or progesterone treatment in ovariectomized gilts. These results suggest that CSF-1 may influence placental and fetal growth, and its differential expression in fetal tissues indicates that CSF-1 may also affect embryonic differentiation and growth of those tissues.

Retinol-binding protein: immunolocalization of protein and abundance of messenger ribonucleic acid in conceptus and maternal tissues during pregnancy in pigs.

Retinol-binding protein (RBP) is a major secretory product of liver as well as uterine endometrium and periimplantation conceptuses of pigs. The present study examined distribution of RBP protein and abundance of RBP mRNA in pig maternal and conceptus tissues throughout pregnancy. A porcine liver RBP cDNA clone was isolated and used as probe in these studies. Northern blot analysis detected a 1.0-1.1-kb transcript in conceptus, trophoblast, yolk sac, placenta, endometrium, myometrium, oviduct, and numerous fetal tissues. Slot blot analyses of RBP mRNA abundance in endometrium, myometrium, conceptus, trophoblast, chorioallantoic placenta, and embryo/fetus indicated differential RBP gene expression in each tissue. Immunocytochemical localization studies indicated the presence of immunoreactive RBP in endometrial surface and glandular epithelium, circular and longitudinal muscle of myometrium, parenchymal cells of fetal liver, and proximal convoluted tubules of fetal kidney. Results suggest an integrated system of RBP gene expression and protein secretion that allows for transport of retinol from the uterine endometrium to the periimplantation conceptus during early pregnancy and to the fetal-placental unit throughout pregnancy in pigs.

Porcine conceptus and endometrial retinoid-binding proteins.

Porcine conceptus secretory proteins were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialysed and concentrated by Amicon ultrafiltration for retinol and retinoic acid (RA) binding studies. Proteins in the 20-kDa range, conceptus-secreted retinol-binding protein (RBP), bound both [3H]retinol and [3H]RA specifically. Cross-competition experiments indicate that [3H]RA was completely displaced with excess cold retinol; however, excess cold RA did not completely displace [3H]retinol, suggesting that conceptus RBP has greater affinity for retinol than RA. Cellular RBP and retinoic acid receptor (RAR)-alpha and RAR-gamma mRNA transcripts (0.7 kb; 3.8 and 2.8 kb; 3.4 kb respectively) were detected in poly (A)+ RNA isolated from Day-15 conceptus, Day-15 pregnant endometrium, late pregnant myometrium and late pregnant fetal tissues of pigs by Northern blot analysis. RAR-alpha and RAR-gamma immunoreactive proteins were detected in extracts of Day-15 conceptus, Day-15 pregnant endometrium and late pregnant fetal tissues by Western blot analysis. Collectively, results indicate that biochemical molecules required for retinoid transport, metabolism and regulatory effects are present in porcine conceptus and endometrial tissues during early pregnancy in swine.

Retinol-binding protein gene expression in cyclic and pregnant endometrium of pigs, sheep, and cattle.

Retinol-binding protein (RBP) is a major secretory product of conceptuses and endometrium of the large domestic animals. The present study examined the abundance of RBP mRNA in cyclic and early pregnant endometrium of pigs, sheep, and cattle and confirmed the presence of RBP mRNA in preiimplantation conceptuses of the large domestic animals. Northern blot analysis, using porcine liver RBP cDNA as the probe, detected a 1.0-1.1-kb transcript in conceptuses and endometrium collected during the preiimplantation period of pregnancy in pigs (Day 15), sheep (Day 16), and cows (Day 18). Slot-blot analysis of RBP mRNA in endometrium of pigs, sheep, and cows indicated differential RBP gene expression across days and statuses in pigs and sheep and across days alone in cows. In pigs, RBP gene expression was low to undetectable at Days 0, 5, and 10 of the estrous cycle and Day 10 of pregnancy. Levels of RBP mRNA increased (p < 0.06) from Days 10 to 12 and further (p < 0.001) to Day 15 across both statuses. At Day 18, RBP mRNA levels decreased (p < 0.01) in cyclic pigs but remained elevated in endometrium of pregnant pigs. In sheep, levels of RBP mRNA increased between Days 10 and 12 and 14 in both cyclic and pregnant ewes; however, between Days 14 and 16, RBP mRNA levels remained elevated in cyclic endometrium but decreased (p < 0.01) in endometrium of pregnant ewes. In a second experiment, RBP mRNA levels increased (p < 0.01) between Days 0-5 and Days 13-15 of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in progesterone and oestrogen receptor mRNA and protein during maternal recognition of pregnancy and luteolysis in ewes.

This study characterized changes in levels of mRNA and protein for endometrial oestrogen receptors (ERs) and progesterone receptors (PRs) during luteolysis and maternal recognition of pregnancy. For cyclic and pregnant ewes, endometrium was collected on days 10, 12, 14, or 16 post-oestrus (4 ewes/day for each status) for the measurement of ER and PR mRNA and protein. The amount of receptor mRNA is expressed in relative units above background, measured from radiographs of dot-blot hybridization of total endometrial RNA with ER and PR cDNAs. At hysterectomy, jugular vein blood samples were collected and assayed for progesterone, total corpus luteum weight was recorded and, in vitro, endometrial oxytocin-stimulated inositol phosphate formation was estimated. In pregnant ewes, plasma progesterone increased gradually between days 10 and 16 (P < 0.01), corpus luteum weight was stable at approximately 0.8 g and oxytocin did not stimulate endometrial formation of inositol phosphates in vitro. In contrast, in cyclic ewes, plasma progesterone decreased from day 10 to day 16 (P < 0.01), corpus luteum weight decreased after day 14 to approximately 0.48 g (P = 0.05) and oxytocin stimulated an increase of approximately 1300% in the endometrial formation of inositol phosphates on day 16. cDNAs specifically hybridized with 1.6 and 3.1 kb transcripts for PR mRNA and a 6.5 kb transcript for ER mRNA. In cyclic ewes, the amount of PR mRNA increased from day 10 to maximum levels on days 14-16. The number of PRs decreased from day 10 (2.25 pmol/mg DNA) to day 12 (0.98 pmol/mg DNA) and then increased from day 14 to day 16 (2.8 pmol/mg DNA). In pregnant ewes, PR mRNA levels were greatest on days 10-12 and decreased by approximately 50% by day 16. In contrast, the number of PRs was relatively unchanged from day 10 to day 16 (1.53 to 1.03 pmol/mg DNA). In cyclic ewes, the amount of ER mRNA was lowest at day 10 and increased fivefold by day 16. The number of ERs remained relatively unchanged from day 10 to day 14 (6.07 pmol/mg DNA) and increased by day 16 (16.12 pmol/mg DNA). In pregnant ewes, ER mRNA decreased by approximately 80% from day 12 to day 16. Similarly, the number of ERs decreased from day 10 to day 16 (5.41 to 2.05 pmol/mg DNA). Correlations between ER mRNA and PR mRNA (r = 0.68), ERs and PRs (r = 0.50) and ER mRNA and ERs (r = 0.50) were high (P < 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine injection of ovine trophoblast interferon.

This study determined whether twice-daily intrauterine injections of ovine conceptus secretory proteins (oCSP) containing type-I trophoblast interferon (25 micrograms/uterine horn) from day 11 to day 15 post-oestrus (oestrus = day 0) could alter the binding capacities of endometrial receptors for oxytocin, progesterone and oestrogen in cyclic ewes when compared with control ewes receiving serum protein (SP) injections. Injections of oCSP on days 11-15 post-oestrus decreased concentrations of oestrogen receptors (P < 0.06), oestrogen receptor mRNA (P < 0.05) and progesterone receptors (P < 0.08) in endometrium on day 16 when compared with SP-infused control ewes, which were undergoing corpus luteum regression on days 14-16. Injection of oCSP also decreased the number (P < 0.10) and affinity (P < 0.06) of oxytocin receptors. Inositol phosphate formation induced in the endometrium on day 16 by 100 nM oxytocin in vitro was highly correlated with the concentration (r > or = 0.93, P < 0.001) and Kd (r = -0.91, P < 0.01) of oxytocin receptors in SP-infused ewes, but was not as highly correlated with concentration (r < or = 0.83, P < 0.06) and Kd (r < or = 0.40, P > 0.40) of oxytocin receptors in oCSP-infused ewes. This indicates that oCSP disrupted the relationship between oxytocin receptor binding and oxytocin-induced activation of its second messenger system. These results indicate that antiluteolytic type-I trophoblast interferon may prevent oxytocin-induced luteolytic pulsatile secretion of prostaglandin F2 alpha during maternal recognition of pregnancy in sheep, by reducing the synthesis and affinity of endometrial oxytocin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in oestrogen receptor protein, mRNA expression and localization in the endometrium of cyclic and pregnant gilts.

Conceptus secretion of oestrogen on Day 11 of gestation is involved with establishment of pregnancy in the pig. Changes in oestrogen receptor (ER) protein, mRNA and cellular localization in the endometrium were evaluated during the oestrous cycle and early pregnancy of the gilt. In nonpregnant gilts, concentration of nuclear ER in the endometrium increased from Days 0 to 12 followed by a decline on Day 15 of the oestrous cycle. In pregnant gilts, changes in endometrial nuclear ER during Days 10, 12, 15 and 18 were similar to that in cyclic pigs. Analysis of endometrial ER mRNA expression did not detect any difference between cyclic and pregnant pigs between Days 10 and 15 postoestrus. Expression of ER mRNA in endometrium of cyclic and pregnant gilts was greatest on Day 10 followed by a decline on Day 15. Endometrial ER mRNA increased on Day 18 of the oestrous cycle, but remained low during pregnancy. Immunocytochemical localization of ER in the endometria of cyclic and pregnant gilts indicated that there was intense staining for ER in stromal cells and moderate to strong staining in surface and glandular epithelial cells during oestrus (Day 0) and Day 18 of the oestrous cycle. However, stromal ER staining was absent from Days 5 to 15 of the oestrous cycle and continued to be suppressed on Day 18 of pregnancy. Immunocytochemical staining of ER in the surface and glandular epithelium was readily detectable from Days 0 to 12 of the oestrous cycle and during pregnancy. Intensity of staining for ER declined in surface epithelial cells on Day 15 in both cyclic and pregnant pigs whereas positive staining for ER in glandular epithelium was absent. Staining for ER on uterine surface epithelial cells increased during pro-estrus (Day 18) of cyclic gilts but remained similar to Day 15 in pregnant gilts. Changes in endometrial ER protein, mRNA and localization in surface epithelium are consistent with a physiological role for conceptus oestrogen secretion in uterine function and maternal recognition of pregnancy in the pig.

Endometrial inositol phosphate turnover in pigs is reduced during pregnancy and estradiol-induced pseudopregnancy.

Three experiments were conducted to examine inositol phosphate (IP) turnover in response to treatments applied in vitro to endometrium from cyclic (CYC), pregnant (PREG) and estradiol-induced pseudopregnant (PSP) gilts. In Exp. 1, treatments (in 25 microliters .1 M NaHCO3) were 1) control (NaHCO3), 2) 125 ng oxytocin, 3) .25 micrograms prolactin, 4) 2.5 micrograms prolactin and 5) 5 micrograms pig conceptus secretory proteins (pCSP). Basal IP turnover on d 14 (estrus = d 0) for CYC was 3.9 to 5.0-fold greater than for PREG gilts and .6 to 1.1-fold greater than for PSP gilts (P less than .05). Oxytocin increased IP turnover 23 to 42% in CYC gilts (P less than .05), but not in PREG or PSP gilts. The treatment x reproductive status interaction (P less than .05) indicated that pCSP increased IP turnover 74 to 140% in PREG gilts but decreased it 18 to 22% in CYC and 17 to 50% in PSP gilts. In Exp. 2, treatments were applied in a 2 x 2 x 2 arrangement: 1) 0 or 125 ng oxytocin; 2) 0 or 2.5 micrograms prolactin and 3) 0 or 5 micrograms pCSP. Basal IP turnover on d 14 was 3.3 to 5.4-fold greater (P less than .05) in CYC than in PSP gilts and was affected by interaction (P less than .05) of pCSP and prolactin. Inositol phosphate turnover was increased by prolactin (12 to 22%) and by pCSP (7 to 34%) but, when combined, the stimulatory effects of each were eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Retinol-binding protein: a major secretory product of the pig conceptus.

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.

Effects of conceptus and conceptus secretory products on uterine development in the pig.

Pregnant gilts (n = 6) were subjected to surgery on Day 3 of pregnancy and uterine horns, within gilts, were assigned to either (1) ligation 15 cm below the uterotubal junction (REST) or (2) ligation proximal to the uterine body (NONREST). On Day 15 of pregnancy gilts were hysterectomized and length and weight of both uterine horns were recorded. NONREST uterine horns were longer (P less than .05) than those of REST, but weights were similar. Conceptuses from the REST uterine horns were retarded in development. Morphometric analysis of surface (SE) and glandular (GE) epithelial cells detected no effect of treatment on height, width, area, nuclear area or percentage of nucleus. Porcine conceptus secretory proteins (pCSP) were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialysed and concentrated by Amicon ultrafiltration for intrauterine infusion. Serum proteins (SP) were obtained from blood collected from a Day 15 pregnant gilt and diluted for intrauterine infusion. Catheters were placed into both uterine horns or cyclic gilts (n = 8) on Day 10 of the oestrous cycle and uterine horns were ligated proximal to the uterine body. Gilts received either oestradiol valerate (E2) (5 mg) or corn oil (CO) on Days 11 and 12. On Days 12-14 each gilt received twice daily infusions of Day 15 pCSP in one uterine horn and SP in the other uterine horn.(ABSTRACT TRUNCATED AT 250 WORDS)