Providing a diet containing only maintenance levels of energy and protein during the latter stages of pregnancy resulted in a prolonged delivery time during parturition in rats.

In mammals, a prolonged delivery time during parturition is dangerous for both mother and fetus, although the mechanisms that prolong delivery are unclear. To investigate whether nutrition affects delivery time, we administered two feeds containing maintenance (L-feed) or higher (H-feed) levels of energy and protein at different points during the latter half of pregnancy and compared the effects of the various treatments on delivery time in rats. After the rats had been maintained on the L-feed and then copulated on pro-oestrus (Day 0), pregnant females were randomly allocated to one of three groups: (1) the no-improvement group, which was fed L-feed throughout gestation; (2) the early group, which was fed L-feed until Day 11 of gestation and then switched to H-feed; and (3) the late group, which was fed L-feed until Day 16 of gestation and then switched to H-feed. There was no significant difference in the number of pups among the three groups. However, delivery time was significantly longer in the no-improvement group (73.7±5.2 min) than the early (46.9±5.6 min) and late (55.4±5.5 min) groups. Consuming a maintenance diet during the latter half of pregnancy resulted in a prolonged delivery time.

Anti-Müllerian hormone and its receptor are detected in most gonadotropin-releasing-hormone cell bodies and fibers in heifer brains.

Circulating concentrations of Anti-Müllerian hormone (AMH) can indicate fertility in various animals, but the physiological mechanisms underlying the effect of AMH on fertility remain unknown. We recently discovered that AMH has extragonadal functions via its main receptor, AMH receptor type 2 (AMHR2). Specifically, AMH stimulates the secretion of luteinizing hormone and follicle-stimulating hormone from bovine gonadotrophs. Moreover, gonadotrophs themselves express AMH to exert paracrine/autocrine functions, and AMH can activate gonadotropin-releasing-hormone (GnRH) neurons in mice. This study aimed to evaluate whether AMH and AMHR2 are detected in areas of the brain relevant to neuroendocrine control of reproduction: the preoptic area (POA), arcuate nucleus (ARC), and median eminence (ME), and in particular within GnRH neurons. Reverse transcription-polymerase chain reaction detected both AMH and AMHR2 mRNA in tissues containing POA, as well as in those containing both ARC and ME, collected from postpubertal heifers. Western blotting detected AMH and AMHR2 protein in the collected tissues. Triple fluorescence immunohistochemistry revealed that most cell bodies or fibers of GnRH neurons were AMHR2-positive and AMH-positive, although some were negative. Immunohistochemistry revealed that 75% to 85% of cell bodies and fibers of GnRH neurons were positive for both AMH and AMHR2 in the POA, ARC, and both the internal and external zones of the ME. The cell bodies of GnRH neurons were situated around other AMH-positive cell bodies or fibers of GnRH and non-GNRH neurons. Our findings thus indicate that AMH and AMHR2 are detected in most cell bodies or fibers of GnRH neurons in the POA, ARC, and ME of heifer brains. These data support the need for further study as to how AMH and AMHR2 act within the hypothalamus to influence GnRH and gonadotropin secretion.

Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells.

Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells (P<0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells (P<0.05). As 10nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.

Kisspeptin-10 stimulates the secretion of growth hormone and prolactin directly from cultured bovine anterior pituitary cells.

Kisspeptins are peptide hormones encoded by the KiSS-1 gene, and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. We recently observed that kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) also has a direct stimulating effect on luteinizing hormone (LH) secretion in bovine anterior pituitary (AP) cells. In the present study, we evaluated the direct effect of kisspeptin-10 on the secretion of other pituitary hormones, growth hormone (GH) and prolactin (PRL), from bovine AP cells. The AP cells, which were prepared from 1- or 8-month-old male calves, were incubated for 2h with the peptides. Kisspeptin-10 at 100 nM (P<0.05), 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10 nM, significantly stimulated GH secretion from the AP cells of 1-month-old calves, while in 8-month-old calves it was significantly (P<0.05) stimulated at 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10nM and 100 nM. The response of GH to 100 nM (P<0.01), 1000 nM (P<0.05) and 10,000 nM (P<0.01) kisspeptin-10 in the AP cells of 1-month-old calves was significantly greater than in those of 8-month-old calves. All tested doses of kisspeptin-10 had no effect on PRL secretion from AP cells of 1-month-old calves. However, 1000 nM (P<0.05) and 10,000 nM (P<0.01), but not lower concentrations, of kisspeptin-10 significantly stimulated PRL secretion from the AP cells of 8-month-old calves. The present study is, as far as we know, the first to examine the direct actions of kisspeptin on the secretion of GH and PRL from the bovine pituitary gland. Further studies are necessary to evaluate the importance of multiple actions of kisspeptin on the pituitary of various animals in vivo.

Short communication: a field study on the relationship between body condition and embryo production in superovulated Holstein yearling heifers.

We conducted a field survey to estimate the relationship between embryo production and the body condition score (BCS) on a 5-point scale, as well as blood concentrations of insulin and glucose, in superovulated Holstein yearling heifers housed in a free-stall barn. They were provided total mixed rations to meet the nutrient requirements. The daily ration was divided between 2 feeding times, utilizing stanchions to separate heifers to avoid social status preventing inferior heifers from having enough feed. The recovered fluid after uterine flushing from heifers (n = 88, 13 mo old) was examined microscopically for the morphological grade and the development stage. The number of heifers in which BCS was 2.75, 3.00, 3.25, and 3.50 was 6, 35, 40, and 7, respectively. The 3.50 BCS heifers produced fewer excellent grade embryos than 3.00 or 3.25 BCS heifers significantly. The 3.50 BCS heifers produced significantly more morula than 2.75, 3.00, or 3.25 BCS heifers. In contrast, 2.75 BCS heifers produced more blastocysts than 3.25 or 3.50 BCS heifers. The 3.50 BCS heifers were hyperinsulinemic. Our results suggested no significant effect of BCS around 3.00 on embryo production, whereas 3.50 BCS heifers may have poorer embryo production.

Influence of brain plasmalogen changes on gonadotropin secretion from the cultured bovine anterior pituitary cells.

We recently discovered that the orphan G-protein-coupled receptor (GPR) 61 colocalized with GnRH receptors (GnRHRs) on the surface of most of bovine gonadotrophs. A recent study suggested that ethanolamine plasmalogen (PI) is a ligand for GPR61 in mouse neuroblastoma. Therefore, this study evaluated the hypothesis that PI alters LH and FSH secretion from cultured bovine anterior pituitary (AP) cells. We prepared bovine AP cells from postpubertal heifers (26 mo old) and cultured the cells for 3.5 d. We treated the cells with increasing concentrations (0, 5, 50, 500, 5,000, 50,000, or 500,000 pg/mL) of phosphoethanolamine PI (PEPI) extracted from the bovine brain, or l-α-lysophosphatidylethanolamine PI (LEPI) extracted from the bovine brain, for 5 min before either no treatment or GnRH stimulation. The medium samples were harvested 2 h after culture for LH and FSH assays. Phosphoethanolamine PI (50-500 pg/mL) stimulated (P < 0.05) the basal secretion of FSH but not LH. Phosphoethanolamine PI at 50 pg/mL also enhanced (P < 0.05) GnRH-induced FSH secretion. However, higher doses (500-500,000 pg/mL) of PEPI suppressed GnRH-induced FSH secretion. Moreover, 50 to 500,000 pg/mL PEPI suppressed GnRH-induced LH secretion. None of the tested concentrations of LEPI showed any effect on basal or GnRH-induced LH or FSH secretion. Pretreatment with Sma and Mad pathway inhibitors suppressed FSH secretion induced by PEPI, whereas an extracellular signal-regulated kinase pathway inhibitor blocked the PEPI-induced suppression of GnRH-stimulated LH secretion. Therefore, PEPI, but not LEPI, extracted from the bovine brain, alters FSH and LH secretion from cultured AP cells. Further studies are required to decide whether PEPI binds to GPR61 and whether PEPI plays an important role in the control of gonadotropin secretion from gonadotrophs.

The introduction of rams induces an increase in pulsatile LH secretion in cyclic ewes during the breeding season.

Application of the ram effect during the breeding season has been previously disregarded because the ewe reproductive axis is powerfully inhibited by luteal phase progesterone concentrations. However, anovulatory ewes treated with exogenous progestagens respond to ram introduction with an increase in LH concentrations. We therefore tested whether cyclic ewes would respond to ram introduction with an increase in pulsatile LH secretion at all stages of the estrous cycle. We did two experiments using genotypes native to temperate or Mediterranean regions. In Experiment 1 (UK), 12 randomly cycling, North of England Mule ewes were introduced to rams midway through a frequent blood-sampling regime. Ewes in the early (EL; n=3) [corrected] and late luteal (LL; n=6) phase responded to ram introduction with an increase in LH pulse frequency and mean and basal concentration [corrected] of LH (at least P<0.05). In Experiment 2 (Australia), the cycles of 32 Merino ewes were synchronised using intravaginal progestagen pessaries. Pessary insertion was staggered to produce eight ewes at each stage of the estrous cycle: follicular (F), early luteal (EL), mid-luteal (ML) and late luteal (LL). In all stages of the cycle, ewes responded to ram introduction with an increase in LH pulse frequency (P<0.01); EL, ML and LL ewes also had an increase in mean LH concentration (P<0.05). In conclusion, ram introduction to cyclic ewes stimulated an increase in pulsatile LH secretion, independent of ewe genotype or stage of the estrous cycle.

Plasma leptin concentrations correlate with luteinizing hormone secretion in early postpartum Holstein cows.

We tested the hypothesis that hypothalamic-pituitary activity, bioassayed by LH pulse frequency, in dairy cattle during early lactation is related to measures of energy status and to circulating profiles of free fatty acids (FFA), insulin, insulin-like growth factor-I (IGF-I), leptin, and growth hormone (GH). On d 14 postpartum, before first ovulation and during the period of negative energy balance (-23.4 +/- 2.4 Mcal/d of metabolizable energy), blood plasma was sampled from 18 multiparous cows at 10-min intervals for 8 h. All samples were assayed for LH and GH and hourly samples were assayed for FFA, insulin, IGF-I, and leptin. Milk yield and composition, body condition score, and energy balance were also measured. Frequency of LH pulses was correlated positively with energy balance (r = 0.51) and plasma leptin concentrations (r = 0.73), and negatively with milk fat content (r = -0.52). Amplitude of the LH pulses was correlated only with leptin (r = 0.53). Frequency of GH pulses was not correlated with any measure of LH secretion, but was correlated negatively with plasma concentrations of insulin (r = -0.62) and IGF-I (r = -0.61). First ovulation was observed 34 +/- 4 d after parturition. These observations reveal an important linkage between pulsatile LH secretion and blood leptin concentrations during the early postpartum period in dairy cows, when their energy balance is negative, and may explain the delay in ovulation.

Relationships between changes in plasma concentrations of leptin before and after parturition and the timing of first post-partum ovulation in high-producing Holstein dairy cows.

During early lactation, dairy cattle are in negative energy balance and the delay to first post-partum ovulation depends on the time taken to recover from this situation. Lactating cows rely heavily on body fat to meet their requirements, leading to the suggestion that leptin, a hormone secreted mainly by adipocytes, is acting as a metabolic signal to sites that control the reproductive axis. The relationship between plasma leptin concentrations and the timing of the first ovulation post partum in 20 high-producing Holstein dairy cows, using a radioimmunoassay based on recombinant bovine leptin was studied. Plasma leptin concentrations declined after parturition, reached a nadir of 0.74 +/- 0.17 ng mL(-1) on 10.1 +/- 2.2 days after parturition (all values are mean +/- SEM). They then increased and became stable near the time of ovulation. Leptin concentrations averaged 1.81 +/- 0.31 ng mL(-1) in the 14 days prepartum, 1.32 +/- 0.21 ng mL(-1) in the post-partum preovulatory period and 1.61 +/- 0.24 ng mL(-1) in the post-ovulatory period. The differences between periods were significant (P<0.01). The interval from parturition to first ovulation averaged 25.9 +/- 2.0 days and was not correlated with the prepartum, preovulatory or post-ovulatory leptin values. However, the interval to first ovulation correlated significantly (r = 0.83 P < 0.0001) with the interval from parturition to the leptin nadir. These results show that plasma concentrations of leptin decrease in dairy cows in the early post-partum period and suggest that a delay in the recovery of leptin secretion increases the delay to the first ovulation.

Reproduction and plasma concentrations of leptin, insulin and insulin-like growth factor 1 in growth-hormone-transgenic female sheep before and after artificial insemination.

The transgenic sheep used in this study expressed an additional copy of the gene for ovine growth hormone (GH), so they had continuously high plasma concentrations of GH. They were used to test whether the GH transgene affected plasma concentrations of the metabolic hormones leptin, insulin-like growth factor 1 (IGF-1) and insulin, and whether these effects were associated with changes in conception, pregnancy or parturition following artificial insemination. Compared with control animals, the GH-transgenic sheep had higher bodyweight, lower body condition score and less subcutaneous fat (P < 0.05). These sheep also had lower plasma concentrations of leptin, higher plasma concentrations of insulin, and higher plasma concentrations of IGF-1 (P < 0.001). A similar proportion of GH-transgenic and control ewes came into oestrus, but the conception rate to artificial insemination was lower in GH-transgenic ewes than in the controls. Only four live lambs were recovered from 12 GH-transgenic ewes (33%) compared with 38 lambs from 43 controls (88%). This outcome was not associated with any difference in plasma progesterone profile in the period leading up to artificial insemination (Day 0). The GH-transgenic ewes had lower concentrations of FSH at all times measured (Day -19, Day -2 and Day 19). These results indicate that appropriate regulation of GH secretion from pituitary or peripheral tissues is necessary for normal reproduction and normal levels of metabolic hormones. Chronically high concentrations of GH were associated with increased levels of IGF-1 and insulin, and decreased levels of leptin.

Relationships between plasma concentrations of leptin and other metabolic hormones in GH-transgenic sheep infused with glucose.

To study the regulation of leptin secretion in sheep, we infused glucose (0.32 g/h/kg for 12 h) into GH-transgenic animals (n = 8) that have chronically high plasma concentrations of ovine GH and insulin, but low body condition and low plasma leptin concentrations, and compared the responses with those in controls (n = 8). In both groups, the infusion increased plasma concentrations of glucose and insulin within 1 h and maintained high levels throughout the infusion period (P < 0.0001). Compared with controls, GH-transgenics had higher concentrations of insulin, IGF-1, GH (all P < 0.0001) and cortisol (P < 0.05), but lower GH pulse frequency (P < 0.0001). Overall, leptin concentrations were lower in GH-transgenics than in controls (P < 0.01). A postprandial increase in leptin concentrations was observed in both groups, independently of glucose treatment, after which the values remained elevated in animals infused with glucose, but returned to basal levels in those infused with saline, independently of transgene status. In both GH-transgenics and controls, glucose infusion did not affect the concentrations of GH, IGF-1, or cortisol. In conclusion, GH-transgenic and control sheep show similar responses to glucose infusion for leptin and other metabolic hormones, despite differences between them in body condition and basal levels of these hormones. Glucose, insulin, GH, IGF-1 and cortisol are probably not major factors in the acute control of leptin secretion in sheep, although sustained high concentrations of GH and IGF-1 might reduce adipose tissue mass or inhibit leptin gene expression.

Effect of a long-lasting opioid receptor antagonist (naltrexone) on pulsatile LH release in early postpartum Holstein dairy cows.

The present trial was the first one to investigate the effect of an intravenous injection of naltrexone, an opioid receptor antagonist that has a longer duration of action than that of naloxone, on the LH pulse in early postpartum Holstein dairy cows. On Day 10 postpartum, blood samples were collected from cows at 10-min intervals for a period of 4 h before (pre-injection period) and a period of 5 h after (post-injection period) an intravenous injection of 10 mL of saline (Control Group, n=5) or 300 mg of naltrexone in 10 mL of saline (Naltrexone Group, n=5). The plasma LH level was assayed by double antibody radioimmunoassay. The number of LH peaks per 1 h, the mean LH level, and the amplitude of LH peaks were analyzed utilizing the Pulsar algorithm, and data were compared by repeated measures ANOVA. No differences were observed in the parameters of LH pulse in the pre-injection period between the Control and the Naltrexone Groups (P>0.10). In the Naltrexone Group, the number of LH peaks per 1 h and the mean LH level were significantly higher in the post-injection period than in the pre-injection period (0.85 +/- 0.29 vs. 1.24 +/- 0.17, P<0.05, and 1.81 +/- 0.70 vs. 2.47 +/- 0.92 ng/ml, P<0.05, respectively), but there was no significant increase in the amplitude of LH peaks (1.48 +/- 0.64 vs. 1.83 +/- 0.82 ng/ml, P>0.10). In contrast, all of the parameters of LH pulse remained unchanged in the Control Group (P>0.10). These results suggested that an intravenous injection of naltrexone activates the LH pulse.

Enhancing effect of acute fasting on ethanol suppression of pulsatile luteinizing hormone release via an estrogen-dependent mechanism in Holstein heifers.

This study was conducted to determine whether acute fasting in Holstein heifers enhances the suppressive effect of an intravenous injection of ethanol on pulsatile LH release (LH pulse) and, additionally, to establish whether or not the mechanism is estrogen-dependent. After estrus synchronization (Day 0 = estrus), 29 heifers were either fasted (fasting group; n = 14) or fully fed as a control (control group; n = 15) from Days 1 to 4. On Day 4, blood samples were taken at 10-min intervals for 4 h before (pre-injection period) and after (post-injection period) an intravenous injection of 1.5 mL of saline, 1.5 mL of ethanol , or 35 mg of tamoxifen dissolved in 1.5 mL of ethanol . We analyzed the mean LH level, the number of LH peaks per 4 h, and the amplitude of LH peaks. No differences were observed in the LH pulse in the pre-injection period between the control and the fasting group. However, in the post-injection period, compared with the saline injected control heifers, ethanol suppression of the LH pulse was observed in the number of LH peaks of the ethanol injected control heifers and in all pulse parameters of the ethanol injected fasting heifers. Furthermore, tamoxifen inhibited suppression of ethanol on LH pulse was observed in the control and fasting heifers injected with tamoxifen dissolved in ethanol. It was concluded that acute fasting in Holstein heifers has an enhancing effect on ethanol inhibition of the LH pulse and that the mechanism may be estrogen-dependent.

Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws.

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P < 0.05) when the embryos were transported in beta-ME supplemented medium (80 to 100%) than when transported without beta-ME (54 %). Blastocysts ranked as A or B after transportation in medium with or without 150 microM beta-ME were nonsurgically transferred to synchronous recipients; 60 d after embryo transfer, 21/36 and 19/35 cows, respectively, were diagnosed as pregnant by palpation per rectum. These results indicate that beta-ME maintains the quality of bovine blastocysts in plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

Freemartinism among singleton bovine females born from multiple embryo transfer.

Heterosexual chimerism among singleton females produced by multiple nonsexed embryo transfer (MNET singleton females) was investigated using chromosome typing and PCR (polymerase chain reaction)-amplification of male-specific DNA (msDNA). Of the 22 animals tested, 21 were classified as normal by both methods (i.e., showing no male cells among 100 metaphase spreads in chromosome typing and being msDNA negative in PCR). No morphological abnormalities of the genital organs were observed among 19 MNET single females. One MNET singleton female was, however, classified as a freemartin by PCR (male-specific DNA positive), but it was classified as normal cytogenetically. This individual probably had a low degree of heterosexual chimerism, and it seems that the chimerism derived from MNET was difficult to diagnose by chromosome typing, although it was detectable by PCR. The genital organs of this individual (15-mo-old Aberdeen Angus) were normal in form (both external and internal) and size. However, a very small structure, resembling seminiferous tubule, was found in the left ovary. It may be concluded that most MNET singleton females are expected to have normal reproductive function.

Twelve oxo-eicosatetraenoic acid induces fetal membrane release after delivery in cows.

Fetal fibroblast cell culture from cotyledons of bovine placenta and animal experiments close to term were used to elucidate afterbirth release and factors missing in the signal transduction mechanism for retained fetal membranes (RFM) after delivery. In cell culture the addition of arachidonic acid (Ara) to the medium caused rapid release to free floating cell in the culture dish, accompanied by matrix metalloproteinase (MMP) activation, being consistent with previous in vivo observations, where a relation between MMP and fetal membrane release had been shown. Ara-induced cell floating was not inhibited by the addition of cyclooxygenase (COX) inhibitor, and not induced by the addition of PGF2α or PGE2 to replace Ara, while 12-lipoxygenase (12-LOX) metabolite of Ara, 12-oxo-eicosatetraenoic acid (12-oxoETE), strongly induced cell floating. In the animal experiments, 12-oxoETE injection to delivery-induced cows (n = 6) using prostaglandin (PG) and dexamethazone resulted in rapid release of fetal membranes. In cows with natural calf delivery, a 12-oxoETE peak (11.7-16.8 ng/ml) was observed in maternal blood plasma prior to release of fetal membranes. This investigation thus gives new indications for that the mediator for fetal membrane release is 12-oxoETE and not PG.

Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines.

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 (n=6) or saline (n=6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period (P<0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle.

Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers.

This study was conducted to estimate the effects of kisspeptin-10 on blood concentrations of LH and GH in prepubertal dairy heifers. Heifers received a single injection of 1 mg kisspeptin-10 (n=5) or saline (n=5) intravenously, and serial blood samples were collected at 15-min intervals to analyze the response curves of both LH and GH after injection. Peak-shaped responses were observed for concentrations of LH and GH, and the peaks were observed at 27+/-3 and 75+/-9 min, respectively, after injection, only in heifers injected with kisspeptin-10. These data suggest various possible important links among kisspeptin, the reproductive axis, and also the somatotropic axis in prepubertal Holstein heifers.

Residues essential for catalytic activity of soybean beta-amylase.

To determine which amino acid residues are essential for the catalytic activity of soybean beta-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in beta-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean beta-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in beta-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50 degrees C to 30 degrees C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.

Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship.

Bioassay-guided fractionation of a methanolic extract of a Thai crude drug, derived from heartwood of Anaxagorea luzonensis A. Gray (Annonaceae), resulted in the isolation of 8-isopentenylnaringenin (1) as an estrogen agonist with a activity of about an order of magnitude greater than genistein. Various flavonoids possessing isopentenyl side chains in the A-ring have been prepared and evaluated for their ability to bind estrogen receptor. In addition, enantiomers of 1 were separated and the respective enantiomers were assayed. These studies have demonstrated that the presence of an 8-isopentenyl group is an important factor for binding. Flavones, flavanones and flavonols having an isopentenyl substituent at C-8 exhibited an appreciable affinity for estrogen receptor. Conversely, isoflavones possessing an 8-isopentenyl substituent at C-8 did not show this activity. Movement of the isopentenyl group from position 8 to 6 resulted in loss of the activity. No significant difference was observed between 2(S)- and 2(R)-enantiomers of 1 in their binding affinity. Prenylflavonoids are reported to possess a wide range of biological activities; however, estrogenic activity has not been described.