Hiroya Kadokawa

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.

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.

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.

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.

Expression of estradiol receptor, GPR30, in bovine anterior pituitary and effects of GPR30 agonist on GnRH-induced LH secretion

G-protein - coupled receptor 30 (GPR30) is an estradiol receptor located on the plasma membrane, and it initiates several rapid, non-genomic signaling events. GPR30 has recently been identified in rat anterior pituitary (AP); however, little is known about the role of GPR30 in controlling luteinizing hormone (LH) secretion from gonadotropes in animals. To fill this research gap, we hypothesized that GPR30 is expressed in bovine AP and mediates estradiol inhibition of gonadotropin-releasing hormone (GnRH)-induced LH release. We confirmed the expressions of GPR30 mRNA and protein by RT-PCR, western blotting, and immunohistochemistry. We cultured bovine AP cells (n=8) for 3 days in steroid-free conditions and then treated them with increasing concentrations (0.001nM, 0.01nM, 0.1nM, 1nM, and 10nM) of estradiol or a GPR30-specific agonist, G1, for 5min before GnRH stimulation. As expected, estradiol at 0.001-0.1nM inhibited the GnRH-stimulated LH secretion. However, we found also that G1 at 0.001nM was able to inhibit this secretion (P<0.05). In contrast, both estradiol and G1 at higher doses were less efficient in suppressing the GnRH-stimulated LH secretion. Neither estradiol nor G1 suppressed GnRH-stimulated follicle-stimulating hormone secretion. In separate experiments, fluorescent immunohistochemistry and immunocytochemistry revealed that approximately 50% of GPR30-positive cells express LH, and about 30% of LH-positive cells express GPR30. In conclusion, GPR30 is expressed in bovine gonadotropes and other AP cells and may partially contribute to rapid negative estradiol feedback of GnRH-induced LH secretion.

Suppressed expression of granulocyte macrophage colony-stimulating factor in oviduct ampullae of obese cows

Obese heifers have been found to produce fewer excellent-grade embryos than lean and normal heifers due to unknown mechanisms. Oviducts synthesize granulocyte macrophage colony-stimulating factor (GMCSF) to promote embryogenesis, and GMCSF expression may be down-regulated in the oviducts of obese cows. The present study evaluated the relationship between the degree of obesity and GMCSF expression in the ampullary or isthmic section of oviducts in lean [n=5; body condition score (BCS) on a 5-point scale, 2.5], normal (n=6; BCS, 3.0), and obese (n=5; BCS, 4.0) Japanese Black cows. GMCSF mRNA and protein expression in the ampulla, measured by real-time PCR and western blotting, respectively, were less (P<0.05) in the obese group than in the normal group. mRNA and GMCSF protein did not differ significantly in the isthmus among the three groups. The obese group had less GMCSF immuno-reactivity in the tunica mucosa, the primary site of GMCSF gene expression, of the ampulla than the normal and lean groups. In conclusion, unlike normal and lean cows, obese cows had suppressed GMCSF gene expression in the ampulla.

Perspectives on improvement of reproduction in cattle during heat stress in a future Japan

Heat stress (HS) causes hyperthermia, and at its most severe form, can lead to death. More commonly, HS reduces feed intake, milk yield, growth rate and reproductive function in many mammals and birds, including the important cattle breeds in Japan. Rectal temperatures greater than 39.0°C and respiration rates greater than 60/min indicate cows are undergoing HS sufficient to affect milk yield and fertility. HS compromises oocyte quality and embryonic development, reduces expression of estrus and changes secretion of several reproductive hormones. One of the most effective ways to reduce the magnitude of HS is embryo transfer, which bypasses the inhibitory effects of HS on the oocyte and early embryo. It may also be possible to select for genetic resistance to HS. Cooling can also improve reproductive performance in cows and heifers, and probably, the most effective cooling systems currently in use are those that couple evaporative cooling with tunnel ventilation or cross ventilation. Its effect on improving reproductive performance in Japan remains to be evaluated.

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.

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations.

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (> 1 nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10 nM (P < 0.05) and 100 nM (P < 0.01) GnRH, but not 0.01-1 nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

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.