Recent progress of interferon-tau research and potential direction beyond pregnancy recognition

Since the discovery of interferon-tau (IFNT) over 30 years ago as the trophectodermal cytokine responsible for the maintenance of the maternal corpus luteum (CL) in ruminants, exhaustive studies have been conducted to identify genes and gene products related to CL maintenance. Recent studies have provided evidence that although CL maintenance, with the up- and down-regulation of IFNT, is important, its regulatory role in the endometrial expression of interferon-stimulated genes (ISGs) is far more important for conditioning the uterine environment for successful conceptus implantation and thereafter. This review initially describes the mammalian implantation process, briefly but focuses on recent findings, as there appears to be a common phenomenon during early to mid-pregnancy among mammalian species.     

Effects of heat stress on the endometrial epidermal growth factor profile and fertility in dairy cows

The endometrial epidermal growth factor (EGF) profile is an indicator of uterine function and fertility in cattle. The present study aimed to investigate the effects of heat stress on the endometrial EGF profile and fertility in lactating Holstein cows. The endometrial EGF profiles of 365 cows in the Hokkaido and Kyushu regions were examined between June and September (heat stress period, n = 211) and between October and January (control period, n = 154). EGF profiles were investigated using uterine endometrial tissues obtained by biopsy 3 days after estrus (Day 3). The proportion of cows with an altered EGF profile was higher between June and September than between October and January (41.2 vs. 16.2%, P < 0.05). The effects of rectal temperature on Days 0 and 3 on the endometrial EGF profile were also assessed in cows (n = 79) between June and September in the Kyushu region. A single embryo was transferred to cow on Day 7 to evaluate fertility (n = 67). Regardless of the rectal temperature on Day 3, the proportion of cows with an altered EGF profile was higher (64.1 vs. 30.0%, P < 0.05) and the pregnancy rate after embryo transfer (ET) was lower (26.7 vs. 51.4%, P < 0.05) in cows with a rectal temperature ≥ 39.5°C on Day 0 than in cows with a rectal temperature < 39.5°C on Day 0. The present results indicate that alterations in the endometrial EGF profile induced by an elevated body temperature on Day 0 contributed to reductions in fertility in lactating dairy cows during the heat stress period.     

The distribution and immunolocalization of fibroblast growth factors (FGFs) in the rat oviduct during early pregnancy and the post-partum period

The mammalian oviduct provides a favourable environment for several reproductive processes, including ovum transport, sperm capacitation, fertilization and pre-implantation embryonic development. This environment is regulated by cyclic ovarian steroids, that is oestrogen, and growth factors. Fibroblast growth factors (FGFs) regulate the differentiation and growth of various cell types in the female genital tract. This study aimed to determine the localization of FGF1, FGF2, FGF receptor 1 (FGFR1) and 2 (FGFR2) in the rat oviduct, by immunohistochemistry, on day 5 of pregnancy and post-partum days 1, 3 and 5, and to demonstrate the possible functions of these proteins during early pregnancy and the post-partum period. On all examination days, cytoplasmic and nuclear FGF1 immunoreactivity was detected in the epithelium lining the infundibulum, ampulla and isthmus of the oviduct. Immunoreactivity was much stronger in the basal bodies of the cilia on the epithelium lining the infundibulum and ampulla. FGF1 immunoreactivity was also detected in stromal cells, myocytes and endothelial cells. Cytoplasmic FGF2 immunoreactivity was observed in the tunica muscularis, vascular myocytes and endothelial cells. While strong cytoplasmic FGF2 immunoreactions were observed in the stromal cells of the lamina propria, the luminal epithelium, some stromal cells and smooth muscle cells displayed a rather weak FGFR1 and FGFR2 immunoreactivity. Immunoreaction intensity did not differ between the periods examined. This study shows that FGF1, FGF2, FGFR1 and FGFR2 are produced by rat oviduct cells during pregnancy and the post-partum period, and reproductive physiology is regulated not only by hormonal mechanisms, but also by growth factors.     

Expression of fibroblast growth factor 1 (FGF1) and FGF7 in mature follicles during the periovulatory period after GnRH in the cow

The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.     

Regulation of endometrial prostaglandin F(2alpha) synthesis during luteolysis and early pregnancy in cattle

Luteal regression is caused by a pulsatile release of prostaglandin (PG) F(2alpha) from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF(2alpha) secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF(2alpha), the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF(2alpha) secretion. It has been recently demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulates PGF(2alpha) output from bovine endometrial tissue not only during the follicular phase but also during the late luteal phase, suggesting that TNF-alpha is a factor in the initiation of luteolysis in cattle. Furthermore, our recent study has shown that IFN-tau suppresses the action of TNF-alpha on PGF(2alpha) synthesis by the bovine endometrium in vitro, suggesting that IFN-tau plays a luteoprotective role by inhibiting TNF-alpha-induced PGF(2alpha) production in early pregnancy. On the other hand, factors other than oxytocin or TNF-alpha have also been suggested to be involved in the regulation of PGF(2alpha) synthesis by bovine endometrium. The purpose of this review is to summarize our current understanding of the endocrine mechanisms that regulate the timing and pattern of uterine PGF(2alpha) secretion during the estrous cycle and early pregnancy.     

Serum fibroblast growth factor 23 (FGF-23): associations with hyperphosphatemia and clinical staging of feline chronic kidney disease

Fibroblast growth factor 23 (FGF-23) is an independent monitor of the progression of chronic kidney disease (CKD) in human medicine, and FGF-23 may have value as a biomarker in feline CKD. We evaluated the relationship between serum FGF-23 and CKD stages, and the effect of age on FGF-23 in normal cats. We measured FGF-23 and intact parathyroid hormone (iPTH) concentrations by ELISA, with intra- and inter-assay CVs ≤ 15%. The percentage recovery of FGF-23 and iPTH remained stable for up to 7 d in samples stored at −20°C and −80°C. We measured FGF-23 in 304 cats, among which 196 were diagnosed with CKD. The 108 clinically healthy cats were divided into 5 subgroups based on growth stage (0–2 y, 3–6 y, 7–10 y, 11–14 y, ≥ 15 y). No statistical difference was found in FGF-23 among age groups (p = 0.15) or by sex in healthy subjects. Using the International Renal Interest Society guideline, 34 cats were defined as CKD stage 1, 74 stage 2, 51 stage 3, and 37 stage 4. FGF-23 was higher in cats in all CKD stages than in controls. Higher serum phosphorus was observed in stage 3 (p = 0.04) and 4 (p < 0.01) compared to controls. iPTH increased as CKD progressed. Pearson analysis indicated a positive linear relationship between FGF-23 and iPTH (control: r = 0.70, p < 0.01; CKD: r = 0.46, p = 0.02). FGF-23 may be a useful biomarker of feline CKD and may precede hyperphosphatemia in advanced feline CKD.     

Expression of fibroblast growth factor 21 in the liver of dairy cows in the transition period and during lactation

Fibroblast growth factor 21 (FGF21) has been identified as a novel hormonal factor involved in the regulation of metabolic adaptations during energy deprivation. The present study aimed to investigate the expression of the FGF21 gene in the liver of dairy cows during the transition from pregnancy to lactation. Therefore, the relative mRNA abundance of FGF21 in liver biopsy samples of 20 dairy cows in late pregnancy (3 weeks pre‐partum) and early lactation (1, 5, 14 weeks post‐partum) was determined. It was observed that hepatic mRNA abundance of FGF21 at 1 week post‐partum was dramatically increased (110‐fold) compared to 3 weeks pre‐partum (p < 0.001). With progress of lactation, mRNA concentration of FGF21 was declining; nevertheless, mRNA abundance at 5 and 14 weeks post‐partum remained 25‐ and 10‐fold increased compared to 3 weeks pre‐partum (p < 0.001). Using a gene array technique, it was found that many genes involved in fatty acid oxidation, gluconeogenesis and ketogenesis were up‐regulated during early lactation compared to late pregnancy. Moreover, there were positive linear correlations between hepatic mRNA concentration of FGF21 and mRNA concentrations of genes involved in ketogenesis as well as carnitine synthesis and carnitine uptake at various time‐points during lactation, indicating that FGF21 could play a role in ketogenesis and carnitine metabolism in the liver of dairy cows (p < 0.05). In overall, the present study shows that expression of the FGF21 gene is strongly up‐regulated during the transition period. It is assumed that the up‐regulation of FGF21 might play an important role in the adaptation of liver metabolism during early lactation in dairy cows such as in other species.     

Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares

The objective of the present study was to evaluate changes in concentrations of free insulin-like growth factor (IGF)-I in follicular fluid (FFL) during follicle development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6–15 mm; n = 54), medium (16–25 mm; n = 23) or large (>25 mm; n = 15) and FFL was collected. Free IGF-I levels in FFL in large follicles of follicular phase mares were greater (P < 0.05) than in large follicles of luteal phase mares and small or medium follicles of luteal and follicular phase mares. Free IGF-I concentrations were greater (P < 0.05) in large follicles of luteal phase mares than small but not medium follicles of luteal phase mares. FFL ratio of estradiol:progesterone paralleled changes in free IGF-I. Free IGF-I concentrations were negatively correlated (P < 0.05) with insulin-like growth factor binding protein (IGFBP)-2, -4 and -5 but not IGFBP-3 levels. In addition, free IGF-I concentrations in FFL were positively correlated (P < 0.01) with FFL estradiol, progesterone, androstenedione, estradiol:progesterone ratio, total IGF-I and total IGF-II. We conclude that increases in intrafollicular levels of bioavailable (free) IGF-I are associated with increased steroidogenesis in developing mare follicles.     

Regulation of fibroblast growth factor 9 on the differentiation of goat intramuscular adipocytes

It has been found that fibroblast growth factor receptor (FGF-FGFR) signaling can regulate the expression of adipocyte differentiation genes. FGF9 is one of the members of FGFs that mainly binds receptors FGFR2 and FGFR3. FGF9 is highly expressed in the adipose tissue of humans and mice, but there are few reports on the role of FGF9 in goat intramuscular adipocyte differentiation. Therefore, this study explored the effect of FGF9 on adipocyte differentiation through cell culture, interference, and overexpression. The expression of receptors FGFR1–FGFR4 in adipocyte differentiation and their effects on differentiation were detected to screen receptor gene of FGF9. Finally, the interaction between FGF9 and the receptor was tested by cotransfection. Our results showed that FGF9 interacts with FGFR2 to inhibit goat intramuscular adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma (PPARγ) and preadipocyte factor 1 (Pref1), which is a data support for subsequent pathway research.     

Localization of fibroblast growth factor I (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis, lactation and involution

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

Intracellular regulation of endometrial PGF(2a) and PGE(2) production in dairy cows during early pregnancy and following treatment with recombinant interferon-tau

Objectives were to examine how the conceptus and recombinant bovine interferon-tau (rbIFN-tau) regulate intracellular components of the PGF(2a) synthetic pathway and to determine if arachidonic acid (AA) is limiting in endometrial tissue of pregnant cows. In Experiment 1, uteri were collected from either cyclic or pregnant dairy cows on Day 17 post-estrus. Intercaruncular explants were dissected and incubated for 60 min to quantify PGF(2a) production in response to oxytocin (10(-6) M), A23187 (10(-5) M), melittin (10(-5) M), and phorbol 12, 13 dibutyrate (PDBu, 10(-6) M). Additional explants from the same cows were incubated for 24 h with and without AA. Oxytocin and A23187 did not stimulate PGF(2a) in explants from either cyclic or pregnant cows. Both PDBu, melittin, and A23187 + melittin stimulated PGF(2a) production in explants of cyclic cows, but not in explants of pregnant cows. The addition of AA to explant cultures for 24 hr did not increase PGF(2a) production during a subsequent 60-min incubation. In Experiment 2, explants were collected from cows that received intrauterine infusions of either BSA (1.9 mg/1.2 ml) or rbIFN-tau (0.2 mg rbIFN-tau + 1.7 mg BSA/1.2 ml) twice a day from Days 14 to 17 of the estrous cycle. Treatments of rbIFN-tau attenuated PGF(2a) secretion induced by in vitro PDBu and A23187 treatments. However, rbIFN-tau treatment in vivo had no effect on the in vitro induction of PGF(2a) secretion by melittin. IFN-tau may regulate the PGF(2a) synthetic pathway by reducing activity of PKC or PKC mediated events.     

牛碱性成纤维细胞生长因子基因在大肠杆菌中的表达及其表达产物的生物学活性

采用巢式PCR方法克隆了牛18ku-bFGF基因完整的编码序列,并构建了原核表达载体pET-28a-bFGF,将其转化大肠杆菌BL21,在25℃低温条件下,用0.5 mmol/L IPTG诱导表达5 h,用Ni-NTA亲和纯化细胞裂解上清液,经Western-blotting检测,结果显示,在特定的诱导条件下,重组牛bFGF基因在大肠杆菌中获得了表达,并且主要以可溶性状态存在于细胞中。经检测,纯化后的重组蛋白能显著促进成纤维细胞的增殖（P〈0.05）,其活性与商品用重组人18ku-bFGF没有差异（P〉0.05）。表明,所获得的可溶性重组牛18ku-bFGF蛋白具有较高的生物学活性,可用于后续研究工作。