A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.     

Endothelin releasing activity in calf serum and porcine follicular fluid

Bovine calf serum (CS) and porcine follicular fluid (PFF) added to bovine aortic endothelial cells (BAEC) in culture stimulate endothelin (ET) release. Sorbent extraction of acidified calf serum and PFF using Bond Elut C18 yields a product with an activity 30% that of the starting material. Fractionation of Bond Elut C18 extracted CS or PFF using HPLC yields peaks of activity with retention times similar to those of TGF beta and activin A, respectively. TGF beta is 10-fold more potent than activin A to stimulate ET release from BAECs. The observation that TGF beta and activin A are dose-additive but not effect-additive to stimulate ET release is compatible with the conclusion that these two substances act through a similar mechanism. Thus, TGF beta and/or activin A-related peptides may contribute to the ET releasing activity observed in acidified Bond Elut C18 extracted CS and PFF. The identity of ET-releasing activity in native CS and PFF remains to be established.     

Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid.

Using competitive ligand-binding studies, ligand blotting, and immunoprecipitation, we have characterized the insulin-like growth factor (IGF)-binding proteins (BPs) of porcine follicular fluid. Competitive ligand-binding studies revealed a preference of ovarian IGFBPs for IGF-II over IGF-I. Follicular fluid from small, 1-3-mm follicles had nearly twice the binding capacity for IGFs as that from large, 6-10-mm follicles. Ligand blots of porcine follicular fluid resolved 5 major bands of IGF-binding activity having apparent molecular sizes of 44, 40, 34, 29, and 22 kDa. The 40-44-kDa bands were immunoprecipitated by an antibody to porcine IGFBP-3, the acid-stable subunit of the 150-kDa growth hormone-dependent IGF-binding protein complex of porcine serum. The 34-kDa band was immunoprecipitated by an antibody to rat IGFBP-2, the major IGF-binding protein found in fetal rat serum. To date we have been unable to immunoprecipitate the 29- and 22-kDa bands with any of the antibodies tested, including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGF-binding protein. The 40-44-kDa species (IGFBP-3) was the predominant form and was equally abundant in fluid from large and small follicles. In contrast, the smaller forms, including IGFBP-2 and the 29- and 22-kDa forms were significantly more prominent in fluid from small follicles. In view of other studies indicating a significant effect of IGFBPs on ovarian cell function, follicular IGFBPs may play an important role in the IGF autocrine/paracrine regulatory system of the ovary.     

Detection of endogenous epidermal growth factor-like activity in the developing chick embryo

Epidermal growth factor-like activity has been detected by radioreceptor assay and radioimmunoassay in the developing chick embryo. Very little activity could be detected prior to Day 8 of embryonic life (hatching is at Day 21). A peak of EGF activity was detectable by both assays over Days 10 to 12. The EGF activity then fell to virtually undetectable levels during Days 14 to 17. A later rise in RRA detectable EGF like activity was then observed over Days 18-20. The EGF activity from a Day 11 embryo chromatographed on high-performance liquid chromatography as a single peak, with very high recovery of activity, at a later elution position than mouse EGF or human EGF.     

Ovarian follicular fluid eicosanoid concentrations during the pre-ovulatory period in humans

Prostaglandins are involved in ovulation and in every mammal studied so far, ovulation has been inhibited by prostaglandin inhibition. Information regarding the role of leukotrienes and thromboxanes in ovulation is more limited. In order to study the production of eicosanoids in human pre-ovulatory follicular fluid, follicular aspiration was timed by means of serial ultrasound scans and human chorionic gonadotrophin (hCG) to be immediately pre-ovulatory. 11 women were studied and the eicosanoids measured by radioimmunoassay (RIA). The follicular fluid was found to contain leukotrienes (LT) B4, LTC4 (D4, E4), prostaglandin (PG) E2, PGF2 alpha 6 keto PGF1 alpha k and thromboxane (TX) B2. This is the first published report of leukotrienes in human follicular fluid in spontaneous cycles, and is one of the few reports showing prostaglandins and thromboxanes. The significance of demonstrating leukotrienes in human follicular fluid is discussed as is the correlation between individual eicosanoids in the human ovary.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14-EGFR complex compared to that of the EGF-EGFR complex; we hypothesized this may be attributed to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant K(D) of 74 microM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling.     

Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo

To evaluate the regulation of ovarian insulin-like growth factor-I (IGF-I) during follicular growth in vivo, we measured the concentration of this peptide in follicular fluid (FFL) of immature gilts during the induction of follicular development by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). FFL concentrations of immunoreactive (i) IGF-I were compared with those of intrafollicular steroids and with concentrations of iIGF-I, estradiol (E2), and porcine growth hormone (GH) in serum. PMSG, administered at Time 0, induced a significant (p less than 0.01), time-dependent increase in intrafollicular iIGF-I that peaked 72 h after administration of the hormone, before the administration of hCG. During the first 72 h, the changes in ovarian iIGF-I paralleled those for progesterone and E2. After the administration of hCG at 72 h, FFL levels of E2 fell, those of iIGF-I remained constant, and progesterone rose. Serum E2 concentrations paralleled those in FFL. Since serum GH and IGF-I levels rise during spontaneous puberty in some species, these levels were also monitored. However, a significant treatment effect on serum GH and iIGF-I was not demonstrated. In summary, ovarian concentrations of iIGF-I are increased by gonadotropic hormones in vivo. The absence of concomitant changes in circulating levels of iIGF-I and GH suggests that the gonadotropin effects are exerted directly on the ovary. These results, together with more abundant data regarding secretion and action of IGF-I in cultured granulosa cells, suggest that IGF-I may function in an autocrine or paracrine fashion to amplify the actions of gonadotropins at an ovarian level.     

Ovarian follicular populations and preovulatory enlargement in Booroola and control Merino ewes

To investigate the factors contributing to the different ovulation rates observed in two strains of sheep (Booroola 5.2, Merino 1.2), in-vivo monitoring of follicular kinetics followed by histological examination of both ovaries was performed during the late luteal and follicular phases. Ewes of both strains were either ovariectomized at Day 13, or had the 3 largest follicles of each ovary ink-labelled at Day 13 and were ovariectomized at Day 15, or had the 3 largest follicles of each ovary ink-labelled at Days 13 and 15 and were ovariectomized 16 h after the beginning of oestrus (N = 6 per time per strain). In another experiment, the age effects on the follicular populations of these two strains were also studied. There were 2-4 times more primordial follicles and 1.5-2 times more preantral follicles in the ovaries of Booroola than in control Merino ewes, although the number of antral follicles was the same. The percentage of normal follicles in this population was higher in Merino than Booroola ovaries. In Booroola ewes, there was no correlation between the number of antral follicles per ovary and the ovulation rate at the previous cycle (r = 0.22). This suggests that follicle numbers do not play a key role in the high ovulation rate of the Booroola strain. The number of follicles initiating growth from the primordial pool, the number of growing follicles disappearing at the preantral stage, the pattern of antrum development, granulosa cell multiplication and appearance of atresia differed between strains. The reasons for the high ovulation rate of the Booroola strain became clear when preovulatory enlargement was followed by ink labelling. An extended period of time during which recruitment of ovulatory follicles takes place, together with a low incidence of selection and the ability of the follicles to wait for ovulation are the features involved in this high ovulation rate.     

Concentrations of insulin-like growth factor-I, estradiol and progesterone in follicular fluid of ovarian follicles during early pregnancy in cattle

To determine if the presence of the developing conceptus is associated with changes in intrafollicular concentrations of insulin-like growth factor-I (IGF-I), estradiol (E2) and/or progesterone during early pregnancy in cattle, either pregnant (n=16) or nonpregnant (n=15) cows were slaughtered on Day 10, 15 or 18 postestrus. Ovaries and follicular fluid were collected. Follicles were grouped by diameter: 1.0 to 3.9 mm (small; n=63), 4.0 to 7.9 mm (medium; n=128), and >/= 8.0 mm (large; n=38). The average diameter of large follicles was greater (P<0.05) in pregnant than in nonpregnant cows on Day 10, but on Day 18 it was greater (P<0.05) in nonpregnant than in pregnant cows (11.3 vs 9.7 mm). Status (pregnant vs nonpregnant) did not affect (P>0.10) follicular fluid progesterone nor IGF-I concentrations. In contrast, the status and days postestrus affected (P<0.05) follicular fluid E2 concentrations. Follicular fluid E2 levels in the three follicle size-categories on Day 10 did not differ (P>0.10) between pregnant and nonpregnant cows. However, on Days 15 and 18 postestrus, follicular fluid E2 concentrations in pregnant cows was lower (P<0.05) in large follicles than in nonpregnant cows. We conclude that the presence of a developing conceptus early in pregnancy may alter follicular growth and inhibit follicular E2 production in cattle. These effects appear to be mediated by factors other than IGF-I.     

Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration

The mechanisms that regulate the transition between the initial priming phase and DNA replication in liver regeneration are poorly understood. To study this transition, we compared events occurring after standard two-thirds partial hepatectomy, which elicits full regeneration, with response to a reduced hepatectomy, one-third partial hepatectomy (1/3PH), which leads to little DNA replication. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between the two procedures, cell cycle progression was significantly blunted in 1/3PH mice. Among the main defects observed in 1/3PH mice were an almost complete deficiency in retinoblastoma phosphorylation and the lack of increase in kinase activity associated with cyclin E. We report that, in two-thirds partial hepatectomy mice, the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) preceded the start of DNA replication and was not detectable in 1/3PH animals. Injection of HB-EGF into 1/3PH mice resulted in a >15-fold increase in DNA replication. Moreover, we show that hepatocyte DNA replication was delayed in HB-EGF knock-out mice. In summary, we show that HB-EGF is a key factor for hepatocyte progression through G(1)/S transition during liver regeneration.     

Ovarian response to injections of charcoal-extracted porcine follicular fluid and porcine follicle-stimulating hormone in gilts fed a progesterone agonist (altrenogest)

This experiment was conducted to compare the negative effects of charcoal-extracted porcine follicular fluid (pFF) and the positive effects of purified porcine follicle-stimulating hormone (pFSH) on growth of follicles and on plasma hormone concentrations. Twenty gilts were fed altrenogest for 18 days (20 mg.day-1.gilt-1) to suppress spontaneous growth of large follicles (greater than 6 mm in diameter). Gilts, assigned at random to receive pFF and pFSH administered in a 2 x 2 factorial arrangement, were injected 9 times at 8-h intervals starting 48 h before the last feeding of altrenogest and ending 8 h before slaughter (24 h after the last feeding of altrenogest). Blood was collected periodically through vena cava catheters. Treatment groups and mean number of medium follicles (3 to 6 mm in diameter)/gilt at necropsy were 1) 20 ml of charcoal-extracted porcine serum i.v. + 4 ml saline i.m., 30.8; 2) 20 ml of pFF i.v. + saline i.m., 0.2; 3) serum i.v. + 8 micrograms of pFSH (USDA-pFSH-B1)/kg BW in saline i.m., 59.0; and 4) pFF i.v. + pFSH in saline i.m., 36.2. Injections of pFF decreased (p less than 0.01) and injections of pFSH increased the number of medium follicles, and the interaction of pFF and pFSH was not significant. Plasma FSH decreased (p less than 0.01) during pFF treatment of saline-injected gilts at a rate of 0.29 ng.ml-1.h-1. During pFSH treatment, plasma FSH increased (p less than 0.05) at statistically identical rates of 0.33 and 0.32 ng.ml-1.h-1 in serum- and pFF-injected gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of inhibins and steroids in follicular fluid during development of dominant follicules in heifers

The objective of this study was to examine changes in intrafollicular concentrations of inhibins and steroids in heifers during growth of dominant follicles. To obtain dominant ovulatory follicles, heifers received injections of prostaglandin (PG) on Day 9 of an estrous cycle and were ovariectomized (OVX) 0, 24, 48, 60, or 72 h after injection. To obtain dominant nonovulatory follicles, heifers were OVX on Day 3, 6, or 9 of a cycle. Follicular size was determined, follicular fluid (FF) was collected from follicles 6 mm or greater in diameter, and RIA was used to quantify concentrations of inhibins, estradiol, and progesterone in FF. During growth of dominant ovulatory follicles, concentrations of estradiol and progesterone increased, whereas inhibins decreased when compared with dominant follicles on Day 9 before PG treatment. Concentrations of inhibins were inversely correlated with size and concentrations of estradiol in dominant ovulatory follicles. As dominant nonovulatory follicles increased in size, concentrations of inhibins, estradiol, and progesterone increased. Concentrations of inhibins were positively correlated with size and with progesterone concentrations in dominant nonovulatory follicles. Concentrations of inhibins were greater in dominant nonovulatory follicles than in atretic follicles. In summary, intrafollicular concentrations of inhibins decreased during growth of dominant ovulatory follicles, but increased during growth of dominant nonovulatory follicles. Because of the well-known suppressive action of inhibins on FSH secretion, we hypothesize that inhibins are involved in growth and atresia of dominant follicles during the bovine estrous cycle.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid

To elucidate the beneficial effects of porcine follicular fluid (pFF) added to maturation medium on the sustenance of cytoplasmic maturation responsible for the subsequent developmental competence after in vitro fertilization (IVF) of porcine oocytes, we focused on the antioxidative role of pFF in its function of protecting oocytes from reactive oxygen species (ROS)-induced cell damage. Porcine follicular fluid collected from small (2-6 mm) follicles had about 7.2-fold higher levels of superoxide dismutase (SOD) activity than that of fetal bovine serum (FBS), and this activity was markedly blocked by the CuZn-SOD inhibitor, diethyldithiocarbamate (DETC). The interruption of meiotic progression and the increasing intracellular glutathione (GSH) content throughout the maturation period, as well as an outbreak of DNA damage in oocytes and cumulus cells were difficult to detect in oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system, whereas cell damage encompassed by ROS was prominent in oocytes cultured with 10% FBS and 10% pFF plus 100 microM DETC. Similarly, significant enhancement to the degree of transformation of the sperm nucleus into the male pronucleus (MPN) after in vitro fertilization was shown by the addition of pFF to the maturation medium. The presence of DETC during in vitro maturation reduced the ability of oocytes to promote MPN formation to the same extent as oocytes matured with FBS. The proportion developing to the blastocyst stage was increased in oocytes that matured with pFF, but this developmental competence was significantly lowered by treatment with DETC (P < 0.05). These findings suggest that pFF plays a critical role in protecting oocytes from oxidative stress through a higher level of radical scavenging activity elicited from SOD isoenzymes, resulting in the enhancement of cytoplasmic maturation responsible for developmental competence postfertilization.     

Purification of a sperm motility stimulator from porcine follicular fluid.

1. We purified a glycoprotein possessing a potent ability to stimulate porcine sperm motility, from porcine ovarian follicular fluid. 2. The protein, of molecular weight 52,000, has an N-terminal sequence similar to that of antithrombin III, indicating that it is identical to or highly homologous to antithrombin III. 3. Thus it is proposed that antithrombin III or its homologue is involved in the mammalian fertilization process.     

Characterization of the gene encoding murine heparin-binding epidermal growth factor-like growth factor

The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I–VI) and five introns (A–E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A–E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A–E of mHB-EGF failed to show significant overall homology with those of hHB-EGF     

Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins

Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)