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PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose-2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is extensively involved in cell proliferation owing to its key role in carbohydrate metabolism. In the present study we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or norgestrel) leads to a rapid increase in Fru-2,6-P2 concentration. Our Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a PRE (progesterone-response element) to which PR (progesterone receptor) binds and thus transactivates PFKFB3 gene transcription. PR expression in the PR-negative cell line MDA-MB-231 induces endogenous PFKFB3 expression in response to norgestrel. Direct binding of PR to the PRE box (-3490 nt) was confirmed by ChIP (chromatin immunoprecipiation) experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK (extracellular-signal-regulated kinase)/RSK (ribosomal S6 kinase) pathway leading to phosphorylation of PFKFB3 on Ser461 is followed by activation of mRNA transcription via cis-acting sequences on the PFKFB3 promoter.

Sixteen female sheep of Degua breed were assigned to receive either the full dose of norgestomet ear implant and injectable solution containing norgestomet and estradiol valerate (n = 8) or half the dose (n = 8). The ear implants were removed in both groups on day 12. All ewes received an intramuscular administration of 500 IU PMSG at implant withdrawal. Synchronized ewes were individually hand mated twice at 48 and 60 hours after implant removal. One ewe in each group however refused mating on both occasions. Pregnancy diagnosis was conducted by bimanual external palpation 90 to 100 days post mating. The conception rates (3/7, 42.85%) and (5/7, 71.42%) were recorded in the two treatment groups, respectively. All eight ewes lambed between 145 to 153 days post mating. In group I ewes carried only singletons (prolificity rate 1.0) whereas in group II two ewes delivered twins, producing 7 lambs with prolificity rate of 1.4 (N.S). From this preliminary investigation it appears that the lower dose of norgestomet ear implants offers better option for estrus synchronization accompanied by higher fertility.

In Exp. 1, 187 lactating beef cows were treated with injections of GnRH 7 d before and 48 h after prostaglandin F2alpha (PGF2alpha; Cosynch) or with Cosynch plus a 7-d treatment with an intravaginal progesterone (P4)-releasing insert (CIDR-B; Cosynch + CIDR). In Exp. 2, 183 lactating beef cows were treated with the Cosynch protocol or with Cosynch plus a 7-d treatment with norgestomet (Cosynch + NORG). In Exp. 1 and 2, blood samples for later P4 analyses were collected on d -17, -7 (first GnRH injection), 0 (PGF2alpha injection), and at timed artificial insemination (TAI; 48 h after PGF2alpha). In Exp. 3, 609 lactating beef cows were treated with the Cosynch + CIDR protocol or were fed 0.5 mg of melengestrol acetate (MGA) per day for 14 d before initiating the Cosynch protocol 12 d after the 14th d of MGA feeding (MGA + Cosynch). Blood samples were collected as in Exp. 1 and 2, plus additional samples on d -33 and -19 before PGF2alpha. In Exp. 4, 360 lactating beef cows were treated with a Cosynch + CIDR protocol, with TAI occurring at either 48 or 60 h after PGF2alpha, while receiving either GnRH or saline to form four treatments. Blood samples were collected as in Exp. 1 and 2. In Exp. 1, addition of P4 reduced the ability of the first GnRH injection to induce ovulation in anestrous cows with low P4 before PGF2alpha but improved (P = 0.06) pregnancy rates (61 vs 66%). In Exp. 2, the addition of NORG mimicked P4 by likewise increasing (P < 0.01) pregnancy rates (31 vs 51%) beyond those after Cosynch. In Exp. 3, the Cosynch + CIDR protocol increased (P < 0.001) pregnancy rates from 46 to 55% compared to the MGA + Cosynch protocol. In Exp. 4, administration of GnRH at TAI improved (P < 0.05) pregnancy outcomes (50 vs 42%), whereas timing of TAI had limited effects. We conclude that a progestin treatment concurrent with the Cosynch protocol improved pregnancy outcomes in all experiments, but pretreatment of cows with MGA was not as effective as the CIDR insert or NORG implants in this Cosynch-TAI model. Most of the improvement in pregnancy rates was associated with the increase in pregnancy rates of anestrous cows, regardless of whether ovulation was successfully induced in response to GnRH 7 d before PGF2alpha. Injection of GnRH at TAI following the Cosynch + CIDR protocol increased pregnancy rates in cycling cows with high P4 before the PGF2alpha injection and in anestrous cows with low P4 before PGF2alpha injection.

The induction of estrus during the non-breeding season was investigated in 100 Egyptian Baladi goats (Capra hircus). All animals assigned to treatments had low progesterone concentrations (<0.5 ng/ml) tested 2 times 10 days apart to confirm anestrous condition. Animals were assigned to three experimental groups. A group of animals received subcutaneous norgestomet ear implant for 11 days and a single i.m. injection of PGF2alpha 24 hr before implant removal (group I; n=40). Second group of animals received subcutaneous norgestomet ear implant for 11 days and a single i.m. injection of PGF2alpha 24 hr before implant removal and gonadotropin releasing hormone 24 hr after implant removal (group II; n=40). Third group of animals received no treatment (control group; n=20). The percentage of goats that showed estrous behavior during the first 72 hr after implant removal was 77.5, 85.0% and 10.0% in group I, group II and control group, respectively. The fertility rate was 57.5, 70.0% and 10.0% in group I, group II and control group, respectively. In conclusion, estrus can be induced in seasonally anestrous Egyptian Baladi goats using norgestomet and PGF2alpha and the injection of GnRH 24 hr after norgestomet implant removal synchronized ovulation in a higher percentage of goats.

Synchronization of estrus and ovulation is essential for AI of ewes during a predetermined time frame, and progestogen-eCG treatments are typically used to prepare the ewes. However, eCG is not readily available in the United States, but P.G. 600 (400 IU of eCG and 200 IU of hCG) is available. Thus, we conducted a study to determine the effects of eCG and P.G. 600 on the timing of estrus and ovulation after progestogen withdrawal. Ewes were assigned to two replicates of an experiment with the following treatments: 1) 3-mg norgestomet implant (i.e., one-half of a Syncro-Mate-B [SMB] implant) for 10 d, plus 2 mL of saline i.m. at SMB removal (n = 11); 2) 3-mg SMB implant for 10 d, plus 400 IU of eCG i.m. at SMB removal (n = 13); and 3) 3-mg SMB implant for 10 d, plus P.G. 600 i.m. at implant removal (n = 9). On d 6 after SMB insertion, PGF2alpha was used to induce luteolysis. Beginning 12 h after implant removal, vasectomized rams were used at 12-h intervals to check for estrus. When a ewe was detected in estrus, each ovary was evaluated ultrasonically. Ovaries were evaluated again 16 h later and then at 8-h intervals until ovulation. Treatment altered the interval from implant removal to estrus (less [P < 0.05] in SMB + eCG than in the other two groups) and to ovulation (greatest [P < 0.05] in SMB). However, the treatment x replicate interaction was significant for the intervals from implant removal to estrus (P < 0.03) and from implant removal to ovulation (P < 0.05). An inconsistent response in the SMB-treated ewes seemed to be primarily responsible for the interaction. The intervals to estrus and to ovulation for the SMB-treated ewes were shorter (P < 0.05) in Replicate 1 than in Replicate 2. Also, both intervals seemed to be less consistent between replicates for the SMB + P.G. 600- than for the SMB + eCG-treated ewes; that is, eCG seemed to increase the predictability of the intervals to estrus and to ovulation. Neither the main effects of treatment and replicate nor their interaction were significant for the interval from estrus to ovulation (38.4 /- 3.3 h), size of the ovulatory follicle (7.7 +/- 0.8 mm), or ovulation rate (1.6 +/- 0.2). We concluded from this experiment that eCG is a better choice than P.G. 600 as the gonadotropin to use at the time of progestogen withdrawal to prepare ewes for AI during a predetermined interval.

Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.

Three experiments were conducted to induce estrus and(or) ovulation in 1,590 suckled beef cows at the beginning of a spring breeding season. In Exp. 1, 890 cows at three locations were allotted to three treatments: 1) GnRH on d -7 + prostaglandin F2alpha (PGF2alpha) on d 0 (Select Synch); 2) GnRH on d -7 + PGF2alpha on d 0 (first day of the breeding season) plus a norgestomet implant (NORG) between d -7 and 0 (Select Synch + NORG); or 3) two injections of PGF2alpha given 14 d apart (2xPGF2alpha). More (P < 0.05) cycling cows were detected to have been in estrus after both treatments that included GnRH, whereas, among noncycling cows, the addition of norgestomet further increased (P < 0.05) the proportion in estrus. Pregnancy rates were greater (P < 0.01) among noncycling cows after treatments that included GnRH. For cows that calved >60 d before the onset of the breeding season, conception rates were greater (P < 0.01) than those that calved < or =60 d regardless of treatment, whereas days postpartum had no effect on rates of detected estrus. When body condition scores were < or =4 compared with >4, rates of detected estrus (P < 0.05) and conception (P = 0.07) were increased. In Exp. 2, 164 cows were treated with the Select Synch + NORG treatment and were inseminated either after estrus or at 16 h after a second GnRH injection (given 48 h after PGF2alpha). Conception and pregnancy rates tended (P = 0.08) to be or were less (P < 0.05), respectively, for noncycling cows inseminated by appointment, but pregnancy rates exceeded 53% in both protocols. In Exp. 3, 536 cows at three locations were treated with the Select Synch protocol as in Exp. 1 and inseminated either: 1) after detected estrus (Select Synch); 2) at 54 h after PGF2alpha when a second GnRH injection also was administered (Cosynch); or 3) after detected estrus until 54 h, or in the absence of estrus, at 54 h plus a second GnRH injection (Select Synch + Cosynch). Conception rates were reduced (P < 0.01) in cows that were inseminated by appointment. An interaction of AI protocol and cycling status occurred (P = 0.05) for pregnancy rates with differing results for cycling and noncycling cows. Across experiments, variable proportions of cows at various locations (21 to 78%) were cycling before the breeding season. With the GnRH or GnRH + NORG treatments, ovulation was induced in some noncycling cows. Conception rates were normal and pregnancy rates were greater than those after a PGF2alpha program, particularly when inseminations occurred after detected estrus.

Beef heifers were assigned to three groups: (1) untreated controls (n=4), (2) Syncro-Mate B(R) (SMB)-treated (n=5), and (3) hysterectomized and SMB-treated (n=4). SMB was administered 8 or 9 days after oestrus, approximately 30 days after hysterectomy. This study was conducted to determine if the uterus was necessary for SMB to induce luteolysis. SMB induced premature luteolysis as only 20% of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone 7 days after the time of SMB treatment, compared to all (100%) of the untreated heifers (p<0.05). By 9 days after the time of SMB treatment, 25% of the untreated heifers and none (0%) of the intact SMB-treated heifers had >/=0.75 ng/ml of progesterone; however, all (100%) of the hysterectomized SMB-treated heifers had >/=0.75 ng/ml of progesterone (p<0.05). Therefore, SMB-induced luteolysis required the involvement of the uterus. The luteolysin, prostaglandin F(2alpha), is probably the secretion from the uterus that mediates the SMB-induced luteolysis. SMB treatment, however, required 7-8 days to induce luteolysis.

Enhanced expression of fatty acid synthase and other lipogenic enzymes has been observed in a subset of breast cancers with poor prognosis. This phenomenon has been related to amplification of a gene on chromosome region 11q13 encoding Spot 14, a putative regulator of lipogenic enzyme expression. In this paper we demonstrate that the induction of lipogenesis by progestins and androgens in the breast cancer cell line T47-D is accompanied by a marked increase in the expression of Spot 14. These data corroborate the correlation between Spot 14 expression and increased lipogenesis. Moreover they show that apart from gene amplification there is another steroid-regulated pathway that may enhance Spot 14 expression and lipogenesis in tumor cells., (Copyright 2000 Academic Press.)

Cycling (n = 16) and noncycling (n = 24), early postpartum, suckled beef cows of three breeds were assigned randomly to three treatments: 1) 100-microg injection of GnRH plus a 6-mg implant of norgestomet administered on d -7 before 25 mg of PGF2alpha and implant removal on d 0 (GnRH+NORG); 2) 100 microg of GnRH given on d -7 followed by 25 mg of PGF2alpha on d 0 (GnRH); or 3) 2 mL of saline plus a 6-mg implant of norgestomet administered on d -7 followed by 25 mg of PGF2, and implant removal on d 0 (NORG). All cows were given 100 microg of GnRH on d +2 (48 h after PGF2alpha). Blood sera collected daily from d -7 to d +4 were analyzed for progesterone and estradiol-17beta, and ovaries were monitored daily by transrectal ultrasonography to assess changes in ovarian structures. Luteal structures were induced in 75% of noncycling cows in both treatments after GnRH, resulting in elevated (P < .01) progesterone on d 0 for GnRH+NORG-treated cows. Concentrations of estradiol-17beta (P < .01) and LH (P < .05) were greater on d +2 after GnRH for cows previously receiving norgestomet implants. Pregnancy rates after one fixed-time AI at 16 h after GnRH (d +2) were greater (P < .05) in GnRH+NORG (71%) than in GnRH (31%) and NORG (15%) cows. Difference in pregnancy rate was due partly to normal luteal activity after AI in over 87% of GnRH+NORG cows and no incidence of short luteal phases. The GnRH+NORG treatment initially induced ovulation or turnover of the largest follicle, induction of a new follicular wave, followed later by increased concentrations of estradiol-17beta and progesterone. After PGF2alpha, greater GnRH-induced release of LH occurred in GnRH+NORG cows before ovulation, and pregnancy rates were greater after a fixed-time AI.

Our objective was to determine whether priming with the progestogen norgestomet for 9 d would enhance estrual and ovulatory responses of prepubertal gilts to PG600 (400 IU eCG + 200 IU hCG). Gilts (140 to 190 d old) were assigned by litter, age, and weight to one of three treatments: 1) 9 d of norgestomet implant with an injection of PG600 after implant removal on d 9 (N+PG; n = 43); 2) no implant and an injection of PG600 on d 9 (PG; n = 36); or 3) neither implant nor PG600 (control; n = 29). Beginning on d 0, gilts were exposed once daily to a boar and checked until estrus was observed or until d 45 after the start of the experiment. Ovaries were examined for number of corpora lutea (CL) after estrus or at 45 d. Greater proportions of N+PG (63%, P < .05) and PG (69%, P < .01) gilts expressed estrus than did controls (34%), but proportions did not differ between N+PG and PG (P > .10). Among gilts in estrus following treatment with N+PG or PG, 100% showed estrus within 6 d after PG600 injection. For gilts that expressed estrus within 45 d, the average age at estrus was reduced (P < .05) by PG to 172 +/- 2 d compared with 182 +/- 4 d for controls. Average age at estrus did not differ (P > . 10) between PG and N+PG (177 +/- 2 d). Greater proportions of N+PG (82%; P < .001) and PG (65%; P < .001) gilts ovulated than controls (13%), but proportions did not differ between N+PG and PG (P > .10). The number of CL (20 +/- 2) was not affected by treatment and ranged from 2 to 71. There was no increase in ovarian cysts in response to treatment. Results indicated that norgestomet before PG600 did not enhance estrus expression or ovulation compared with PG600 alone, but use of PG600 increased the proportions of gilts that expressed estrus and ovulated compared with controls.

Progestogens and follicular stimulants have proved reasonably successful for estrus synchronization, but time of ovulation relative to removal of the progestogen is not clearly established. We monitored time of ovulation in ewes following synchronized estrus. Ovaries of 40 Dorset and Rambouillet x Dorset ewes were evaluated during the spring and fall (20/replicate). Ewes were randomly assigned to one of two treatment groups (n = 20/group): implant-only (I) ewes received a norgestomet implant for 10 d; and implant + PMSG (PI) ewes received a norgestomet implant for 10 d with an i.m. injection of 500 IU of PMSG at implant removal. Time of estrus onset was detected with the HeatWatch Estrus Detection System. Following the initiation of estrus, ovaries were monitored via rectal ultrasonography every 6 h to determine the interval from implant removal to ovulation (OVUL), and onset of estrus to ovulation (INT). Four I and PI ewes that lost implants before scheduled removal on d 10 were removed from the study. Estrus was detected in 13 of 16 (81%) I and 14 of 16 (88%) PI ewes within 108 h after implant removal. The interval from implant removal to estrus (EST), INT, and OVUL were not affected by replicate. The INT was not different in I (33.8 h) and PI (35.0 h) ewes. Mean OVUL was greater (P < .01) in I (79.8 h) than in PI ewes (68.6 h), and mean EST was also greater (P < .01) for I (46.0 h) than for PI ewes (32.6 h). The present data indicate that ovulation occurs on average 70 to 80 h after implant removal in ewes treated with norgestomet, and PMSG reduces the interval from implant removal to ovulation.

The aims of this study were to determine: (1) if short-term treatment of Bos indicus heifers with progesterone (P4) while implanted with a s.c. norgestomet implant for 17 days would influence the time interval to oestrus and increase fertility of the synchronised oestrus, and (2) whether the response to treatment with P4 would differ between heifers treated with a norgestomet implant for 17 vs. 11 days when short-term treatment with P4 is applied 3 days prior to implant removal. B. indicus heifers at two separate sites (A and B) were allocated to three groups at each site. Heifers in two groups (NG and NGP4 groups) were given a single s.c. norgestomet implant on the first day of treatment (day 0) while heifers in a third group (NGP4PG group) were implanted on day 6. A single P4 releasing Controlled Internal Drug Release device (CIDR) was inserted on day 14 in heifers in the NGP4 and NGP4PG groups and was removed 23.5 +/- 0.07 h later (day 15). Heifers in the NGP4PG group were administered an analogue of prostaglandin F2 alpha (PGF2 alpha) at the time of CIDR removal to regress corpora lutea. Implants were removed from all heifers on the same day (day 17) and a 400 IU of equine chorionic gonadotrophin (ECG) was administered s.c. Animals were artificially inseminated 11.1 +/- 0.17 h after detection of oestrus, using frozen semen from one bull at site A and one of five bulls at site B. Inseminations were carried out by one of two technicians. Treatment with P4 delayed oestrus and reduced the synchrony of oestrus at site A (hours to oestrus +/- SD: NG group, 39.0 +/- 13.7; NGP4 group, 66.3 +/- 24.4; NGP4PG group, 58.9 +/- 20.5 h; P < 0.05) but not at site B (41.4 +/- 15.2, 42.5 +/- 10.1, 45.4 +/- 10.3 h; P > 0.05). Pregnancy rates 6 weeks after insemination were found to be significantly associated with bull (P < 0.001), treatment group (P = 0.013) and insemination technician (P = 0.033). Pregnancy rates were greater in the heifers in the NGP4 group than heifers in the NG group [50.3% (78/155) vs. 36.4% (60/165); odds ratio = 1.83, 95% CI = 1.14 to 2.96] and similar between heifers in the NGP4 and NGP4PG groups [50.3% (78/155) vs. 51.1% (63/117); odds ratio = 1.06, 95% CI = 0.67 to 1.69]. It was concluded that acute treatment with P4 can improve pregnancy rates in B. indicus heifers treated for 17 days with norgestomet implants. Reducing the duration of norgestomet treatment to 11 days and administration of PGF2 alpha at the time of ending treatment with a CIDR device resulted in no differences in fertility, mean intervals to oestrus or synchrony of oestrus.

The aim of the present study was to develop a treatment protocol for the precise synchronization of oestrus that would avoid the development of persistent dominant ovarian follicles. Bos indicus heifers, in which oestrous cycles had been presynchronized, were allocated randomly, according to the day of their oestrous cycle, to one of five treatment groups. All heifers received a subcutaneous ear implant containing 3 mg of norgestomet for 17 days starting on day 0 and an injection of an analogue of prostaglandin F2 alpha on days 0 and 4. Heifers in group 1 (control group; n = 7) received no other treatment, while heifers in groups 2 (n = 8), 3 (n = 7), 4 (n = 7), and 5 (n = 7) received a single progesterone-releasing controlled internal drug release device (CIDR) for 24 h on days 10, 12, 14 and 16, respectively. Treatment with a single CIDR delayed the mean time of ovulation and the day of emergence of the ovulatory follicle in heifers treated on days 14 and 16 compared with control heifers (P < 0.05). There was less variation in the interval to ovulation in heifers treated on day 10 compared with other treated heifers (P < 0.05). The variation among heifers in the day of emergence of the ovulatory follicle and the age of the ovulatory follicle at ovulation was less for all groups treated with a CIDR than for the control group (P < 0.05). The duration of dominance and variation in the duration of dominance of the ovulatory follicle was less in heifers treated with a CIDR device on days 10 and 16 than for control heifers (P < 0.05). Mean age (days from emergence to ovulation) of the ovulatory follicle did not differ among treatment groups (P > 0.05). Concentrations of LH and oestradiol decreased coincident with increased concentrations of progesterone on the days of CIDR treatment in treated compared with control heifers (P < 0.02) but increased again after removal of the CIDR. A smaller proportion of follicles in the growing phase of follicular development at the time of CIDR treatment become atretic compared with follicles that had reached a plateau phase of follicular growth (14.3% (1/7) versus 90.5% (19/21), respectively; P < 0.001). It was concluded that acute treatment with progesterone can influence the growth pattern of ovarian follicular development. However, the effect varies with the stage of ovarian follicular development. Short term treatment with progesterone 7 days before the end of a 17 day period of norgestomet treatment resulted in precise synchrony of ovulation without the ovulation of a persistent dominant ovarian follicle.

The efficacy of erodible norgestomet implants for preventing pregnancy in postpubertal heifers was evaluated in two experiments at five locations each. Heifers (n = 896) within each study location were stratified by weight and allotted randomly to receive an ear implant containing either 0, 24, 36, or 48 mg of norgestomet (d 0). Heifers were exposed to fertile bulls immediately after implantation for 75 d (d 0 to 74) in Exp. 1 (n = 476) or for 80 d (d 75 to 154) in Exp. 2 (n = 420). Weights were recorded on d 0 and 74 (Exp. 1 and 2) and d 154 (Exp. 2). Each heifer was palpated rectally for pregnancy at the end of each experiment. Pregnancy rates were higher (P < .01) for control heifers (0 mg implant) than for heifers that received 24, 36, or 48 mg of norgestomet. In Exp. 1, pregnancy rates were 96, 29, 6, and 4% for heifers that received 0, 24, 36, and 48 mg implants of norgestomet, respectively. In Exp. 2, pregnancy rates were 85, 36, 19, and 9% for heifers that received 0, 24, 36, and 48 mg implants of norgestomet, respectively. Estrous activity during the first 3 wk of bull exposure was reduced (P < .05) among heifers that received norgestomet implants compared to control heifers but was not completely abolished at any dosage in Exp. 1. During the first 75 d of Exp. 1 and 2, heifers treated with 36 or 48 mg norgestomet implants gained weight faster (P < .05) than control heifers. Combined across both experiments, ADG during the first 74 d were .53, .56, .59, and .60 kg/d for heifers treated with 0, 24, 36, and 48 mg implants of norgestomet, respectively. These data indicate that norgestomet implants increased rate of weight gain, reduced estrous activity, and reduced the occurrence of pregnancy in heifers on pasture.

Crossbred heifers (n = 75) fed for rapid (R; .82 kg/d) or slow-then-rapid (SR; .41 kg/d for 90 d then .82 kg/d) postweaning gain were used to examine the effects of age or pattern of gain on induction of puberty by a progestin. At 9.5, 11.0, and 12.5 mo of age, 12 prepuberal heifers from each growth treatment received progestin (a 6-mg Norgestomet implant for 10 d) or control treatments. Induction of puberty, LH secretory profiles, and ovarian follicular characteristics were assessed in Norgestomet-treated and control heifers. Body weights of R heifers were greater (P < .01) than those of SR heifers at all ages. At 12.5 mo, more Norgestomet-treated heifers exhibited a puberal estrus within 5 d after implant removal compared with controls (82% vs 9%, respectively), but Norgestomet did not induce puberty at 9.5 or 11 mo of age (progestin x age, P < .05) in heifers of either gain pattern. Norgestomet increased (P < .01) LH pulse frequency at all ages, whereas Norgestomet increased only mean LH concentrations at 12.5 mo of age (progestin x age, P < .03). Norgestomet treatment altered (P < .01) ovarian follicular characteristics at all ages. Gain pattern did not affect (P > .1) LH secretory profiles, ovarian characteristics, or induction of puberty by Norgestomet. We conclude that progestins induce puberty by hastening the normal cascade of endocrine and ovarian events associated with spontaneous puberty. Furthermore, age, but not pattern of gain, seems to be the critical factor influencing the efficacy of progestins to induce puberty in heifers.

One hundred fifty beef heifers and 403 beef cows suckling calves were administered norgestomet implants (8 d) and alfaprostol, a PGF2 alpha analogue, approximately 28 h before implant removal. Thirty hours after implant removal, females were administered either GnRH via injection, GnRH via implantation, or no GnRH. The dosage of GnRH was 250 micrograms, and implants prolong the induced LH surge. Ovulation response, incidence of short cycles, and calving rate were analyzed as a 2 x 2 x 3 completely randomized factorial design with female (heifers and cows), estrous cycles (with or without), and GnRH as the main effects. There were no interactions (P > .10), and because heifers and cows had responses that did not differ (P > .25), they were summarized together. Females with estrous cycles had a higher (P < .05) ovulation response, fewer (P < .01) short luteal phases, and a higher (P < .01) calving rate than females without estrous cycles. Gonadotropin-releasing hormone treatment increased the ovulation response (P < .01) and the calving rate (P < .05), and these responses were not affected (P > .10) by the method of GnRH administration. Based on these data, the increased ovulation response to GnRH may account for 29% of the increase in calving rate observed in the GnRH-treated females. In summary, in norgestomet- and alfaprostol-synchronized females, GnRH enhanced calving rate regardless of how it was administered. This increase was due to more than an increased ovulation rate.

The ultimate aim of any estrus synchronization method is to allow artificial insemination at a predetermined time after the end of treatment. This requires a very tight synchronization of estrus which is not observed in goats after administration of the usual fluorogestone acetate (FGA)/prostaglandin (PG) F2 alpha/equine chorionic gonadotrophin (eCG) treatment. The possibility to improve the synchronization of estrus and luteinizing hormone (LH) peak with different progestagens (FGA versus norgestomet) and routes of administration (vaginal sponge versus subcutaneous ear implant) was evaluated in two experiments where goats received one of three progestagen treatments: (1) a vaginal sponge impregnated with 45 mg of FGA, (2) a half-implant of norgestomet, or (3) a whole implant containing 3 mg of norgestomet. The progestagens were left in place for 11 days and intramuscular injections of 400 or 500 IU of eCG (according to milk yield) and 50 micrograms of an analogue of PGF2 alpha (cloprostenol) were given 48 h prior to progestagen removal. In Experiment 1, 117 cycling goats were checked for the time of onset of estrus, preovulatory LH peak and ovulation rate following estrus synchronization treatment. Goats treated with half-implants came into estrus earlier than those receiving vaginal sponges (27.8 +/- 5.0 h vs. 33.0 +/- 6.6 h, respectively; P < 0.05). No effect of progestagen priming was observed on the variability of the onset of estrus. However, the interval between the time of onset of estrus and LH peak was more variable (P < 0.05) in goats treated with half-implants. In Experiment 2, 170 non-cycling goats were monitored for the time of onset of estrus, percentage of females ovulating, fertility and prolificacy after estrus induction treatment and artificial insemination with frozen-thawed semen performed 24 h after the onset of estrus. No effect of progestagen treatment was observed either on the time or the variability of onset of estrus. The percentage of goats ovulating and overall fertility rate were higher (P < 0.05) in goats receiving vaginal sponges (98.2% and 75.0%, respectively) than those treated with half-implants (81.8% and 45.5%, respectively). However, no significant difference was observed, for the same parameters, in animals receiving implants (86.3% and 58.8%, respectively). In conclusion, estrus synchronization with a norgestomet implant or half-implant did not reduce the variability in the onset of estrus and LH peak. The fertility tended to be lower in goats treated with a whole implant and was significantly decreased in goats which received a half-implant.

Three experiments were conducted to evaluate the effects of prostaglandin F2 alpha treatments 5, 9, and 14 d before Syncro-Mate B on calving rates of 1,072 beef cows from inseminations at a pre-set time. The administration of prostaglandin F2 alpha 5 d before Syncro-Mate B decreased (P < .01) calving rates. This reduction was associated with lower (P < .05) calving rates in cows administered Syncro-Mate B in the first half of the estrous cycle. Calving rates were unaffected n cows administered prostaglandin F2 alpha 9 d before Syncro-Mate B. The administration of prostaglandin F2 alpha 14 d before Syncro-Mate B reduced (P < .05) calving rates in previously anestrous cows. In summary, prostaglandin F2 alpha should not be administered to postpartum cows 5 to 14 d before Syncro-Mate B synchronization.

Two experiments were designed to investigate the endocrine mechanisms by which progestin administration induces puberty in heifers. In experiment 1, prepubertal heifers were randomly assigned by age to receive either a single norgestomet implant for 10 days (NORG; n = 15) or to serve as unimplanted controls (CONT; n = 14). Serial blood samples were obtained on Days -0.5, 8.5, and 10.5 (Day 1 = day of implant insertion) and were analyzed for concentrations of LH. On days 9 and 11, 4 heifers in each treatment were slaughtered, and the reproductive tracts were obtained. Weekly progesterone analyses were performed to estimate the day of puberty in heifers not slaughtered. Puberty was induced in 6 of 7 heifers in the NORG treatment, resulting in an earlier (p < 0.05) day of puberty in the NORG than in the CONT treatment. The frequency of LH pulses was higher (p < 0.05) on Days 8.5 and 10.5 in the NORG as compared to the CONT treatment. Although no difference (p > 0.10) was observed between treatments in follicular development, uterine weight was greater (p < 0.05) in NORG than in CONT heifers on Day 11. In experiment 2, prepubertal heifers (n = 47) were administered either 1 (1NORG; n = 16), 3(3NORG; n = 16), or 0 (CONT; n = 15) norgestomet implants for 10 days, and serial blood samples were obtained as described for experiment 1. Transrectal ultrasonography was used to determine the diameter of the largest follicle on Day 9. Plasma samples were obtained after each serial sample collection period and were analyzed for estradiol concentrations. Puberty was induced in 75% (12 of 16) and 81% (13 of 16) of heifers in the 1NORG treatments, respectively. Four heifers in the 1NORG treatment, from which serial blood samples were collected, ovulated before removal of the progestin implant, and the LH data for this treatment were deleted. In the 3NORG treatment, LH pulse frequency was suppressed (p < 0.05) on Day 8.5, but was greater (p < 0.05) 12 h after removal of the progestin (Day 10.5) than in the CONT treatment. We conclude that progestin administration hastens puberty by increasing LH secretion after progestin withdrawal and propose that progestin administration induces puberty by accelerating the peripubertal decrease of estradiol negative feedback on LH secretion.

The expression and secretion of cathepsin D by ZR-75-1 human breast cancer cells, and tamoxifen-resistant (ZR-75-9a1) and estrogen-independent (ZR-PR-LT) variants was examined by electrophoresis of labeled proteins and Western blotting. Secreted proteins of 160 kDa, 52 kDa and 34 kDa were identified, and in ZR-75-1 cells, they were shown to be estrogen-inducible. Treatment of ZR-75-9a1 cells with 17 beta-estradiol (E2) and the progestin ORG 2058 increased secretion of the 52 kDa protein; ZR-PR-LT cells were unaffected. Western blotting showed that each cell line expressed high levels of the 52 kDa and 34 kDa forms of cathepsin D but that relatively little was being secreted. Each cell line secreted 52 kDa procathepsin D, but 34 kDa mature-cathepsin D was not detected as a secreted protein. ZR-75-1 cells expressed and secreted the highest levels of cathepsin D while ZR-75-9a1 cells expressed and secreted the least.

We have investigated the expression of insulin-like growth factor I receptors (IGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen-resistant (ZR-75-9a1) and oestrogen-independent (ZR-PR-LT) variants. ZR-75-1 cells expressed 6633+/-953 receptors per cell,(K(d) 0.24+/-0.06 nM). IGFR expression was reduced in ZR-75-9a1 cells (1180+/-614 receptors per cell, K(d) 0.13+/-0.05) and increased in the ZR-PR-LT cell line (18 430+/-3210 receptors per cell, K(d) 0.24+/-17). A comparison of these data with previously published findings for epidermal growth factor receptor (EGFR) expression by these cell lines revealed that IGFR and EGFR expression are inversely related in the variant lines whereas ZR-75-1 cells express similar numbers of both receptors. Since the changes in IGFR expression observed are associated with changes in steroid hormone receptor status, we also investigated the effects of oestradiol, the synthetic progestin ORG 2058 and dexamethasone on IGFR expression. Oestradiol increased IGFR expression only in the ZR-75-1 cell line. Low concentrations of ORG 2058 increased IGFR levels in the two cell lines positive for progesterone receptor (ZR-75-1 and ZR-PR-LT). High concentrations of ORG 2058 increased IGFR expression in all cell lines, as did dexamethasone. These data suggest that EGFR and IGFR expression may be linked in breast cancer, and that EGFR/IGFR ratios in breast cancer may be a more sensitive prognostic indicator than EGFR expression alone. Regardless of basal IGFR expression by the cell studied, ORG 2058 increased IGFR expression, possibly via both the progesterone and glucocorticoid receptors.

Two experiments were conducted on postpartum suckled beef cows synchronized with Syncro-Mate B and artificially inseminated approximately 48 h after implant removal. In Exp. 1, cows (> or = 42 d postpartum at the timed AI) were randomly assigned to treated (n = 101) and control (n = 85) groups on d 12 after the timed AI. Treated cows received norgestomet/silicone implants that were left in situ for 9 d. Norgestomet treatment had no effect (P > .25) on the calving rates from the initial timed AI or from the return estrus. Nonpregnant norgestomet-treated cows returned to estrus in a more (P < .05) synchronized manner than the nonpregnant control cows. In Exp. 2, early postpartum cows (< 42 d postpartum at the first AI; n = 30) were included and all 118 cows (88 cows were > or = 42 d postpartum) received norgestomet/silicone implants as in Exp. 1. Of the 30 early postpartum cows, eight (19 to 41 d postpartum at the time of the first AI; mean = 29.3 d) calved to the first AI and nine calved to the second synchronized estrus. The calving dates at the next calving season for these 17 cows (57% of the cows in this group) was advanced an average of 46 d (319-d calving interval). The calving rates for the two timed insemination periods were similar (P > .25) for early and later (> or = 42 d postpartum) postpartum cows. Treatment with norgestomet implants on d 12 through 21 had no detrimental effects on established or subsequent pregnancy, synchronized the return estrus of nonpregnant cows, and was efficacious in establishing pregnancy early postpartum.

Two commercially available progestogen products for cattle, melengestrol acetate (MGA) and norgestomet (SMB), were evaluated for their ability to induce synchronized estrus in anovulatory ewes. Seasonally anestrous ewes (n = 232) were randomly assigned to one of seven treatments: 1) control (C); 2) MGA only (OMGA; .3 mg of MGA/d); 3) MGA+zeranol (RMGA; 2.5 mg of zeranol); 4) MGA+PG-600 (PMGA; 400 IU of PMSG + 200 IU of hCG in a 5-mL dose); 5) SMB only (OSMB); 6) SMB + zeranol (RSMB); and 7) SMB + PG-600 (PSMB). Treatments were initiated 10 d before breeding. Immediately preceding progestogen treatment, RMGA and RSMB ewes were given a single i.m. injection of zeranol. Concomitant to progestogen removal, PMGA and PSMB ewes were given a single i.m. injection of PG-600. On May 4, 1992, all treatment groups were combined into one breeding group for a 30-d breeding period. Mating at synchronized estrus was greater (P < .001) for progestogen-treated ewes. Within progestogen treatments, more (P < .001) SMB ewes than MGA ewes were marked within the first 5 d of breeding. Overall, there were no treatment differences in estrus response for the 30-d breeding period. Progestogen-treated ewes had a shorter (P < .001) mean interval from ram introduction to lambing. Fertility and prolificacy were not different for C, MGA, or SMB ewes. Ewes primed with zeranol before MGA or SMB treatment had fertility and intervals from ram introduction to lambing similar to those of ewes receiving an injection of PG-600 after progestogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone receptors (PgR) of human breast cancer T47-D cells grown in an estrogenic environment (presence of phenol red, natural estrogens of foetal calf serum and insulin) were found to be present in considerable amounts (1-3 pmol/mg protein and 20 pmol/mg DNA), and to specifically bind progestins with a high affinity characterized by a Kd around 3 nM for ORG2058, and 4 nM for nomegestrol acetate (NOM; 17 alpha-acetoxy-6-methyl-19-nor-pregna-4,6-diene-3, 20-dione), when measured under equilibrium conditions. Both compounds formed an highly stable ligand-receptor complex with a dissociation constant (k-1) around 1 x 10(-5) s-1. At high pharmacological concentrations, NOM, ORG2058 and other synthetic progestins including promegestone (R5020), medroxy-progesterone acetate and norethindrone acetate (NOR), induced a dose-dependent inhibition of cell proliferation as measured by [3H]thymidine incorporation. Dexamethasone, which did not bind to PgR, did not reproduce this inhibitory effect. NOM, R5020 and NOR treatments of T47-D cells at concentrations around Kd resulted in an 80% decrease in PgR content. Our data on NOM as compared to other progestins are consistent with their antiproliferative effects on human breast cancer cells grown in estrogenic conditions.

Crestar consists of an ear implant containing 3 mg norgestomet combined with an intramuscular injection of 3 mg norgestomet and 5 mg oestradiol valerate. Its effectiveness for synchronising oestrus in embryo transfer recipients was evaluated in comparison with a progesterone-releasing intravaginal device (PRID) and prostaglandin regimen, using 334 maiden heifers. The treatment devices were inserted on day 1, prostaglandin was administered to the PRID-treated heifers on day 8 and the devices were removed on day 10. High proportions of the heifers were seen in oestrus within five days of the removal of the devices after both the PRID prostaglandin (90.4 per cent) and Crestar (86.2 per cent) treatments. The interval from the removal of the device to the onset of oestrus was significantly shorter for Crestar than for PRID prostaglandin-treated heifers (45 vs 51 hours, P < 0.001), and the duration of oestrus was significantly longer (13 vs 10 hours, P < 0.01). The PRID prostaglandin treatment resulted in a higher degree of synchrony than the Crestar treatment (74.1 per cent vs 61.8 per cent, P < 0.05). There were no significant differences between the treatments in the proportions of the heifers selected as embryo transfer recipients or in the proportions which became pregnant after embryo transfer.

Our hypothesis was that conception in bovine females would be enhanced if the corpus luteum was present during the period of progestin treatment to synchronize estrus. In this study, 67 heifers (one replicate) and 124 cows (two replicates) were randomly assigned to one of two treatment groups. Seven days after estrus (Day 0), all animals were implanted with norgestomet and the implant remained in place for 10 days. All implants were removed on Day 17. Cows and heifers in one group received prostaglandin F2 alpha (PGF2 alpha) on Day 7 of the estrous cycle (PG 7; norgestomet without corpus luteum), and animals in the second group received PGF2 alpha on Day 17 (day of implant removal; PG 17; norgestomet with corpus luteum). All heifers and cows exhibiting behavioral estrus were artificially inseminated 12 h after estrus was detected during a 7-day period following removal of norgestomet. Blood samples were collected from cows of replicate 1 to determine serum concentrations of progesterone and 17 beta-estradiol. Percentage of females that had calves as a result of artificial insemination was greater (p < 0.01) in the PG 17 group (87% and 78% cows [two replicates] and 58% heifers) compared to the PG 7 group (31% and 44% cows [two replicates] and 41% heifers).(ABSTRACT TRUNCATED AT 250 WORDS)

Sixteen suckled beef cows were used to determine effects of Syncro-Mate-B (SMB) on the development and function of corpora lutea (CL) and LH release. Twelve cows were treated 2 d after estrus with the standard SMB treatment regimen, a 6-mg Norgestomet ear implant (in situ 9 d) and a 2-mL i.m. injection of 3 mg of Norgestomet and 5 mg of estradiol valerate at time of implant insertion. Four cows served as untreated controls. In cows treated with SMB with subsequently nonfunctional CL (n = 5), mean concentrations of progesterone (P4) were lower on d 9, 12 (implant removal), and 14 of the treated cycle than in control cows or in those cows treated with SMB that developed functional CL (P < .01). In cows treated with SMB that developed functional CL (n = 7), mean concentrations of P4 were lower only on d 12 of the treated cycle than those in control cows (P < .01). In cows treated with SMB with subsequently nonfunctional CL, CL were smaller from d 9 through 14 of the treated cycle than in control cows or in those cows treated with SMB that developed functional CL (P < .01). In cows treated with SMB that developed functional CL, CL were smaller on d 9 and 11 of the treated cycle than in control cows (P < .01). Regardless of subsequent CL function, mean concentrations of LH and numbers of LH pulses were lower in cows treated with SMB than in control cows on d 3 and 4 of the treated cycle (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of luteolysis on myometrial activity in late pregnant cows was studied by measuring electromyographic (EMG) activity and concentrations of progesterone and 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in maternal arterial plasma of five cows. Application of two ear implants containing progestagen at day 261 of gestation was followed by an injection of a luteolytic dose of a prostaglandin F2 alpha (PGF2 alpha) analogue (PG) on day 264. Calving was initiated by removal of the implants and a single injection of 5 mg flumethasone on day 270. All calves were born alive at a mean interval of 36 h. After 4 h of hyperactivity immediately following the injection of PG, myometrial activity was almost completely absent for 20 h; before luteolysis the total duration of EMG activity was 7.21 +/- 0.31 min h-1 (mean +/- SEM), during the 4 h after PG 23.61 +/- 3.40 min h-1, and during inhibition 2.70 +/- 0.87 min h-1. After the last period, EMG activity recurred and reached values of 14.52 +/- 3.07 and 11.17 +/- 2.87 min h-1, on days 265 and 269, respectively. Concentrations of progesterone in maternal plasma decreased from 4.17 to 1.14 ng ml-1 within 12 h after PG, whereas concentrations of PGFM in plasma remained low, varying from 15 to 30 pg ml-1. After removal of the implants and injection of flumethasone on day 270, EMG activity increased without an inhibitory phase and reached maximum values around the time of expulsion of the calves.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of progestin and luteinizing hormone (LH) pulse frequency on dynamics of dominant follicle growth during the first follicular wave after oestrus was examined in non-lactating Holstein cows by ultrasonography. On day 8 of the cycle, cows (n = 8) received a luteolytic dose of prostaglandin F2 alpha (PGF2 alpha; 25 mg) and an ear implant of Norgestomet (6 mg). On day 18, cows were assigned to a crossover design in which the implants were retained (T1) or replaced with a new implant (T2). All implants were removed on day 23. After oestrus, cows underwent a normal intervening oestrus cycle. On day 8 of the third cycle, T1 and T2 were reversed among cows. Ultrasonography and blood sampling were performed on alternate days throughout the experiment. On days 10 and 19 of the third cycle, blood was sampled every 15 min for 8 h in concert with an additional control group (n = 3) sampled on day 10 of the cycle. Progesterone concentration on day 8 before PGF2 alpha was 6.5 +/- 0.5 ng ml-1. Dominance of the first wave dominant follicle was extended beyond day 18 in 15 of 16 cows for T1 and T2 periods. The original dominant follicle ovulated in five of eight T1 and none of eight T2 periods (P < 0.01). New dominant follicles were detected on day 24 +/- 1 in T1 (n = 3) and on day 20.6 +/- 1 in T2 (n = 8; P < 0.01) cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Norgestomet is the progestational component present in Syncro-Mate-B, which is used to synchronize estrus in cattle. In post-partum cows, luteal phases anticipated to be short following first ovulation are of normal length when cows are pretreated with norgestomet. Because Syncro-Mate-B is used experimentally as a progestogen to affect uterine function, these studies were conducted to investigate how norgestomet acts at the level of the uterus. Receptor binding assays and a sensitive estrogen bioassay in tissue culture were used to address the possibility that some effects of norgestomet might be mediated through interaction of this compound with steroid hormone receptors other than the progesterone receptor (rP). The source of receptors was high-speed cytosol, prepared from bovine uterine endometrium, which was obtained from cyclic cows. Results of single-point and complete competition analyses comparing norgestomet and progesterone indicated that norgestomet competed even more effectively than did progesterone for specific binding of [3H]progesterone to rP. Results of similar studies, which compared the abilities of norgestomet and diethylstilbestrol to compete with [3H]estradiol for binding by uterine endometrial estrogen receptors (rE), provided no evidence for norgestomet competitive binding to rE. In MCF-7 breast cancer cell bioassays, norgestomet showed weak estrogenic activity, but only at concentrations greater than 1 micromolar. Finally, norgestomet did not compete with [3H]triamcinolone acetonide when present in an 100-fold excess, and only competed with [3H]dexamethasone for binding by endometrial glucocorticoid receptors (rG) when present in the micromolar range. We conclude that, at the concentrations used in synchronizing estrus, norgestomet interacts with bovine endometrium as a progestogen and that its biological actions occur through binding of this compound to rP present in target tissues.

This study investigated whether treatment with progestagen, which improves fertility after early weaning in postpartum cows, altered concentrations of gonadotrophins or development and function of follicles. Patterns of luteinizing hormone (LH), oestradiol and follicle-stimulating hormone (FSH) and of follicular growth before first postpartum ovulation were compared in two experiments. At 17 to 25 days post partum, suckled anoestrous beef cows received an ear-implant containing 6 mg progestagen (norgestomet) for 9 days or served as untreated controls. Calves were weaned from all cows 7 days after initiation of treatment. Cows were observed for oestrous behaviour twice a day until 10 days after weaning. As expected, the proportion of anoestrous cows that formed a corpus luteum with a normal lifespan was greater (P < 0.01) in cows treated with norgestomet (Expt 1, 17 of 24; Expt 2, 18 of 22) than in control cows (Expt 1, 2 of 16; Expt 2, 3 of 18). In general, patterns of secretion of LH and oestradiol and of final growth of the preovulatory follicle did not differ between control and norgestomet-treated cows. However, there was a transient rise in FSH in association with weaning in control cows that did not occur in norgestomet-treated cows. In addition, mean LH increased more rapidly and mean oestradiol was higher during the 3 days immediately before the preovulatory surge of LH in the norgestomet-treated cows (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Our working hypothesis was that the low concentrations of progesterone (P4) and synthetic progestins administered in hormonal regimens to control estrous cycles of cows would have similar effects on secretion of LH and 17 beta-estradiol (E2). In addition, we hypothesized that concentrations of exogenous P4 typical of the midluteal phase of the estrous cycle and the corpus luteum (CL) would have similar effects on LH and E2, and the effects would be different from those of synthetic progestins and low concentrations of P4. Cows (n = 29) were randomly assigned to one of five treatment groups: 1) one Progesterone Releasing Intravaginal Device (1PRID; n = 6); 2) two PRIDs (2PRID; n = 6); 3) norgestomet, as in Syncro-Mate-B regimen (SMB; n = 6); 4) melengestrol acetate (MGA; 0.5 mg/day; n = 5); and 5) control (CONT; n = 6). Treatments were administered for 9 days (Day 0 = initiation of treatment). All cows from 1PRID, 2PRID, SMB, and MGA groups were injected with prostaglandin F2 alpha (PGF2 alpha) on Days 2 and 5 of the treatment period to regress CL. Cows in the 1PRID and SMB groups were also administered exogenous estrogen according to the respective estrous synchronization protocol for these products. Daily blood samples were collected from Day 0 to 35 to determine concentrations of P4. On Day 8, blood samples were collected at 15-min intervals for 24 h to determine pattern of LH secretion. On Day 9, all treatments ceased and cows in the CONT group received injections of PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Prepubertal Angus crossbred heifers (n = 24) between 8 and 10 mo of age were used to determine if progestogen treatment would enhance jugular concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) after oxytocin (OT) injections. Heifers were stratified by age and weight and allotted to randomized treatments in a 2 x 2 factorial arrangement. Heifers were treated with either a norgestomet (NOR) implant (6 mg) for 9 d or no implant (0 mg; BLK). On d 8 of NOR treatment, jugular veins were catheterized and, on d 9, blood samples were collected every 15 min for 165 min. The first four samples were used to determine basal PGFM concentrations (an indirect measure of uterine PGF2 alpha release). After collection of the fourth sample, either OT (100 IU) or saline (0 IU; SAL) was injected via the jugular catheter. After the 165-min sample was collected, NOR implants were removed. Beginning 48 h after implant removal, a second 165- min blood sampling period was initiated. Average progesterone concentrations were less than 1 ng/ml during both bleeding periods. Within treatment, PGFM concentrations were similar between the first and second sampling periods; therefore, data within treatment were combined. Basal PGFM concentrations were higher (P < .01) in NOR-treated than in BLK heifers. Oxytocin did not increase PGFM concentrations in BLK-OT heifers; however, a marked increase in PGFM was detected in the NOR-OT heifers in response to oxytocin. Average PGFM concentration was greatest (P < .0001) in NOR-OT heifers, and PGFM profiles differed (P < .0001) between NOR-OT and each of the other treatment groups. Results from this study indicate that NOR increases basal PGFM and may "condition" the uterus to respond to OT in prepubertal heifers.

In an experiment replicated over 2 yr, 149 suckled beef cows were administered Syncro-Mate-B (SMB), a 6-mg Norgestomet (NOR) ear implant (in situ 9 d) in conjunction with an i.m. injection of 5 mg of estradiol valerate (EV), and either 3.0, 4.5, or 6.0 mg of NOR, 2 d after estrus. All cows were artificially inseminated at 48 h (timed insemination; TI) after implant removal (IR) and cows were reinseminated at any estrus subsequent to 24 h of TI through 30 d. Blood samples collected before treatment, every 3 d through IR, and at TI were assayed for progesterone (P4). At TI, 44, 39, and 12% of cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively, had serum concentrations of P4 greater than 1 ng/mL (3.0 and 4.5 mg vs 6.0 mg, P less than .01). Fifty-eight, 63, and 84% of cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively, exhibited a synchronized (within 5 d of IR) estrus (3.0 and 4.5 mg vs 6.0 mg, P less than .05). Pregnancy rates for the 5-d synchronized period were 38, 45, and 66% for cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively (3.0 and 4.5 mg vs 6.0 mg, P less than .05). First-service pregnancy rates were 66, 71, and 79% for cows treated with 3.0, 4.5, or 6.0 mg of NOR, respectively (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)

In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP. This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis. The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506% +/- 100 of the control levels, while that of PAP was decreased to 26% +/- 3, in 7 days. These effects were dependent on the concentration of the steroid in the growth medium. The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels. LNCaP cells were found to have very different capacities for secreting PAP and PSA. Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium. PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells. Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells. Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes. The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

Plasma luteinizing hormone and progesterone concentrations, time to onset of estrus, and pregnancy rates were determined in nonlactating anestrous does given 1 of 4 treatments: subcutaneous ear implants containing 3 mg of norgestomet for 9 days (NOR; n = 6); subcutaneous administration, using osmotic minipumps, of 250 ng of gonadotropin-releasing hormone (GnRH)/h for 48 hours (GnRH; n = 6); 3 mg of NOR for 9 days, followed immediately by 250 ng of GnRH/h for 48 hours (NOR + GnRH; n = 6); or no treatment (control; n = 6). During the 72-hour period after removal of NOR or insertion of GnRH pumps, 6 of 6, 0 of 6, 6 of 6, and 3 of 6 does were observed in estrus at a mean (+/- 13.8) hours in groups NOR, GnRH, NOR + GnRH, and control, respectively. Time from end of treatment to peak concentrations of luteinizing hormone were 56 +/- 4.0, 28 +/- 4.7, 34 +/- 4.3, and 41 +/- 9.7 hours (mean +/- SE) for NOR, GnRH, NOR +/- GnRH, and control, respectively. Peak concentrations of luteinizing hormone were significantly greater and occurred significantly later in does given NOR. Progesterone concentrations in does that became pregnant increased to concentrations greater than or equal to 1.0 ng/ml 3 to 5 days after breeding and remained high. Functional corpora lutea (CL) was found in 6 does that did not become pregnant, 1 CL was associated with pseudopregnancy and 1 CL was associated with ovulation prior to placement of the GnRH pumps. Functional CL failed to form in 10 of the 12 doses in groups GnRH and control.(ABSTRACT TRUNCATED AT 250 WORDS)

Seventy-four heifers in four separate breeding groups were allocated into two treatment groups for oestrus synchronisation. Group 1 was given a combination of an initial injection of norgestomet and oestradiol valerate together with a norgestomet ear implant left in place for nine days. Group 2 was given two injections of the synthetic prostaglandin luprostiol 10 days apart. The animals in group 1 were artificially inseminated once 48 hours after the removal of the implant and those in group 2 were inseminated once 72 hours after the second injection of luprostiol. Subsequently any returns were rebred by either AI or natural service. There was no significant difference between the numbers of animals in the two treatment groups which were diagnosed pregnant 33 to 35 days after insemination, although the implant treatment gave a higher overall proportion of pregnancies (70 per cent, 51 per cent). It also gave significantly smaller numbers of 'open' days over the whole of the breeding period. A study of individual animals by progesterone assay and investigation of ovarian structures by real-time ultrasound showed that some of them had unusual progesterone profiles but nevertheless became pregnant. It would appear that a corpus luteum may be responsive to prostaglandins even though it is secreting only a low level of progesterone.

The effects of chronic treatment with norgestomet on follicular dynamics, corpus luteum growth and function as well as the temporal relationships among body temperature, oestrous behaviour, the luteinizing hormone (LH) surge and ovulation following implant removal were studied in 16 Holstein heifers. Oestrous cycles of the heifers were initially synchronized using 2 injections of prostaglandin F-2 alpha (PGF-2 alpha) 12 days apart. The heifers were then implanted with a norgestomet ear implant for 9 days, beginning either at the middle of the synchronized cycle (dioestrus) or at the end of the synchronized cycle (pro-oestrus). Follicular dynamics, corpus luteum growth and regression, and plasma progesterone were not affected by norgestomet treatment at dioestrus. The dominant follicle present at the time of norgestomet implantation in the pro-oestrus group was maintained during the 9-day implant period of 6 of 8 heifers and ovulated after implant removal. Time from implant removal to onset of standing oestrus and time to LH peak following implant removal were highly correlated with the time of ovulation (r = 0.92 and 0.96, respectively). Onset of standing oestrus and the LH peak and the onset of standing oestrus and peak vaginal and rectal temperatures were also highly correlated (r = 0.96, 0.82 and 0.81, respectively). It is concluded that any decrease in pregnancy rates following treatment with norgestomet is not due to asynchrony among oestrus, the LH surge and ovulation.

The role of PGF-2 alpha in determining the lifespan of corpora lutea in the post-partum beef cow was investigated. In control cows (N = 5) induced to ovulate at Day 28 to 36 post partum by injection of 1000 i.u. hCG, corpora lutea had an average lifespan of only 8 days. In cows pretreated with 6 mg implants of a progestagen (norgestomet, N = 4) for 9 days, with implant removal 2 days before injection of hCG, luteal lifespan averaged 17.5 days. Concentrations of PGF-2 alpha in 9 hourly samples of plasma collected from the posterior vena cava via indwelling catheters were higher on Days 4 through 9 after injection of hCG (P less than 0.05) in the cows with short-lived corpora lutea. Greater release of PGF-2 alpha could therefore be a major factor in premature luteal regression. Concentrations of PGFM and oxytocin did not differ between cows with corpora lutea of short or normal lifespan. In a second experiment, concentrations of PGF-2 alpha in plasma from the posterior vena cava were examined during treatment with norgestomet (N = 8) or in contemporary controls (N = 7). In progestagen-treated cows, PGF-2 alpha was higher than in control cows (P less than 0.05), beginning on Day 3 of treatment and peaking on Day 5. It is concluded that the post-partum uterus increases secretion of PGF-2 alpha very early after first exposure to endogenous or exogenous progestagen.

The characteristics of binding (Kinetic and equilibrium binding analysis) of nomegestrol acetate (NOM, 17 alpha-acetoxy-6 alpha-methyl-19-nor-pregna-4.6-diene-3.20-dione) to the progesterone receptor (PgR) in rat uterine cytosolic fraction were determined in comparison to progesterone (P), to fully appreciate the amplitude and specificity of the induced biological response. Since an appropriate radio-labelled form of this steroid molecule was not available, competition studies were performed against the synthetic progestin: [3H]-Organon 2058 [( 3H]-ORG). This allowed a direct comparison between the unlabelled forms of NOM and P, the kinetic constants of which were respectively: Inhibition constant (Ki): 22.8 and 34.3 nM; Association rate constant (k1): 0.39 X 10(3) and 0.21 X 10(3) M-1.s-1; Dissociation rate constant (k-1): 1.81 X 10(-5) and 2.16 X 10(-5) s-1. These results are much more informative than the mere determination of relative binding affinities which only reflect the specificity of the PgR. It was concluded that NOM behaves like the natural hormone in the cytosol of rat uterus.

To examine effects of norgestomet pretreatment on development of follicles and their response to administration of gonadotropin releasing hormone (GnRH), 45 pluriparous suckled beef cows were assigned at random to receive a 6-mg implant of norgestomet for 9 d (inserted 24 d postpartum) or serve as untreated controls. Ovaries were obtained 48 h after removal of implants or 10 to 11 or 20 to 22 h after im administration of 150 micrograms GnRH at 48 h after removal of the implant. The largest follicle (F1) and all follicles within 3 mm in diameter of the F1 were dissected from the ovaries. Theca, granulosa and follicular fluid were separated and assayed for steroids and prostaglandins. Diameters and weights of F1 and weights of follicular components remained unchanged in control cows, but increased by 10 h and declined by 20 h in norgestomet-pretreated cows (treatment X time, quadratic, P less than .05). Ovarian volume and numbers of follicles at the surface of the ovary did not differ with treatment, but the diameter of the second-largest follicle (F2) was smaller (P less than .05) in norgestomet-pretreated cows than in controls (6.0 +/- .9 vs 8.2 +/- .7 mm). The F1 were embedded in the ovary in fewer norgestomet-pretreated than control cows (2/22 vs 8/23; P less than .05). Changes in steroids in F1 paralleled those in size (treatment X time, quadratic, P less than .05). Overall, F1 from norgestomet-pretreated cows had higher (P less than .05) contents of estradiol. Contents of prostaglandins in F1 follicles did not differ with treatment, but increased (P less than .05) following treatment with GnRH. The F2 had lower contents of estradiol than F1. It is suggested that norgestomet effected the maturation of a single follicle which produced more estradiol.

The effects of estradiol and two synthetic progestins (ORG2058 and R5020) on the expression of the high-affinity, metastasis-associated laminin receptor in two human breast carcinoma cell lines were examined. The T47D cell line contains estrogen and progesterone receptors, but the MDA-MB 231 cell line lacks both receptors. Treatment of T47D cells with 10(-9) M estradiol alone results in a three-fold increase (P less than or equal to .05) in the steady-state level of laminin receptor mRNA determined by RNA blot analysis as well as in cell-surface, laminin receptor expression that is evaluated by immunofluorescence. No effects of estradiol on the receptor-negative MDA-MB 231 cells were observed. Untreated and steroid-treated MDA-MB 231 cells had higher levels of laminin receptor mRNA than did untreated or estradiol-treated T47D cells. A more dramatic increase (five-fold; P less than or equal to .005) of mRNA and cell-surface expression in T47D cells was observed after treatment with estradiol plus 10(-8) M progestin or with progestin alone. Estradiol treatment also increased chemotaxis and haptotaxis of T47D cells but not of MDA-MB 231 cells to laminin; it had no effect on the attachment of these latter cells to laminin. Interestingly, treatment with estradiol plus progestin or progestin alone significantly increased the attachment of T47D cells to laminin but did not have an effect on either haptotaxis or chemotaxis to laminin. These results suggest that the various cell-laminin interactions are mediated by different mechanisms. The augmentation of laminin receptor mRNA by estrogen and progesterone treatment in hormone receptor-positive cells, but not in cells that lack these receptors, may relate functionally to the difference in the clinical aggressiveness between classes of breast cancers.

The purpose of these studies was to investigate the pattern and timing of preovulatory endocrine events, estrus and ovulation in Brahman X Hereford (F1) heifers synchronized with norgestomet and estradiol valerate. In Exp. 1, 66 nulliparous and 191 primiparous Brahman X Hereford (F1) heifers were used to estimate the interval from norgestomet implant removal to onset of estrus. The mean interval from implant removal to onset of estrus was 29.8 +/- .5 h, with 80.9% exhibiting estrus within 48 h. Endocrine and reproductive characteristics were examined in detail during Exp. 2 with 37 primiparous heifers. Continuous observation for estrus, 6-h or 2-h blood sampling and ovarian palpation per rectum were employed. All animals were artificially inseminated 48 h after implant removal. Mean interval from implant removal to onset of estrus and to onset of the luteinizing hormone (LH) surge were closely related (r = .91; P less than .0001). Mean intervals from implant removal to ovulation, onset of estrus to ovulation and onset of LH surge to ovulation were 59.1 +/- 2.5 h, 23.3 +/- 1.4 h and 23.1 +/- 1.6 h, respectively. Approximately 73% of heifers exhibited estrus within 54 h after implant removal (optimal timing); conception rate was 59.3% in this subgroup. Conception rate of heifers that did not exhibit estrus within 54 h after implant removal or exhibited an LH surge later than 12 h after estrus (delayed timing) was 10%. Assessment of plasma estradiol-17 beta concentrations suggested that retarded selection and(or) maturation of the preovulatory follicle following implant removal delayed estrus and lowered conception in up to 28% of females timed-inseminated at 48 h.