Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus.

It is well established that estrogen and progesterone are critical endogenous hormones that are essential for implantation and pregnancy in females. However, the distribution of estrogen receptor α (ERα) and progesterone receptor (PR) in female reproductive tracts is elusive. Herein, we report that after serial treatments with pregnant mare's serum gonadotrophin (PMSG) with or without anti-PMSG (AP), mice could regulate the distribution of ERα and PR in the murine ovary, oviduct and uterus and the level of estradiol in serum. ERα and PR regulation by PMSG and anti-PMSG was estrous cycle-dependent and critical for promoting the embryo-implantation period. Furthermore, our results suggested that AP-42 h treatment is more effective than the other treatments. In contrast, other treatment groups also affected the distribution of ERα and PR in mouse reproductive tracts. Thus, we found that anti-PMSG has the potential to restore the distribution of ERα and PR, which could effectively reduce the negative impact of residual estrogen caused by the normal superovulation effect of PMSG in mice.

Selective modulation of follicle-stimulating hormone signaling pathways with enhancing equine chorionic gonadotropin/antibody immune complexes.

The injection of equine chorionic gonadotropin (eCG) in dairy goats induces the production of anti-eCG antibodies (Abs) in some females. We have previously shown that Abs negatively modulate the LH and FSH-like bioactivities of eCG, in most cases, compromising fertility in treated females. Surprisingly, we found out that some anti-eCG Abs improved fertility and prolificity of the treated females, in vivo. These Abs, when complexed with eCG, enhanced LH and FSH ability to induce steroidogenesis on specific target cells, in vitro. In the present study, we analyzed the impact of three eCG/anti-eCG Ab-enhancing complexes on two transduction mechanisms triggered by the FSH receptor: guanine nucleotide-binding protein alphaS-subunit/cAMP/protein kinase A (PKA) and beta-arrestin-dependent pathways, respectively. In all cases, significant enhancing effects were observed on ERK phosphorylation compared with eCG alone. However, cAMP production and PKA activation induced by eCG could be differently modulated by Abs. By using a pharmacological inhibitor of PKA and small interfering RNA-mediated knock-down of endogenous beta-arrestin 1 and 2, we demonstrated that signaling bias was induced and was clearly dependent on the complexed Ab. Together, our data show that eCG/anti-eCG Ab-enhancing complexes can differentially modulate cAMP/PKA and beta-arrestin pathways as a function of the complexed Ab. We hypothesize that enhancing Abs may change the eCG conformation, the immune complex acquiring new "biased" pharmacological properties ultimately leading to the physiological effects observed in vivo. The modulation of ligand pharmacological properties by Abs opens promising research avenues towards the optimization of glycoprotein hormone biological activities and, more generally, the development of new therapeutics.

Four years of induction/synchronization of estrus in dairy goats: effect on the evolution of eCG binding rate in relation with the parameters of reproduction.

Ninety-eight Alpine goats of two herds were followed over 4 years in a program of annual artificial insemination after estrus induction/synchronization, including progestagen administration (vaginal sponge) followed by prostaglandin analog and equine chorionic gonadotrophin (eCG) 48 h before sponge removal. Goats were sampled every 4 hours from the 16th to the 56th following sponge removal, for determination of LH surge and tested for estrus by the presence of a buck. Seven days after AI, endoscopic examination of the ovaries was performed to determine the number of corpus lutea. Pregnancy diagnosis was performed at day 21-22 post AI by determination of plasma progesterone and at day 40-45 by ultrasonography. Parturition, number and sex of kids were recorded. All the goats were sampled before and after each treatment, for anti-eCG antibodies screening. Statistical analysis of the results clearly established a significant effect of the treatments on anti-eCG antibodies. Time of estrus and LH surge were significantly different between herd. The antibodies significantly delayed the time of coming out of estrus as well as the time of LH surge. Two antagonistic effects were evidenced: first, the delayed of time of estrus and time of LH surge in relation with the immune reaction to eCG; secondly, the ahead of time of estrus and time of LH surge during the years of treatment, identical to both herd. The antibodies negatively influenced the percentage of ovulating females as well as kidding rate. Finally, no effect of antibodies on prolificacy was found.

Mechanism of induction of follicular atresia after equine chorionic gonadotrophin (eCG) antisera treatment in hypophysectomized eCG-injected hamster model: possible involvement of c-myc and cdc25.

An intracellular mechanism of induction of apoptosis of granulosa cells implicated in the initiation of experimentally induced atresia of ovarian follicle in hypophysectomized eCG-injected hamster followed by eCG-antisera treatment is proposed. Induction of atresia after withdrawal of injected eCG by eCG-antisera treatment is possibly caused by inadequate level of the gonadotropin-induced growth factor that results in apoptosis of granulosa cells associated with the activation of c-myc requiring cdc25A.

Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin.

Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2-3 x 10(5) viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/micrograms DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations.

In vitro and in vivo evidence on the site of neutralization of equine chorionic gonadotrophin (eCG) by an eCG antiserum.

This study was designed to determine whether the major site of eCG neutralization by an antiserum to the hormone is at the peripheral or ovarian level. Hamsters hypophysectomized at oestrus were injected s.c. with 25 iu eCG. Three days later, preovulatory follicles were dissected and cultured for 5 h and the medium was changed every hour. At the end of the first hour of incubation, oestradiol and androstenedione accumulation was high, with a sharp drop over the next 4 h, whereas progesterone concentrations did not change over the entire period. Addition of eCG antiserum to the incubated follicles did not affect steroidogenesis. Addition of 1.0 iu eCG in the second hour or every hour sustained oestradiol production at supraphysiological amounts. However, addition of eCG plus eCG antiserum every hour eliminated the stimulatory effects of eCG on oestradiol production. In another experiment, hamsters injected with eCG were treated 3 days later by i.p. injection of eCG antiserum and groups of animals were killed over the next 8 h. Serum samples before and after injecting eCG antiserum were incubated overnight with a goat anti-rabbit immunoglobulin to separate free, unbound eCG from bound eCG. At time zero (before injecting the antiserum) free eCG was increased, but within 1 h after eCG antiserum there was an eightfold decrease of the hormone, and these concentrations were maintained over the next 7 h. The fall in unbound eCG in vivo coincided with the decay in serum oestradiol and androstenedione.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of neutralization of pregnant mares' serum gonadotrophin (PMSG) shortly before or at the preovulatory LH surge in PMSG-superovulated heifers on follicular function and development.

Normally cyclic heifers (n = 34) received 2500 iu pregnant mares' serum gonadotrophin (PMSG) i.m. at day 10 of oestrus, and 15 mg prostaglandin (PG) i.m. at day 12. Thereafter, a monoclonal antibody against PMSG was administered i.v. before (n = 24), at (n = 6) or shortly after (n = 4) the preovulatory LH surge. Peripheral blood concentrations of LH and oestradiol were compared; follicular development was monitored by daily ultrasound scanning; and the numbers of preovulatory-sized follicles and ovulations were counted 96 h after injection of PG following death. Anti-PMSG treatment before the LH surge inhibited the LH surge in 16 heifers (67%). In these heifers, the initial increase in oestradiol concentration upon PMSG stimulation to 167.5 +/- 35.0 pmol l-1 was terminated immediately after anti-PMSG treatment and decreased rapidly to basal values, while the number of preovulatory-sized follicles remained constant until 68 h after PG injection; on average 0.4 +/- 0.1 ovulations were counted. In the remaining eight heifers, five animals showed an immediate, but temporary, 20-60% drop in oestradiol concentration after anti-PMSG treatment. In all eight heifers 25% of the preovulatory-sized follicles ovulated. Treatment with anti-PMSG at or shortly after the LH surge did not affect the pattern of oestradiol concentration, but a significantly higher ovulation rate was observed in the animals treated shortly after the LH surge: 20.3 +/- 2.6 versus 6.3 +/- 2.3 in animals treated at the LH surge, which corresponded to 76% and 24% of the preovulatory-sized follicles monitored shortly before the period of multiple ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Superovulation in the cow with pregnant mare serum gonadotrophin: effects of dose and antipregnant mare serum gonadotrophin serum.

The effects of pregnant mare serum gonadotrophin (PMSG) dose and PMSG antiserum on superovulation in crossbred beef cows were studied. In experiment I, three groups were treated with 1200, 2400 or 3600 IU of PMSG and 48 h later with prostaglandin (PGF). The mean numbers of corpora lutea (CL), unovulated follicles, and total ova/embryos collected increased as the PMSG dose increased. The percent of fertilized ova and transferable embryos was lowest in the highest dose group (p < 0.05). In experiment II, all cows received 2500 IU of PMSG; groups 1 and 2 were treated with sheep anti-PMSG serum at 48 h or 60 h after PGF; group 3 cows were PMSG-only controls. The number of CL was lowest and the number of unovulated follicles highest in the PMSG-only group (p < 0.05). The number of CL was higher in group 2 (anti-PMSG at 60 h) than in the control group, with the anti-PMSG at 48 h not different from the other groups. Numbers of total ova/embryos, fertilized ova, and transferable embryos were higher (p < 0.05) in both antiserum-treated groups relative to the PMSG-only group. We conclude that superovulation of beef cows with PMSG and treatment with PMSG antiserum will induce a higher superovulatory response and will result in higher CL numbers and fewer unovulated follicles. Further, the variability in the superovulatory response to PMSG treatment was still evident when PMSG antiserum was administered.

Morphological and hormonal changes after superovulation in cows treated with Neutra-PMSG.

Morphological changes in ovaries and hormonal changes as well as changes in Na, K, Ca, Mg, and P blood plasma levels were observed after superovulation induced by the administration of PMSG and the neutralization of this hormone with monoclonal antibodies Neutra-PMSG administered either 72 or 108 h later. The introduction of Neutra-PMSG 108 h after PMSG injection clearly decreases the number of surviving non-ovulated follicles with a diameter > 10 mm (1.7 +/- 0.8 vesicle in an individual cow on the average) in comparison to the group without Neutra-PMSG (19.4 +/- 9.5). The efficiency of ovulation in the group treated with Neutra-PMSG in the 108th h of the experiment (8.2 +/- 4.6 corpora lutea per cow on the average), did not differ statistically from the group treated with PMSG only (12.4 +/- 10.6). Early administration of Neutra-PMSG (72h), totally inhibits superovulation. Observations showed, that injection of Neutra-PMSG in the 108th h, caused a considerable decrease in the estradiol level, beginning with the 120th h of the experiment. Determination of the progesterone blood plasma level reflects the number of corpora lutea and can be helpful in evaluating the effects of superovulation. Superovulation did not effect the level of Na, K, Ca, Mg, and P in the blood plasma.

Yield of embryos in PMSG-superovulated cows treated with anti-PMSG six or 18 hours after the peak of luteinising hormone.

One hundred and forty-six Dutch cross Friesian cows were selected from a local slaughterhouse and synchronised with norgestomet. The 134 cows with a normal progesterone pattern after the removal of the norgestomet implant were treated intramuscularly with 3000 iu pregnant mare's serum gonadotrophin (PMSG) on day 10 followed by 22.5 mg prostaglandin 48 hours later. Blood samples were collected daily and at hourly intervals from 30 to 54 hours after the prostaglandin. The 113 cows with a pre-ovulatory peak of luteinising hormone (LH) were divided into three groups: 37 control cows (group 1) received a placebo six hours after the LH peak; 42 cows (group 2) received anti-PMSG six hours after the LH peak and 34 cows (group 3) received anti-PMSG 18 hours after the LH peak. All the cows were inseminated 10 hours after the LH peak. Six or seven days after insemination the cows were slaughtered and the embryos were evaluated after flushing the ovaries, and the numbers of corpora lutea, cysts and follicles on the donor ovaries were counted. Treatment with anti-PMSG had no significant effect on the numbers of corpora lutea or the numbers of embryos compared with the control group. The mean (+/- sem) numbers of corpora lutea were 14.7 +/- 1.4, 16.3 +/- 1.4 and 16.6 +/- 1.4 for groups 1, 2 and 3, respectively. The numbers of transferable embryos were 3.5 +/- 0.6, 4.1 +/- 0.7 and 5.0 +/- 0.7 for groups 1, 2 and 3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of follicular development in silver foxes (Vulpes vulpes) with equine chorionic gonadotrophin (eCG) and antibodies against eCG.

Experiments were conducted that demonstrated that 1000 iu equine chorionic gonadotrophin (eCG) was effective in induction of follicular development and ovulation in silver foxes during anoestrus. This treatment resulted in large, unovulated follicles; thus, trials in which the effects of eCG were reduced or abrogated by antibodies against eCG were carried out. Passive immunization against eCG on days 3 and 4 after eCG treatment interfered with subsequent follicular development and prevented ovulation. Treatment with eCG antibodies on days 5 or 7 after eCG treatment did not prevent ovulation and neutralization beginning on day 5 appeared to provide for the best ovulatory yield. The results suggest that combinations of eCG and anti-eCG antibody may provide a useful means of inducing ovarian activity in anoestrous foxes.

The use of bovine anti-PMSG serum in beef cattle after PMSG-superovulation.

Thirteen beef cows were superovulated using 4,000 i.u. of pregnant mare serum gonadotrophin (PMSG) on days 9 to 14 of the estrous cycle, followed by two injections of 500 micrograms prostaglandin F2 alpha analogue (PGF2 alpha) 48 and 55 hrs later. Seven of them were injected intramuscularly with bovine anti-PMSG serum 12 hrs after the first signs of estrus. The remaining 6 cows were served as controls and received no antiserum. Peripheral blood concentrations of progesterone (P) and estradiol-17 beta (E2) were compared in relation to the superovulatory responses. The injection of anti-PMSG serum did not significantly affect the numbers of the corpora lutea (CL), the anovulatory follicles and the transferable embryos at 7 to 8 days after superovulatory estrus, but increased the ratio of embryos classified as excellent or good quality. Although the plasma P concentration showed no significant differences between the anti-PMSG-treated and control cows, the plasma E2 concentration displayed a characteristic difference, suppressing the second E2 peak in the anti-PMSG-treated cows. It is concluded that the use of bovine anti-PMSG serum for PMSG/PGF2 alpha-treated cows at 12 hrs after the beginning of the estrus improves the quality of embryos recovered, probably due to inhibition of high estrogenic environment following ovulation.

Enzyme immunoassay (EIA) for equine chorionic gonadotropin/pregnant mare serum gonadotropin (eCG/PMSG).

A simple, accurate, sensitive enzyme immunoassay (EIA) has been developed that permits the measurement of equine Chorionic Gonadotropin activity in pregnant mare plasmas or serums as well as in commercial and highly-purified preparations. This assay is specific for eCG and eLH which share the same polypeptide structure but differ in their oligosaccharidic chains. The more important result is that this EIA has been found to be give data in very close agreement with the in vivo assay. Therefore this very rapid and convenient assay can be used to measure the activity of eCG/PMSG in pregnant mares serums in in-field conditions as well as in crude or highly-purified preparations.

PMSG profiles in superovulated and anti-PMSG antiserum treated mice and heifers with enzymeimmunoassay.

A sandwich enzymeimmunoassay (EIA) for pregnant mare serum gonadotropin (PMSG) using a microtiter plate was developed. Sensitivity of the assay to PMSG was 15.6 mIU/ml (0.2 ng/well). The PMSG levels in serum were measured with the EIA in superovulated and anti-PMSG rabbit antiserum treated mice and heifers. In mice, the PMSG blood level was measurable in the serum 4-6 days after intraperitoneal injection of 5-30 IU of PMSG. The administration of anti-PMSG antiserum at the same dose level as PMSG caused a rapid decrease in the PMSG blood level, declining to undetectable levels within 17 hours. In heifers, the PMSG level was measurable at 10-11 days after the injection of 2500 or 3000 IU of PMSG. When antiserum was injected 48 hours after the PMSG injection, the clearance rate of PMSG was affected by the route of the administration. The administration of 3000 units of anti-PMSG antiserum intravenously caused a rapid decline and the disappearance of circulating PMSG within 17 hours. When 3000 units of anti-PMSG antiserum was injected intra-muscularly, the PMSG blood level also decreased and became unmeasurable 24 hours after administration; however, it was still detectable for up to 17 hours. These results indicate that the administration of anti-PMSG antiserum at the proper timing and dosage could lead to successful superovulation through the improvement of hormonal conditions.

Construction of a bovine-murine heteromyeloma cell line; production of bovine monoclonal antibodies against rotavirus and pregnant mare serum gonadotrophin.

Bovine-murine heteromyeloma cell lines were prepared by fusing lymphoid cells from a bovine leukemia virus (BLV)-infected cow with mouse myeloma cells. Selection of hybrid cell colonies was based on the ratio of bovine and murine chromosomes, the presence of cell-surface immunoglobulins and growth characteristics. First-generation fusion partners were compared for fusion efficiency and the number of antigen-specific antibody-producing clones generated. Hybrid cell colonies that initially secreted antibodies were selected from first-generation heteromyelomas to function as second-generation fusion partners. Although fusion efficiencies for both generations did not differ, the second-generation heteromyelomas yielded a higher number of specific antibody-producing clones. Fusion of hteromyelomas with either lymph node cells or splenocytes indicated that fusion with lymph node cells results in a higher number of specific antibody-producing clones, whereas fusion efficiency was found to be higher with splenocytes. The optimal time intervals between the final booster injection and fusion were found to be 4 days for splenocytes and 7 days for lymph node cells. Finally, the characterization of bovine monoclonal antibodies against bovine rotavirus and pregnant mare serum gonadotrophin and their neutralizing capacities in vitro are described.

Effect of tyrosine modification on the biological and immunological properties of equine chorionic gonadotropin.

The tyrosine residues of equine chorionic gonadotropin have been nitrated with tetranitromethane and the resulting effects on the biological and immunological activities of the hormone studied. All of the tyrosine residues in equine chorionic gonadotropin were found to react with tetranitromethane when a 100-fold molar excess of reagent was used or with an 8.6 molar excess in the presence of 5 M guanidine hydrochloride. Complete nitration abolished the biological activities and decreased the immunological activity of the hormone. The nitration of one tyrosine residue resulted in the loss of 70% of the LH activity of equine chorionic gonadotropin; the FSH activity declined in a similar fashion. Maximal nitration resulted in the loss of about 50% of the immunological activity of the native hormone. Nitrated derivatives of equine chorionic gonadotropin were unable to compete with the native hormone in the rat Leydig cell assay for LH. The results indicate that the tyrosine residues of equine chorionic gonadotropin play an important role in the manifestation of both the FSH and LH activity of the hormone.

Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat.

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration, respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24-48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin aslo had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and t 1/2 of 0.078 h-1 and 8.9 h for FSH receptors and 0.086 h-1 and 8.0 h for the LH receptors, respectively.

Steroid production by isolated theca and granulosa cells after initiation of atresia in the hamster.

Hypophysectomized PMSG-primed hamsters were injected with PMSG antiserum and the theca and granulosa cells of the resulting atretic follicles were incubated in vitro. In the absence of added hormone, 17 alpha-hydroxyprogesterone and oestradiol production was not detectable in granulosa cells collected and incubated at 0, 12 and 24 h after antiserum. Progesterone production was not detected in control incubations at 0 h but was measurable with cells collected at 12 h after PMSG antiserum. When incubated with androstenedione or pregnenolone (10 ng/ml for each) 17 alpha-hydroxyprogesterone and progesterone production by granulosa cells were significantly increased at 0, 12 and 24 h after antiserum. Granulosa cells were capable of aromatizing androstenedione to oestradiol at all times examined. At 0 and 12 h after antiserum to PMSG, isolated thecal shells produced androstenedione. LH stimulation caused increased androstenedione production in all thecae at 0 h, in 50% of the thecae at 12 h and in none at 24 h after antiserum. Thecal shells produced 17 alpha-hydroxyprogesterone in response to LH at 0, 12 and 24 h after antiserum, and produced progesterone at all times examined. Thecae also responded to LH with increased progesterone production up to 72 h after antiserum. These experiments demonstrate that one important steroidogenic event in atresia may be the loss of activity of C 17,20 lyase in the theca leading to loss of substrate (androstenedione) for granulosa cell aromatization, although aromatase activity is present until at least 24 h after the induction of atresia.

Implantation and later fetal development in immature rats given a superovulatory dose of pregnant mare's serum gonadotropin, later neutralized by antiserum.

In an earlier experiment, 29-day-old female rats were superovulated with 40 IU pregnant mare's serum gonadotropin (PMSG) and very few blastocysts were recovered from the uterus on Day 5. Administration of a PMSG antiserum (a/s) prior to ovulation resulted in recovery of blastocysts in all rats and the present set of experiments was undertaken to investigate the later development of these blastocysts. Implantation was found to occur in only approximately 50% of superovulated (SOV) a/s-treated animals regardless of whether or not estrogen was given on Day 4. It is suggested that the exposure to high preovulatory estrogen and an imbalance in the progesterone/estrogen ratio during the early preimplantation period are possible causes of the loss of blastocysts. The majority of SOV a/s-treated rats in which implantation occurred carried fetuses to Day 20, although there was a further small loss of pregnancy between Days 8 and 20. On Day 8 steroid concentrations in both serum and ovaries were similar to those observed on Day 5 in earlier experiments. By Day 20 serum and ovarian progesterone concentrations were significantly higher than on Day 8 in all superovulated rats, which may reflect increased production by the larger number of corpora lutea in these animals. The present set of experiments shows that in a significant percentage of SOV a/s animals blastocysts are capable of implanting and continuing to develop into normal fetuses by Day 20. However, at least 50% of SOV a/s-treated rats fail to maintain pregnancy between Days 5 and 8 and this could be a result of a failure in the uterus to provide a suitable environment for implantation or to abnormalities in the blastocysts.