Ovarian Ascorbic Acid Depletion Assay Research Articles

Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats

BackgroundThe current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection.MethodsThe biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2°C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion.ResultsFSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2°C for not more than 1 h prior to injection.ConclusionMixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.

Is an immunoassay available for the measurement of bioactive LH in serum?

An in vitro bioassay for luteinizing hormone (LH) is in our opinion the "gold standard" bioassay. The rodent interstitial cell testosterone assay (RICT) is specific for bioactive LH and very sensitive, accurate, and reproducible. Diverse LH standards consistently display parallel dose-response characteristics. Sera also manifest parallel dose-response characteristics throughout reproductive life, with the exception of basal samples from prepubertal children. This indicates that all known hormones with LH bioactivity have a similar bioactive site. The in vivo bioassays for LH used for calibration of World Health Organization standards are more cumbersome and less precise and accurate than the in vitro bioassay. The ovarian ascorbic acid depletion assay corresponds better than the seminal vesicle weight assay with in vitro bioassay. Variation in the ratio of bioactive to immunoreactive LH (B/I) principally reflects variation in LH immunoassay dose-response characteristics, rather than a change in the bioactive moiety of LH. The varying B/I ratio is due to molecular heterogeneity at multiple levels. Different LH standards contain different proportions of nonbioactive but immunoreactive material. The immunoreactive LH isoforms in serum contain different proportions of bioactive material and the isoform distribution differs with reproductive status. Furthermore, the antibodies comprising the various immunoassay systems detect heterogeneous epitopes on LH, which are not necessarily bioactive. B/I ratio disparities indicate lack of specificity of immunoassays for bioactive LH. Polyclonal antibody-based radioimmunoassay requires the use of purified reagents, including a bioactive tracer, in order to achieve high specificity for bioactive LH. The new generation of monoclonal antibody-based immunometric assays yields results that are lower than, but correlate with, LH measured by the in vitro bioassay. The purest of standards, even a recombinant standard, yields results that differ up to 50% or more from one immunoassay to another. Serum LH levels also differ up to two-fold among assays. The immunometric assays have the advantage of being more sensitive and more specific for low levels of LH in serum than radioimmunoassays, but B/I ratio discrepancies remain great. An immunoassay specific for the bioactive "docking site" of human LH isoforms is still needed.

Purification of human pituitary LH and thyrotrophin by hydrophobic chromatography

A new preparative procedure is described for the efficient purification of LH and TSH from frozen human pituitary glands. LH and TSH were isolated simultaneously from crude preparations by hydrophobic-interaction chromatography followed by separation and purification by reverse-phase high-performance liquid chromatography in a single step. Highly purified hormones were prepared in good yields (45 mg LH and 20 mg TSH/1000 glands) and with high biological activities; the potencies of purified LH and TSH were 5.8 x NIH-LH-S1 equivalent in an ovarian ascorbic acid depletion assay and 7.1 U/mg against human TSH MRC Research Standard (T1 70/9) in the McKenzie assay respectively. Cross-contamination by other glycoprotein hormones was low.

Purification and characterization of guinea-pig chorionic gonadotrophin.

A human chorionic gonadotrophin-like protein (GF-1, 1.0 g) from the placentae of 50 guinea-pigs killed at Day 26 of gestation was purified by pH and ammonium salt fractionation followed by column chromatography on DEAE-Sephadex and filtration on Sephadex G-100. Relative to the Second International hCG standard (MRC 61/6) GF-1 had an immunological potency of 21 000 i.u./mg as measured in a specific hCG-beta radioimmunoassay and, using the ovarian ascorbic acid depletion assay, an apparent biological potency of 24 064 i.u./mg. Isoelectric focusing yielded 6 bands between pH 4.4 and 5.7 and the material comprised two non-covalently linked subunits. The Stokes' radii were 3.40 nm for the native preparation, and 2.38 nm and 3.15 nm for GF-1-alpha and GF-1-beta subunits respectively. The guinea-pig placenta therefore produces a chorionic gonadotrophin which on purification has physicochemical, biological and immunological properties similar to those of hCG.

A pitfall in the ovarian ascorbic acid depletion assay (OAAD): effect of unilateral ovariectomy, hCG and asialo-hCG.

The estimation of the effect of desialylation on the biological activity of human chorionic gonadotrophin (hCG), as determined in the ovarian ascorbic acid depletion assay (OAAD), according to the modification in which the animals are used twice, appeared to be dependent on the assay conditions. Animals which received hCG in the first assay (OAAD-1) gave higher potency estimates for hCG and asialo-hCG in OAAD-1. The results of the experiments indicate that the ovarian sensitivity for hCG and asialo-hCG is increased after administration of hCG and unilateral ovariectomy and decreased after administration of asialo-hCG.

OVARIAN BINDING AND INTRINSIC BIOLOGICAL ACTIVITY OF DESIALYLATED HUMAN CHORIONIC GONADOTROPHIN (ASIALO-hCG) IN IMMATURE SUPEROVULATED RATS

The present study was undertaken to investigate the effect of desialylation on the intrinsic in vivo biological activity of hCG. To block the hepatic uptake of asialo-hCG we investigated asialo-orosomucoid. The biological activity of hCG and asialo-hCG was estimated with the ovarian ascorbic acid depletion assay (OAAD). The uptake studies were performed with [125I]hCG and [125I]asialo-hCG. Desialylation of hCG increased its hepatic uptake, almost completely abolished its ovarian uptake, and reduced its in vivo biological activity to less than 1%. Administration of asialo-orosomucoid caused a reduction of the hepatic uptake of asialo-hCG to the level observed for hCG. The ovarian uptake increased, but was lower than observed for hCG, due to a considerably higher renal accumulation of asialo-hCG. The in vivo biological activity of asialo-hCG was approximately 1/5 of the activity of hCG when both were tested in the presence of asialo-orosomucoid. To exclude the interference of renal accumulation, binding and biological activity were also investigated after bilateral nephrectomy. The uptake of radioactivity in the ovaries following injection of [512I]asialo-hCG and asialo-orosomucoid was then almost the same as observed after injection of [125I]hCG alone. When both preparations were now tested in the presence of asialo-orosomucoid, the in vivo biological activity of asialo-hCG appeared to be approximately 1/3 of the value obtained for hCG. It is concluded that the intrinsic in vivo biological activity of hCG is lowered by desialylation.

RABBIT LUTROPIN: PREPARATION, CHARACTERIZATION OF THE HORMONE, ITS SUBUNITS AND RADIOIMMUNOASSAY

The purification of rabbit lutropin is described. A product with a potency of 1.53 X NIH-LH-Sl was obtained as assayed by the ovarian ascorbic acid depletion assay. In a homologous radioimmunoassay, which is described, rabbit lutropin has a potency 4.83 X NIH-LH-Sl. In a radioligand assay, utilizing labeled ovine lutropin as the trace, the relative potency was 0.47 X NIH-LH-Sl measured by 50% inhibition comparison since rabbit lutropin response in this system did not parallel ovine lutropin. A counter-current distribution procedure for separation of rabbit lutropin subunits is described. Amino acid composition of the isolated subunits and intact rabbit lutropin was determined. The carbohydrate composition of the latter is presented; only amino sugar determinations are available for the subunits. The NH2-terminal amino acids are phenylalanine (alpha subunit) and alanine (beta subunit). Preliminary data on COOH-terminal amino acids are provided.

Effect of diverse mammalian and nonmammalian gonadotropins on isolated rat Leydig cells

The isolated rat Leydig cell assay was employed to study species specificity in biological action. Eight mammalian luteinizing hormones (LHs), human chorionic gonadotropin, and pregnant mare serum gonadotropin stimulated testosterone production in this assay with a 250-fold potency range. A high potency correlated with a high carbohydrate content. Comparison of the potencies of these hormones in the Leydig assay, the ovarian ascorbic acid depletion assay, and the Xenopus ovulation assay showed the results of the two rat assays to be the most similar, demonstrating that the receptor as well as the hormone play a role in determining species specificity. The LH specificity of the assay was reexamined and follicle-stimulating hormone (FSH) was found to have a small intrinsic activity. Nonmammalian LHs, which generally possess little or no activity in in vivo mammalian assays, were able to stimulate rat Leydig cells, at 1000x mammalian LH doses. LH specificity was retained with nonmammalian LHs but the potency differences between nonmammalian LHs and FSHs were much less than mammalian LHs and FSHs.

Species specificity in the response of an in vitro amphibian ( Xenopus laevis) ovulation assay to mammalian luteinizing hormones

Relative potencies of pituitary LH and pregnancy gonadotropins (hCG and PMSG) from numerous mammalian species estimated by an in vitro ovulation bioassay in the anuran Xenopus laevis correlated poorly with estimates obtained by various mammalian bioassays. For example, potencies of seven preparations of pituitary LH which varied by about 5-fold in the in vivo mammalian ovarian ascorbic acid depletion (OAAD) bioassay, differed by 140-fold in potency in the anuran bioassay. The ranking of hormonal potencies was also very different in these two assays; e.g., human LH was the most potent in the rat OAAD assay and among the least potent in Xenopus (ovine LH was most potent in Xenopus). Results of the OAAD assay corresponded closely to those observed in several in vitro assays based on steroid production by mammalian (rat and rabbit) tissues. Thus, pronounced species specificity is evident in the response of Xenopus ovaries to gonadotropins from mammalian origin; these findings have important implications for interpreting comparative data on LH purification and biological actions of hormones.

Effect of selective nitration of ovine lutropin on the subunit association and biological activity of the hormone.

Different nitrated lutropin (LH) subunits were tested for their abilities to recombine with their counterpart subunits. (A detailed description of the preparations tested is presented in the preceding paper (Burleigh, B. D., Liu, W.K., and Ward, D.N. (1976) J. Biol. Chem. 251, 308-315).) The results indicated that the fully nitrated beta subunit of ovine LH (oLHBeta) (diatyrosyl oHbeta) can combine with the alpha subunit of ovine LH (oLHalpha) as wen which tyrosine residues alpha21, alpha41, alpha93 are modified) recombined partially with oLHbeta yielding about 40% as recombined product under specified recombination conditions. The fully nitrated oLHalpha (pentaatyrosyl oLH) combined poorly with oLHbeta and only 20 to 25% was obtained as the recombined product. The various nitrated LH and recombined LH derivatives containing separately nitrated subunits were tested for their potency in an in vitro radioligand assay and their in vivo biological potency in the ovarian ascorbic acid depletion assay. In the radioligand assay, the monoatyrosyl oLH and the oLHalpha + diatyrosyl oLHbeta were the most active derivatives and had about the same competitive inhibtion curves as that of oLHalpha + oLHbeta. Diatyrosyl oLH had intermediate native potency and the triatyrosyl oLH, tetraatyrosyl oLH + OLHbeta, and pentaatyrosyl oLHalpha + oLHbeta were the least active derivatives in this series. No tetranitromethane-modified LH derivative in our study was completely devoid of the specific binding activity in the radioligand assay. At a dosage of 1000-fold excess over the 125I-labeled oH, all the preparations tested in this series completely inhibited the specific binding of 125I-labeled oLH to the hormone receptors in rat testis preparations. In the in vivo ovarian ascorbic acid depletion assay, both the monoatyrosyl oLH and oLHalpha + diatyrosyl oLHbeta retained about 50% of its original activity; the diatyrosyl oLH and triatyrosyl oLH had about 10% of the activity and the recombined products of tetraatyrosyl oHalpha + oLHbeta and pentaatyrosyl oLHalpha + oLHbeta showed virtually no activity. Based on the results obtained from this paper and the preceding paper, the role of the individual tyrosine residues in oLH is assessed.

The Role of the Amino Group in Subunit Association and Receptor Site Interaction for Ovine Luteinizing Hormone as Studied by Acylation

Abstract The positively charged amino groups (in physiological pH range) of ovine luteinizing hormone (oLH) and its subunits (oLHα and oLHβ) were acylated to produce negatively charged (maleylated) or neutral groups (carbamylated) in their place. In both reactions, the number of modified e-lysine amino groups in luteinizing hormone (LH), LHα, and LHβ was 10, 9, and 2, respectively. The NH2 termini were also completely acylated in both reactions. Maleylation or carbamylation did not alter the hydrodynamic volume of oLH or oLHβ significantly, although a greater Stokes radius was obtained with the oLHα derivatives compared to native oLHα as judged by Kav values from molecular exclusion chromatography. Maleylated LHβ combined with native LHα as well as the native subunits recombine, but maleylated LHα recombined with native LHβ less effectively. Efficiency of recombination was judged by the quantity of material with a hydrodynamic volume similar to LH under specified recombination conditions. Maleylated LHα and maleylated LHβ recombined very poorly. The series of recombination products with carbamylated subunits were all obtained in yields comparable to those observed with the recombination of the native subunits. Biological activities of the various recombined products were examined by both the in vivo ovarian ascorbic acid depletion assay and the in vitro radioligand receptor assay and the results agreed. Intact oLH which had been carbamylated or maleylated, as well as the recombined subunit derivatives from these reactions were completely inactive. Either the carbamylated or maleylated oLHα plus native oLHβ, and native oLHα plus maleylated oLHβ gave a low biological activity with response characteristics different from that of the native hormone. The recombined native subunits, or native oLHα plus carbamylated oLHβ, gave products that were slightly less potent than native oLH, with response characteristics that paralleled those of the native hormone. These results suggest that the charges on lysine e-amino groups in the oLHα play a role in the recombination with oLHβ, but that the charge on the oLHβ amino groups is not crucial for subunit-subunit interaction. It may also be concluded that the charge on the free amino groups of oLHβ must be positive or neutral for receptor site interaction. At least a portion of the amino groups on the oLHα must carry a positive charge for maximum hormone-receptor site interaction.

The specificity of an ovine pituitary incubation system linked with gonadotrophin assays for detecting gonadotrophin releasing activity.

SUMMARY A sheep pituitary incubation system was developed which may be used to study the release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as assayed biologically. Specificity was examined by adding at high concentration various materials present in the hypothalamus, a crude acid extract of sheep cerebral cortex or crude or partly purified extracts of ovine hypothalamic tissue. Of the materials tested for LH and FSH releasing activity, oxytocin, adrenaline, synthetic lysine vasopressin and cerebral cortex extract failed to influence LH or FSH release. Natural vasopressin containing unknown proportions of the lysine and arginine forms did not affect FSH release but produced a significant increase in the LH content of the medium, the reason for which was not clear. Noradrenaline failed to influence FSH release but produced an apparent depression in the LH content of the medium due to an action on the LH released or upon the ovarian ascorbic acid depletion assay since the addition of noradrenaline to standard ovine LH reduced the potency in the assay to an almost identical degree. Extracts of ovine hypothalamic tissue consistently increased the LH and FSH content of the medium. The method described may be applicable to the detection and estimation of LH and FSH releasing activity in ovine hypothalamic tissue extracts and to detecting changes in the responsiveness to hypothalamic stimulation of sheep pituitary tissue.

Immunologic and biologic behavior of hCG and bovine LH subunit hybrids.

Bovine LH subunits were combined with hCG subunits. The hybrids were characterized biologically and immunologically using the ovarian ascorbic acid depletion assay and specific homologous radioimmunoassays for hCG and its subunits. Hybrids comprised of complementary subunits formed biologically active preparations. The immunologic behavior of the hCG subunits was unaltered by combination with complementary bovine LH subunits. One would expect the biologically active hybrids to have similar conformations, assuming the latter to be a requisite for biologic activity. Consequently, it would appear that our radioimmunoassay systems were not dependent on the conformation necessary for biologic activity since the immunologic and biologic behaviors of the hybrids were disparate. (Endocrinology 92: 411, 1973)

FSH and LH of the pituitary gland of gonadectomized snowshoe hares.

Experiments were designed to examine effects of bilateral gonadectomy on pituitary FSH and LH and serum LH of snowshoe hares (Lepus americanus) during the breeding season. Male and female hares were gonadectomized and then sacrificed at 10-, 20-, 30-, 45- and 60–day intervals, and the pituitaries were assayed for LH and FSH using modifications of the Parlow Ovarian Ascorbic Acid Depletion Assay (OAAD) and Steelman and Pohley HCG augmentation assays, respectively. In both males and females, pituitary LH and FSH increased progressively between the 10th and 60th day after gonadectomy. By the 60th day pituitary LH in the female had increased to 3 times that observed in the male; however, the male pituitary contained more FSH. Serum LH increased dramatically to the 20th day, and continued to increase at a much slower rate thereafter. (Endocrinology 92: 344, 1973)

Effect of progesterone on the time of ovulation in immature rats pretreated with pregnant mare's serum gonadotrophin (PMSG).

ABSTRACT The time of ovulation in immature rats primed with 3 IU PMSG on day 22 of age was between 1:00 and 4:00 a. m. on day 25 as determined by the presence of ova in the oviducts. When 0.5 mg progesterone was given at 10:00 a. m. on day 24, ovulation was between 1:00 and 2:00 a. m. on day 25. However, ovulation was advanced to 11:00 p. m. of day 24 when 0.5 or 2.0 mg progesterone was injected at 5:30 a. m. on day 24. In animals pretreated with 12 IU PMSG. ovulation occurred between 10:00 p. m. of day 24 and 2:00 a. m. of day 25 when the same steroid (2.0 mg) was given at 5:30 a. m. on day 24. Ovulation was inhibited with 4.5 mg phenobarbital injected at 10:30 a. m. or 11:00 a. m. on day 24 in progesterone-injected rats which had been pretreated with 3 or 12 IU PMSG, respectively, indicating that the critical period has been shifted forward by 3 hours. The highest plasma LH occurred at 3:00 and 8:00 p. m. on day 24 in rats primed with 3 IU PMSG alone, whereas the highest plasma LH in rats injected with 3 IU PMSG and progesterone occurred at 10:00 a. m. and 3:00 p. m. of day 24, as determined by a modified method of the ovarian ascorbic acid depletion (OAAD) assay. It is concluded that progesterone advanced the time of ovulation by shifting forward the critical period and causing an adequate release of ovulatory gonadotrophins between 10:00 a. m. and 3:00 p. m. on the day preceding ovulation.

Effects of dehydroepiandrosterone and cyano-ketone on ovarian weight, cholesterol content, and ultrastructure in PMS-HCG treated, immature rats.

Immature rats were given the PMSHCG treatment recommended by Parlow for the preparation of rats for the ovarian ascorbic acid depletion (OAAD) assay of LH. At 30 days of age one group of 5 rats received two 5 mg injections of cyano-ketone (a potent inhibitor of δ5- 3β-hydroxysteroid dehydrogenase) and two other groups were given cyano-ketone (CK) followed by daily injections of 15 or 20 mg of dehydroepiandrosterone (DHA) for seven days. Finally, one group was given the daily DHA treatment (20 mg) but received no CK. At autopsy, ovaries were weighed and either analyzed for total cholesterol content or prepared for ultrastructural study. The CK treatment did not significantly alter either the ovarian weight or cholesterol content. All three groups given DHA showed a remarkable enlargement of the heavily luteinized ovaries and a significant reduction in ovarian cholesterol concentration as well as in total ovarian cholesterol. These changes were accompanied by ultrastructural signs of heightened secretory ac...

A bioassay for progesterone and gonadotropins based on the meiotic division of Xenopus oocytes in vitro

A sensitive and rapid quantal bioassay has been developed which may be used to measure progesterone and related steroids or gonadotropic hormones. The technique is based on the meiotic division of Xenopus oocytes in vitro, and procedures for obtaining sensitive test oocytes are described, together with criteria used in detecting the response. For the assay of progesterone the main advantages of the test over alternative bioassays lie in its sensitivity, rapidity, and the ease with which the results can be evaluated statistically. The main drawback is the low specificity of the response, which can best be overcome by combining the test with a specific extraction and purification procedure. However determinations of the progestin in plasma from pregnant rats were of the same order of magnitude as those made by other workers using an established technique, and it is felt that this test may prove more reliable than the Hooker-Forbes bioassay. Gonadotropic hormones also induce meiosis, and the test is particularly sensitive to LH, being as sensitive as the ovarian ascorbic acid depletion assay for LH, though less specific. In addition the test is extremely sensitive to Xenopus pituitary material; corticotropin and prolactin were ineffective, and the merits of the test for the assay of amphibian gonadotropin are stressed.

Stimulation and inhibition of synthesis of ovulating hormone (OH) in organ culture by subcellular fractions of rat medial basal hypothalami.

Subcellular fractions of rat medial basal hypothalami were examined for their ability to stimulate the synthesis of ovulating hormone (OH) in a pituitary organ culture system. The hypothalamic stimulator(s) of OH synthesis (HOHS) was associated with the 1–15,000 × g participates and was not retained by a 0.5 μ (diameter pore size) Nuclepore filter. This fraction appeared to contain many of the electron dense vesicles present in this region of the hypothalamus. Sepharose 2B gel filtration of these participates revealed not 1 but 2 zones of HOHS activity separated by a clear, distinct area containing an inhibitor of OH synthesis. Similar data were obtained using the Bogdanove- Gay ovarian ascorbic acid depletion assay for LH in place of the ovulating hormone assay. That the hypothalamus may, in addition to producing gonadotropin releasing factors, be producing gonadotropin inhibitory substances is discussed. Such inhibitory substances provide a means whereby gonadotropin production could be suppressed. (End...

Progesterone synthesis in response to luteinizing hormone by rat ovarian tissue in vitro.

SUMMARY A bioassay for luteinizing hormone (LH) activity is described. The method depends on progesterone synthesis by pooled, sliced rat ovarian tissue in vitro, the ovaries being obtained from immature rats pretreated with pregnant mare serum gonadotrophin. The main advantage of the method is its increased sensitivity compared with the commonly used ovarian ascorbic acid depletion assay, its high degree of precision, and the fact that it allows the use of large numbers of well randomized samples from a small group of animals.

The effects of desialylation on the biologic and immunologic activity of human pituitary luteinizing hormone

A highly purified preparation of human pituitary luteinizing hormone was treated with neuraminidase resulting in 94% removal of the sialic acid moiety. This led to a marked loss of biologic activity as measured by the ventral prostate weight and ovarian ascorbic acid depletion assays, and enhancement of the radioimmunologic activity.