Ovarian Ascorbic Acid Depletion Research Articles

Ovarian ascorbic acid depleting factors in the chicken hypothalamus.

A substance active in the ovarian ascorbic acid (OAA) depletion assay was found in acid extracts of chicken hypothalamus but not in extracts of cerebral cortex. The depletions evoked by these extracts were not proportional to the logarithm of the dose, and the extracts were equally active in intact and hypophysectomized assay rats. This effect was not due to the presence of LH in the extracts for the active substance had no effect in the 32P assay, and it passed through a dialysis membrane with an exclusion limit of under 12,000 molecular weight. The activity of this small molecular weight fraction was destroyed by incubation with thioglycollate. Purification of this fraction on a column of Sephadex G-25 produced a single peak with OAA depleting activity. This fraction also contained vasopressor activity, and the elution volume of the fraction was the same as that of synthetic arginine vasotocin (AVT). Because of the many similarities of AVT and the active compound in the extracts, it was concluded that AVT was the primary active substance of the extracts. The possible relationships of AVT to chicken luteinizing hormone-releasing factor are discussed. (Endocrinology85: 113, 1969)

Sex difference in the release of luteinizing hormone evoked by progesterone.

SUMMARY Long-term gonadectomized female and male rats showed increased plasma luteinizing hormone (LH) activity (as measured by the ovarian ascorbic acid depletion method). A single injection of 20 μg. oestradiol benzoate or 2·5 mg. testosterone propionate after 3 days reduced the raised values significantly. Progesterone injected into gonadectomized rats primed with gonadal steroids exerts a positive feed-back action on the release of LH. In ovariectomized rats the injection of progesterone 3 days after a single injection of oestradiol or testosterone induced, a few hours later, a significant increase in plasma LH. In contrast, castrated male rats responded to progesterone with an increase in plasma LH only when they had been primed 3 days before with testosterone. This difference was not eliminated by gonadectomy on the first day of life. Androgen-sterilized ovariectomized rats failed to show an increase in plasma LH after progesterone when primed with testosterone but did so after priming with oestradiol. In male rats treated with testosterone in the early neonatal period, the normal LH release induced by progesterone in testosterone-primed animals was abolished. It is concluded that separate female and male patterns of LH release evoked by progesterone exist in the rat, and the difference between them is not determined by a postnatal differentiation in the neural mechanism which controls the secretion of LH.

A substance resembling arginine vasotocin in the anterior pituitary gland of the cockerel.

Fractionation by ultrafiltration of acid extracts of cockerel adenohypophyses revealed 2 components which depleted ovarian ascorbic acid (OAA) in the appropriately pretreated assay rat. Avian LH was retained in the large molecular weight (over 12,000) fraction. In the 32P assay, this fraction evoked a response curve which was parallel to that of the NIH-LH standard. The other fraction, which contained molecules with a molecular weight of less than 12,000, caused depletion of OAA in both intact and hypophysectomized assay rats, but was inactive when tested for its ability to increase 32P uptake by the chick testis. Thus, its OAA depleting activity was due neither to luteinizing hormone (LH) nor to LH-releasing factor. Fractionation of the small molecules on a column of Sephadex G-25 revealed a potent OAA depleting compound with the additional following characteristics: 1) same elution volume as synthetic arginine vasotocin (AVT), 2) potent vasopressor activity, 3) inactivated by thioglycollate. These data ...

EFFECT OF FREEZING AND THAWING AND OF DILUENT ON THE POTENCY OF HUMAN CHORIONIC GONADOTROPHIN IN THREE METHODS OF BIOASSAY

ABSTRACT In a study designed as a factorial experiment, the biological activity of standard solutions of human chorionic gonadotrophin in distilled water (A), saline (B), 1 % bovine serum albumin (C), 0.5 % gelatin (D), and borate buffer of pH 9 (E) was investigated under four different conditions of freezing and thawing, using the following three methods of bioassay: ovarian ascorbic acid depletion in rats (OAAD), uterine weight in mice (UW), and ovarian hyperaemia in rats (OH). Repeated freezing and thawing and prolonged storage at -15°C did not affect the potency in any test. In the OAAD test, the potency was increased 4–5fold by D, and 2–3fold by C. In the OH test, E augmented the potency 2–3fold. These findings are of interest in the practice of bioassay, in studying mechanisms of response, and regarding administration for therapeutic purposes. Diluents which possess augmenting properties could be used to improve the sensitivity of a bioassay if standard and unknowns showed the same degree of augmentation.

Plasma and pituitary LH and FSH in the castrated rat following short-term steroid treatment.

The effects of short-term steroid treatment on the blood and pituitary LH and FSH levels of previously castrated adult male and female rats were examined, using ovarian ascorbic acid depletion (OAAD) and HCG-augmentation bioassays for LH and FSH activities. Pituitary and plasma FSH activities, 3 days after an injection of 1) testosterone propionate (TP) or 2) estradiol benzoate plus progesterone (E + P), were compared in 5 experiments. Concomitant changes in plasma and pituitary LH levels were studied in 3 of these experiments and 2 others. In 2 further studies in spayed rats, both stored and circulating LH and FSH were examined after 7 days of TP treatment. In addition, pituitary FSH and plasma LH were studied 24 hr after single injections of TP. Both steroid treatments (TP and E + P) decreased plasma LH levels without altering pituitary LH concentrations detectably. The TP effect on plasma LH was evident after only 24 hr. Thus, both treatments apparently reduced the rate of LH release. Because of the re...

Studies on the disappearance of LH and FSH in the rat; a quantitative approach to adenohypophysial secretory kinetics.

The disappearances of circulating endogenous and exogenous FSH and endogenous LH in the rat were compared, using HCG-augmentation and ovarian ascorbic acid depletion assays. The studies of endogenous LH and FSH were done in previously castrated male and female rats. Exogenous FSH was studied after iv injection of NIH-FSH (ovine) into several types of rats: 1) long-term castrates of both sexes (with and without prior hypophysectomy); 2) intact males and females, and hypophysectomized males; 3) rats orchidectomized, spayed or sham-castrated only 2–3 min prior to injection; 4) intact and previously castrated rats, treated chronically with estrogen or androgen and hypo-physectomized 2–3 min before they were injected. The following findings emerged. 1) Endogenous LH activity disappears from the circulation of the castrated rat at least 4 times as rapidly as endogenous FSH activity. Half-lives averaged 30 (19–38) and 149 (96–239) min, respectively. 2) The exogenous FSH preparations disappeared more rapidly than...

On the isolation of human pituitary follicle-stimulating hormone.

A homogeneous preparation of follicle-stimulating hormone was isolated from human pituitary glands by a procedure involving ion exchange chromatography, exclusion chromatography, and density gradient electrophoresis. The hormone has over 300 times the activity of NIH-FSH-Sl in the ovarian HCG-augmentation assay, thus confirming, in contradistinction to a number of other reports, the high potency estimates obtained by Roos and Gemzell for highly purified preparations of human follicle-stimulating hormone. The luteinizing hormone potency of this same preparation is less than 0.009 times that of NIH-LH-S1 in the ovarian ascorbic acid depletion assay. (Endocrinology84: 953, 1969)

Pituitary levels of FSH and LH at various intervals after ovariectomy in the rat.

Summary. The pituitary contents of fsh (hcg augmentation method of Steelman & Pohley) and lh (ovarian ascorbic acid depletion method of Parlow) were determined at 0, 2, 9, 13 and 25 weeks and at 0, 2 and 13 weeks, respectively, after bilateral spaying. The fsh concentration (μg/mg) increased up to 9 weeks with no changes thereafter, but the fsh content (μg/gland) and also lh levels increased up to 13 weeks.

The ovarian ascorbic acid depletion test for luteinizing hormone. Lack of specificity.

ABSTRACT Urinary extracts from two women hypophysectomized for metastasizing mammary adenocarcinoma and from 11 children of either sex between 5 and 9 years were found to produce marked ovarian ascorbic acid depletion in the Parlow test for luteinizing hormone. As these urines cannot contain significant amounts of luteinizing hormone, it is concluded that the depletion must be due to unspecific substances, probably proteins in the urines. From the literature other indications of non-specificity of the Parlow test are mentioned, and the main conclusion of the present investigation is that for the assay of luteinizing hormone in extracts of human urine the test is unreliable.

Studies on the site of action of oral contraceptive steroids. I. Effect of antifertility steroids on plasma LH levels and on the response to luteinizing hormone-releasing factor in rats.

ABSTRACT The effects of ovulation inhibiting steroids on plasma luteinizing hormone (LH) levels and on the response to LH-releasing factor (LRF) were studied in an attempt to learn whether these compounds exert part of their effect on the pituitary or on the hypothalamus. Ovariectomized rats were injected subcutaneously for 5 days with estrogens, progestins, and combinations thereof. Plasma LH levels were measured by the ovarian ascorbic acid depletion assay. The progestins: chlormadinone acetate (0.05–10 mg), ethynodiol diacetate (2 mg), norethynodrel (20 mg), norethindrone (5 mg), medroxyprogesterone (10 mg), norgestrel (1 mg) and dimethisterone (2.5–20 mg) given daily in doses indicated significantly depressed plasma LH. Administration of mestranol (0.001–0.2 mg) and ethinyl estradiol (0.1 mg) also significantly lowered plasma LH. The progestin/estrogen combinations: chlormadinone acetate/mestranol (C-Quens, 10.4 μg), ethynodiol diacetate/mestranol (Ovulen, 5.5 μg), norethynodrel/mestranol (Enovid, 12....

Gonadotropic activity of stalk median eminence extracts in hypophysectomized rats.

Standard preparations of crude or partially purified porcine LH-RF (prepared by molecular sieving on Sephadex-G25) elicited the same depletion of ovarian ascorbic acid in intact and hypophysectomized rats. At doses of 1/16, 1/4, 1 and 4 stalk median eminence equivalents (SME), crude extracts elicited similar depletions in intact and hypophysectomized rats, which increased with dose. At these same doses, purified extracts evoked a linear dose response in intact rats and nonsignificant responses at the lower 3 doses in hypophysectomized rats. The high dose effected similar responses in both intact and hypophysectomized rats. An assay which depends on release of pituitary LH into the blood stream, under the influence of high doses of LH-RF was not able to distinguish between LH and LH-RF. Both compounds evoked higher activities in the plasma of intact than hypophysectomized rats. The partially purified preparations of LH-RF used in these studies were not contaminated with LH since ultrafiltration of LH through a dialysis membrane removed the LH activity in hypophysectomized assay rats, while filtration of LH-RF did not. The highest doses of the partially purified extracts also contained less than 35 mU of vasopressor activity. This level of vasopressin contamination was too low to affect the depletion of ovarian ascorbic acid. (Endocrinology83: 225, 1968)

Human and porcine thyrotropins: A comparison of electrophoretic and immunological properties with the bovine hormone

The electrophoretic and immunochemical properties of purified human and porcine thyrotropins (TSH) have been compared with those of the bovine hormone. Preparations from all three species show similar polymorphism in gel electrophoresis. Human TSH exhibits more electronegative character than either porcine or bovine TSH due to the presence of about 1.5% sialic acid whereas the bovine and porcine material are essentially free of sialic acid. Removal of the sialic acid by neuraminidase does not affect the hormonal activity or immunochemical properties of human TSH. Immunological cross reactivity was found in double diffusion between the TSH of all three species with antisera to any of the three. Anti-human TSH serum completely neutralized the biological activity of the bovine and porcine hormones. These studies also demonstrated the bovine and porcine hormones to be quite similar, a conclusion further supported by peptide maps. In addition, human TSH appears to be closely related immunologically to denatured bovine TSH when the two are compared against anti-bovine TSH serum. The separation of the closely related glycoprotein, luteinizing hormone (LH), from TSH has been studied by biological and immunochemical criteria. Rechromabovine tography of TSH on diethylaminoethyl cellulose yielded preparations whose LH content as assayed by depletion of ovarian ascorbic acid was about 2% by weight. Absorption by an anti-LH serum further reduced the LH activity to values representing less than 1% contamination by weight. Examination of immunodiffusion properties of LH and TSH with antisera to each hormone yielded no evidence of cross reactivity between either bovine or human TSH and the homologous luteinizing hormones.

GONADOTROPHIN PATTERNS IN MALE AND FEMALE RATS:

ABSTRACT The effects of various doses of testosterone propionate (TP) upon the release of luteinizing hormone (LH or ICSH) from the hypophysis of a gonadectomized male or female rat were compared. Prostate weight in hypophysectomized male parabiotic partners was used to evaluate the quantity of circulating LH. Hypophyseal LH was measured by the ovarian ascorbic acid depletion method. Males castrated when 45 days old secreted significantly more LH and had three times the amount of pituitary LH as ovariectomized females. Administration of 25 μg TP daily reduced the amount of LH in the plasma, and increased the amount in the pituitary gland, in both sexes. Treatment with 50 μg caused a further reduction in plasma LH in males, but not in females, while pituitary levels in both were equal to that of their respective controls. LH fell to the same low level in partners of males or females receiving 100 μg TP. When gonadectomized at 39 days, males and females had the same amount of plasma LH, but males had more stored hormone. Pituitary levels were unchanged from controls following treatment with 12.5, 25 or 50 μg TP daily, but plasma values dropped an equal amount in both sexes with the latter two doses. Androgenized males or females, gonadectomized when 39 days old, were very sensitive to the effects of TP and plasma LH was significantly reduced with 12.5 μg daily. Pituitary LH in androgenized males was higher than that of normal males but was reduced to normal by small amounts of TP. The amount of stored LH in androgenized females was not different from that of normal females and it was unchanged by any dose of TP tested. Results are consistent with the conclusion that the male hypothalamic-hypophyseal axis is at least as sensitive as the female axis to the negative feedback effects of TP. Androgenization increases the sensitivity to TP in both males and females.

A new approach to the biological determination of the luteinizing hormone.

ABSTRACT The response of the uterine weight of immature mice to FSH and LH preparations with low contamination by each other was studied. The new approach to the biological assay of LH, which could be designated the »uterine-augmentation« test, was based on injecting intact immature mice with a constant dose (4.44 IU) of a biologically pure FSH preparation plus increasing amounts of extracts containing LH. A dose as little as 0.068 IU of LH is capable to increase the uterine weight when injected together with 4.44 IU of biologically pure FSH. A significant uterine response can be obtained when animals are injected with 29.4 μg of 2nd IRP-HMG (equivalent to 0.128 IU-LH and 0.128 IU-FSH). When a constant dose of 4.44 IU of biologically pure FSH is added to each dose level of the 2nd IRP-HMG, a significant uterine response is obtained with 14.7 μg of this reference preparation. The end point was the mouse uterine weight. Some determinations of the LH activity in urinary extracts have been performed according to this new method and from the results it seems that the LH activities, as determined by the ovarian ascorbic acid depletion method, agree with those determined by the uterine-augmentation test. It also seems that the uterine weight response is proportional to the LH activity and not dependent on the FSH-LH ratio of the samples assayed.

Studies on the purification of ovine prolcatin-inhibiting factor.

After several extractions, hypothalamic extract was subjected to gel filtration on long columns of Sephadex G-25. Prolactin-inhibiting activity of the fractions was estimated by their ability to block suckling-induced decline in pituitary prolactin. This inhibitory activity was recovered in each of 5 fractionations with Sephadex. In each instance the prolactininhibiting activity was found to be closely associated with luteinizing hormone (LH)-releasing activity as estimated by ovarian ascorbic acid depletion; however, in 3 of 5 experiments there was a tendency for the prolactin-inhibiting activity to be eluted from the column just prior to the emergence of the peak of the LH-releasing activity. When the zones with prolactin-inhibiting and LH-releasing activity from several columns were pooled, lyophilized and eluted from a carboxymethylcellulose column by application of ammonium acetate buffers of increasing pH and ionic strength, prolactin-inhibiting activity was again recovered from the column in fractions which also possessed LH-releasing activity. The peptide content in the highly purified prolactininhibiting factor (PIF) was estimated to be < 3 μg. Clearly, a manyfold purification of PIF was achieved without obtaining a clear-cut separation from the LH-releasing factor (LH-RF). The PIF was partially separated from follicle stimulating hormone-RF and removed from the zones which contained growth hormone-RF, melanocyte stimulating hormone-RF, thyrotropin-RF and vasopressin. (Endocrinology82: 1236, 1968)

Failure of inhibitors of protein synthesis to affect the LH-releasing action of hypothalamic extracts in vitro.

SummaryAnterior pituitary halves from adult male rats were incubated in vitro for 6 hours following a preincubation of 30 min in tissue culture medium 199. The LH release from the glands was determined by the ovarian ascorbic acid depletion assay. Neither puromycin nor actinomycin in doses of 10-80 μg/ml interfered with the “basal” release of LH from the pituitaries. Purified LH-releasing factor (LHRF) enhanced the release of LH, and this enhancement was not influenced by the presence of either of the antibiotics. The LHRF can thus act when protein synthesis is blocked and must facilitate release of preformed LH from the cell. The present results with LHRF are different from those previously obtained with FSHRF whose action in increasing FSH release was blocked by the antibiotics and therefore appeared to require protein synthesis.

The effect of hysterectomy on parameters of luteinizing hormone secretion in pseudopregnant rats.

ABSTRACT The possibility that the luteotrophic effect of hysterectomy in the pseudopregnant rat is due to a decreased release of luteinizing hormone (LH) was investigated. The problem was approached indirectly by measuring pituitary LH concentration (Ovarian Ascorbic Acid Depletion Assay) (OAAD) in intact and hysterectomized pseudopregnant rats killed on various days of pseudopregnancy. Values obtained in the two groups were not significantly different. The size of the interstitial cells in the ovaries of the two groups of pseudopregnant rats was also not significantly different. The results do not offer support to the hypothesis that the uterus affects the secretion of LH, but rather suggest that it has a direct effect on the corpora lutea.

Adenohypophyseal and serum gonadotropins in male rats treated neonatally with estradiol benzoate.

This study investigated the effect of 100 μg of estradiol benzoate injected during the 5th day of life on pituitary and serum concentrations of gonadotrophins in intact and orchidectomized male rats. The ovarian ascorbic acid depletion method of Pariow was used for the assay of pituitary and serum luteinizing hormone (LH). The adenohypophyseal and serum concentration of follicle stimulating hormone (FSH) was determined by means of the augmentation method of Steelman and Pohley. Neonatal administration of estradiol benzoate to male rats resulted in animals with significantly smaller testes, ventral prostate and seminal vesicles. No differences in pituitary LH concentration were observed between estrogen-treated and non-injected controls of 60 days of age, but 180-day-old estrogen-injected males showed a significantly higher LH concentration value than their controls. Determination of pituitary FSH concentrations showed no significant differences between estrogen-treated and control animals of 60 and 180 da...

DEPLETION OF RAT OVARIAN ASCORBIC ACID BY A FACTOR OTHER THAN LUTEINIZING HORMONE PRESENT IN THE BLOOD OF THE PIG

The ovarian ascorbic acid depletion (oaad) assay of Parlow (1958) is an established method for the measurement of luteinizing hormone (lh). Tests using purified preparations of other anterior pituitary hormones have shown the bio-assay to be specific for lh (McCann & Taleisnik, 1960; Schmidt-Elmendorff & Loraine, 1962) but vasopressin possesses appreciable activity (McCann & Taleisnik, 1960). Pelletier (1964) has reported that hypophysectomized ewe plasma caused a significant response when assayed by this method. Evidence has been produced suggesting that the plasma acted by releasing lh from the pituitary glands of the assay rats (Pelletier, 1965). Recently, determinations of the pituitary content of lh in the sow at various reproductive stages and after ovariectomy have been carried out in this laboratory. A preliminary report of this work has

Ovulation, corpora lutea development and pituitary LH activity in rabbits with intra-uterine devices.

4 experiments were performed on 68 New Zealand white rabbits (showing typical lordosis when exposed to males) to study pituitary LH content ovulation time and corpus luteum development in the presence of IUDs in both horns. In the 1st experiment (1) 20 rabbits (7-9 months) were divided into 4 groups of 5 each: 1) IUD inserted into each horn nonmated; 2) sham operated nonmated controls; 3) IUD inserted in each horn mated; 4) sham operated mated controls. The polyethylene plastic spiral (IUD) was inserted into the upper end of each uterine horn. Mated animals were killed 24 hours after mating and non-mated on the same day relative to surgery. Anterior lobes of the pituitary glands were weighed and frozen for LH assay and corpora lutea (CL) dissected and weighed. In the second experiment (2) 8 rabbits (7-9 months) were divided into 2 groups: 4 fitted with IUDs in each horn mated Day 4 following insertion and 4 sham operated mated controls. 11 hours after mating all were laparotomized and ovulation points and follicles 1 mm or more were counted. Animals were sacrificed 24 hours after mating and CL weighted. 3rd experiment (3) consisted of 20 rabbits (10-13 months) treated as in (1). 4th (4) experiment had 20 rabbits (10-13 months) divided into 2 groups 1 receiving an IUD in each horn and mated the other sham operated and mated. Both groups were killed 11 hours after mating. Relative pituitary LH activity was measured by the ovarian ascorbic acid depletion method of Parlow using Holtzman rats. Doses of homogenized glands of 1 2 and 4 mg were used in (1) and 0.75 1.5 and 3.0 mg in (3) and (4). Analysis of variance and Duncans multiple range test were used. Pituitary LH activity was lower in the mated groups than nonmated (P<.05). CL of IUD mated rabbits were lighter than control mated (P<.05) with less luteiniztion under histological examination of the IUD mated group. Differences in the numbers of ovulations at 11 hours between the treated and controls suggest delayed ovulation in the treated group as there were no differences at 24 hours. IUD treated mated rabbits had higher relative potency of pituitary LH activity than control mated (P<.05). This was not observed in nonmated groups. It is suggested that IUDs in mated rabbits delay LH release and thus ovulation time also.