Ovarian Ascorbic Acid Depletion Method Research Articles

Extraction and purification of a substance with luteinizing hormone releasing activity from the leaves of Avena sativa.

Attempts were made to purify the LH-releasing substance extracted from the leaves of Avena sativa by means of two-step chromatographic procedures using a weakly acidic ion-exchange resin (CG-50,type II) and DEAE-Sephadex A-25 (coarse) with successful results. For preliminary fractionation of such starting materials as dried leaves, fresh leaves, and acetone-extracted powder(crude extracts), 5% acetate-buffered active carbon proved to be more effective than starch zone electrophoresis. From its behavior on chromatography with weakly acidic ion-exchange resins as well as Sephadex gel filtration, the active fraction extracted from the leaves of Avena saliva was assumed to be different from the LH-RH present in the hypothalamus. This partially purified material, however, was demonstrated to have an LH-releasing activity by the ovarian ascorbic acid depletion method using Wistar-Imamichi strain rats. Evidence was presented that its site of action is in the adenohypophysis.

Prostaglandin-induced ovarian ascorbic acid depletion.

The object of this study was to determine whether prostaglandins (PG's) have an LH-like action measurable by the ovarian ascorbic acid depletion method. PGE1, E2, or F2α produced a highly significant decrease in the ovarian content of ascorbic acid in intact and hypophysectomized immature rats with luteinized ovaries due to previous treatment with pregnant mares′ serum (PMS) and human chorionic gonadotropin (hCG). Ten min after a single iv injection of PGE1 E2 or F2α in immature rats with luteinized ovaries, serum LH levels were increased significantly as shown by radioimmunoassay, but LH levels in the control groups were undetectable. Ovulation was induced by hCG treatment in 100% of hypophysectomized immature rats treated with PMS, while it was not induced after injection of PGE2 or F2α in similarly treated rats. Histologically, massive luteinization of the ovaries of hCG-treated rats was observed, but the formation of corpora lutea was never observed in the rats treated with PGE2 or F2α. From these observations, we conclude that PG's have a dual effect on corpus luteal function as expressed by ovarian ascorbic acid depletion: an indirect effect by inducing LH secretion, and a direct effect manifested in hypophysectomized rats. The latter is not due to an LH-like action of PG's, because PG's failed to induce ovulation and luteinization in hypophysectomized immature rats pretreated with PMS. (Endocrinology95: 417, 1974)

Changes in the luteinizing hormone releasing activity of the hypothalamus, and in pituitary gland and plasma luteinizing hormone during the oestrous cycle of the sheep.

SUMMARY Thirty ewes with regular oestrous cycles were divided into six groups for slaughter relative to the time of first acceptance of the ram. The slaughter times were: 0 h (in practice within 40 min of onset of oestrus), 6, 12 and 36 h after onset and days 10 and 16 of the cycle. The hypothalamus was removed and the luteinizing hormone (LH) releasing factor activity extracted with 0·1 m-HCl. The extracts were tested for LH releasing activity by adding them to the medium in which anterior pituitary tissue from castrated male sheep was incubated. The LH content of the medium was measured by the ovarian ascorbic acid depletion method (Parlow, 1958). The activity of the extract from the group slaughtered on day 16 of the cycle was high (minimal effective dose (MED) = 0·00625 hypothalamic equivalents (HE)). The potency declined with the onset of oestrus and remained low at 6 and 36 h after onset (MED in each case 0·025 HE) with intermediate potencies at 12 h and 10 days after onset (MED in each case 0·0125 HE). These changes are compared with changes in the LH content of the pituitary gland (bioassay) and of the plasma (radioimmunoassay) and with parameters of the ovarian activity of the animals.

Pituitary luteinizing hormone content of unmated, pseudopregnant, pregnant and progesterone-treated rabbits.

Summary. The lh content of the pituitary of rabbits in various reproductive states was assayed by the ovarian ascorbic acid depletion method. It was highest in unmated rabbits and decreased significantly (P<0·01) during both pseudopregnancy and pregnancy. Administration of progesterone (20 mg/day for 10 days) reduced pituitary lh in unmated rabbits to levels similar to those found during pseudopregnancy and pregnancy. It is inferred that the capacity of the pituitary gland to accumulate lh during pseudopregnancy and pregnancy decreases mainly as a result of secretion of progesterone.

Gonadotrophic secretion in male rats subjected to an acute noxious stimulus

This study evaluated the effect of an acute noxious stimulus, electric shock, on the pituitary concentration of gonadotrophins and on plasma luteinizing hormone (LH) activity in intact and orchidectomized rats. The ovarian ascorbic acid depletion method of Parlow was used for the assay of pituitary and plasma LH. The adenohypophyseal concentration of follicle stimulating hormone (FSH) was determined by means of the augmentation method of Steelman and Pohley. Administration of an electric shock of moderate intensity which was effective in inducing a marked increase in plasma corticosterone did not result in significant changes in pituitary LH and FSH in intact and orchidectomized animals. Plasma LH significantly increased 5 min after the stressful procedure in orchidectomized rats and returned to control levels at 20 min. No significant differences in plasma LH activity were defected in intact rats.

FSH and ICSH in pituitary and plasma of castrate protein-deficient rats.

Pituitary gonadotropic function was investigated in young male rats 1 month after gonadectomy and after the elimination of protein from the diet. Gonadectomized control rats were maintained on a purified diet complete in all known essential nutrients. Hypophyseal FSH content was determined by the ovarian weight-augmentation test, and plasma was bioassayed for its capacity to stimulate follicular growth in ovaries of immature hypophysectomized rats. ICSH potency of pituitary and plasma was measured by the ovarian ascorbic acid-depletion method. Interpretations of the results were based on relative estimates of gonadotropic activity, derived by comparing the responses of assay rats to donor plasma and pituitary glands. Protein deprivation limited the postgonadectomy rise of both FSH and ICSH in the pituitary gland, although the differences between castrate protein-deficient rats and castrate control animals disappeared when gonadotropic potency was expressed per milligram of pituitary tissue. Likewise, strikingly similar castration changes were found in the pituitary glands of these animals. One milliliter of plasma from castrate protein-depleted rats was estimated to have twice as much FSH activity and the same ICSH potency as 1 ml of control rat plasma. However, when allowance was made for differences in blood volume between rats of the two groups, only ICSH content was significantly reduced in the plasma of protein-deficient animals.

The luteinizing hormone releasing activity of extracts of blood from the hypophysial portal vessels of rats.

1. A method of acid ethanol extraction and gel filtration was used to obtain a ;luteinizing hormone (LH)-free' fraction of blood collected from the cut pituitary stalk of rats (termed hypophysial portal blood).2. The ;LH-free' fraction of hypophysial portal plasma from hypophysectomized and from ovariectomized rats caused a greater depletion of ovarian ascorbic acid in immature rats, pretreated with gonadotrophins, than a similar fraction of systemic plasma obtained from the same donor animals.3. The ;LH-free' fraction of hypophysial portal plasma from ovariectomized rats evoked a rise in the level of LH in the systemic plasma of ovariectomized, oestrogen and progesterone treated, rats. This fraction also caused ovulation in rabbits when infused directly into the anterior pituitary gland of these animals. The activity of the ;LH-free' fraction of systemic plasma was considerably less than that of portal plasma in either of these assay systems.4. The results of these experiments suggest the presence of a factor in the ;LH-free' fraction of hypophysial portal plasma which is capable of causing the release of luteinizing hormone from the anterior pituitary gland. The molecular weight of this factor, as assessed by its behaviour on filtration through ;Sephadex G-25', is probably less than 5000.5. The LRF activity of the ;LH-free' fraction of hypophysial portal plasma obtained from rats at various phases of the oestrous cycle was measured by the ovarian ascorbic acid depletion method. There appears to be a decrease in the level of activity at oestrus. However, a rise in LRF activity, which was expected to occur at the ;critical period' of prooestrus, was not evident. The significance of these findings is discussed.

Short- and Auto-Feedback Control of Pituitary FSH Secretion

In order to clarify a short- and auto-feedback control of FSH release from the anterior pituitary, changes in plasma and pituitary FSH and LH were investigated in rats with FSH implanted into the ME of the hypothalamus and into the anterior pituitary. FSH was bioassayed by the Igarashi-McCann method and LH by the ovarian ascorbic acid depletion method. The following results were obtained. 1. Stereotaxic implantation of FSH (NIH-FSH-ovine) into the ME of ovariectomized rats for 7 days significantly decreased plasma FSH levels in each of 3 experiments and significantly reduced pituitary FSH in 2 out of 3 experiments. Neither pituitary content nor plasma levels of LH showed any significant change in the same rats. 2. Stereotaxic implantation of FSH into the anterior pituitary for 7 days inovariectomized rats did not induce any constant change in plasma FSH levels, butsignificantly decreased pituitary FSH content in all 3 experiments. In 2 out of 3experiments, there was also a significant decrease in plasma LH levels. Pituitary LHcontent remained unchanged. 3. Stereotaxic implantation of FSH into the ME of intact adult female rats for 7 days significantly decreased both plasma and pituitary FSH, but the plasma and pituitary LH did not change significantly. 4. These results clearly demonstrate two different mechanisms of FSH secretion control: the short-feedback and the auto-feedback. The term ‘short-feedback control’ is used to characterize the ability of FSH to inhibit both FSH release and synthesis at the hypothalamic level, while the term ‘auto-feedback control’ is reserved to characterize the ability of FSH to inhibit its own synthesis at the anterior pituitary level.

Influence of progesterone on the pituitary levels of LH in the male rat.

Effects of progesterone on pituitary gonadotrophin stores have been more extensively studied in the female than in the male rat (Rothchild, 1965). Van Rees (1959) reported that the ability of pituitary glands from male rats treated with progesterone (1 or 6 mg/day for 26 days) to stimulate ventral prostatic weights of hypophysectomized rats was lower than that of pituitary glands from controls. Ryan & Philpott (1967), however, noted variable effects of this steroid on pituitary lh stores ofcastrated rats while Kar, Kamboj & Setty (1967) observed no alterations in the pituitary 'total' gonadotrophin content (mouse uterine weight method). In view of the variable results of these workers, the pituitary lh stores of the male rat were measured in the present study by a specific assay method using a reference standard. Male rats of the Holtzman strain were housed under standardized conditions of light (14 hr of artificial illumination) and temperature (72 to 75° F), and permitted free access to Purina chow and tap water. Progesterone (Sigma Chemical Co., St. Louis) was dissolved in corn oil and injected subcutaneously at the rate of 4 mg/day in 0-2 ml oil/injection for 13 days into 45-day-old (pre¬ puberal, Experiment 1 ) rats, and for 10 days into 55-day-old (puberal, Experi¬ ment 2) rats (Table 1). At autopsy, seminal vesicles and anterior pituitary glands were weighed. The latter were pooled within each group and kept frozen. Subsequently glands were bio-assayed for lh by the ovarian ascorbic acid depletion method of Parlow (1961) employing oestrogenized assay rats (Bogdanove & Gay, 1967). The details of the assay have been described else¬ where (Labhsetwar, 1969). The results show that progesterone administration resulted in atrophy of seminal vesicles (Table 1). This was associated in both experiments with over 40% decrease in the pituitary lh stores (Table 1). These results contrast with

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.

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.

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.

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...

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.

DAILY MEASUREMENT OF PITUITARY LH CONTENT DURING PREGNANCY IN THE RAT: DO CYCLIC CHANGES PERSIST?

Summary. Daily measurements of pituitary lh content during pregnancy in the rat, by the ovarian ascorbic acid depletion method, reveal no evidence of the regular changes manifested during the non-pregnant oestrous cycle. Instead, lh content rises from typical oestrous values on Day 1 to levels characteristic of cyclic pro-oestrus on Day 8. Pituitary lh content then exceeds pro-oestrous levels throughout the remainder of pregnancy until Day 21. Ovarian weight first decreases up to Day 10, then rises until the end of pregnancy. Ovarian histology reveals the presence of follicles of all sizes on each day, accompanied by increasing size of the corpora lutea of pregnancy. The significance of these observations and previous data in the literature concerning the pituitaryovarian axis during pregnancy are discussed from the point of view of the question of persistence of some manifestations of the oestrous cycle during pregnancy.

Luteinizing hormone concentration in pituitary and in blood plasma during the estrous cycle of the rat.

Simultaneous measurement of luteinizing hormone (LH) in the pituitary and in circulating blood was carried out by the ovarian ascorbic acid depletion method during the estrous cycle in rats showing a regular 4-day cycle under the controlledenvironment of 12 hrs. light (8a.m.-8p.m.) and 12 hrs. darkness. The pituitary LH content was relatively high in the morning and early afternoon of proestrus as compared with that in the diestrus, but it depleted suddenly at 5 p.m. of proestrus and continued to decline until the next morning. The plasma LH was detectable only in the afternoon of proestrus and in the morning of estrus, and the maximal concentration was detected at 7 p.m. of proestrus. The rats hypophysectomized before 4 p.m. of proestrus did not ovulate at all, but some of the rats hypophysectomized at 5 p.m. or 6 p.m. did ovulate and all the rats operated at 7 p.m.or later ovulated completely. The concentration of ovarian ascorbic acid in normal rats depleted markedly at 7 p.m. of proestrus. These results indicate that the ovulatory surge of LH occurs in the late afternoon of proestrus and its critical period is around 5 to 7 p.m. under our laboratory conditions. The relationship between the LH release and the ovarian steroids secretion is discussed.

RELEASING FACTORS IN HUMAN HYPOTHALAMIC AND NEUROHYPOPHYSIAL EXTRACTS

ABSTRACT Human hypothalamic tissues were defatted with acetone, extracted with 2N acetic acid, subjected to prolonged boiling and lyophilized. Acetone defatted human neurohypophysial tissues which included the pituitary stalk were extracted with acetic acid and lyophilized. The polypeptide materials in such extracts were concentrated by glacial acetic acid extraction, gel filtration on Sephadex and phenol extraction. Multiple assays were performed with these human hypothalamic extracts and purified human neurohypophysial preparations in order to determine their content of hypothalamic releasing factors. Luteinizing hormone-releasing factor (LRF) activity was determined by injection of test material into ovariectomized rats pretreated with estrogen and progesterone. The plasma of those animals was assayed for LH by the ovarian ascorbic acid depletion method. Thyrotropin-releasing factor (TRF) activity was estimated by measuring the release of 131I from the thyroids of mice pretreated with codeine and 1/μg ...

Luteinizing hormone activity of the hen's pituitary during the egg-laying cycle

Anterior pituitaries of hens in a regular laying cycle with 1-day pause between clutches were removed at various times in the period from ovulation of the terminal ovum (C t) of the clutch to 8 hours after ovulation of the second ovum (C 2). They were assayed for LH activity using the rat ovarian ascorbic acid depletion method. Two peaks of LH activity were found during the period between any ovulations. In the period from ovulation of the initial ovum (C 1) of the clutch to the C 2 ovulation, the first peak appeared 6 hours after the C 1 ovulation (20 hours before the C 2 ovulation) and the second peak 18 hours after the C 1 ovulation (8 hours before the C 2 ovulation). In the period between the C t and C 1 ovulations, the first peak appeared 2 hours after the C t ovulation (38 hours before the C 1 ovulation) and the second peak 18 hours after the C t ovulation (22 hours before the C 1 ovulation). This second peak was maintained at a fairly uniform level until 10 hours before the C 1 ovulation. The decrease in the LH level from the second peak may be closely related to ovulation. The delay of the decrease from the second peak may cause an inhibition of ovulation after the C t oviposition which results in a pause in the egg-laying cycle.

Effects of norethynodrel and mestranol on pituitary luteinizing hormone in the female rat.

The effects on the pituitary lh levels of norethynodrel (20 \g=m\g/ratorapproximately 0\m=.\1mg/kg daily) and mestranol (1 \g=m\g/rator approximately 5 \g=m\g/kgdaily) administered subcutaneously singly or in combination for 10 days to intact female rats have been studied. lh was assayed by the ovarian ascorbic acid depletion method of Parlow (1958). Norethynodrel caused a significant increase in hypophysial lh stores without causing ovarian atrophy or reducing compensatory hypertrophy in unilaterally ovariectomized post-pubertal rats. Mestranol administration, on the other hand, resulted in a significant ovarian atrophy without significant changes in the pituitary lh level, and also resulted in an inhibition ofcompensatory hypertrophy. The combined treatment resulted in effects essentially similar to those observed in the mestranoltreated group. No evidence for potentiation between the two steroids was obtained. Data support the conclusion that when the two steroids are administered together in the dosages used, the oestrogenic component is primarily involved in the pituitary lh inhibition.