Darhl Foreman

A modification of the Roe procedure for determination of fructose in tissues with increased specificity

Abstract A new modification of the procedure of Roe for determination of fructose concentrations in tissues is described. The modification shows no reaction with glucose or glucose phosphates. Tissues are homogenized in dilute perchloric acid, filtered, and reacted with alcoholic resorcinol and 30% HCl for 1 hr at 80°C. As little as 0.33 μg/ml of fructose may be reliably determined. Fructose phosphates react in this test but to a lesser extent than pure fructose.

DIABETES PREVENTS THE NORMAL RESPONSES OF THE OVARY TO FSH

FSH in vitro stimulates increased oxygen uptake by isolated follicular granulosa cells from immature rats treated with diethylstilbestrol (DES) when substrates are present (glucose, glutamate, pyruvate or fumerate) or are completely absent. However, when glucose is the only substrate or when any single substrate is omitted from the buffer, FSH has no effect. FSH in vitro also increases the uptake of glucose and the formation of 14CO2 from [1-6 14C]-glucose. Granulosa cells from diabetic immature rats treated with DES did not show increased oxygen uptake with in vitro FSH. Diabetic cells had similar receptor binding of FSH to that of control non-diabetic cells. The addition of both insulin and FSH in vitro to buffer with diabetic granulosa cells gave increased oxygen uptake over that of control cells from diabetic rats. The insulin stimulation of oxygen uptake by FSH in cells from diabetic rats was not duplicated by either epidermal growth factor (EGF) or insulin-like growth factor I (IGF-1). Follicle counts of ovaries from diabetic and control immature rats treated with DES showed increased atresia in the diabetic ovaries after only 44 hr. of diabetes. Follicle counts of ovaries from adult diabetic rats showed increased atresia in 24 hours after induction of diabetes at proestrus. Follicle counts of pseudopregnant rats showed increased atresia by 3 days after diabetes was induced. We conclude that diabetes prevents normal follicle growth stimulated either by exogenous DES or by endogenous hormones secreted during proestrus.

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle.

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones.

Plasma progesterone levels and corpus luteum morphology in the female prairie dog (Cynomys ludovicianus).

Plasma progesterone levels in female prairie dogs were determined by a radioimmunoassay specific for progesterone. Plasma progesterone levels were determined in samples taken before estrus, at estrus, during the luteal phase, and during anestrus from females maintained all year in the laboratory. Progesterone levels were also determined in plasma samples taken in the laboratory from two pregnant and three postparturient females captured in the field. Progesterone levels were low before estrus and continued low during estrus. They rose on the first week after estrus to 0.8 ng/ml or above and continued at or above this level for 9-14 weeks following estrus. Gestation in prairie dogs is 35 days in this species. Progesterone levels of three postparturient females were above 1.0 ng/ml for 7 weeks after their arrival in the laboratory. These females all had uterine scars showing that they had delivered their litters before they were captured. Two females were determined to be pregnant at the time of their capture. These females later reabsorbed their fetuses (determined by laparotomy). Progesterone values of samples from these females were all above 1.0 ng/ml except for one low value in one female which occurred 3 weeks after her capture and after reabsorption of her fetuses was in progress. The cells of the corpus lutea (CL) of nonpregnant, pregnant, and postparturient females had well-developed rings of cytoplasmic basophilia but as the CL regressed this pattern became disorganized and disappeared. The function of this basophilia is not known. The long luteal phase found in female prairie dogs is compared to those found in other species of mammals. This is the first annually breeding rodent reported to have a longer luteal phase that the period of gestation.

Effects of E2 levuglandins on the contractile activity of the rat uterus

The effects of E2 levuglandins on the contractile activity of rat uterine horns were studied. LGE2, AnLGE2, delta 9-LGE2 and the synthetic epimer, 8-epi-delta 9-LGE2 all induced contractions in a dose-response fashion. AnLGE2 gave decreased responses with increased bath concentrations. Paired comparisons showed potent and selective inhibitory effects of AnLGE2 on the uterotonic activity of prostaglandins. AnLGE2 inhibited the uterotonic activity of PGE2 at a 0.1:1 ratio, of PGD2 at a 1:1 ratio, but did not inhibit the activity of PGF2 alpha. Exposure of spontaneously contracting uteri to high concentrations of AnLGE2, or prolonged exposure to lower concentrations, suppressed contractions.

Anhydrolevuglandin D2 inhibits the uterotonic acivity of prostaglandins F2α and D2

Abstract Anhydrolevuglandin D 2 (AnLGD 2 ), which is produced from PGH 2 by a water-induced rearrangement and subsequenct dehydration, is uterotonic. However, increasing concentrations caused decreased responses of the uterine horns. AnLGD 2 inhibited responses of uteri to stimulation by specific prostaglandins. PGF 2α was inhibited at an AnLGD 2 :PGF 2α ratio of 0.05:1 with 5 to 25 pg/ml concentrations of PGF 2α . The response to PGD 2 was inhibited at an AnLGD 2 :PGD 2 ratio of 0.05:1 with PGD 2 concentrations of 5 to 75 pg/ml. In contrast, the uterotonic effects of PGE 2 werer not inhibited by AnLGD 2 . When AnLGD 2 was added to baths with contracting uteri it inhibited contractions less if the exposure period was 5 min than if it was 10 min. The longer exposure times produced prolonged inhibition of contractile activity with bath concentrations of AnLGD 2 as little as 2.5 pg/ml.

Inhibin is present in the corpus luteum of pseudopregnant rats and can interact with progesterone to inhibit resumption of estrous cycles.

The corpora lutea (CL) of ovaries of adult pseudopregnant (PSP) rats were stained by immunohistochemistry for inhibin alpha subunit using sheep alpha-chain1-27 antibody as a measure of inhibin content. Staining was light in estrous cycle CL (stage III), increased in Day 2 and Day 4 PSP CL and reached maximal density on day 6 PSP when both large cells and many small cells stained. Staining was decreased in CL from 8 and 10 day PSP. Granulosa cells of growing follicles showed light stain on days 8 and 10 of PSP. Adult female rats were treated for 5 days beginning at estrous cycle Stage III (cornified masses). In Experiment 1, three groups of rats were used: 1. implanted with 30mm progesterone capsules and injected with saline, 2. implanted with 30mm progesterone capsules and injected with 10 ng rh inhibin A daily, 3. implanted with empty capsules and injected with 10 ng rh inhibin A daily. Both groups of rats with progesterone implants had significantly slower return of cycling after removal of the capsules than did the rats injected with 10ng rh inhibin A alone. In Experiment 2, the same treatments were given except that 20ng of rh inhibin A (10ng 2x/day) was given to both inhibin treatment groups. Rats treated with progesterone and 20ng inhibin had a significantly slower return of cyclic activity (4.4 days) over that found in the other two treatment groups. We conclude that the absence of cyclic activity in PSP rats is possibly due to the secretion of both inhibin and progesterone by the CL.

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH in vitro, but not LH, increased the O2 uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O2 uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O2 uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP in vitro increased O2 uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O2 uptake in cells from 23 day old rats treated with DES alone.

The effects of gonadotrophins on the metabolism of ovaries from prairie dogs (Cynomys ludovicianus)

Abstract The oxygen uptake of ovaries from prairie dogs was significantly higher during Stage III of the estrous cycle than during the long anestrous period. Injections of FSH into anestrous prairie dogs increased the oxygen consumption of their ovaries. Hemiovariectomy of anestrous animals for 1 week during the anestrous period did not increase either the weight or oxygen consumption of their remaining ovaries. The injection of FSH into hemiovariectomized prairie dogs increased both the weight and oxygen consumption of the remaining ovary. The addition of substrates of the tricarboxylic acid cycle in vitro increased the oxygen consumption of ovaries from anestrous animals. Armour FSH added alone or two preparations of NIH FSH added in the presence of substrates in vitro increased the oxygen consumption of anestrous ovaries. NIH LH S1 added in vitro in the presence of substrates did not increase oxygen consumption. NIH FSH and NIH LH B1 acted together to increase the oxygen consumption in the absence of substrates.

Evidence that the prostaglandin E2 receptor and the anhydrolevuglandin E2 receptor in the rat uterus are the same receptor

Anhydrolevuglandin E2 (AnLGE2) is closely related to prostaglandin E2 (PGE2) and has been found to inhibit the uterotonic activity of PGE2. The binding of PGE2 and its inhibition by AnLGE2 has been determined in rat uterine membrane fractions. AnLGE2 inhibited the binding of 3HPGE2 in a dose related fashion. 3HAnLGE2 also binds to rat uterine membrane fractions and its binding is inhibited by PGE2 in a dose related fashion. These data support previous physiological observations that AnLGE2 inhibits the actions of PGE2 by acting at the PGE2 receptor. Thus, AnLGE2 appears to be a specific inhibitor of PGE2 actions at its uterine receptors.