Donald C. Johnson

Phospholipase A2 activity in the rat uterus: Modulation by steroid hormones

Phospholipase A2 (PLA2), an enzyme which provides free arachidonic acid for the synthesis of prostaglandins (PG), has been studied in the rat uterus under various experimental conditions. Uterine PLA2 activity increased 14 fold in hypophysectomized rats implanted with Silastic capsules containing estradiol-17 beta as compared to those treated with oil vehicle. Dexamethasone treatment reduced the PLA2 activity induced by estrogen by 78%. Hypophysectomized animals treated with progesterone (2mg/day) for 5 days had low levels of uterine PLA2 activity but a single injection of estradiol (10 micrograms/rat) given 24 h after the last injection of progesterone increased activity 5 fold within 12 h. Administration of the protein synthesis inhibitor cycloheximide in the rats treated with progesterone, before and after injection of estradiol, prevented the stimulating action of the estrogen on PLA2 activity. If the estrogen was given at the time of the last injection of progesterone, PLA2 activity did not increase until 22 h late and the level was much less than when progesterone was absent. The results are consistent with the view that estrogen stimulates uterine prostaglandin production because of its effect upon PLA2; this effect can be greatly reduced by a glucocorticoid. Progesterone may modulate the PLA2 stimulating effect of estrogen in order to direct the production of specific PGs by regulating the amount of arachidonic acid available for PG synthetase.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estrous cyclicity and ovulation in female Sprague-Dawley rats

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on estrous cyclicity and ovulation in Sprague-Dawley rats was examined. TCDD at a single oral dose of 10 micrograms/kg resulted in irregularity of cycles, characterized mainly as prolonged periods of diestrus. In rats cycling normally this dose of TCDD reduced the ovulatory rate and the number of ova recovered. The results suggest that TCDD is a reproductive toxin also in female rats, the mechanism of which is as yet unknown.

A review of mechanisms controlling ovulation with implications for the anovulatory effects of polychlorinated dibenzo-p-dioxins in rodents

Polychlorinated dibenzo-p-dioxins (PCDDs) can impinge on female fertility by preventing ovulation. In this review, the aspects of normal ovulatory physiology most relevant to our current understanding of PCDD action on the ovary are briefly reviewed. This is followed by a comprehensive assessment of data relevant to the effects of PCDDs during ovulation in the rat. PCDDs interrupt ovulation through direct effects on the ovary in combination with dysfunction of the hypothalamo-hypophyseal axis.

Ovulation, Ovarian 17α-Hydroxylase Activity, and Serum Concentrations of Luteinizing Hormone, Estradiol, and Progesterone in Immature Rats with Diabetes Mellitus Induced by Streptozotocin

Abstract Immature female rats were injected with streptozotocin (60 mg/kg, iv) 3 to 4 days prior to the injection (iv) of 20 IU of pregnant mares serum gonadotropin (PMS). Animals were killed at various intervals and the serum levels of estradiol, luteinizing hormone (LH), and progesterone were determined by radioimmunoassays. The ovarian steroid 17α-hydroxylase activity was determined by a tritium exchange assay using pregnenolone as the substrate. Ovulation was determined 72 hr after PMS by flushing of the oviducts. The diabetes mellitus induced by the drug reduced the number of animals ovulating and in some animals the number of ova shed when compared to controls. However, a surge in LH, which reached a peak at 60 hr, was seen in the diabetic animals; a larger peak with the same timing was found in the controls. Changes in ovarian 17α-hydroxylase also indicated that an increase in LH release occurred in the diabetic animals at about 60 hr. Estradiol levels were higher, but progesterone levels lower, in diabetic than control animals. Administration of 1 mg of progesterone to diabetic animals 48 hr after PMS resulted in an increase in the number of animals ovulating and the number of ova shed. The results indicate that hyperglycemia induced by streptozotocin is not inconsistent with production of an LH surge or with ovulation following ovarian stimulation by PMS. However, the lowered production of progesterone, which may be a cause or a result of lowered LH output, appears to be a primary factor in the reduced ovulatory rate.

Differential Effects of Dichlorodiphenyltrichloroethane Analogs, Chlordecone, and 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Establishment of Pregnancy in the Hypophysectomized Rat

Abstract Many of the organochlorine pesticides have been shown to elicit estrogenic responses in laboratory animals. Two estrogenic actions, initiation of implantation and maintenance of pregnancy, were examined in progesterone-primed, delayed-implanting, hypophysectomized rats exposed to several polychlorinated hydrocarbons. The insecticide P,P′-dichlorodiphenyltrichloroethane (DDT) was nearly devoid of estrogenic activity for initiating implantation, as was a dichloro analog, 1.1-dichloro-2-[p-chlorophenyl],2-[o-chlorophenyl]ethane (O,P′-DDD), but another such analog, 1,1-dichloro-2-(p-chlorophenyl),2-(o-chlorophenyl)ethylene (O.P′-DDE), was nearly as estrogenic as the O,P′-DDT isomer of DDT and the methoxylated analog methoxychlor. The latter three compounds not only initiated implantation, but maintained pregnancy when given in large (200 mg/kg) and repeated doses. Another insecticide, chlordecone (Kepone) was more estrogenic than any of the DDT analogs and maintained pregnancy with a single dose of 50 mg/kg. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a toxic contaminant of herbicide production, did not induce implantation at a dose of 125 μg/kg, but inhibited the implantation initiated by estrone in 35% of the animals. The mechanism of this anties-trogenicity is unknown but most probably does not involve direct action via the classical estrogen receptor. The possible interference with the normal blastocyst-uterine interactions of these polychlorinated xenobiotics may be an important factor in their being considered reproductive toxins.

Temporal Augmentation of LH by Prolactin in Stimulation of Androgen Production by the Testes of Hypophysectomized Male Rats

Summary Sex accessory organ weight was used as an index of androgen production by the testes of hypophysectomized male rats treated with ovine LH for 5 days. Prolactin augmented the testicular response to LH to a greater extent when both hormones were given simultaneously, but at different sites, early in the dark period than early in the light period. The effect of testosterone upon the accessories was not augmented by prolactin. The results suggest that the sensitivity to tropic hormone stimulation is rhythmic and that gradients in function may have a temporal relationship to the periodicity of tropic hormone release by the pituitary. The ovine LH and prolactin used were kindly supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases, NIH.

Endogenous versus exogenous glucocorticoid responses to experimental bacterial sepsis

Although lack of adrenals dramatically reduces resistance against sepsis generally, the value of glucocorticoid levels above those normally produced by stress remains controversial. An early and long‐held concept is that glucocorticoid protection against lipopolysaccharides in animal models is important. Supporting this concept, C3H/HeJ mice, lacking Toll‐like receptor‐4 (TLR‐4), and consequently, endotoxin hyporesponsive, have recently been shown to be resistant to glucocorticoid protection against live Escherichia coli. Effective antibiotic intervention, as an additional parameter and with concomitant administration of glucocorticoid, not only allows for expected antibiotic protection but also for glucocorticoid protection against E. coli or Staphylococcus aureus of mice sensitized to tumor necrosis factor α, regardless of the status of the TLR‐4 receptor. TLRs, including but not limited to TLR‐2, may be involved in glucocorticoid protective efficacy against Gram‐positive and Gram‐negative sepsis. Overlapping and possibly endotoxin‐independent signaling may become important considerations.

A method for the preparation of steroid-protein antigens for use in immunoassay of steroids

Abstract A general method for the preparation of steroid-protein conjugates coupled through carbon 6 of Δ 4 steroids was demonstrated using 6β Br-testosterone acetate and 6β Br-progesterone covalently bound to sulfhydryl-group-enriched bovine serum albumin. The antigenicity of the conjugates and the specificity of the antibodies were evaluated. With anti-progesterone the assay accuracy was determined to be 10.3%, the intra-assay precision was 8.7% and the inter-assay precision was 12.6%. The material was used to assay the progesterone content of normal human male and female serum as well as the changes in this hormone following corpora lutea induction in constant-estrus androgenized female rats.

Release of prostaglandins and leukotrienes from the rat uterus is an early estrogenic response.

The early estrogenic responses are considered to be involved in inducing embryo implantation in a progesterone (P4)-primed uterus. Because of their involvement in the process of implantation and decidualization, prostaglandins (PGs) and leukotrienes (LTs) could be the mediators of early estrogenic responses in a P4-primed uterus. Therefore, temporal effects of estrogen on the production and/or release of PGF2, PGF2 alpha, LTB4 and LTC4 by the P4-primed uterus of hypophysectomized rats were examined. Hypophysectomized mature female rats were injected for 4 days with P4 (2 mg/rat, s.c.) or with P4 plus a single injection of estradiol-17 beta (E2) (100 ng or 200 ng/rat, i.v.) on the last day of P4 treatment. In one set of experiments, animals were killed at 0.5, 2, 4, 8, 12 and 30th after the last steroid treatment. The production of PGs and Lts by uterine homogenates was measured by radioimmunoassays (RIAs). The production of PGE2 and PGF2 alpha in P4-treated animals showed peaks at 2, 6 and 12h. The superimposition of E2 on P4 treatment induced a higher production rate of PGE2 and PGF2 alpha at 0.5h and abolished the peaks induced by P4 at 2h, but not the peaks at 6 or 12h. Irrespective of the kind of steroid hormonal treatments, uterine production of LTs showed a rapid decline between 6 and 8h followed by a sharp rise at 12h. The superimposition of E2 on P4-treatment again increased the production rates of LTB4 and LTC4 at early hours, i.e. at 0.5 and 2h, respectively, as compared to P4 treatment only.

The effects of intrauterine position on competition and behavior in the mouse

The purpose of the present study was to examine the effect of intrauterine position on: (1) competition for limited food resources when the animals were previously deprived of food; (2) social preference for a male during various phases of the estrous cycle; and (3) social and sexual behavior when the animals were provided with like-treated females or receptive females, respectively. Females developing in utero between two males (2M females) and females developing between two females (0M females) were different on only one measure recorded during the competition for limited food, i.e., gaining control of the food pellet, with the 0M females outcompetiting the 2M females. When given a choice between a male or a female neither 2M or 0M females in estrus showed a preference for the male. The results also indicated that 2M females were significantly more aggressive and more likely to show male sexual behavior when compared to 0M females.