Oestrogen and progesterone interactions in the control of gonadotrophin and prolactin secretion

Oestrogen and progesterone have marked effects on the secretion of the gonadotrophins and prolactin. During most of the oestrous or menstrual cycle the secretion of gonadotrophin is maintained at a relatively low level by the negative feedback of oestrogen and progesterone on the hypothalamic-pituitary system. The spontaneous ovulatory surge of gonadotrophin is produced by a positive feedback cascade. The cascade is initiated by an increase in the plasma concentration of oestradiol-17 beta which triggers a surge of luteinizing hormone releasing hormone (LHRH) and an increase in pituitary responsiveness to LHRH. The facilitatory action of oestrogen on pituitary responsiveness is reinforced by progesterone and the priming effect of LHRH. How oestrogen and progesterone exert their effects is not clear but the facilitatory effects of oestrogen take about 24 h, and the stimulation of LHRH release is produced by an indirect effect of oestradiol on neurons which are possibly opioid, dopaminergic or noradrenergic and which modulate the activity of LHRH neurons. In the rat, a spontaneous prolactin surge occurs at the same time as the spontaneous ovulatory gonadotrophin surge. The prolactin surge also appears to involve a positive feedback between the brain-pituitary system and the ovary. However, the mechanism of the prolactin surge is poorly understood mainly because the neural control of prolactin release appears to be mediated by prolactin inhibiting as well as releasing factors, and the precise role of these factors has not been established. The control of prolactin release is further complicated by the fact that oestradiol stimulates prolactin synthesis and release by a direct action on the prolactotrophes. Prolactin and gonadotrophin surges also occur simultaneously in several experimental steroid models. A theoretical model is proposed which could explain how oestrogen and progesterone trigger the simultaneous surge of LH and prolactin.     

Luteinizing and ovulatory action of oestradiol in the pregnant rat

Oestradiol injection on Day 10 of pregnancy in rats, resulted in either ovulation or luteinization in 50% of cases on Day 12. Cytological data showed that the number of pituitary LH cells decreased significantly on Day 11 in all oestradiol-treated animals whether responsive or not to oestrogen by ovarian modifications, while the number of pituitary FSH cells only decreased significantly in females with characteristic ovarian signs of preovulation. Bioassay of pituitary FSH confirmed the cytological data. It is concluded that ovulation and luteinization only occurred in the pregnant rat when oestradiol triggered off a synchronous release of LH and FSH.     

Proceedings: Action of chronically administered oestradiol on the uterus of the rat

Oestradiol induces increased synthesis of RNA and DNA in the uterus of ovariectomized rats. The effects of continuously administered oestradiol on nucleotide synthesis in the uterus of the rats are reported. Ovariectomized rats were given 2 Mug oestradiol-17 beta, subcutaneously, every 8 hr until autopsy at various times 1 to 7 days after the first injection of oestradiol. (3H) uridine or (3H) thymidine was administered intraluminally 15 min before beath. Uteri were processed for autoradiography. The number of labelled cells and the average number of grains/cell were counted. (3H) uridine labelling reached a peak at 6 to 54 hr and then decreased steadily thereafter. DNA synthesis was maximal at 48 hr in all regions and minimal at 144 hr. These results indicate that oestrogen caused maximum stimulation of RNA synthesis in the rat uterus at 30 and 48 hr respectively but activity was reduced thereafter. The uterine epithelium and stroma were hypertrophied and hyperplastic when RNA and DNA synthesis were minimal. This could be due to refractoriness of the specific target tissues to continued hormonal stimulation.     

Inhibitory effects of Celiptium on thymidine kinase synthesis induced in the uterus by 17 beta-estradiol

Inhibitory effects of Celiptium on the thymidine kinase synthesis induced by oestradiol-17 beta in the rat uterus. In the rat uterus, the synthesis of thymidine kinase specifically induced by oestradiol-17 beta was inhibited by Celiptium. The synthesis was totally inhibited when the drug was administered before the oestrogen and partially when it was administered after. These facts suggested that Celiptium was competitive to the acceptor sites for oestradiol-receptors and inhibited the expression of the thymidine kinase gene.     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

OESTROGEN EFFECTS ON BRAIN AND PITUITARY ENZYME ACTIVITIES

Abstract— Ovariectomized female rats were treated daily with oestradiol-17β benzoate for intervals up to one week and enzyme activities were measured in the pituitary and various brain regions. Brain regions were selected for study on the basis of their previously demonstrated content of putative oestradiol receptor sites. (1) Pituitary showed oestrogen-dependent increases in glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and lactic dehydrogenase (LDH), and no change in NADP⁺-dependent isocitric dehydrogenase (ICDH), NADP⁺-dependent malic dehydrogenase (MDH) or hexokinase (HK). MDH and ICDH were elevated in whole hypothalamus. Enzyme activities did not change significantly in whole amygdala, cerebral cortex, or hippocampus. (2) Sub-regions of the preoptic area, hypothalamus and amygdala were dissected to obtain more highly concentrated populations of cells containing putative oestrogen receptor sites. In the basomedial sub-region of hypothalamus, activities of MDH, ICDH and G6PDH were elevated by oestrogen treatment. In the corticomedial sub-region of amygdala, MDH and ICDH were elevated by oestrogen treatment. No change was observed in any of the six enzymes in medial preoptic area. (3) Increases in enzyme activities were related to the total in vivo dose of oestradiol benzoate given. (4) Hypophysectomy or adrenalectomy did not prevent the enzymatic responses to oestrogen. (S) Oestrogen added directly to the enzyme incubation medium did not change enzyme activities. (6) Weight loss in ovariectomized rats due to reduced food intake did not increase enzyme activities. (7) In the pituitary, good correlation was obtained between the known receptor binding properties of various oestrogenic and non-oestrogenic steroids and the elevation in G6PDH activity. The results indicate that oestradiol acts directly to cause changes in activities of some brain and pituitary enzymes. The possibility is discussed that these changes may result from oestrogen interaction with putative receptor sites found in pituitary and certain brain regions.     

CHANGES OF THYMIDINE KINASE IN THE DEVELOPING RAT BRAIN

Abstract— Thymidine kinase (ATP: thymidine-5'-phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6-day-old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM-ATP and 2.5 mM-MgCl₂ for maximum activity. The activity was completely inhibited by addition of 1.8 mM-TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. K_m was 2.8 × 10⁻⁶ M for [6-³H]thymidine.
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity.     

3H]Oestradiol metabolism by 18-day rat interstitial cells in culture and the effect of FSH: presence of 16 alpha-hydroxylase

The metabolism of [3H]oestradiol by interstitial cells in culture prepared from 18-day old rat testes was investigated. Interstitial cells were able to convert [3H]oestradiol to [3H]oestriol as confirmed by recrystallization of oestriol to constant specific activity from samples containing cells and not from controls. This demonstrated for the first time the presence of 16 alpha-hydroxylase in rat testicular interstitial cells. The effect of in vitro FSH treatment on the cells in culture was also investigated. FSH failed to affect 16 alpha-hydroxylase activity since we could not demonstrate a significant difference between treated and untreated preparations. The 16 alpha-hydroxylation of phenolic steroids is widely regarded as the major pathway of oestrogen metabolism in mammals. This metabolic step significantly reduces the biological activity of oestradiol. The presence of 16 alpha-hydroxylase in interstitial cells suggests that it may play a role in inactivating the oestradiol that is produced in the Leydig cells and thus prevent its intracellular accumulation. Such activity may conceivably play a role in the overall local "fine tuning" of androgen biosynthesis.     

Regulation and role of the insulin-like growth factor I system in rat luteal cells.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor beta mRNA contents were determined by semiquantitative RT-PCR. The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor beta. Furthermore, whereas IGF-I upregulated oestrogen receptor beta expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor beta. Luteal cells with increased oestrogen receptor beta can respond fully to oestradiol, leading to cell hypertrophy.     

Catechol oestrogens stimulate and direct prostaglandin synthesis

We have tested the action of a catechol oestrogen -2,3,17β- trihydroxy oestra-1,3,5 (10)-triene (2-OH oestradiol) in stimulating prostaglandin (PG) production by an homogenate of rat uterus. Marked and dose dependent stimulation was observed in PGF_2α and PGE₂ production using 20–250 μM concentrations of catechol oestrogen; a concentration of 250 μM 2-OH oestradiol resulted in a 23 fold increase in PGF_2α production with a 50% reduction in the synthesis of 6-keto PGF_1α. Tryptophan, catechol and glutathione were without effect on PGF_2α and PGE₂ production whereas adrenalin stimulated the production of all PGs, although the increase was less than that seen with 2-OH oestradiol. Oestradiol had a slight stimulatory action on PGF_2α production which reached a maximum at around 40 μM but had a more marked stimulation of 6-keto PGF_1α formation. Stimulation of prostaglandin production by oestradiol and 2-OH oestradiol showed no variation at different stages of the rat oestrous cycle. The use of 5 to 100 mg of tissue/ml gave similar product distribution although the effect of catechol oestrogen both in terms of stimulation of E and F formation (expressed per mg of tissue) and in its action on product distribution was more marked at lower concentrations of tissue.     

The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle.

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.     

Effect of neonatal exposure to the antioestrogens nafoxidine and CI-628 upon the development of the uterus in the prepubertal rat

Neonatal Sprague-Dawley rats were injected with the antioestrogens nafoxidine or CI-628 on Day 3 of life alone or in combination with oestradiol benzoate 24 h later. Oestrogen-stimulated glucose oxidation and cytoplasmic oestrogen binding sites of the uteri were assessed at 21-23 days of age. Neither antioestrogen antagonized the prepubertal uterine impairments produced by neonatal oestradiol treatment. Both antioestrogens administered alone produced deficits which mimicked those produced by neonatal oestrogenization. However, the agonist property of each antioestrogen was differentially expressed: treatment with CI-628 reduced prepubertal oestrogen binding sites in the uterus, but nafoxidine exposure decreased the sensitivity of the uterus to oestradiol stimulation of glucose oxidation. It is postulated that CI-628 directly affects the uterus to reduce production of oestrogen receptor protein, while nafoxidine affects the development of the uterine phosphogluconate oxidative pathway indirectly through impaired ovarian function. However, antioestrogens blocked the neonatal oestradiol-induced reduction in the oestrogen-stimulated production of actomyosin in the adult uterus. Therefore, while both CI-628 and nafoxidine are clearly agonists in the neonatal rat, each appears to exhibit cell-specific agonist and antagonist properties.     

Thymidine kinase of rat liver. Activation by phospholipase C and subcellular distribution in the liver

1. The thymidine kinase activity of rat liver is greatly enhanced on addition of phospholipase C to the assay mixture. 2. Most of the thymidine kinase activity of the liver is recovered in the mitochondrial and in the impure ;nuclear' fractions. No activity was detected in purified nuclei prepared in high-density sucrose. 3. A substantial thymidine kinase activity could be detected, with the aid of phospholipase C, in all rat tissues examined.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Both GPER and membrane oestrogen receptor‐α activation protect ventricular remodelling in 17β oestradiol‐treated ovariectomized infarcted rats

Clinical and experimental studies have established that gender is a factor in the development of ventricular hypertrophy. We investigated whether the attenuated hypertrophic effect of oestradiol was via activation of phosphatidylinositol 3‐kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) through non‐genomic action. Twenty‐four hours after coronary ligation, female Wistar rats were randomized into control, subcutaneous oestradiol treatment or a G‐protein coupled oestrogen receptor (GPER) agonist, G‐1 and treated for 4 weeks starting from 2 weeks after bilateral ovariectomy. Ventricular hypertrophy assessed by cardiomyocyte size after infarction was similarly attenuated by oestradiol or G‐1 in infarcted rats. The phosphorylation of Akt and eNOS was significantly decreased in infarcted rats and restored by oestradiol and G‐1, implying the GPER pathway in this process. Oestradiol‐induced Akt phosphorylation was not abrogated by G‐15 (a GPER blocker). Akt activation was not inhibited by actinomycin D. When a membrane‐impermeable oestrogen‐albumin construct was applied, similar responses in terms of eNOS activation to those of oestradiol were achieved. Furthermore, PPT, an ERα receptor agonist, activated the phosphorylation of Akt and eNOS. Thus, membrane ERα receptor played a role in mediating the phosphorylation of Akt and eNOS. The specific PI3K inhibitor, LY290042, completely abolished Akt activation and eNOS phosphorylation in infarcted hearts treated with either oestradiol or oestradiol + G‐15. These data support the conclusions that oestradiol improves ventricular remodelling by both GPER‐ and membrane‐bound ERα‐dependent mechanisms that converge into the PI3K/Akt/eNOS pathway, unveiling a novel mechanism by which oestradiol regulates pathological cardiomyocyte growth after infarction.     

Rapid and sensitive detection of oestrogen receptors in cells and tissue sections by autoradiography with 125I-oestradiol

Summary The presence of receptors for steroid hormones in individual cells and tissue sections was assessed within 4–24 h using dry mount autoradiography with radio-iodinated oestradiol. Low affinity and nonspecific binding of steroids were significantly reduced by washing the cells or sections with diluted antiserum to oestradiol.For cells of the MCF-7 cell line variations in grain density were observed, indicating that cells of the MCF-7 cell line are heterogenous with respect to their cellular receptor concentrations of oestrogen receptors. Receptor-negative cells, such as peritoneal macrophages, did not retain oestradiol label.In tissue sections of rat and calf uterus, predominant labelling was observed on the endometrial gland cells and stroma.Oestradiol receptor binding in the uterus cytosol for both radio-iodinated and tritiated oestradiol showed the same qualitative characteristics as determined by sucrose gradient sedimentation profiles and a comparable amount of binding sites was found for both labels. The relative binding affinity of¹²⁵I-oestradiol compared to [³H]oestradiol is about 70–80%.The dry mount autoradiographic technique as presented can be used for rapid screening of heterogeneiety in oestrogen receptor distribution in cells and tissue sections, since this technique reveals differences in receptor concentrations on the single cell level.     

Regulation of follicular development by diethylstilboestrol in ovaries of immature rats

Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.     

Oestrogen synthesis by the foetal rat testis in organ culture

Testes from 19- to 21-day old rat foetuses were bisected and cultured in the presence of tritiated testosterone, androstenedione or dehydroepiandrosterone as precursors for oestrogen biosynthesis. Oestrone and oestradiol formed were identified by recrystallization to constant specific activity, and their conversion rate was determined after isotopic dilution. Both oestrogens formed from either precursor, their conversion rate being about 0.05% for oestradiol and 0.015% for oestrone.     

Differences in the induction of thymidine kinase isozymes in estrogen-treated immature and adult rats

Thymidine kinase activity in immature and castrated adult rat uterus has been examined in respose to estrogen treatment. Following estrogen administration. it was found that immature uterine thymidine kinase activity was increased 30-fold after 24 h, but almost no effect was produced on castrated or non-castrated adult uterus. Uterine thymidine kinase activity was separated into three peaks (peak 1, 2 and 3) by means of DEAE-cellulose column chromatography. In response to estrogen, the thymidine kinase isozymes differed in adult and immature uteri. In immature uteri, marked and selective increase of the activity was found in peak I, whereas in adult only a slight increase in peak 2 activity was observed. The thymidine kinase activity in peak 1 and peak 2 were found to have different enzymatic properties and molecular weight, as determined by gel filtration of 125 000 for peak 1 and 100 000 for peak 2.From these results, it is suggested that estrogen induces specific thymidine kinase isozyme in immature uterus and that the isozyme may be involved in DNA synthesis. Such a induction mechanism seems to be lost during the development.     

Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia.

The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.