Effects of septal lesions on the lordotic behavior of weanling male and female rats

Twenty-four-day-old weanling male and female rats were either lesioned in the septal area, gonadectomized, lesioned and gonadectomized, or not treated. Tests for lordotic behavior were carried out at 27 and 28 days of age after priming with estradiol alone and with estradiol plus progesterone. A second set of tests for lordotic behavior was carried out at 47 and 48 days of age. In the interim period, some of the animals were given chronic estrogen treatment. In tests given at 27 days of age, septal lesions facilitated lordotic responding after estrogen priming; no differences were observed between male and female animals. At 47 and 48 days of age, however, unless male rats had been exposed to chronic estrogens following septal lesioning, no facilitation of estrogen-induced lordotic behavior occurred. In addition, it was found that female rats gonadectomized at 24 days of age and given no exposure to estrogens between the tests at 27 and 28 days and those at 47 and 48 days of age showed reduced sensitivity to estrogens, as compared to normal or estrogen-treated females, whether lesioned or not.     

Immunohistochemical evidence for the presence of progesterone receptor in rat submandibular glands.

Immunohistochemical analysis of progesterone receptor was carried out in rat submandibular glands. Immunoreactivity to progesterone receptors was found in cell nuclei of the intralobular duct system within male and female rat submandibular glands. The female glands contained more immunoreactive cells than the male glands. In ovariectomized rats progesterone receptor-containing cells decreased in number while testectomized glands revealed an increase. When estradiol was administered to gonadectomized rats of both sexes, the immunoreactivity in cells of the intralobular duct system markedly increased. These results suggest the possibility that progesterone may modulate various metabolic functions in the rat submandibular glands.     

Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat

The effect of gonadectomy and treatment with sex-steroids on renal prostaglandin 9-ketoreductase activity in 10-11 week old male and female rats was determined. Rats were gonadectomized or subjected to sham operation at 3 weeks of age. During week 7, rats were injected s.c. twice over a 6-day interval with vehicle (peanut oil, 0.5 ml X kg-1) or with depot forms of testosterone (5 mg X kg-1), estradiol (0.02 mg X kg-1), progesterone (5 mg X kg-1), or estradiol and progesterone combined. Renal prostaglandin 9-ketoreductase activity was about 50% higher in female rats than in males. Gonadectomy decreased 9-ketoreductase activity in females, but not in males, and eliminated the gender difference in enzyme activity. Treatment with estradiol elevated 9-ketoreductase activity in males and females, while treatment with testosterone or progesterone was without effect. Progesterone did, however, antagonize the elevation in 9-ketoreductase activity produced by estradiol.     

Effects of estradiol benzoate and progesterone on superoxide dismutase activity in the thymus of rats

The activity of mitochondrial superoxide dismutase (MnSOD) and cytosol superoxide dismutase (CuZnSOD) was measured in corresponding subcellular fractions prepared from the thymi of intact and chronically gonadectomized (GX) rats of both sexes, as well as of GX male and female rats injected subcutaneously with a single dose of 5 microg estradiol benzoate (EB) and/or 2 mg progesterone (P). Animals were sacrificed 2 h or 24 h following hormone treatment. In the females, the activity of MnSOD in the thymus was stable during the estrous cycle and did not change after ovariectomy. Treatment of GX females with estradiol benzoate resulted 2 h later in a significant elevation of MnSOD activity, whereas 24 h later the activity returned back to control values. On the other hand, treatment of GX females with progesterone had no effect on the MnSOD activity. However, combined hormone treatment, in which EB injection preceded progesterone injection by one hour, enhanced the effect on MnSOD activity similar to that of estradiol benzoate alone. The activity of CuZnSOD in cycling rats was increased in proestrus, whereas removal of the ovaries kept the values at low diestrus and estrus levels. Contrary to MnSOD, CuZnSOD activity did not change after EB treatment of GX females, while progesterone increased the enzyme activity at 2 h and 24 h after hormone treatment. However, combined EB+P treatment proved to be ineffective. In the males, neither MnSOD nor CuZnSOD activity was affected by the removal of testes or by progesterone treatment of GX animals. Only EB injection to GX rats significantly increased CuZnSOD activity 24 h later.     

Sex-related differences in the hepatobiliary transport of phenolsulfonphthalein in the rat

Sex-related differences in the hepatobiliary transport of phenolsulfonphthalein (PSP) were investigated in male and female Wistar rats. Maximal biliary excretion of unconjugated PSP was significantly higher in females while the excretion of the conjugated dye and liver UDP-glucuronosyltransferase activity toward PSP were higher in male animals. Orchidectomy decreased enzyme activity and excretion of the conjugate, whereas ovariectomy produced the opposite effect. Both in gonadectomized males and females maximal biliary excretion of the unconjugated dye was significantly reduced. Testosterone treatment increased the excretion of both conjugated and unconjugated PSP and transferase activity in orchidectomized males. Combined treatment of gonadectomized females with estradiol plus progesterone led to excretions of both conjugated and unconjugated PSP and UDP-glucoronosyltransferase activities similar to those found in control rats. These data indicate the existence of sex-related differences in the conjugation and biliary excretion of PSP in the rat and its modulation by sex hormones.     

Long-term ovariectomy and hormone-induced sexual behavior, progestin receptors, and hypothalamic morphology in female rats

Long-term ovariectomy reduces the ability of estradiol and progesterone treatment to induce sexual receptivity in female rats. Previous researchers suggested that this effect may be due to a decreased induction of neural progestin receptors by estradiol in the long-term ovariectomized rats. The present study was designed to replicate and extend this finding, and to search for neuroanatomical correlates by measuring the volume of the ventromedial nucleus (VMN) of the hypothalamus, a putative site of action of estradiol and progesterone for the induction of female sexual behavior. Long-term ovariectomy (5 to 6 weeks) as compared to short-term ovariectomy (1 week) reduced the ability of estradiol-17 beta and progesterone treatment to induce sexually receptive and proceptive behaviors. Consistent with previous reports, our data show that the reduced levels of cytosol progestin receptors after long-term ovariectomy and estradiol treatment are related to a reduced ability of estradiol to induce the receptors. Long-term ovariectomy did not affect the concentration of cytosol progestin receptors in the preoptic area, suggesting a neuroanatomical specificity to this effect. Contrary to our predictions, long-term ovariectomy did not affect the volume of the VMN. In fact, estradiol treatment, while blocking the effect of long-term ovariectomy on sexual behavior, decreased the volume of the VMN. Therefore, the measurement of the volume of the VMN is not a good predictor of the responsiveness to steroid hormone induction of sexual behavior.     

Influence of photoperiod and gonadal steroids on hibernation in the European hamster

Torpor was monitored daily in adult male and female European hamsters (Cricetus cricetus) induced to hibernate by exposure to a cold environment (6 degrees C). The effect of photoperiodic manipulations or administration of exogenous gonadal steroids was examined in gonadectomized or intact hamsters. 1. Gonadal regression occurred in all short day, but only in some long day, cold-exposed hamsters. Entry into hibernation was not observed until reproductive regression had occurred. Thus, gonadal atrophy appears to be a necessary precondition for hibernation. 2. Castrated hamsters in the short day cold condition showed a significantly greater incidence of torpor than those in the long day cold condition. Hence, photoperiod affected torpor independently of its effect on the gonadal cycle. 3. Testosterone, when administered via silastic capsules at near physiological levels, completely inhibited torpor in gonadectomized male and female hamsters hibernating in the short day cold condition. 4. In ovariectomized females, torpor was unaffected by progesterone treatment, but partially inhibited by estradiol. A greater inhibition of torpor was observed when estradiol-primed females were administered both estradiol and progesterone simultaneously. Thus, the effect of both hormones may be functionally comparable to that of the single testicular hormone. 5. Estradiol inhibited torpor to a greater extent in intact and ovariectomized female hamsters hibernating in long days than those in short days, suggesting an effect of photoperiod on responsiveness to estradiol. These results indicate an inverse relationship between the gonadal and hibernation cycles, and a probable role for gonadal steroids to influence the timing of the hibernation season. However, non-gonadal factors must also be involved in controlling hibernation, since photoperiod affected the incidence of torpor in gonadectomized animals and because hamsters were able to terminate hibernation in the absence of gonadal hormones.     

Participation of progesterone receptors in plus-maze behavior in female mice

The current study tested delayed effect pf progesterone on the anxiety level of female mice. The elevated plus maze (EPM) behavior was assessed in ovariectomized mice injected for 7 days with estradiol benzoate and progesterone or progesterone alone after 6 hrs of the last treatment. One group of ovariectomized mice was injected with progesterone receptor blocker Mifepristone before 2 hrs of the last treatment. The immunocytochemistry method was used to visualize cells in different brain areas having immunoreactivity (ir) for progesterone receptors. In the EPM, progesterone administration significantly increased the anxiety levels of ovariectomized mice as compared with estradiol benzoate and progesterone administration. The participation of nuclear progesterone receptors in anxiety levels regulation is confirmed by high correlation of the change of progesterone receptor-ir cell number in some brain areas and anxiety levels. Mifepristone decreased anxiety levels and progesterone receptor-ir cell number in both groups of mice that suggests involvement of genomic mechanisms in anxiety regulation in female mice.     

Role of sex steroids in development of anxiety in female mice

Level of sex steroid hormones being changed throughout an estrous cycle influences physiological and behavioral features of female subjects. To test how estrogen and progesterone affect the anxiety level in mice the ovariectomy (OVX) followed by hormone treatment was carried out. After 1 week of recovery period estradiol benzonate (20 micrograms, s/c) was injected once a day during 7 consequent days. By the 7th day in addition to EB injection progesterone (500 micrograms, s/c) was also injected. Four hours later the mice were tested in elevated plus-maze to measure the anxiety level. Control animals were treated with sesame oil only. Behavioral data obtained demonstrate that the hormonal treatment altered anxiety state in experimental animals. In plusmaze paradigm, it has been demonstrated that progesterone-treated mice revealed the lowest level of open arm activity. In contrast, these mice showed the highest grooming activity as compared to other experimental groups. Immunohistochemical data on progesterone receptor (PR), immunoreactivity in brain have shown that the manipulation with different hormonal treatments modified the number of PR-ir cells in many brain areas. Our data suggest that sex steroid hormones play an important role in induction of anxiety level, as measured by elevated plus-maze, and this action might be partially mediated through the classical steroid receptors.     

Mouse epidermal growth factor concentrations are altered by gonadectomy and treatments with estradiol and progesterone

In adult male and female mice we compared the epidermal growth factor (EGF) concentrations after gonadectomy and studied the effects of postgonadectomy treatments with estradiol and progesterone. In gonadectomized mice the mean concentration of EGF in the submandibular salivary gland (SMG) was (7-fold) higher in the females than the males. In the kidneys the males had (1.3-fold) higher levels of EGF than the females. Yet, gonadectomized males had higher plasma EGF levels and females higher urinary EGF concentrations. Estradiol treatment clearly decreased the EGF concentration in the SMG and increased it in urine and kidneys. Progesterone decreased male kidney EGF. Combined treatment with estradiol and progesterone increased the EGF concentration in the male urine and SMG, and decreased it in male kidneys.     

Role of ovarian steroids on the catecholamine synthesis and release in female rat adrenal: in vivo and in vitro studies

In a previous report, we describe the existence of an effect of ovarian steroids on the adrenal medulla activities of the enzymes involved in catecholamine (CA) catabolism. To complete that study, we have now examined the adrenal medulla activity of tyrosine hydroxylase (TH), the rate limiting enzyme of the CA synthesis, as well as the in vitro release of CAs from incubated adrenal medullas. The study has been performed with adrenal medullas from female rats with physiological (estrous cycle) or pharmacological (steroid treatment) alterations in their circulating levels of estrogens and progesterone. The in vitro release of CAs from incubated adrenal medullas of estradiol-treated rats was lower than that obtained in vehicle-treated animals. In consequence, the preovulatory increase of estradiol would be the responsible of the low in vitro release of CAs observed during the estrous phase of ovarian cycle. However, this steroid does not seem to affect the CA synthesis, since the adrenal medulla activity of TH was not altered after the estradiol treatment nor during the estrous cycle. On the contrary, progesterone treatment increased TH activity 24 h after the steroid injection. This effect was independent of estradiol. However, an estrogen-dependent increase in TH activity occurred short-time after the steroid administration. Although progesterone by itself failed to modify the in vitro release of both CAs, it was able to reverse the estradiol-induced decrease in epinephrine release. In summary, estradiol seems to decrease the ability of the adrenal medulla to release CAs to the peripheral blood, without affecting the CA synthesis, whereas progesterone mostly affects TH activity, being its effects temporary and partially depending on estrogens.     

Sex hormone regulation of anti-bacterial activity in rat uterine secretions and apical release of anti-bacterial factor(s) by uterine epithelial cells in culture

In mature female rats, sex hormones regulate the reproductive (estrous) cycle to optimize mating and fertility. During the part of the estrous cycle when mating occurs, and when estrogen is the dominant sex hormone, the uterus is susceptible to infection with bacteria that can be deleterious for survival and fertility. The present study investigated whether sex hormones regulate innate immunity in the female reproductive tract by affecting the secretion of an anti-bacterial factor(s) in the rat uterus. Uterine fluids from intact rats at the proestrous stage of the estrous cycle significantly inhibited Staphylococcus aureus growth. When ovariectomized rats were treated with estradiol, anti-bacterial activity against both S. aureus and Escherichia coli increased in uterine secretions with hormone treatment. In contrast, rats injected with either progesterone and estradiol or progesterone alone displayed no bactericidal activity indicating that progesterone reversed the stimulatory effect of estradiol on anti-bacterial activity. In other studies, isolated uterine epithelial cells from intact animals were grown to confluence and high transepithelial resistance on cell inserts. Analysis of apical secretions indicated that a soluble factor(s) is released by polarized epithelial cells which inhibits bacterial growth. These results demonstrate that sex hormones influence the presence of a broad-spectrum bactericidal factor(s) in luminal secretions of the rat uterus. Further these studies suggest that epithelial cells which line the uterine lumen are a primary source of anti-bacterial activity.     

Sex differences in adrenocortical structure and function. III. The effects of postpubertal gonadectomy and gonadal hormone replacement on adrenal cholesterol sidechain cleavage activity and on steroids biosynthesis by rat adrenal homogenates.

In rats postpubertal orchiectomy results in an increase in the adrenal weight, testosterone replacement restores the adrenal weight to the normal level. Neither ovariectomy (8 weeks of duration) nor estradiol replacement has an effect on adrenal weight in female rats. Pregnenolone synthesis as well as corticosterone and blue tetrazolium-positive steroids secretion is significantly higher in homogenates of adrenals from female rats than from males. Orchiectomy results in a marked increase in pregnenolone biosynthesis, testosterone replacement restores the value to the normal levels. Neither ovariectomy nor estradiol replacement has an effect on pregnenolone synthesis in v i t r o. In both sexes gonadectomy causes a marked decrease in corticosterone output by adrenal homogenates, concomitantly the increase in the adrenal 5alpha-reductase activity is observed. The ratio of secreted corticosterone to pregnenolone is significantly lower in gonadectomized rats of both sexes than in control animals. Estradiol or testosterone replacement inhibits the adrenal 5alpha-reductase activity and restores the corticosterone output as well as corticosterone/pregnenolone ratio to the normal values. The above described findings show that the sex differences in steroids secretion by the rat adrenal are partially conditioned by a cholesterol sidechain cleavage activity. Testosterone inhibits this activity while estradiol under applied experimental conditions has no effect on the cholesterol sidechain cleavage activity.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Membrane-bound progesterone receptor expression in human aortic endothelial cells.

Observational studies demonstrate that estradiol and progesterone affect vasoreactivity. In animal studies, progesterone treatment causes immediate relaxation of precontracted arteries with inhibition of calcium influx in vascular endothelial and smooth muscle cells, suggesting a non-genomic mechanism of action. In this study we investigated the presence of novel membrane-bound progesterone receptors in human aortic endothelial cells and correlated the expression with cell-cycle stage. Western blotting analysis with an antibody directed to the hormone-binding domain of the classic progesterone receptors shows predominant bands at 100 and 60 kD, whereas analysis with an antibody to the DNA-binding region shows only the 100-kD band. In contrast, classic nuclear progesterone receptors B and A are identified at 116 and 94 kD in similarly processed T47D cells. Both novel bands localize to the membrane fraction after differential centrifugation. Plasma membrane-bound progesterone receptor was further shown with immunofluorescent antibody and ligand-binding studies in a small percentage of human aortic endothelial cells. Fluorescent activated cell sorting demonstrated that approximately 8% of the human aortic endothelial cells expressed a plasma membrane progesterone receptor and that a greater percentage of the expressing cells were in the G2/M-phase of the cell cycle. Treatment with progesterone conjugated to BSA did not show any significant cell-cycle changes. Plasma membrane-bound progesterone receptor in vascular endothelial cells may regulate the non-genomic actions of progesterone, and expression of the receptor appears to vary with cell cycle stage.     

Properties and regulation of 17β-hydroxysteroid oxidoreductase of OVCAR-3, CAOV-3, and A431 cells: Effects of epidermal growth factor,estradiol, and progesterone

Although there is a growing body of evidence that 17β-hydroxysteroid oxidoteductase plays a role the regulation of steroid levels in epithelial tumors of the endometrium and breast, out knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17β-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17β-hydroxysteriod oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basic of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGE, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGE decreased the activity of CAOV-3 cells slightly. In contrast, EGE stimulated A431 17β-hydroxysteriod oxidoreductase activity 7–8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGE. With EGE, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGE alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGE to low-affinity receptors on A431 cells. © 1995 Wiley-Liss, Inc.     

The role of the uterus in regulation of heat duration in cycling rats

These experiments examined the effects of hysterectomy on heat duration and on the reinduction of estrous behavior by progesterone (P) following the termination of spontaneous heat in 4-day cycling rats. Hysterectomy did not affect the onset of estrus but prolonged heat duration. The average duration of sexual receptivity for hysterectomized (H) and sham-hysterectomized (SH) rats was 18.2 and 13.0 hr, respectively. Furthermore, H animals injected with either 0.5 mg P within 2 hr, or 4.0 mg P 24 hr following the termination of natural estrus showed significantly higher lordosis and solicitation responses than SH rats similarly treated. These behavioral findings were correlated with the level of hypothalamic progestin receptors. That is, H animals had a significantly higher concentration of progestin receptors than SH rats immediately following the termination of spontaneous heat and also 24 hr later. Both in estrous-cycling rats and in gonadectomized animals treated with estradiol benzoate (EB), hysterectomy resulted in higher serum estradiol (E2) levels. The results of these experiments suggest that prolongation of the period of sexual receptivity and the facilitated behavioral responses to P following the cessation of estrus in hysterectomized animals may be due to a lowered clearance rate of circulating estradiol which presumably enhances the estrogen conditioning of the neural substrates.     

Role of aromatization in sexual differentiation: Effects of prenatal ATD treatment and neonatal castration

Pregnant female rats were administered either the aromatization inhibitor ATD (1,4,6-androstatriene-3,17-dione) or propylene glycol from Days 10 to 21 of gestation. On the day of birth one-half of the offspring from each group were gonadectomized. The remaining offspring were gonadectomized 35 days after birth. When adult the animals were given eight weekly mating tests following treatment with 2 or 8 μg of estradiol benzoate (EB) and 25 or 200 μg of progesterone (P). The probability of lordotic behavior as well as the frequency of ear-wiggle and hop and dart responses was measured. Prenatal ATD treatment resulted in a slight increase in lordotic behavior in the males. Lordotic potential was greatly facilitated by castration at birth. ATD treatment also increased the frequency of proceptive behaviors in males and combined ATD treatment and neonatal castration produced a dramatic increase in these behaviors. Prenatal ATD treatment and neonatal ovariectomy had only modest effects on the display of receptive and proceptive behaviors in females. Two weeks after the last test for female mating behaviors, the animals received daily injections of 200 μg of testosterone propionate. Four weekly tests for male-typical responses were given starting 1 week after the first injection. Prenatal ATD treatment did not markedly affect masculine behavior in the males. Castration at birth eliminated the ejaculatory response and reduced the frequency of mounting and intromission behavior. Prenatal ATD treatment and ovariectomy at birth had no appreciable effects on the display of male-typical behaviors in the females. Testosterone-stimulated masculine behavior of the female was similar to that of the male castrated at birth.     

Cell adhesion in neoplastic cells and syngeneic fibroblasts of the rat: effect of progesterone and estradiol]

Recent advances in the study on the importance of steroid receptors in the treatment of hormone-responsive tumors established the relationship between plasma membrane and the first steps of the cellular response to the endocrine stimulus. We have therefore found interesting to study the influence of estrogen and progesterone on cell adhesion of neoplastic (SGS/3A) and normal syngeneic (FG) cells. We demonstrated the presence of a nuclear estrogenic and a cytosolic progestin receptor in the SGS/3A cells. In syngeneic fibroblasts both receptors are absent. Cell adhesion kinetics obtained determining the percent of single labeled cells with 3H-leucine adhering to confluent monolayer (cell-cell adhesion) showed that the physiological concentrations of estradiol and progesterone induce an inhibition of cell-substratum adhesion (25-30%) in neoplastic cells, but do not influence cell-cell adhesion. In FG cells the two hormones cause an increase of cell-substratum adhesion (35-40%), but do not influence cell-cell adhesion. Results suggest that FG cells, although lacking cytoplasmic or nuclear receptors for estradiol and progesterone, probably have other steroid-receptive molecules involved in adhesive processes on their cell-surface.