The in vivo regulation of the progesterone "receptor" in guinea pig uterus: dependence on estrogen and progesterone

An assay for quantifying the high affinity progesterone binding protein in guinea pig uterine cytosol was developed using Florisil to separate bound and free steroid. The activity of the progesterone binding protein increased between 4–12 hours following estrogen administration and by 4 days of treatment was 10-fold higher than castrate controls. When estrogen administration was discontinued the progesterone binding activity declined slowly with a half-life of 3 days. By contrast, progesterone treatment caused a rapid decline of binding activity within 3 hours. These studies suggest that the antagonistic actions of estrogen and progesterone determine the quantity of available progesterone binding sites in guinea pig uterine cytosol.     

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.     

Depression of estrogen-induced uterine peroxidase in alloxan-diabetic rats

The influence of alloxan diabetes on reproductive function and the estradiol-stimulated increase in uterine peroxidase was investigated. Alloxan monohydrate in a dose of 75 mg/kg body weight effectively produced permanent diabetes. In adult rats, 20 days of diabetes resulted in cessation of the estrous cycle and a significant reduction in the gain of body weight, the weights of anterior pituitary gland, ovary, uterus, the level of serum progesterone and the activity of the estradiol-stimulated uterine peroxidase (P less than 0.05). After 10 days of insulin treatment, the ovarian weight, the estrous cycle and the level of ovarian hormones were restored to normal whereas the uterine weight and the estradiol-stimulated uterine peroxidase activity were only partially recovered. Persistent depression of the uterine response in the insulin-treated diabetic rats to both endogenous and exogenous ovarian hormone stimulation suggests that the uterus was directly affected by diabetes. The direct effect of diabetes upon the uterus was further demonstrated in the ovariectomized immature rat in which diabetes depressed the stimulatory action of estradiol on both uterine weight and uterine peroxidase activity.     

Heme oxygenase-carbon monoxide (HO-CO) system in rat uterus: effect of sexual steroids and prostaglandins

Pregnancy maintenance is a very complex phenomenon, and the mechanisms that allow the survival of the fetus within the maternal uterus are still poorly understood. Our objectives were to analyze heme oxygenase (HO) activity and expression in the pregnant rat and to study its association with steroid hormones and prostaglandins. Uterine tissues were obtained from non-pregnant and from time-mated rats at days 5, 13, 18-22 of pregnancy and postpartum. HO activity was significantly higher at days 5 and 20 while HO-1 protein levels measured by Western blot, were significantly elevated from days 19 to 22. In ovariectomized rats, estrogen and progesterone in estrogenized animals increased HO activity and expression. Cyclooxygenase inhibitors augmented HO activity and HO-1 expression. Pre-incubation with prostaglandin F2alpha (PGF2alpha) diminished the enzymatic activity in ovariectomized rat uterus. Tin protoporphyrin IX, an HO inhibitor, significantly decreased uterine cGMP accumulation. Bilirubin decreased uterine thiobarbituric acid substances levels (an index of lipid peroxidation). These results demonstrate a uterine gestational pattern of HO activity and expression in the rat. In addition, these results suggest that uterine HO activity could regulate uterine quiescence in pregnancy via cGMP and it may contribute to the defense against oxidative stress.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30-80 days gestation) contains a factor which inhibits phospholipase A2 (PLA2) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA2 assay system. Inhibitory activity was associated with a 35-45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA2-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA2 activity was of a non-competitive nature (Ki = 4.1 +/- 0.7 micrograms/ml, ca 118 nmol/l). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Inhibition of lymphocyte proliferation by uterine fluid from the pregnant ewe

Immunosuppressive molecules in uterine fluid from the nonpregnant uterine horn of unilaterally pregnant ewes at Days 60, 100, and 140 of gestation were examined. Uterine fluid from all days inhibited [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Inhibitory activity increased with advancing gestation. Uterine fluid also inhibited lymphocyte proliferation caused by other mitogens or by mixed lymphocyte reactions. Inhibitory activity was found in both salt volume (Mr less than 1000) and void volume (Mr greater than 5000) fractions of uterine fluid resolved by Sephadex G-25 desalting columns. Only activity in the void volume was sensitive to pronase. Several fractions containing inhibitory activity were resolved when dialyzed uterine fluid was fractionated into acidic and basic components by cation-exchange chromatography and further resolved by gel filtration using Sepharose CL-6B. The most active fractions (inhibition/micrograms protein) for both acidic and basic components eluted at the void volume of Sepharose CL-6B (Mr greater than 4 x 10(6). The inhibitory factor in the basic component that eluted at the void volume of Sepharose CL-6B was rich in carbohydrate, slightly cytotoxic, and partially sensitive to digestion with trypsin or oxidation with periodate. In conclusion, uterine fluid of unilaterally pregnant ewes is enriched in molecules that inhibit lymphocyte proliferation in vitro. Among these are low molecular weight, non proteinaceous factors and a very high molecular weight (Mr greater than 4 x 10(6) fraction that contains protein and carbohydrate.     

Myometrial activity around estrus in sows: spontaneous activity and effects of estrogens,cloprostenol, seminal plasma and clenbuterol

A new, nonsurgical, open-end catheter technique was used to study spontaneous uterine activity around estrus in sows, and the effects of estrogens, seminal plasma, cloprostenol, and clenbuterol on uterine activity. In the first experiment, uterine activity was studied in 14 multiparous, cyclic sows, during one or more estrous cycles, from day -4 to day 4 of the cycle (day 0: first day of standing estrus). From a few days before estrus until estrus, the percentage of sows showing any uterine contractions increased from 55 to 100%, and frequency and mean amplitude of uterine contractions for these sows increased from 15 to 22/h, and from 20 to 40 mmHg on average. After estrus, uterine activity decreased. There were large differences between sows in uterine activity, which were consistent over the days of the cycle. In the second experiment, 11.5 microg of estrogens in 100 ml saline (n = 17), 100 ml seminal plasma (n = 5), 1 mg cloprostenol in 100 ml saline (n = 10), 0.30 mg clenbuterol in 100 ml saline (n = 11), or 100 ml saline (n = 5) was infused IU, after recording spontaneous activity. Infusion with saline or seminal plasma did not affect uterine activity. Estrogens increased frequency of contractions. Cloprostenol increased both frequency and amplitude of contractions. Clenbuterol reduced both frequency and amplitude of contractions. In conclusion, this study shows that spontaneous uterine activity in sows is increased around estrus, and it supports the role of estrogens in boar seminal plasma in affecting uterine activity around mating. Further, this study has yielded possible tools to study the relation between uterine activity and sperm transport.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

Artificial insemination in the anoestrous and the postpartum white rhinoceros using GnRH analogue to induce ovulation

The objective of this study was to develop AI and to achieve first time pregnancy in a nulliparous rhinoceros. For this, one 24-year-old irregular cycling female white rhinoceros was selected, which had never been mated. The endocrine function was monitored by faecal and serum pregnane analysis. Ultrasound determined the optimal day for AI by measuring follicle sizes of 2.0, 2.6, 3.0, 3.2 cm on days -6, -4, -1, 0 of the induced oestrous cycle, respectively. AI was performed and ovulation induced when a pre-ovulatory-sized follicle was present using GnRH analogue, deslorelin. Fresh semen was deposited in the uterine horn using a patented AI catheter overcoming the hymeneal membrane and torturous cervical folds non-surgically. Moreover, ultrasound monitoring of the uterine involution and ovarian activity on days 16, 26, 30 postpartum facilitated the induction of and the AI on the first postpartum oestrous in a rhinoceros using GnRH analogue. Two consecutive pregnancies were achieved by AI for the first time in the rhinoceros. Pregnancies were diagnosed by elevated serum and faecal 20-oxo-pregnane concentrations. In addition ultrasound measured biometric parameters of the two foetuses on days 86 and 133 of gestation. Two female calves were born after 490 and 502 days of gestation, yet one calf was stillborn. AI in rhinoceros might now be used as assisted reproduction technology tool to boost critically small captive rhinoceros populations.     

Structural changes in the luminal epithelium of the porcine uterus between days 10 and 19 of the estrous cycle

Light and electron microscopy were used to study the morphology of uterine luminal epithelium from 4 or 5 gilts slaughtered on each of days 10, 13, 16, and 19 of the estrous cycle. Ultrastructural evidence indicated that metabolic activity and accumulation of glycogen by the uterine epithelium increased between days 10 and 16 of the estrous cycle. This phase of high synthetic activity had been terminated by day 19, as evidenced by the reduction or absence of glycogen deposits and decreased incidence of organelles associated with synthetic activity. Diffuse degeneration of epithelial cells occurred throughout the period of study but was maximal between days 16 and 19. Mitotic activity indicated that cell replacement also occurred between day 16 and day 19.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: III. Stimulation of uterine weight by dextrose, sucrose and corn starch

We have shown previously that mice fed the American Institute of Nutrition (AIN-76A) purified diet experience a significant increase in uterine:body weight (U:BW) ratios when compared to the U:BW ratios of mice fed a closed formula natural ingredient diet (Certified Rodent Chow #5002) for 7 days. The AIN-76A purified diet contains 5% corn oil and 65% carbohydrates with 50% of the carbohydrates coming from sucrose or dextrose and 15% from corn starch. The objective of this study was to determine whether the fat and carbohydrate content contributed to the unexpected uterine growth promoting activity observed in mice fed the AIN-76A diet. Estrogen bioassays were performed using CD-1 mice weaned at 15 days of age and assigned randomly to the negative control diet (Certified Rodent Chow #5002) or to the positive control diet (#5002) containing 4 or 6 ppb DES for comparison or to the test diets. The test diets were prepared by adding sucrose, dextrose, corn starch, corn oil or soybean oil to the #5002 negative control diet at 10% w/w concentration. Uterine:BW ratios were determined at 7 days post-feeding. The uterine weights and the U:BW ratios of mice fed the test diets containing dextrose, corn starch, or corn oil, were increased significantly (P less than 0.05) over those of mice fed the negative control diet. The uterine weights and U:BW ratios of mice fed the test diets containing sucrose or soybean oil also were increased over those of mice fed the negative control diet. These increases in uterine weights and U:BW ratios were similar to the increases in uterine weights and U:BW ratios of mice fed the positive control diet containing 4 ppb DES. It was concluded that the fats and carbohydrates caused preferential increases in uterine weights and in U:BW ratios and may account for the estrogen-like uterine growth promoting activity observed in mice fed the AIN-76A purified diet.     

Alteration of the timing of implantation by in vivo gene transfer: delay of implantation by suppression of nuclear factor kappaB activity and partial rescue by leukemia inhibitory factor

Nuclear factor kappaB (NF-kappaB) is activated in the murine endometrium during implantation period [Am. J. Reprod. Immunol. 51 (2004) 16]. Transient transfection of IkappaBalpha mutant (IkappaBalphaM) cDNA into the mouse uterine cavity using hemagglutinating virus of Japan envelope vector suppressed uterine NF-kappaB activity less than half of that observed in control on days 3.5 and 4.5 p.c. IkappaBalphaM cDNA transfection led to significant delay of implantation. After IkappaBalphaM cDNA transfection, LIF mRNA expression in the uterus was significantly suppressed on days 3.5 and 4.5 p.c. Co-transfection of LIF cDNA with IkappaBalphaM cDNA in the uterus partially rescued the delay of implantation induced by suppression of NF-kappaB activity. Taken together, these findings indicate that NF-kappaB activation determines the timing of the implantation, at least in part, via control of LIF expression.     

Epidermal growth factor (EGF) in the goat uterus: immunohistochemical localization of EGF and EGF receptor and effect of EGF on uterine activity in vivo

This study examined the distribution of immunoreactive epidermal growth factor (EGF) and EGF receptor (EGF-R) in the uterus and the effects of EGF on uterine activity in goats. Immunohistochemistry of EGF and EGF-R in the uteri showed distinct staining in the luminal and glandular epithelium and slight to moderate staining in the stromal and myometrial cells. To examine possible roles of the EGF system in the regulation of uterine activity, pressure changes in the intrauterine balloon were determined after intraluminal infusion of EGF into the uterine horn. Either at estrus or diestrus (9 to 14 days after the first day of estrus), treatment with 1 or 5 microg of EGF gradually reduced uterine activity, whereas infusion of the vehicle alone had no effect. The maximum reduction in uterine activity was seen 4 h after the treatment with 1 microg of EGF (40% to 45% reduction in the area surrounded by the contraction curve and its baseline), and the activity slowly returned thereafter. These results suggest that EGF in the uterus may play a role in regulating uterine activity in goats.     

The stimulatory effect of testosterone propionate and 17 beta-estradiol on 5'-methylthioadenosine phosphorylase activity in rat target tissues

The effect of castration and subsequent administration of 17 beta-estradiol and testosterone propionate on 5'-methylthioadenosine phosphorylase activity in rat target tissues was studied. Castration 34 days earlier resulted in a 95% reduction in ventral prostate 5'-methylthioadenosine phosphorylase activity and 16 days earlier in a 67% reduction in uterine 5'-methylthioadenosine phorphorylase activity. Four days of testosterone propionate administration stimulated ventral prostate 5'-methylthioadenosine phosphorylase activity 32% above castrate levels, which represented more than 50% of the intact control levels. 17 beta-Estradiol on the other hand stimulated uterine 5'-methylthioadenosine phosphorylase activity 35% above castrate controls within 24 h and with 3 days of continuous hormone treatment to within 97% of the intact control levels. However, castration and subsequent 17 beta-estradiol administration did not affect 5'-methylthioadenosine phosphorylase activity in rat liver and lung. Both prostate and uterine 5'-methylthioadenosine phosphorylase were shown to metabolize 5'-methylthioadenosine to 5-methylthioribose through a 5'-methylthioribose 1-phosphate intermediate. The data suggest aht 5'-methylthioadenosine is not allowed to accumulate in rat target tissues even under conditions which are known to stimulate polyamine synthesis.     

The postpartum buffalo: I. Endocrinological changes and uterine involution

To maintain a calving interval of 13-14 months in buffaloes, successful breeding must take place within 85-115 days after calving. Disturbances during this period due to delay of uterine involution or resumption of estrous activity are likely to prolong the calving interval and reduce the lifetime reproductive and productive efficiency. In this article literature on endocrinological changes in the peripartum period and on factors affecting uterine involution are reviewed. The available information indicated that although the availability of releasable FSH does not appear to be a limiting factor for resumption of postpartum cyclicity a substantial increase of releasable LH and replenishment of pituitary stores occurred around Day 20 in dairy and Day 30 in swamp buffaloes. There is evidence that follicular activity is resumed early (15-30 days) in the postpartum period. However, the factors which initiate release of appropriate LH pulses, follicular maturation and ovulation in the postpartum buffalo need further studies. The mean interval to complete uterine involution varied widely between 19 and 52 days. Assessment of cervical and uterine horn diameters by rectal palpation alone is not satisfactory to diagnose delayed uterine involution and possible subclinical uterine infection. Vaginal inspection can be included as a fundamental part of postpartum genital examination for diagnosis of such case. Uterine involution, however, does not seem to be a limiting factor for achievement of satisfactory fertility in the postpartum buffalo but the main determinant is resumption of estrous activity.     

Effect of alpha-adrenergic blocking agents on uterine activity in the ewe at the end of gestation

The electromyographic activity (EMG) of the uterus was recorded in vivo in 8 unanaesthetized ewes from the 140th day of gestation up to parturition. The effects on uterine activity of treatments with an alpha 1-receptor blocker (prazosin) and an alpha 2-receptor blocker (yohimbine) were studied. During the last days of gestation, EMG activity consisted of periodic active phases (1-2/h). During the last 16-17 hours, uterine activity increased sharply; this period was referred to a labour. Intravenous perfusion of prazosin (0.03 mg/kg/mn over 1 h) or intravenous injections (1 mg/kg) did not modify uterine activity either before or during labour. Intravenous perfusion of yohimbine (0.03 mg/kg/mn during 1h) inhibited uterine activity before and during labour. In all cases, lambing occurred between the 142nd and 145th day of gestation, which corresponds to the normal lambing period. These results suggest that, in the ewe, uterine alpha 2-receptors are important for normal uterine activity at the end of gestation and in. parturition.     

Development of myometrial electrical activity during the first half of pregnancy in the sheep

Uterine myoelectrical activity was recorded in seven pregnant sheep covering the period between 13 and 75 days post-coitum. Activity in the myometrium was present at day 13 and took the form of intermittent spikes of low amplitude. Bursts of spikes of irregular duration became noticeable between days 25 and 40 but most were not coordinated throughout the myometrium. Coordinated bursts of myoelectrical activity, which could be recorded at several sites simultaneously, first appeared between 40 and 65 days. These bursts had similar characteristics to the myoelectrical activity associated with uterine contractions during the last third of gestation. The myoelectrical activity showed a progressive increase in amplitude during the first half of gestation. There was no relationship between plasma progesterone levels and the increase in amplitude or appearance of coordinated bursts of uterine activity.     

A study of prostaglandin F2alpha as the lutbolysin in swine: IV An explanation for the luteotrophic effect of estradiol

This study evaluated effects of estradiol valerate on synthesis, secretion and direction of movement of immunoreactive prostaglandin F2alpha (PGF) in swine. Gilts were randomly assigned to provide uterine flushings representing days 11, 13, 15, 17 and 19 of the estrous cycle (three gilts/day). The same gilts then were allowed one estrous cycle for recovery. During the second postoperative estrous cycle they were treated with estradiol valerate (EV) (5mg/day, SC) on days 11 through 15 and uterine flushings again were obtained on the same respective days with the same gilts represented within each day. Total recoverable PGF per uterine horn increased from day 11 (X - 1.98 ng) to day 17 (X = 210.20 ng) and then declined to day 19 (X = 66.20 ng) during the control period. Following EV treatment average total recoverable PGF was the control period. Following EV treatment average total recoverable PGF was 1.9, 4,144.3 and 4,646.7 ng on the same respective days. EV treatment also resulted in maintenance of elevated levels of total protein and acid phosphatase activity in uterine flushings. These data suggest that estradiol may exert its luteotrophic effect by preventing the release of PGF from the uterine endometrium into the uterine venous system (endocrine secretion) while maintaining the movement of endometrial secretions into the uterine lumen (exocrine secretion).     

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiol's activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.