Estrous Stage Research Articles

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Peripheral oxytocin in female baboons relates to estrous state and maintenance of sexual consortships

The neuro-hypophysial hormone oxytocin (OT) has been implicated in female reproductive and maternal behaviors and in the formation of pair bonds in monogamous species. Here we measure variation in urinary OT concentrations in relation to reproductive biology and socio-sexual behavior in a promiscuously breeding species, the chacma baboon (Papio hamadryas ursinus). Subjects were members of a habituated group of baboons in the Okavango Delta, Botswana. We collected behavioral data and urine samples from n=13 cycling females across their estrous cycles and during and outside short-term, exclusive sexual consortships. Samples were analyzed via enzyme immunoassay (EIA) and we used linear mixed models (LMM) to explore the relationship between peripheral OT and a female's estrous stage and consortship status, her previous reproductive experience and fertility. We also used a Pearson's correlation to examine the relationship between OT concentrations of consorting females and their extent of behavioral coordination with their consort partners. The results of the LMM indicate that only estrous stage had a significant influence on OT levels. Females had higher OT levels during their periovulatory period than during other stages of their estrous cycle. There were no differences in the OT levels between consorting and non-consorting periovulatory females. However, among consorting females, there was a significant positive relationship between urinary OT levels and the maintenance of close proximity between consort partners. Our results suggest that physiological and behavioral changes associated with the initiation and maintenance of short-term inter-sexual relationships in baboons correspond with changes in peripheral OT.

Fertility and estrogenic activity of Turraeanthus africanus in combination with Lepidium meyenii (Black maca) in female mice

Aim of the studyThe effect of Black maca (Lepidium meyenii) and Turraeanthus africanus on fertility and estrogenic activity in female mice was investigated. Materials and methodsThirty female mice were treated daily with extracts of Black maca plus T. africanus (BM+TA), T. africanus alone (TA) or vehicle (control) at dose 1g/kg BW for 30days. ResultsThe length of the estrous cycle inside the group of (BM+TA)-treated mice significantly (p<0.001) shorten (4.08±0.06 days) when compared to control (4.53±0.05). On the proestrous stage, the oestradiol level for animals treated with TA (27.3±0.5pg/ml) and (BM+TA) (36.0±0.9pg/ml) significantly increased when compared to control (24.0±0.4pg/ml), while in the estrous stage only the estradiol level of (BM+TA) treated animal (28±2pg/ml) significantly increased (p<0.01). The uterine weight of ovariectomized of TA-treated animals was higher (p<0.05) than those observed in controls while no change in the (BM+TA) group was observed. The weight of uterus of non pregnant mice of both treated TA and (BM+TA) extract significantly increased (p<0.001) in the proestrous and estrus stage when compared to control.The number of fetuses in the uterus after (BM+TA) treatment (14.66±0.25) was significantly higher (p<0.05) than the TA treated alone (13.60±0.45) and the control (9.5±1.10) one at day 15 of pregnancy.No significant difference between groups (p>0.05) was observed for the two treatments concerning the Fertility index. ConclusionsTA can affect the number of embryo implantation and the uterine weight. This effect increased in association with BM. BM could regulate estrous cycle.

Estrous cycle phase does not influence myocardial infarction but may alter arrhythmogenicity in isolated rat hearts

The cardioprotective effects of estrogen are well documented in humans and animals. Although gain/loss‐of‐function studies show estrogen as a cardioprotective hormone, it is not known if physiological fluctuations during the estrous cycle influences cardiac ischemia/reperfusion (I/R) injury. We conducted this study to determine if the extent of I/R injury varies as a function of normal estrous cycling. Estrous cycle stage was determined by serum hormone levels and vaginal cytology immediately before heart excision. Isolated rat hearts underwent 25 min ischemia and 2 h reperfusion. No baseline differences were seen in heart rate, left ventricular pressure, or coronary flow. Infarct size was not affected by estrous stage (42±6, 49±4, 40±9, and 43±8% zone at risk for Proestrus (P), Estrus (E), Metestrus (M), and Diestrus (D) respectively; P&gt;0.05). Infarct size was not correlated with serum levels of estradiol or progesterone. There was a trend for reperfusion arrhythmia fluctuations during estrous (arrhythmia scores 3.6±0.3, 4.3±0.5, 4.9±0.3, 3.3±0.6 for P, E, M, and D respectively; P=0.08). Restoration of coronary flow during reperfusion also varied with phase (P&lt;0.05). This study demonstrates that studies using female rats have potential confounding effects of ovarian activity on cardiac I/R injury.

Spinal Cord Injury Induced Arrest in Estrous Cycle of Rats Is Ameliorated by S‐nitrosoglutathione: Novel Therapeutic Agent to Treat Amenorrhea

Amenorrhea following spinal cord injury (SCI) has been well documented. There has been little research on the underlying molecular mechanisms and therapeutics. The purpose of the present study was to investigate the effect of GSNO in ameliorating SCI-induced amenorrhea through affecting the expression of CX43, NFkB, and ERβ protein. SCI was induced in female SD rats at the T9-T10 level. Estrous stage was determined by vaginal smear. GSNO (50 µg/kg body weight) was gavage fed daily. Animals were sacrificed on day 7 and 14 post SCI. Ovaries were fixed for histological and biochemical studies. Expression levels of ERβ, CX-43, and NFkB were analyzed by Western blot and immunofluorescence. GSNO hastens resumption of the estrous cycle following SCI-induced transient arrest. Resumption of estrous cycle was hastened by GSNO. Atretic and degenerating follicles seen in the ovary of SCI rats on day 14 post-SCI were decreased in GSNO treated animals. The increased CX43 expression observed with SCI ovary was decreased by GSNO. ERβ expression decreased significantly on day 7 and 14 post-SCI and was restored with GSNO treatment. Following SCI, NFkB expression was increased in the ovarian follicles and the expression was reduced with GSNO administration. The number of terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling positive follicular and luteal cells was increased after SCI. GSNO-treated animals had significantly fewer apoptotic cells in the ovary. SCI-induced amenorrhea is accompanied by an increase in CX43 expression and a decrease in ERβ expression. SCI animals treated with GSNO resumed the estrous cycle significantly earlier. These results indicate a potential therapeutic value for GSNO in treating amenorrhea among SCI patients.

Expression of the luteinizing hormone receptor (LHR) gene in ovine non-gonadal tissues during estrous cycle

Luteinizing hormone (LH) is an important glycoprotein hormone that regulates gonadal function in mammals and in turn regulates physiological status changes during the estrous cycle. The function of LH is mediated by luteinizing hormone receptor (LHR). In order to examine the expression patterns of the LHR gene in non-gonadal tissues during the 4 phases of the ovine estrous cycle, tissues from healthy non-pregnant adult Hu sheep were examined according to the estrous cycle for normal ovaries using real-time fluorescence quantitative PCR and ELISA methods with GAPDH as the reference gene. LHR mRNA expression levels were significantly correlated with protein concentrations and the LHR gene was abundantly expressed in olfactory bulb, hypothalamus, rumen, small intestine, kidney, and uterine tissues. When comparing the expression levels of LHR during the 4 estrous phases in particular tissues, the results showed that LHR expression levels were significantly different and relatively lower at the estrous stage in a number of non-gonadal tissues. The trends of change in LHR expression levels were highly significantly correlated between hypothalamus and rectum, hypophysis and oviduct, ileum and uterus, and among jejunum, olfactory bulb, and kidney (P < 0.01), and there was also significant correlation between duodenum and oviduct, hypothalamus and medulla oblongata, jejunum and uterus, omasum and abomasum, and reticulum and colon (P < 0.05). These results indicate that the ovine LHR gene (or LH) might control important mechanisms in non-gonadal tissues and that the level of LH activity in some tissues may be influenced by hormonal status during the estrous cycle.

Telemetric Assessment of Referred Vaginal Hyperalgesia and the Effect of Indomethacin in a Rat Model of Endometriosis

Symptoms of endometriosis (ENDO), among others, include pelvic/abdominal and muscle pain. Non-steroidal anti-inflammatory agents are first-line treatment for this pain. Similar to women, rats with surgically induced ENDO, but not its surgical control, exhibit vaginal hyperalgesia, which in rats is evidenced by a decreased threshold for the visceromotor response (VMR) induced by vaginal distention. Here we assess the VMR in rats with implanted probes that telemetrically transmit EMG activity from the abdominal muscle. The feasibility and sensitivity of this technique for monitoring the VMR threshold across the estrous cycle and the influence of Indomethacin on ENDO-induced vaginal hyperalgesia were evaluated. VMR thresholds in response to vaginal distention with an infusion pump were measured in different estrous stages. Indomethacin (5 or 10 mg/kg i.p. or s.c.) was injected in proestrus rats and 40–60 min later the VMR threshold was measured. The VMR threshold varied across the estrous cycle only in ENDO rats, being lowest in proestrus. Indomethacin increased this threshold in proestrus ENDO rats. These results show that telemetric assessment of the VMR is a sensitive tool, suitable for long-term studies in conscious rats. The results with this technique also suggest that ENDO-associated vaginal hyperalgesia involves COX activity, the feature that also underlies inflammatory pains.

Antifertility effect of chronically administered Tabernaemontana divaricata leaf extract on female albino mice

ObjectiveTo study the antifertility activity of Tabernaemontana divaricata leaf extract on female albino mice. MethodsAnimals were divided into three groups consisting of five animals in each group. One group served as control and received vehicle orally for 21 days. The other two groups received dried ethanolic extract of leaves orally at a dose of 250 and 450 mg/kg animal body weight per day. After 21 days of treatment, extract was withdrawn from the mice and estrous cycle was studied for another 21 days. ResultsThe results of this study revealed that treatment of mice with extract of 250 and 450 mg/kg body weight for 21 days caused a prolonged eastrous cycle with significant increase in the duration of diestrus phase and elongation of estrus stage in treatment with higher dose (450 mg/kg body weight) and simultaneously decrease in the luteinizing hormone (LH) in both the treated groups and follicle stimulating hormone (FSH) in higher dose of treatment compared to the control animals may indicate the disturbance of estrous cycle and ovulation through suppression of FSH. ConclusionsIt concludes that disturbance on the estradiol secretion with significant decrease during estrous stage of the cycle observed with the extract treatment may be due to impairment of LH and FSH.

Gender Specific Pelvic Pain Severity in Neurogenic Cystitis

Interstitial cystitis/painful bladder syndrome is a chronic bladder inflammatory disease of unknown etiology that is often regarded as neurogenic cystitis. The condition is associated with focal inflammation, urothelial lesions, voiding dysfunction and pain in the pelvic/perineal area. Approximately 90% of patients with the condition are women, suggesting the possibility of hormonal involvement in interstitial cystitis/painful bladder syndrome symptoms. We examined the basis of gender specific pelvic pain in a murine model of neurogenic cystitis that recapitulates features of interstitial cystitis/painful bladder syndrome and in which pelvic pain is mediated by mast cell histamine. Murine neurogenic cystitis was induced by tail base inoculation of C57BL/6 or BALB/c mice with the Bartha strain of pseudorabies virus. Pelvic pain behavior was assessed by quantifying tactile allodynia in response to mechanical stimulation with von Frey filaments. Bladder mast cells were quantified by flow cytometry. Female mice of each genetic background showed significantly greater pelvic pain behavior than males, although responses were greater in BALB/c females. Gender specific pelvic pain behavior did not correspond to increased bladder inflammation or barrier dysfunction. Modulating reproductive hormonal status by ovariectomy and subsequent estrogen replacement had no effect on the magnitude of pseudorabies virus induced pain. The number of mast cells was associated with pelvic pain severity in female mice but it did not correlate with gender specific pelvic pain. These data suggest that pelvic pain in mice with murine neurogenic cystitis is mediated by gender specific responsiveness to mast cells while pelvic pain severity is modulated by genetic factors.

Hypothyroidism caused by phenobarbital affects patterns of estrous cyclicity in rats

We found that repeated treatment with phenobarbital (PB), a thyroid modulator, resulted in a persistent estrous stage in the present study. Although the effects of PB in blocking the surge release of luteinizing hormone (LH), inducing anovulation and prolonging the diestrous period has been well established, there is still no research describing the appearance of persistent estrous states in normal cycling rats dosed with PB. To further study this phenomenon, female rats exhibiting regular estrous cycle were administered an oral dose of PB for 14 consecutive days. Consecutively, vaginal smears were observed and rats from all the groups were sacrificed and serum hormone levels for prolactin, progesterone, estradiol, triiodothyronin (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) were measured. Pituitary, thyroid, liver, uteri and ovaries were excised, weighed and further subjected to histological observations. We found that PB induced irregular estrous cycles, especially persistent estrus in rats. Histopathologically, the persistent estrous stages are characterized by persistent vaginal cornification in the vagina, cystic follicles and anovulation in the ovaries. Endocrinologically, serum T3 and T4 levels were significantly lower, and TSH was higher in treated-female rats compared to control females. The serum estradiol level and the estradiol/progesterone ratio tend to increase in treated-females. Furthermore, PB-treated animals with irregular estrous cycle were reduced by T4 replacement. Our data indicate that treatment with PB resulted in hypothyroidism and irregular estrous cycle, particularly a persistent estrous stage in normal cycling female rats.

The newly formed corpora lutea of normal cycling rats exhibit drastic changes in steroidogenic and luteolytic gene expressions

In normal estrous cycling rats, corpora lutea (CL) regress over several cycles; however, the period during which they secrete progesterone (P4) is strictly limited. In the present study, we clarified the function of CL in normal cycling rats. We especially focused on expression levels of four steroidogenic and two luteolytic genes in the two different populations of the CL (new and old CL) at each estrous stage. The ovaries of female rats at each estrous cycle were collected, and new and old CL were separated with laser microdissection and analyzed for mRNA expression. In the new CL, the expressions of scavenger receptor class B type I (SR-BI), steroidogenic acute regulatory protein (StAR), and P450 cholesterol side-chain cleavage (P450scc) mRNA reached their highest levels at metestrus, and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA gradually increased from estrus to diestrus. Meanwhile, 20α-hydroxysteroid dehydrogenase (20α-HSD) and prostaglandin F2 alpha receptor (PGF2α-R) mRNA levels were remarkably low from estrus to metestrus and gradually increased thereafter. These gene levels in new CL corresponded to serum P4 levels during the estrous cycle. In the old CL, all steroidogenic and luteolytic gene levels were consistently high throughout the estrous cycle. These results provide clear evidence that new CL at metestrus have strong steroidogenic activity and through inhibition of luteolysis, maintain P4 production in normal cycling rats. The elevation of 20α-HSD and PGF2α-R levels in new CL at diestrus may be a trigger of functional luteolysis.

Proliferation and apoptosis in normal bitch mammary tissues in relation to progesterone level

The objective of this study was to investigate the proliferating activity and apoptosis in canine normal mammary tissues in different cycle stages. Mammary tissues were collected from five beagle bitches at six different estrous stages: anestrus, proestrus, estrus, early diestrus, mid diestrus, and late diestrus. The expression of Ki-67 protein and apoptotic bodies was evaluated by avidin–biotin–peroxidase complex (ABC) method and TUNEL assay, respectively. Percentage of Ki-67 positive cells and apoptotic bodies was calculated. The significant highest percentage of Ki-67 positive cells and apoptotic bodies was observed at mid diestrus. Positive correlation was found mainly in the alveolar epithelium between progesterone levels and percentage of Ki-67 positive cells, as well as between progesterone levels and apoptosis. In conclusion, the results from the present study suggested that progesterone may influence on proliferation and apoptosis in canine mammary tissue.

Distribution of estrogen receptor alpha and progesterone receptor, and leukocyte infiltration in the cervix of cyclic bitches and those with pyometra

The objectives were to localize estrogen receptor alpha (ERα) and progesterone receptor (PR), and enumerate leukocyte infiltration in cervical tissue of normal bitches during various stages of the estrous cycle (n = 35), as well as in those developing open (n = 22) or closed-cervix pyometra (n = 19). Each pyometra group was subdivided into anestrus and diestrus. Cervical tissues were collected after ovariohysterectomy. Receptor expressions were determined by immunohistochemistry and leukocyte infiltration was evaluated in histological sections stained with haematoxylin-eosin. The assessment was performed in two parts of cervical sections: the uterine part in four tissue layers (surface epithelium (SE), lamina propria (LP), glandular epithelium (GE), and tunica muscularis (M)), and the vaginal part in three layers (SE, LP and M). An immunohistochemical total score consisted of the addition of both the intensity and proportional scores. The ERα and PR scores differed between groups (P < 0.05) and between layers (P < 0.05), but were not significantly different between uterine and vaginal parts. The ERα score was lowest in the open-cervix pyometra bitches at anestrus and in closed-cervix pyometra bitches at diestrus. For all types of immune cells, there were no significant differences among stages of the estrous cycle in normal bitches, whereas neutrophils were lower in both sub-groups of closed-cervix versus open-cervix pyometra (P < 0.05). In conclusion, distributions of ERα and PR were similar along the longitudinal axis of the canine cervix. We inferred that cervical dilation in normal bitches and bitches with uterine pathology was likely controlled by different mechanisms. Receptor expressions were influenced by stage of the estrous cycle in normal bitches, whereas neutrophil infiltration in cervical tissue appeared to be involved in cervical dilation in bitches with pyometra, regardless of estrous stages.

Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

In vitro comparison of myometrial contractility induced by aglepristone-oxytocin and aglepristone-PGF2alpha combinations at different stages of the estrus cycle in the bitch

The aim of this in vitro study was to compare the uterokinetic activity of oxytocin and dinoprost, the natural PGF2α, with or without aglepristone, in canine myometrial fibers. Thirty-three bitches were allocated into one of four groups, depending on their estrous stage and whether or not they had received a treatment with aglepristone (metestrus aglepristone, n = 5; metestrus without treatment, n = 9; anestrus aglepristone, n = 9; anestrus without treatment, n = 10). After hysterectomy, longitudinal and circular uterine strips were mounted in organ baths. Oxytocin or PGF2α (10 nmol/l to 10 micromol/l) were applied non-cumulatively. A linear mixed effects models theory was used to compare the fiber effect, the aglepristone effect, and the treatment effect, from the area under the curves calculated from the contractile effect/concentration curves for each drug. Oxytocin and PGF2α induced concentration-dependent myometrial contractions in longitudinal (LF) and circular myometrial fibers (CF), indicating the presence of functional contractile oxytocin- and PGF2α-receptors in metestrus and anestrus. The contractile response to oxytocin was greater in LF than in CF in all of the groups; the response to PGF2α was greater in LF than in CF in non-treated bitches in anestrus and in treated bitches in metestrus. These results suggest that there is a difference in sensitivity or a heterogeneous distribution of oxytocin and PGF2α-receptors in the myometrial layers, which is independent of hormonal impregnation. The contractile response to oxytocin and PGF2α was significantly increased after aglepristone treatment in LF during metestrus, suggesting that the progesterone withdrawal induced by aglepristone has a role to play. The longitudinal myometrial layer also appeared to be the target for the two drugs at this stage. This study provides new information about canine uterine contractile activity, notably the differing behavior of myometrial CF and LF; in vivo studies are required to test the use of a combination of aglepristone and oxytocin in the treatment of canine pyometra.

Antinociception and sedation following intracerebroventricular administration of Δ 9-tetrahydrocannabinol in female vs. male rats

Systemically administered cannabinoids produce greater antinociceptive and sedative effects in female compared to male rats. Sex differences in the brain endocannabinoid system have also been reported. The aim of this study was to determine whether sex differences in antinociceptive and motoric effects of a cannabinoid can be attributed to supraspinal mechanisms. Vehicle or Δ 9-tetrahydrocannabinol (THC, 100 μg) was administered i.c.v., and behavioral effects were compared between gonadally intact male and female rats, and among females in different estrous stages (early proestrus, late proestrus, estrus and diestrus). Antinociception on the tail withdrawal and paw pressure tests after i.c.v. THC was slightly but not significantly greater in females (pooled across estrous stages) compared to males. THC suppressed locomotor activity similarly in all groups, with the exception that only males showed hyperlocomotion at 4 h post-injection. When females in the four estrous stages were compared, females in late proestrus showed significantly greater THC-induced antinociception than females in estrus (and males). These results suggest that supraspinal mechanisms may contribute to greater systemic THC effects in females compared to males, and to estrous stage-dependent differences in THC effects among females.

Cyclic Morphological Changes in the Beagle Mammary Gland

Interpretation of chemical-induced effects on the female beagle mammary gland can be difficult owing to the wide variation of normal glandular morphology. In this retrospective study, morphological features of the gland in four (proestrus, estrus, diestrus, and anestrus) phases of the cycle are described. The gland was quiescent (inactive) in proestrus and estrus. In diestrus, with the rise of progesterone, four (I-IV) distinct morphological changes were evident. In phase I, there was exuberant stromal and ductal proliferation. In phase II, there was early lobular development with branching ducts and alveolar proliferation. In phase III, there was an abundance of glandular tissue with large lobules containing secretory material, whereas phase IV had features of early regression, increased interlobular connective tissue, and eosinophilic secretions in distended ducts and acini. In early anestrus, ducts were distended, with eosinophilic secretions with alveolar regression, whereas regression was complete in late anestrus. Glandular morphology was slightly variable in the mammary chain within the same dog. Progesterone receptor expression was prominent in estrus and early diestrus, and peak estrogen receptor expression was noted in diestrus II. Expression of proliferation marker ki-67 was highest in diestrus I, followed by diestrus II. There was excellent concordance between the estrous stage and the glandular morphology.

Association between serum cortisol and progesterone concentrations and the infiltration of immune cells in the endometrium of gilts with vaginal discharge

The objective of the study was to investigate an association between serum cortisol and progesterone (P4) concentrations and the distribution of immune cells in the endometrium of the gilts with vaginal discharge. Genital organs from 39 Landrace×Yorkshire crossbred gilts culled owing to vaginal discharge problem were collected from two commercial swine herds in Thailand. The estrous stage and gross pathology were examined. Blood samples were collected from the jugular vein prior to being slaughtered. Serum P4 and cortisol were analyzed by means of enzyme immunoassay. The samples observed were in inactive (n = 4), follicular (n = 10), and luteal (n = 25) phases. They, afterwards, were processed in hematoxylin and eosin sections. The endometrium of the gilts was histologically divided into three layers, i.e., epithelial, subepithelial connective tissue, and glandular connective tissue layers. Immune cells, i.e., lymphocytes, neutrophils, eosinophils, macrophages, and plasma cells, in each layer were quantified under a light microscope (×400). The results revealed that mean serum cortisol was 430.6 ± 68.3 nmol/l. Serum P4 varied by ovarian status. Serum P4 of the gilts in the luteal phase was higher than those in the follicular phase (88.3 ± 7.7 versus 20.6 ± 6.2 nmol/l, P < 0.05). As for the endometrium condition, the gilts were classified into acute/subacute endometritis (n = 13), chronic endometritis (n = 9), and normal endometrium (n = 17). Neutrophils were the main local immune cells in the epithelial layer. Lymphocytes were the dominant population in the subepithelial and glandular connective tissue layers. Generally, the serum cortisol tended to negatively correlate with lymphocytes in the subepithelial connective tissue layer (r = −0.28, P = 0.081). In the gilts with acute/subacute endometritis, no correlation among serum cortisol, P4, and immune cells was observed. In chronic endometritis gilts, only a negative correlation was remarked between P4 and epithelial lymphocytes (r = −0.83, P = 0.010), epithelial neutrophils (r = −0.79, P = 0.019), and subepithelial neutrophils (r = −0.73, P = 0.025). In the gilts with normal endometrium, P4 negatively correlated with subepithelial neutrophils (r = −0.55, P = 0.022) while positively correlated with subepithelial macrophages (r = 0.54, P = 0.024) and subepithelial eosinophils (r = 0.60, P = 0.011).

Optimizing the time of doxil injection to increase the drug retention in transplanted murine mammary tumors

Sex hormonal milieus during the female fertility cycle modulate the tumor vascular permeability of breast cancer. It has been proposed that the liposomal formulated doxorubicin (ie, Doxil), given at the menstrual/estrous stage with the predicted highest tumor vascular permeability, allows significantly increased drug retention in the breast tumor. In the current study, syngeneic murine 4T1 mammary tumors were established on the backs of female BALB/c mice and Doxil was administered at particular mouse estrous cycle stages. The results indicated that Doxil administration during certain times in the mouse estrous cycle was crucial for drug retention in 4T1 tumor tissues. Significantly higher drug concentrations were detected in the tumor tissues when Doxil was administered during the diestrus stage, as compared to when the drug injection was given at all other estrous stages. Our study also showed that the tumor-bearing mice exhibited nearly normal rhythmicity of the estrous cycle post drug injection, indicating the feasibility of continual injection of Doxil at the same estrous cycle stage. By using 4T1 cells cultured in vitro, we showed that progesterone (P4) significantly inhibited cell proliferation and the production of six tumor-derived cytokines, eg, sTNF-RI, CXCL-16, GM-CSF, MIP-1alpha, MIP-1gamma, and Flt3-L. Some of these factors have been shown to be vascular modulators in diverse tissues. In this report, we demonstrated that the concentration of P4 in the plasma and/or estrous cycle stage of 4T1 tumor-bearing mice can be used to select the best time for administrating the liposomal anticancer drugs.

Neuronal Activity in the Hypothalamic Paraventricular Nucleus Varies Across the Estrous Cycle in Anesthetized Female Rats: Effects of Dopamine Receptor Agonism

The central nervous system plays a pivotal role in sexual behavior. The role of the paraventricular nucleus (PVN) of the hypothalamus in female sexual behavior is poorly characterized. In males, there is a correlation between PVN neuron activity and erectile activity, and in mediating dopamine receptor agonist-induced sexual arousal. To understand the role of the PVN in female sexual function, baseline PVN neuronal activity and responses to dopamine receptor agonism were assessed in anesthetized rats. Single unit recordings were used to assess the firing properties of individual PVN neurons; and local field potentials quantified PVN network activity (combined activity of large numbers of PVN neurons). Baseline and apomorphine-stimulated activity was measured across the estrous cycle. Baseline firing rates of single units were found to differ across the stages of the estrous cycle with metestrus showing the highest firing rate (3.7 vs. 0.9 Hz in diestrus). Apomorphine administration caused significant increases in firing rate in 29% of neurons, and significant decreases in 71%. Basal local field potentials also varied between estrous stages and in response to apomorphine; significant differences in the total power of alpha1 and beta1 bands were observed in both cases. This study shows that PVN neuronal activity varies with hormonal state, and these neurons are differentially affected by apomorphine, suggesting two different populations. These data are indicative of a critical role for the PVN in female sexual function. It is important that the hormonal state should be considered when investigating sexual physiology and the effect of pharmacological agents.