Fluctuations of hippocampal neuronal protein levels over the estrous cycle in the rat

Hippocampal function is known to be estrous-cycle-dependent but information on estrous-cycle-dependent protein expression is limited. It was therefore the aim to study protein levels of the neuronal network over the estrous cycle in the hippocampus of female rats and in males showing protein chemical neuroanatomy in this area. Female and male OFA Sprague-Dawley rats were used and females were grouped to proestrous, estrous, metestrous and diestrous by using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Spot volumes were quantified with specific software. A Synapsin-1 expression form was differentially regulated between proestrous and diestrous, a Synapsin IIa expression form was differentially regulated between proestrous and metestrous, the sum of ERC-2 proteins organizing the cytomatrix at the nerve terminals active zone was showing sex-dependent levels in the proestrous phase and Neurofilament triplet L protein was differentially expressed between the estrous phase and males. The findings may represent estrous-cycle-dependent hippocampal synaptic function that has been shown already in terms of electrophysiology and neuroanatomy. Neurofilament changes over the estrous cycle may reflect endoskeleton changes over the estrous cycle. We learn from this study, although increasing complexity of protein knowledge, that the estrous cycle and not only the sex per se has to be taken into account for design of future studies and interpretation of previous work at the protein level.     

Vaginal smear cycle in 4-day cyclic Chinese hamsters, Cricetulus griseus]

The contents of vaginal smear of 4-day cyclic Chinese hamster (Cricetulus griseus) was investigated every 3 hours for 5 days. A light-dark cycle of 14--10 hr was used with the lights turned on at 6 : 00 a.m. Estrous cycle of the Chinese hamster determined by vaginal smears can be divided into 6 periods. The proestrous phase started at about 0 : 00 of day 1, the day of the proestrous phase was designated as day 1 of the estrous cycle. In the afternoon of the same day 1, nucleated epithelial cells gradually increased in number (proestrus : I), and the vaginal contents became to consist solely of nucleated epithelial cells at about 18 : 00 to 21 : 00 (estrus : II). At about 0 : 00 of day 2, however, nucleated epithelial cells were superseded suddenly by cornified epithelial cells, and this phase lasted for 9 to 12 hr (metestrus I : III). Towards the end of the cornified stage, nucleated cells appeared in short duration (metestrus II : IV). And then, in the evening of day 2, leucocytes gradually increased in number with degeneration of nucleated cells (diestrus I : V-1). On day 3, vaginal smear contained a large amount of mucus as well as degenerated nucleated cells and leucocytes (diestrus II : V-2). At about 21 : 000 of day 4, some cornified epithelial cells were seen and then proestrous stage was returned. The females were mated with 3 to 5 males in the evening of day 1, copulation was confirmed in 83.7% females in the next morning,thus the copulation in the Chinese hamster may be thought to occur during the vaginal smear stage of nucleated epithelial cells (estrous phase), i.e. about 18 : 00 to 24 : 00 of day 1.     

Characterization of depression-like behavior in prenatally stressed female rats during ovary cycle

The aim of the present work was a comparative analysis of dynamics of depression-like behavior in prenatally stressed and non-prenatally stressed female rats in the key phases of the ovary cycle. It was found that non-stressed female rats demonstrated high level of depression-like behavior in proestrous phase as compared to the diestrous phase, whereas these rats showed low level of depression-like behavior in estrous phase in Porsolt's test. On the contrary, there were no significant differences in extent of depression-like behavior between prenatally stressed rats in the diestrous and proestrous, although in the phase of estrous in these animals an increase in level of depression-like behavior was noted. Thus, the results of this study indicated pronounced effects of prenatal stress on the character of depression-like behavior of females in different phases of ovary cycle. This study revealed leveling and reversed action of prenatal stress on depression-like behavior in key phases of sexual cycle in female rats.     

Changes of estrous cycles with aging in female F344/n rats.

Changes of estrous cycles with aging of F344/N rats between 1 and 30 months of age (M) were monitored by vaginal smear cytology. The vaginal opening and first cornified cell phase were identified at 1.3 +/- 0.1 M and 1.5 +/- 0.2 M, respectively. Thereafter, estrous cycles showed about 5-day intervals, and ceased at 16.4 +/- 1.2 M. Thereafter irregular appearance of single cornified cell phases without the preceding of nucleated cell phases interspersed with a predominant leukocyte phase was seen in vaginal smears until 26.9 +/- 0.5 M. Growing and mature follicles as well as corpora lutea persisted until at least 30 M, and characterized the post reproductive aging of F344/N females. The F344/N rats seem to resemble humans in that the cessation of estrous cycles occurs at approximately half their entire lifespan. However, other aging characteristics are unknown in postmenopausal women. Therefore, we must be careful when extrapolating the aging changes of reproduction in F344/N rats to human beings.     

Functional anatomy of the female genital organs of the wild black agouti (Dasyprocta fuliginosa) female in the Peruvian Amazon

This study examined anatomical and histological characteristics of genital organs of 38 black agouti females in the wild in different reproductive stages, collected by rural hunters in the North-eastern Peruvian Amazon. Females in the follicular phase of the estrous cycle had greater antral follicle sizes than other females, the largest antral follicle measuring 2.34mm. Antral follicles in pregnant females and females in luteal phase of the estrous cycle had an average maximum diameter smaller than 1mm. In black agouti females in follicular phase, some antral follicles are selected to continue to growth, reaching a pre-ovulatory diameter of 2mm. Mean ovulation rate was 2.5 follicles and litter size was 2.1 embryos or fetuses per pregnant female, resulting in a rate of ovum mortality of 20.8%. Many follicles from which ovulation did not occur of 1-mm maximum diameter luteinize forming accessory CL. The constituent active luteal tissues of the ovary are functional and accessory CL. Although all females had accessory CL, transformation of follicles into accessory CL occurred especially in pregnant females, resulting in a contribution from 9% to 23% of the total luteal volume as pregnancy advances. The persistence of functional CL throughout pregnancy might reflect the importance for the maintenance of gestation and may be essential for the continuous hormonal production. The duplex uterus of the agouti female is composed by two completely independent uterine horns with correspondent separate cervices opening into the vagina. In pregnant females, most remarkable observed uterine adaptations were induced by the progressive enlargement caused by the normal pregnancy evolution. The wild black agouti showed different vaginal epithelium features in accordance with the reproductive state of the female.     

Reproductive behavioral changes during the ovarian cycle of lesser bushbabies (Galago moholi) in captivity.

Behavioral patterns were quantified in seven heterosexual lesser bushbaby (Galago moholi) pairs during the estrous cycle to determine the relative significance of behavioral and nonbehavioral components of female sexuality in mate attraction. Increases in the occurrence of several male behaviors indicating female attractiveness were initiated during vaginal swelling when the female was sexually nonreceptive. Female behavioral estrus, as indicated by intromission, was confined to a portion of vaginal opening coinciding with proestrous and vaginal estrous smears. Female attractiveness was maintained for much of the period of vaginal opening, whereas female receptivity ended a day or two earlier than attractiveness. Female receptive and proceptive behaviors were not well defined or extensive, and few female behaviors exhibited significant changes during the cycle. Scent-marking behaviors, such as urine washes, and male grooms, were generally elevated outside the behavioral estrous period. In G. moholi, male sexual arousal appears to be elicited primarily by female attractiveness, while behavioral components of female sexuality appear to be less important in influencing mate attraction.     

Isolation accelerates reproductive senescence and alters its predictors in female rats

The process of reproductive senescence in female rats (Rattus norvegicus, Sprague-Dawley strain) was altered by their social environment during adulthood. The incidence of constant estrus (CE), which marks the end of estrous cyclicity, was nearly twice as high in females living in isolation as it was in females living in groups. Isolated females also entered CE at three times the rate of group-housed females. In addition, the characteristics of a rat's estrous cycle when she was young predicted whether or not she would enter CE during reproductive senescence. However, the characteristics of the cycle that predicted if a rat would enter CE were different for isolated females than for females living in groups. In isolated rats, entry into CE was predicted by a pattern of regular cycles followed by irregular cycles, a lordosis reflex of consistently high intensity during irregular cycles, and an absence of spontaneous pseudopregnancies. Furthermore, a long duration of CE was predicted by early cessation of estrous cycles. In rats living in groups, increased estrogenization of the vaginal smears during irregular cycles was the only predictor of entry into CE. In both environments, the onset of an acyclic lordosis reflex predicted the timing of CE. The potential role of ovarian steroids as mediators of these effects is discussed.     

The antiepileptic effect of sodium valproate during different phases of the estrous cycle in PTZ-induced seizures in rats

Catamenial epilepsy is a form of epilepsy which is related to the menstrual cycle. Cyclic variation in the levels of ovarian hormones plays a pivotal role in its pathogenesis. Sodium valproate (VPA) is one of the oldest antiepileptic drugs (AEDs) which inhibits hepatic metabolizing enzymes. The aim of this study was to evaluate the antiepileptic effects of VPA during different phases of the estrous cycle in rats. 72 adult female Wistar rats in three groups (control, 75 and 100 mg/kg VPA), each with four subgroups (proestrous, estrous, metestrous and diestrous) were used (n = 6). Initially, puberty was assessed using vaginal smears and rats with two regular cycles were selected. VPA with doses 75 and 100 mg/kg was administered intraperitoneally (i.p) in the treatment groups followed by i.p. injection of 80 mg/kg pentylentetrazol (PTZ) in the treatment and control groups. After induction of seizure by PTZ, initiation time of myoclonic seizures (ITMS), initiation time of tonic–clonic seizures (ITTS), seizures duration (SD) and mortality rate (MR) were recorded for 30 min. Data were presented as mean±SD, one-way ANOVA followed by Tukey–Kramer multiple comparison post hoc test were used for analysis of data (P < 0.05). The results of this study showed that VPA significantly improved antiepileptic parameters including ITMS, ITTS, SD, and MR, in which they were significantly more prominent during the luteal phase than the follicular phase (P < 0.05). In addition, there was no significant difference neither between proestrous and estrous nor between metestrous and diestrous in each separately group of rats (P > 0.05).     

Physiological pineal effects on female reproductive function of laboratory rats: prenatal development of pups, litter size and estrous cycle in middle age

The present study investigates whether and how the pineal or its hormone melatonin influences female reproductive functions, namely the litter size, prenatal development of offsprings, and estrous cyclicity, especially its age-related cessation in a non-seasonal breeder, the laboratory rat. Wistar rats were maintained under a 24 h light-dark (12Lratio12D) cycle. Female rats were divided into 3 groups: non-operated (NO), sham-operated (SX), and pinealectomized (PX). Surgeries were performed in 35-40 day-old females. Starting at an age between 70 days and 7 months, female rats of all 3 groups were repeatedly mated with intact males. PX mothers more frequently delivered pups with malformations (e.g., taillessness, hydronephrosis, 7 out of 1263 pups) than control rats (0/1323; p<0.007). In the first delivery at 3 months of age, but not at later ages, PX mothers delivered more pups of lower body weight than control animals (p<0.001). Examination of vaginal smears showed that almost all female rats of the NO, SX, and PX groups had 4-day estrous cyclicity when they were young-between 60 days and 5 months of age. At an age of 17 to 18 months, most female rats of the NO and SX groups showed irregular, continuously diestrous or pseudopregnancy-like patterns, and 4-day estrous cyclicity was found in only 10% of the NO or SX animals. In contrast, about 50% of the PX rats showed 4-day estrous cyclicity at this older age (p< 0.001). Melatonin, when added to drinking water (0.4 mg/L) for 16 days during the dark phase increased the frequency of diestrous phase, except in continuously diestrous rats and very few others. This melatonin effect was strong in PX rats but relatively weak in SX rats. In conclusion, the pineal hormone appears to influence various reproductive functions and developmental processes, especially pregnancy and the timing of reproductive aging in rats. The effects of pinealectomy are more prominent at an age of 60 to 80 days (i.e., shortly after puberty) and at the beginning of the cessation of cycles in middle-aged females.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

Reproductive functional anatomy and oestrous cycle pattern of the female brush-tailed porcupine (Atherurus africanus,Gray 1842) from Gabon

In the present study, we examined certain features of the functional anatomy of the female genital tract of the wild brush-tailed porcupine (Atherurus africanus) to obtain data on the reproductive biology of this African forest rodent. Two consecutive experiments were performed. The aim of the first was to establish macroscopic and microscopic features of the genital organs, and to explore correlations between predominant ovarian structures and vaginal contents in 20 wild, mature females. In the second experiment, we inspected the external genitalia and vaginal smears of a further 10 females in captivity on a daily basis for 90 days. The uterus of the brush-tailed porcupine is bicornuate and composed of two separated uterine horns, a uterine body and cervix. The genital tract does not present a vaginal vestibule. Thus, there is no portion common to genital and urinary tracts. Females in the follicular phase of the oestrous cycle showed increased cornification of the vaginal epithelium and a high density of eosinophilic cells in vaginal smears. The vulva and vaginal opening were open, reddish and tumefacted. In luteal phase or in pregnancy, epithelial cornification and eosinophilic features were notably reduced and the vagina presented a pale, non-tumefacted vulva and a vaginal closure membrane. Females in captivity showed spontaneous cycles, a polyoestrous reproduction pattern and, based on features of the external genitalia and vaginal smears, their oestrous cycle length was 27.1+/-6.4 days (n=12).     

Anterior pituitary leptin expression changes in different reproductive states: in vitro stimulation by gonadotropin-releasing hormone.

This study was designed to learn more about the changes in expression of rat anterior pituitary (AP) leptin during the estrous cycle. QRT-PCR assays of cycling rat AP leptin mRNA showed 2-fold increases from metestrus to diestrus followed by an 86% decrease on the morning of proestrus. Percentages of leptin cells increased in proestrus and pregnancy to 55-60% of AP cells. Dual labeling for leptin proteins and growth hormone (GH) or gonadotropins showed that the rise in leptin protein-bearing cells from diestrus to proestrus was mainly in GH cells. Only 10-20% of leptin cells in male or cycling female rats coexpress gonadotropins. In contrast, 50-73% of leptin cells from pregnant or lactating females coexpress gonadotropins and only 19% coexpress GH, indicating plasticity in the distribution of leptin. Leptin cells expressed GnRH receptors, and estrogen and GnRH together increased the coexpression of leptin mRNA and gonadotropins. GnRH increased cellular leptin proteins three to four times and mRNA 9.8 times in proestrous rats and stimulated leptin secretion in cultures from diestrous, proestrous, and pregnant rats. These regulatory influences, and the high expression of AP leptin during proestrus and pregnancy, suggest a supportive role for leptin during key events involved with reproduction.     

Role of nipple stimulation in the suppression of estrous cyclicity during extended lactation in the rat

The role of nipple stimulation in the suppression of the estrous cycle during extended lactation was studied in rats subjected to either total, partial, or sham excision of the nipples. Each female cohabited with 4 pups, 4-14 days old, over a period of 70 days postpartum, during which vaginal smears were recorded daily. Initially, regardless of the presence of nipples, all rats exhibited a postpartum diestrus that lasted for 12-20 days. Intact females (bearing 6 pairs of nipples) continued to exhibit successive prolonged diestrous phases over 70 days of lactation. A comparable result was obtained with females bearing only the anterior pair of nipples, which, in a separate experiment, was found to be the most frequently suckled pair. However, females devoid of nipples resumed regular (4-day) estrous cycles between Days 12 and 27 postpartum, in spite of their continuous contact with pups. Thus, when lactation is prolonged beyond the normal time of weaning (Day 21 postpartum), stimulation of the nipples by sucking becomes indispensable for the continued arrest of the estrous cycle. The possible mechanisms underlying this phenomenon are discussed.     

Nonphotic phase shifting in female Syrian hamsters: interactions with the estrous cycle

Nonphotic phase shifting of circadian rhythms was examined in female Syrian hamsters. Animals were stimulated at zeitgeber time 4.5 by either placing them in a novel running wheel or by transferring them to a clean home cage. Placement in a clean home cage was more effective than novel wheel treatment in stimulating large (> 1.5 h) phase shifts. Peak phase shifts (ca. 3.5 h) and the percentage of females showing large phase shifts were comparable to those found in male hamsters stimulated with novel wheels. The amount of activity induced by nonphotic stimulation and the amount of phase shifting varied slightly with respect to the 4-day estrous cycle. Animals tended to run less and shift less on the day of estrus. Nonphotic stimulation on proestrus often resulted in a 1-day delay of the estrous cycle reflected in animals' postovulatory vaginal discharge and the expression of sexual receptivity (lordosis). This delay of the estrous cycle was associated with large phase advances and high activity. These results extend the generality of nonphotic phase shifting to females for the first time and raise the possibility that resetting of circadian rhythms can induce changes in the estrous cycle.     

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.     

Effect of a 20 kHz sawtooth magnetic field exposure on the estrous cycle in mice

Female mice post weaning were exposed to 20 kHz sawtooth electric and magnetic fields (EMF) with 6.25 microT peak intensity for 6 weeks. Estrous cycles were checked using vaginal smears over the last 10 days of the experimental period. The vaginal smears from EMF-exposed mice revealed an increase in the frequency of one or two phases persisting. The number of estrous cycles less than 1 was more in the EMF-exposed group than in the sham control group. Furthermore, in the EMF-exposed group, the duration of proestrous and metestrous stages of the estrous cycle was significantly increased compared with the control group. In conclusion, our results suggest that exposure to 20 kHz sawtooth EMF may affect normal cycling of the estrous cycle by disrupting the female reproductive endocrine physiology. We should not disregard the possible adverse reproductive effect of the 20 kHz sawtooth EMF generated under the occupational exposure situation in females.     

Effect of consumption of sucrose and saccharin on passive avoidance learning in female Wistar rats

Influence of consumption of 32% sucrose or 0.1% saccharin solutions on passive avoidance acquisition in female Wistar rats was studied at different stages of estrous cycle. Under conditions of weak electrical shock, rats of the control and saccharin groups showed no acquisition at any stage of estrous cycle. Under the same conditions females of the sucrose group acquired the avoidance behavior if trained on the first day of diestrous and metestrous but not proestrous and estrous. The stage of estrous cycle on the second day did not influence the latency during testing session.     

An abnormal pattern of embryonic development during early pregnancy in aging rats

There is recent evidence that a decline in fertility and litter size precedes the cessation of regular estrous cyclicity in middle-aged female rats. This decline in litter size is related to a decrease in the number of normal blastocysts that are present on Day 5 of gestation, immediately prior to implantation. Thus, the pattern of embryonic development during the first 5 days of pregnancy may be altered in middle-aged rats, resulting in fewer implanting embryos and smaller litter sizes. The present study examined the ovulation rates, fertilization rates, and the patterns of embryonic development in regularly cyclic, young and middle-aged females during the first 5 days of pregnancy. Examination of the numbers of ovulated ova revealed that the ovulation rate was significantly reduced in 12- to 14-mo-old females (13 mo; 9.0 +/- 1.0/rat), but not in 9- to 11-mo-old females (10 mo; 12.2 +/- 0.8/rat), as compared to that in young animals (12.8 +/- 1.0/rat). However, there was no decrease in fertilization rate in either the 10-mo or 13-mo group. While the total numbers of embryos present on Days 2-5 were similar among all 3 groups, embryos from 10-mo females displayed a delayed pattern of development and an increased incidence of morphological abnormalities. These changes in embryo development were even more pronounced in the 13-mo group. By Day 5 of pregnancy there was a significant reduction in normal blastocysts in 10-mo (7.3 +/- 1.2/rat) and 13-mo (6.0 +/- 1.6/rat) rats, as compared to young females (10.6 +/- 0.9/rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the estrous cycle by pheromones from pregnant and lactating rats

Female rats were exposed to odors from females that went through a birth cycle of pregnancy, birth and lactation. The estrous cycle was shortened by odors from pregnant rats and lengthened by odors from lactating rats and their pups. Changes in estrous cycle length were accompanied by changes in the length of the luteal phase (metestrus-early diestrus). A log survivor plot demonstrated that variation in cycle length was the result of different rates of ovulation within the population under each condition. Neither pregnancy nor lactation odors affected the intensity or timing of the lordosis reflex. These odors could modulate fertility and coordinate birth cycles within a group of rats.