Premature Ovarian Insufficiency (POI) Induced by Dynamic Intensity Modulated Radiation Therapy via P13K-AKT-FOXO3a in Rat Models.

This study is aimed to investigate the mechanisms of radiation-induced mouse models of premature ovarian insufficiency (POI). Wistar female rats were grouped into the control, 3.2 Gy, 4.0 Gy, and 4.8 Gy groups. Overall ovarian functions were assessed with the H&E staining and ELISA. Proinflammatory cytokine secretion was analyzed ELISA, and the reactive oxygen species (ROS) levels were analyzed with immunohistochemistry. Protein expressions were analyzed by Western blot analysis. The 4.0 Gy and 4.8 Gy groups had significantly lower ovarian weight coefficients than the control and 3.2 Gy groups (after only one irradiation therapy). The 3.2 Gy radiation group induced periodic disturbance and hormone change at 4 weeks after radiation. In the 4.0 Gy and 4.8 Gy groups, the preantral follicles and antral follicles were decreased, while Atresia follicles were increased. E2 was decreased, while FSH and LH secretions were increased. The ovaries in the 4.0 Gy group were not completely atrophied, and some preantral follicles remained. Ovarian atrophy and follicular Atresia were found in the 4.8 Gy group. Inflammatory and oxidative markers were upregulated. PI3K and AKT were downregulated in the 4.0 Gy and 4.8 Gy groups, while FOXO3a was upregulated. Ovarian injuries may lead to oxidative damages and inflammatory injuries, downregulate the expression of P13k and Akt, upregulate the expression of FOXO3a, and lead to follicular atresia in the ovary.

Rapid Jetlag Resetting of Behavioral, Physiological, and Molecular Rhythms in Proestrous Female Mice.

A gradual adaptation to a shifted light-dark (LD) cycle is a key element of the circadian clock system and believed to be controlled by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Endocrine factors have a strong influence on the regulation of the circadian clock network and alter acute photic responses of the SCN clock. In females, endocrine function depends on the stage of the ovarian cycle. So far, however, little is known about the effect of the estrous cycle on behavioral and molecular responses to shifts in the LD rhythm. Based on this, we investigated whether estrous state affects the kinetics of phase shift during jetlag in behavior, physiology, and molecular clock rhythms in the SCN and in peripheral tissues. Female mice exposed to an advanced LD phase at proestrous or metestrous showed different phase-shift kinetics, with proestrous females displaying accelerated adaptation in behavior and physiology. Constant darkness release experiments suggest that these fast phase shifts do not reflect resetting of the SCN pacemaker. Explant experiments on SCN, adrenal gland, and uterus confirmed this finding with proestrous females showing significantly faster clock phase shifts in peripheral tissues compared with the SCN. Together, these findings provide strong evidence for an accelerated adaptation of proestrous compared with metestrous females to new LD conditions that is accompanied by rapid behavioral, physiological, and molecular rhythm resetting. Not only do these findings open up a new avenue to understand the effect of estrous cycle on the clock network under changing environmental conditions but also imply a greater susceptibility in proestrous females.

Delay of constant light-induced persistent vaginal estrus by 24-hour time cues in rats.

The normal ovarian cycle of female rats is typically replaced by persistent estrus when these animals are housed under constant light. Evidence presented here shows that the maintenance of periodicity in the environment can at least delay (if not prevent) the photic induction of persistent vaginal estrus. Female rats in constant light were exposed to vaginal smearing at random times or at the same time every day. In another experiment, female rats were exposed to either constant bright light, constant dim light, or a 24-hour photic cycle of bright and dim light. The onset of persistent vaginal estrus was delayed in rats exposed to 24-hour time cues even though the light intensities were the same as or greater than those for the aperiodic control groups. The results suggest that the absence of 24-hour time cues in constant light contributes to the induction of persistent estrus.

Regulation of the MAPK signaling pathway by miR-421-5p in rats under light pollution.

The present study aimed to explore the difference in the expression profiles of ovarian microRNA sequences in rats in a light pollution environment and rats in a normal light environment. Rats in the control group were exposed to 12­h light/dark cycles, while rats in the model group were continuously exposed to 24­h light. The ovaries were extracted from the two groups of rats, and Illumina HiSeq 2500 high­throughput sequencing technology was used to detect the differences in microRNA (miRNA) expression among the two groups. Fluorescence quantitative reverse transcription­polymerase chain reaction was used to verify the differential expression of miRNA. The present study was designed to experimentally validate the interaction between miR­421­5p and mitogen­activated protein kinase (MAPK) 7 by using the dual­luciferase reporter system, and to explore the expression of proteins in the MAPK signaling pathway with a lentiviral vector­mediated small hairpin RNA interference against microRNA­421­5p. The expression of 45 miRNAs was significantly different. In total, 13 miRNAs were upregulated, of which 5 miRNA sequences were known and 8 were predicted. Furthermore, 32 miRNAs were downregulated, of which 11 miRNA sequences were known and 21 were predicted. The results of the luciferase reporter assay confirmed the targeting association between miR­421­5p and MAPK7. The expression levels of MAPK and genes in its downstream signaling pathways, including c­Fos, CREB and c­Myc, were downregulated when miR­421­5p was overexpressed and upregulated when miR­421­5p was silenced. The differential expression of miRNAs may serve an important role in the development of the ovary in a light pollution environment. miR­421­5p may regulate ovarian growth and development by targeting the MAPK signaling pathway in light polluted rat ovaries.

[Effect of light regimens on the age dynamics of estrous function and serum prolactin level in rats].

The effect of different light regimens (standard 12 h. light : 12 h. darkness LD; 24-hour constant light LL, light deprivation DD, natural light regimen of the North-West of Russia NL) on the age dynamics of estrous function and on the serum prolactin in female LIO rats has been studied for two years. The rats were maintained at the one of above-mentioned regimes from the age of 25 days. The cytological monitoring of the estrous cycle was conducted every three months. The level of prolactin was studied at the age of 6, 12, 18 and 24 months. It has been found out that the regimen of constant light (LL) and the spring-summer period lead to earlier maturation, premature age-related changes of the rats' estrous cycle and to the increase of the level of serum prolactin in comparison with those in rats kept at the LD regimen. Light deprivation (DD) leads to the opposite changes. It has been noted that the exposure to constant light during the whole year is accompanied by more expressed and earlier developed age-related changes of estrous cycle in comparison with the natural light of the North-West of Russia with its peculiar year photoperiodicity (short day in autumn-winter period and white nights in spring-summer period).

Reproductive performance of Lacaune dairy sheep exposed to artificial long days followed by natural photoperiod without and with additional progestagen treatment during the nonbreeding season.

This study compared the reproductive performance of Lacaune dairy ewes exposed to a light program and subsequent male introduction without (n = 36) or with (n = 36) an additional 6-day progestagen treatment during the nonbreeding season. All ewes were exposed to extended day length (16 hours light and 8 hours darkness) for 77 days during winter (December 15 until March 2) followed by increasing natural photoperiod. At the end of the photoperiodic treatment, three blood samples were collected 6 days apart for progesterone (P4) analysis to determine cyclic activity. One half of the ewes were additionally subjected to a 6-day progestagen treatment in combination with PGF2α and eCG at insert withdrawal. Rams fitted with marking harnesses were introduced to females for 45 days and marked ewes recorded. Ewes exposed to the light program only were joined 40 days after the end of photoperiodic treatment, and ewes with additional progestagen treatment were joined 1 day after insert removal (40-44 days after the end of photostimulation). Lambing data were recorded and fertility (percentage of ewes lambing, lambing rate, and litter size) assessed to the first service period and overall. Mean serum P4 concentrations were similarly (P > 0.05) low in both groups (0.4-0.7 ng/mL vs. 0.4-0.6 ng/mL). On the basis of elevated P4 levels (>1 ng/mL), evidence of luteal activity was found in 27.8% of the ewes at the end of the light program. Estrus response was equally high (97.2%) and estrus distribution highly synchronized in progestagen-treated ewes (91.7% within 4 days). In ewes exposed to the light program only, estrous activity was recorded within 4 days (six ewes), from Day 8 to Day 17 (17 ewes) and from Day 19 to Day 25 (12 ewes) after joining. The percentage of ewes that lambed to the first service period was higher (P < 0.05) in ewes exposed to the light program only than that in the group additionally treated with progestagen/PGF2α/eCG (94.4% vs. 69.4%). Overall, the percentage of lambing ewes was similar in both groups (97.2% and 94.4%), and lambing rates (1.4-1.9) and litter sizes (1.9-2.1) were high and not influenced (P > 0.05) by the treatment. In conclusion, this study reports that exposition of Lacaune ewes to artificial long days followed by natural day length and male introduction is highly effective to induce fertile estrous activity during the nonbreeding season and offers a reliable and practical alternative to hormonal manipulation for out-of-season breeding in sheep.

Early short photoperiod effects in female LSH/SsLak Syrian hamsters.

Previous experiments showed that LSH/SsLak female hamsters cease ovarian cyclicity after 2-4 weeks of short photoperiod (SP) treatment. This study was designed to reveal early hormonal and histological changes on day 1 of the estrous cycle (estrus) in SP-treated animals and changes brought about by the SP-induced acyclic condition. Groups of 8-10 animals were killed on day 1 after 0, 4, 8, 12, and 16 days of SP treatment; sera and pituitaries were saved for hormone determinations, and ovaries were prepared for histological analysis. Intraatrial cannulae were inserted into anovulatory animals, and blood samples were withdrawn on the first and second postoperative days; the animals were killed on the third postoperative day. PRL levels were significantly reduced in cycling animals after 16 days of SP exposure and diminished further in acyclic animals. Pituitary PRL did not change in cycling animals, although it was highly depressed in SP-treated acyclic animals. The estrous FSH surge, serum LH levels, and pituitary gonadotropin contents were not affected by SP in cycling animals; in anovulatory animals, pituitary gonadotropin contents were significantly increased, and daily afternoon gonadotropin surges were observed. Uterine weights were reduced in cycling animals and underwent a further reduction after cyclicity ceased. Ovarian analysis revealed that all cycling animals had the same number of recruited follicles, yet significantly fewer corpora lutea and an apparent increase in atretic antral follicles were observed after 16 days of SP exposure. These results suggest that SP-induced cessation of estrous cyclicity occurs abruptly. SP does not alter the secondary FSH surge, but might adversely affect maintenance of follicular growth and depress serum PRL levels.

Melatonin rhythms time photoperiod-induced puberty in the female lamb.

Photoperiod is an important environmental cue timing puberty in the domestic female sheep, a seasonal breeder. Because the effects of photoperiod on reproductive function in the adult sheep are mediated by the pineal melatonin rhythm, the present study determined whether the pineal through its secretion of melatonin is involved in the pubertal process. Neonatal denervation of the pineal by removal of the superior cervical ganglia (SGX) abolished the nocturnal melatonin rhythm and delayed the onset of reproductive cycles beyond 1 yr of age in female lambs reared under natural conditions. Puberty was also delayed in SGX lambs reared under an artificial photoperiod sequence of long days (experienced at 17-22 weeks of age) followed by short days. By comparison, intact lambs under this same photoperiod treatment began cycles at the normal age. Another group of SGX lambs was treated each night with melatonin; the infusion replicated the duration of the nighttime rise in serum melatonin occurring in intact lambs exposed to 5 weeks of long days followed by short days (9-h or 15-h infusions, respectively). Melatonin treatment of these SGX lambs restored puberty to the normal age. These findings suggest that the pineal melatonin rhythm is an essential component of the photoperiodic mechanism timing puberty in the female sheep.

Rate of reproductive involution following either exposure to short days or daily administration of melatonin is faster in inbred than in random-bred female Syrian hamsters.

The onset of cessation of oestrous cyclicity and associated organ and hormonal changes were compared in random-bred (RB) and inbred (IB) female Syrian hamsters kept either under short days (8 h light:16 h darkness; 8L:16D) or long days (14L:10D) and given daily afternoon injections of 25 micrograms melatonin. In response to short-day treatment, 100% of the IB hamsters exhibited vaginal acyclicity within 35 days; by comparison, none of the RB animals were acyclic at this time. The IB hamsters also exhibited other changes associated with exposure to short days, including increased body weight, enlarged ovaries, regressed uteri, elevated pituitary concentrations of FSH, and depressed pituitary and plasma concentrations of prolactin. At this time, only the pituitary FSH levels were increased in the RB animals kept under the same short-day conditions. In a second experiment, RB and IB female Syrian hamsters were maintained under long days (14L:10D) and the rate of reproductive regression in response to daily afternoon injections of melatonin was compared. After 8 weeks of melatonin injections, 80% of the IB females were anoestrous, while all RB hamsters were still exhibiting 4-day oestrous cycles. Other changes associated with melatonin administration in the IB females included a marked drop in uterine weight and a depression in pituitary and plasma prolactin levels. The RB hamsters, although they were all still cyclic after 8 weeks, had increased body and ovarian weights, increased pituitary concentrations of FSH, and lower pituitary and plasma prolactin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of acetyl-L-carnitine on the hypothalamo-pituitary-gonadal system in female rats.

Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus.

Cytogenetic effects of X-rays in the guinea pig female germ cells. II. The maturing oocyte.

In a previous study, we showed that the guinea pig constitutes one of the best models to evaluate the genetic risk associated with an irradiation of the human female germ cells. Herewith, experiments were undertaken to evaluate the chromosomal radiosensitivity of oocytes of this species at two different stages of follicular development, separated by only 1 week. Female guinea pigs were X-irradiated on the ovaries, at either the beginning (day 3) or the middle (day 10) of the 17-day oestrous cycle. The doses delivered were 1 or 2 Gy. Meiotically competent oocytes were collected 1 week after irradiation (day 3) or immediately thereafter (day 10), and they were cultured to the metaphase of the first meiosis (MI) and examined for the presence of chromosome aberrations. Our data demonstrated a dramatic increase in the radiosensitivity of the oocyte during this short time interval: oocytes irradiated at the beginning of the oestrous cycle had a low frequency of chromosome aberrations, while those irradiated at the middle of the oestrous cycle (when growing Graafian follicles are clearly visible at the surface of the ovaries) exhibited heavy chromosome damage. However, we also found that oocytes irradiated at the middle of the oestrous cycle were eliminated from the ovaries in a few days, after their evolution to the MII stage. The stimulation of the first meiotic division by radiation required less than 24 h after doses of 1 or 2 Gy and was probably due to a rapid atresia of the large follicles containing the oocytes. On the basis of these results, it can be concluded that the radiosensitivity of the nearly mature guinea pig oocyte (1 week before ovulation) is clearly much higher than that of the corresponding stage in the mouse, both in terms of sensitivity to killing and to induction of chromosome aberrations.

Effects of far-infrared ray on reproduction, growth, behaviour and some physiological parameters in mice.

The effects of chronic exposure to far-infrared ray (FIR) on reproduction, growth, behaviour, survival time and some related parameters were examined in SHN mice. The reproductive parameters differed slightly between the females on the normal racks and those on the FIR racks, which emitted FIR from the ceiling. The age and body weight on the day of vaginal opening was lower in the experimental mice born and maintained on the FIR rack than in the control on the normal rack. In both sexes, the levels of urinary components in the experimental group was significantly higher than the control at 6-7 months of age. Spontaneous motor activity of females during the light and dark phases were higher and lower, respectively, in the experimental group than the control. The survival rate was significantly higher in the experimental group than the control. These findings suggest that FIR has 'normalization effects' on the organisms.

Proposal of an experimental model for the study of polycystic ovaries.

In view of the correlation of the circadian system with the estrous cycle in the rat, some investigators have proposed the induction of polycystic ovaries by exposing rats to continuous light. However, there is divergence with respect to time of exposure and light intensity. In the present paper we propose a simple and efficient experimental model for the study of polycystic ovarian physiopathology in rats. Twenty female Wistar rats weighing 200-220 g were initially submitted to a 14-h light/10-h dark cycle. Of the animals with regular estrous cycles, 9 were exposed to continuous light (600 lux) for 74 days (group 1) and 6 remained under the initial lighting conditions. A daily vaginal smear was used as indirect evidence for the changes in the estrous cycle. Plasma gonadotropin measurement and histological ovarian analysis showed that 90% of the animals in group 1 developed cystic follicles with the absence of the corpus luteum, despite the lack of reduction in ovarian weight commonly observed under 24-h lighting. This observation may be explained by the reduced time of stress to which the animals were submitted, since polycystic ovarian development occurred within less than 75 days of continuous exposure to 600-lux light, a time relatively shorter than commonly reported in the literature.

Effects of interrupted photoperiods on the induction of ovulation in anestrous mares.

The ability of interrupted photoperiods to induce early estrus and ovulation was examined. Horse mares were exposed to long (16 h light) or short (10 h light), noninterrupted photoperiods, ambient light, or various interrupted photoperiod treatments from December 1 to April 15 (135 d). Follicular development was assessed by rectal palpation and estrous behavior was determined by teasing with a stallion. Serum concentrations of progesterone were used as an indicator of corpus luteum function. Differences among the light treatment groups were compared for the following behavioral and ovarian characteristics: days to first detectable 3-cm follicle, days to first estrous behavior, days to first ovulation, the number of mares ovulating within the treatment period, and the number of ovulations within the treatment period per mare. Compared with the ambient and 10L:14D (L = h of light and D = h of darkness) photoperiod treatments, ovulation was advanced to the greatest extent by a photoperiod of 16L:8D and the interrupted photoperiod 10L:8D:2L:4D. These two stimulatory photoperiod treatments were characterized by the presence of light 8 to 10 h after dusk. Therefore, the present data are consistent with an external coincidence model for the induction of seasonal breeding in horses, with the photoinducible phase occurring within the period 8 to 10 h after dusk.

Effect of light regimen under various ambient temperatures on sow and litter performance.

Primiparous Large White females were exposed to one of two light regimens characterized by an increase (from 12 to 16 h/d, LONG treatment) or a decrease (from 12 to 8 h/d, SHORT treatment) in photoperiod during gestation. Two successive replicates were conducted, and farrowings occurred in January and July. During the last month of gestation, during lactation and after weaning, ambient temperatures varied between 18 and 25 degrees C and between 25 and 38 degrees C for the first and second replicates, respectively. Weaning occurred at 3 wk postpartum. Percentage of sows in estrus within 10 d after weaning was higher (P < .05) in the SHORT than in the LONG group for both replicates and when farrowing occurred in January compared with July for both photoperiod groups (SHORT-January: 92%, LONG-January: 53%, SHORT-July: 32%, LONG-July: 14%). Loss of live weight in sows during lactation was higher in July than in January and higher in the SHORT than in the LONG photoperiod (P < .05). Live weight of pigs at birth was higher in July than in January, whereas growth rate of pigs until weaning was higher in January (P < .05). Temperature may have a greater influence on sow and litter performance than photoperiod.

Reduction in age of puberty in gilts consuming melatonin during decreasing or increasing daylength.

Two experiments were conducted to determine whether oral administration of melatonin alters the onset of puberty in gilts during naturally increasing or decreasing daylength. In Exp. 1, 20 crossbred prepubertal gilts weighing 77.5 +/- .5 kg at 171.8 +/- 1.0 d of age were assigned randomly to receive either a daily oral dose of 3 mg of melatonin (MEL) or ethanol vehicle (ETH) at 1530 from August 31 to December 1, 1987 (decreasing daylength). Gilts were exposed to mature boars for 20 min thrice weekly and blood samples were collected twice weekly. Serum concentrations of progesterone were used to establish age at puberty and length of estrous cycle. In Exp. 2, 20 crossbred prepubertal gilts weighing 67.7 +/- .7 kg at 143.8 +/- 1.1 d of age received either MEL or ETH treatment from February 1 to May 15, 1988 (increasing daylength). Age of puberty was less in gilts that received MEL than in gilts that received ETH in both Exp. 1 (198 +/- 3 vs 228 +/- 7 d; P less than .01) and Exp. 2 (183.8 +/- 2.7 d vs 194.3 +/- 3.3 d; P less than .05). Gilts that received MEL reached puberty at a lighter weight than gilts that received ETH in Exp. 1 (95.6 +/- 2.1 vs 112.4 +/- 3.9 kg; P less than .01) and Exp. 2 (88.1 +/- 1.5 vs 96.0 +/- 1.8 kg; P less than .01). Serum concentrations of LH and FSH, length of estrous cycles, and percentage of muscle of carcasses were similar between MEL and ETH gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Light intensity and the oestrous cycle in albino and normally pigmented mice.

The effects of light intensity (15-20 lux & 220-290 lux) on the oestrous cycle of albino and normally pigmented mice were examined. The oestrous cycle of both types of mice was shorter at the lower intensity but the difference was significant only with the black mice. The proportion of albino mice from which embryos were recovered was significantly smaller than the proportion of black mice at 15-20 lux but not at 220-290 lux. No significant differences due to strain or light intensity were found in the number of embryos recovered. We conclude that pigmented mice respond in the same way as albino mice to changes in light intensity within the range normally found in laboratory animal accommodation. That is, increased light intensity prolongs the oestrous cycle and the period of vaginal cornification.

Effects of environmental lighting and tryptophan devoid diet on the rat vaginal cycle.

Cerebral serotonin level influences luteinizing hormone release and, consequently, ovulation. The present study evaluated the effects of precooked maize meal (polenta), a diet almost devoid of tryptophan the serotonin precursor on the alterations of the estrus cycle as measured by vaginal smears analysis in Wistar rats. Several conditions of environmental lighting were used in order to modify ovarian cycle: 1) natural alternating light/dark cycle; 2) continuous darkness; 3) continuous light by sodium steams: 4) continuous light by fluorescent neon tubes. Rats bred in continuous lighting showed estrus-proestrus rate significantly greater than rats bred in normal lighting or in continuous darkness. The feeding with precooked maize meal suppressed persistent estrus in rats bred in continuous lighting, and significantly cut down the estrus-proestrus frequency in any condition of environmental lighting. Our results lead to hypothesize that polenta diet, for its low tryptophan content, cutting down both tryptophan plasma content and serotonin neuronal synthesis, promotes luteinizing hormone peak.

Effect of photic changes and olfactory impairment on reproduction in female south Indian gerbil.

Effect of exposure to constant light (CL), blinding and olfactory bulbectomy (OBX) on reproduction of adult and weanling female Tatera indica cuvieri was investigated. In adult females, CL induced changes in estrus cyclicity. Weanlings subjected to CL showed reduced ovarian weight. Blinding did not bring about changes in estrus cyclicity and reproductive organ weight (ovary and uterus) of either adults or weanlings. Estrus cyclicity of both adults and weanlings were affected consequent to OBX. In weanlings, OBX lowered the ovarian and uterine weight.