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Neuroscience Jul 2018Hindbrain-derived stimuli restrain the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) reproductive neuroendocrine axis during energy...
Hindbrain 5'-Adenosine Monophosphate-activated Protein Kinase Mediates Short-term Food Deprivation Inhibition of the Gonadotropin-releasing Hormone-Luteinizing Hormone Axis: Role of Nitric Oxide.
Hindbrain-derived stimuli restrain the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) reproductive neuroendocrine axis during energy insufficiency. Interruption of food intake, planned or unplanned, is emblematic of modern life. This study investigated the premise that the hindbrain energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibits reproductive neuroendocrine function in short term, e.g. 18-h food-deprived (FD) estradiol (E)-implanted ovariectomized female rats. Intra-caudal fourth ventricular administration of the AMPK inhibitor Compound C (Cc) reversed FD-induced inhibition of rostral preoptic (rPO) GnRH protein expression and LH release in animals given E to replicate proestrus (high-E dose-, but not metestrus (low-E dose)-stage plasma steroid levels. FD caused Cc-reversible augmentation or diminution of preoptic norepinephrine (NE) activity in high- versus low-E rats, respectively, and AMPK-independent reductions in hypothalamic NE accumulation in the latter. Nitric oxide (NO) and kisspeptin are key stimulatory signals for the preovulatory LH surge. Here, FD inhibited rPO neuronal nitric oxide synthase protein expression in high-, but not low-E-dosed animals. Lateral ventricular delivery of the NO donor 3-morpholinosydnonimine (SIN-1) reversed inhibitory GnRH and LH responses to FD in high-E rats, and normalized rPO Vglut2, anteroventral periventricular KiSS1, and dorsomedial hypothalamic RFRP-3 mRNA and/or protein profiles. Data show that FD curtails reproductive neuroendocrine outflow by hindbrain AMPK-dependent mechanisms in the presence of peak estrous cycle E levels. Results indicate that neural networks linking this sensor to GnRH neurons likely involve NO signaling, which may function upstream of one or more neurotransmitters identified here by SIN-1-reversible inhibitory responses to FD.
Topics: Adenylate Kinase; Animals; Estradiol; Female; Food Deprivation; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Neurosecretory Systems; Nitric Oxide; Rats; Rats, Sprague-Dawley; Rhombencephalon
PubMed: 29746990
DOI: 10.1016/j.neuroscience.2018.04.040 -
International Journal of Reproductive... Feb 2018The Wingless-type (Wnt)/β-catenin signaling pathway controls cell homeostasis. Reproductive tissues are dynamic in response to steroidal hormone changes. Steroidal...
BACKGROUND
The Wingless-type (Wnt)/β-catenin signaling pathway controls cell homeostasis. Reproductive tissues are dynamic in response to steroidal hormone changes. Steroidal hormones are known to control the Wnt/β-catenin pathway, but their role in reproductive tissues remains unknown.
OBJECTIVE
The present study aims to investigate the expression patterns of Wnt/β-catenin target genes in mouse reproductive tissues during the normal estrous cycle.
MATERIALS AND METHODS
In this experimental study, 16 adult NMRI mice were grouped as proestrus, estrus, metestrus, and diestrus according to vaginal smear and histological evaluation of uterine and ovarian tissues. Uterine horns and ovarian tissues were collected. Reverse transcription quantitative polymerase chain reaction was performed to evaluate the expression of Wnt/β-catenin target genes ( and ) at different stages of the estrous cycle.
RESULTS
The expression levels of , , and in uterine tissue were significantly higher at the proestrus phase than at the other stages. Meanwhile, expression in uterine tissue was significantly higher at the metestrus stage than at the other stages. Furthermore, expression was significantly higher at the diestrus stage than at the estrus and metestrus stages. In the ovarian tissue, the highest expression of , , and was detected at the proestrus stage, whereas the highest expression of and was observed at the estrus stage.
CONCLUSION
This study showed that Wnt/β-catenin target genes profiles are different among estrous cycle. It seems that different hormonal profiles during estrous cycles play a key role in the expression pattern of Wnt/β-catenin target genes in ovarian and uterine tissue.
PubMed: 29675490
DOI: No ID Found -
Frontiers in Molecular Neuroscience 2018Periodic oscillations of gonadal hormone levels during the estrous cycle exert effects on the female brain, impacting cognition and behavior. While previous research...
Periodic oscillations of gonadal hormone levels during the estrous cycle exert effects on the female brain, impacting cognition and behavior. While previous research suggests that changes in hormone levels across the cycle affect dendritic spine dynamics in the hippocampus, little is known about the effects on cortical dendritic spines and previous studies showed contradictory results. In this imaging study, we investigated the impact of the estrous cycle on the density and dynamics of dendritic spines of pyramidal neurons in the primary somatosensory cortex of mice. We also examined if the induction of synaptic plasticity during proestrus, estrus, and metestrus/diestrus had differential effects on the degree of remodeling of synapses in this brain area. We used chronic two-photon excitation (2PE) microscopy during steady-state conditions and after evoking synaptic plasticity by whisker stimulation at the different stages of the cycle. We imaged apical dendritic tufts of layer 5 pyramidal neurons of naturally cycling virgin young female mice. Spine density, turnover rate (TOR), survival fraction, morphology, and volume of mushroom spines remained unaltered across the estrous cycle, and the values of these parameters were comparable with those of young male mice. However, while whisker stimulation of female mice during proestrus and estrus resulted in increases in the TOR of spines (74.2 ± 14.9% and 75.1 ± 12.7% vs. baseline, respectively), sensory-evoked plasticity was significantly lower during metestrus/diestrus (32.3 ± 12.8%). In males, whisker stimulation produced 46.5 ± 20% increase in TOR compared with baseline-not significantly different from female mice at any stage of the cycle. These results indicate that, while steady-state density and dynamics of dendritic spines of layer 5 pyramidal neurons in the primary somatosensory cortex of female mice are constant during the estrous cycle, the susceptibility of these neurons to sensory-evoked structural plasticity may be dependent on the stage of the cycle. Since dendritic spines are more plastic during proestrus and estrus than during metestrus/diestrus, certain stages of the cycle could be more suitable for forms of memory requiring formation and elimination of spines and other stages for forms of memory where retention and/or repurposing of already existing synaptic connections is more pertinent.
PubMed: 29615867
DOI: 10.3389/fnmol.2018.00083 -
Toxicological Sciences : An Official... May 2018Multidrug resistance protein 1 (MDR1), a phase III drug transporter that exports substrates out of cells, has been discovered in both cancerous and normal tissues. The...
Multidrug resistance protein 1 (MDR1), a phase III drug transporter that exports substrates out of cells, has been discovered in both cancerous and normal tissues. The over expression of MDR1 in cancer cells contributes to multiple drug resistance, whereas the MDR1 in normal tissues protects them from chemical-induced toxicity. Currently, the role of MDR1 in the ovary has not been entirely understood. Our objective is to determine the function of MDR1 in protecting against chemotherapy-induced ovarian toxicity. Using both the in vivo transgenic mouse model and in vitro follicle culture model, we investigated the expression of MDR1 in the ovary, the effect of MDR1 deficiency on doxorubicin (DOX)-induced ovarian toxicity, and the ovarian steroid hormonal regulation of MDR1. Results showed that the MDR1 was expressed in the ovarian epithelial cells, stroma cells, theca cell layers, endothelial cells, and luteal cells. The lack of MDR1 did not affect female ovarian function and fertility; however, its deficiency significantly exacerbated the DOX-induced ovarian toxicity in both in vivo and in vitro models. The MDR1 showed significantly higher expression levels in the ovaries at estrus and metestrus stages than those at proestrus and diestrus stages. However, this dynamic expression pattern was not regulated by the ovarian steroid hormones of estrogen (E2) and progesterone (P4) but correlated to the number and status of corpus luteum. In conclusion, our study demonstrates that the lack of MDR1 promotes DOX-induced ovarian toxicity, suggesting the critical role of MDR1 in protecting female ovarian functions during chemotherapy.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Antibiotics, Antineoplastic; Apoptosis; Doxorubicin; Female; Fertility; Mice, Knockout; Ovary; Superovulation
PubMed: 29462422
DOI: 10.1093/toxsci/kfy038 -
Molecular Human Reproduction Mar 2018Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females?
STUDY QUESTIONS
Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females?
SUMMARY ANSWER
OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility.
WHAT IS KNOWN ALREADY
Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence.
STUDY DESIGN, SIZE, DURATION
Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP.
MAIN RESULTS AND THE ROLE OF CHANCE
TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction.
LARGE SCALE DATA
None.
LIMITATIONS REASONS FOR CAUTION
Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-.
WIDER IMPLICATIONS OF THE FINDINGS
Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics.
STUDY FUNDING AND COMPETING INTEREST(S)
The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.
Topics: Animals; Calcium-Transporting ATPases; Fallopian Tubes; Female; Humans; Mice; Microscopy, Electron, Transmission; Oviducts; Plasma Membrane Calcium-Transporting ATPases; Premenopause; Sperm Capacitation; Sperm Motility
PubMed: 29370405
DOI: 10.1093/molehr/gay003 -
Endocrinology Feb 2018Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females...
Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females with prepregnancy hyperandrogenemia and impaired ovulation, however, has not been examined. We developed a mouse model of hyperandrogenemia by implanting a low-dose dihydrotestosterone (DHT) pellet 15 days before conception. Female offspring born to dams with hyperandrogenemia (DHT daughters) had delayed puberty (P < 0.05) with first estrus on postnatal day (PND) 41 compared with daughters from dams with physiological levels of DHT (non-DHT daughters, PND37.5). Serum follicle-stimulating hormone (FSH) levels in the DHT daughters were fourfold higher (P < 0.05) on PND21, and anti-Müllerian hormone levels were higher (P < 0.05) on PND26 than in non-DHT daughters (controls). DHT daughters showed an extended time in metestrus/diestrus and a shorter time in the proestrus/estrus phase compared with non-DHT daughters (P < 0.05). To examine ovarian response to gonadotropins, superovulation was induced and in vitro fertilization (IVF) was performed. Fewer numbers of oocytes were retrieved from the DHT daughters compared with non-DHT daughters (P < 0.05). At IVF, there was no difference in rates of fertilization or cleavage of oocytes from either group. There were fewer (P < 0.01) primordial follicles (6.5 ± 0.8 vs 14.5 ± 2.1 per ovary) in the ovaries of DHT daughters compared with non-DHT daughters. Daughters from hyperandrogenemic females exhibited elevated prepubertal FSH levels, diminished ovarian response to superovulation, impaired estrous cyclicity, delayed onset of puberty, and reduced ovarian reserve, suggesting that fetal androgen exposure had lasting effects on female reproductive function.
Topics: Animals; Cells, Cultured; Chronic Disease; Female; Fertility; Hyperandrogenism; Male; Mice; Mice, Inbred C57BL; Ovarian Reserve; Pregnancy; Prenatal Exposure Delayed Effects; Primary Ovarian Insufficiency; Sexual Maturation
PubMed: 29315373
DOI: 10.1210/en.2017-03078 -
Sheng Li Xue Bao : [Acta Physiologica... Dec 2017Careful analysis of follicular morphology and numbers of different follicular maturation stages, in combination with the measurements of gonadotropic and sex hormone...
Careful analysis of follicular morphology and numbers of different follicular maturation stages, in combination with the measurements of gonadotropic and sex hormone profiles, provide an accurate and rapid evaluation system of the ovarian function. The aim of this study is to improve the existing methods of ovarian tissue section preparation and staining methods, and to establish a fast and easy method to observe and evaluate follicular maturation stage and numbers using mouse ovary samples. Ovaries were collected at menstrual phases of proestrus, oestrus, metestrus and diestrus from C57BL/6J female mice. Then the ovaries were fixed in 4% paraformaldehyde, dehydrated with graded sucrose, embedded with OCT, frozen-sectioned at 7 μm thick, and subjected to quick haematoxylin & eosin staining for observation. The results showed that the present method was able to distinguish secondary, preantral, antral, and preovulatory follicles. Although our method was unable to discriminate and distinguish primordial follicles and primary follicles, the results were comparable to those from more complicated techniques. We conclude that this improved and quick method can be used in combination with hormone analysis to investigate ovarian development and function in different mouse models.
Topics: Animals; Estrus; Female; Frozen Sections; Gonadal Steroid Hormones; Mice; Mice, Inbred C57BL; Ovarian Follicle; Staining and Labeling
PubMed: 29270594
DOI: No ID Found -
Journal of Pharmacy & Pharmaceutical... 2017Monocarboxylate transporters (MCTs) are involved in the transport of monocarboxylates such as ketone bodies, lactate, and pharmaceutical agents. CD147 functions as an...
PURPOSE
Monocarboxylate transporters (MCTs) are involved in the transport of monocarboxylates such as ketone bodies, lactate, and pharmaceutical agents. CD147 functions as an ancillary protein for MCT1 and MCT4 for plasma membrane trafficking. Sex differences in MCT1 and MCT4 have been observed in muscle and reproductive tissues; however, there is a paucity of information on MCT sex differences in tissues involved in drug disposition. The objective of the present study was to quantify hepatic MCT1, MCT4 and CD147 mRNA, total cellular and membrane protein expression in males, over the estrous cycle in females and in ovariectomized (OVX) females.
METHOD
Liver samples were collected from females at the four estrous cycle stages (proestrus, estrus, metestrus, diestrus), OVX females and male Sprague-Dawley rats (N = 3 - 5). Estrus cycle stage of females was determined by vaginal lavage. mRNA and protein (total and membrane) expression of MCT1, MCT4 and CD147 was evaluated by qPCR and western blot analysis.
RESULTS
MCT1 mRNA and membrane protein expression varied with estrous cycle stage, with OVX females having higher expression than males, indicating that female sex hormones may play a role in MCT1 regulation. MCT4 membrane expression varied with estrous cycle stage with expression significantly lower than males. MCT4 membrane expression in OVX females was also lower than males, suggesting that androgens play a role in membrane expression of MCT4. Males had higher membrane CD147 expression, whereas there was no difference in whole cell protein and mRNA levels suggesting that androgens are involved in regulating CD147 membrane localization.
CONCLUSIONS
This study demonstrates hepatic expression and membrane localization of MCT1, MCT4 and CD147 are regulated by sex hormones. Sex differences in hepatic MCT expression may lead to altered drug disposition, so it is critical to elucidate the underlying mechanisms in the sex hormone-dependent regulation of MCT expression. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Topics: Animals; Basigin; Biological Transport; Cell Membrane; Female; Gonadal Steroid Hormones; Male; Monocarboxylic Acid Transporters; RNA, Messenger; Rats; Rats, Sprague-Dawley
PubMed: 29249221
DOI: 10.18433/J3CH29 -
Behavioural Brain Research Feb 2018Females are an under-represented research model and the mechanisms through which sleep loss impairs cognition are not clear. Since levels of reproductive hormones and...
Females are an under-represented research model and the mechanisms through which sleep loss impairs cognition are not clear. Since levels of reproductive hormones and the estrous cycle are sensitive to sleep loss and necessary for learning and memory, we hypothesized that sleep deprivation impacts learning and memory in female mice by interfering with the estrous cycle. We used the object recognition task to assess learning and memory in female mice during separate phases of the estrous cycle and after sleep loss. Mice in metestrus/diestrus attended to sample objects less than mice in proestrus/estrus during object acquisition, the first phase of the object recognition task. Subsequently, during the recognition phase of the task, only mice in proestrus/estrus displayed a preference for the novel object. Sleep deprivation for 12h immediately before the object recognition task reduced time attending to sample objects and novel object preference for mice in proestrus/estrus, without changing length of the estrous cycle. These results show that sleep deprived mice in proestrus/estrus had learning deficits and memory impairments, like mice in metestrus/diestrus. Since sleep deprivation did not disrupt the estrous cycle, however, results did not support the hypothesis. Cognitive impairments due to acute sleep loss were not due to alterations to the estrous cycle.
Topics: Animals; Behavior, Animal; Estrous Cycle; Female; Learning; Memory; Metestrus; Mice, Inbred C57BL; Proestrus; Sleep Deprivation
PubMed: 29180134
DOI: 10.1016/j.bbr.2017.11.033 -
Scientific Reports Sep 2017Female house mice produce pheromone-carrying major urinary proteins (MUPs) in a cycling manner, thus reaching the maximum urinary production just before ovulation. This...
Female house mice produce pheromone-carrying major urinary proteins (MUPs) in a cycling manner, thus reaching the maximum urinary production just before ovulation. This is thought to occur to advertise the time of ovulation via deposited urine marks. This study aimed to characterize the protein content from the house mouse vaginal flushes to detect putative vaginal-advertising molecules for a direct identification of reproductive states. Here we show that the mouse vaginal discharge contains lipocalins including those from the odorant binding (OBP) and major urinary (MUP) protein families. OBPs were highly expressed but only slightly varied throughout the cycle, whilst several MUPs were differentially abundant. MUP20 or 'darcin', was thought to be expressed only by males. However, in females it was significantly up-regulated during estrus similarly as the recently duplicated central/group-B MUPs (sMUP17 and highly expressed sMUP9), which in the mouse urine are male biased. MUPs rise between proestrus and estrus, remain steady throughout metestrus, and are co-expressed with antimicrobial proteins. Thus, we suggest that MUPs and potentially also OBPs are important components of female vaginal advertising of the house mouse.
Topics: Animals; Estrous Cycle; Female; Lipocalins; Mice; Pheromones; Vagina
PubMed: 28916783
DOI: 10.1038/s41598-017-12021-2