Mechanism of kisspeptin neuron synchronization for pulsatile hormone secretion in male mice.

The mechanism by which arcuate nucleus kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to generate pulsatile hormone secretion remains unknown. An acute brain slice preparation maintaining synchronized ARNKISS neuron burst firing was used alongside in vivo GCaMP GRIN lens microendoscope and fiber photometry imaging coupled with intra-ARN microinfusion. Studies in intact and gonadectomized male mice revealed that ARNKISS neuron synchronizations result from near-random emergent network activity within the population and that this was critically dependent on local glutamate-AMPA signaling. Whereas neurokinin B operated to potentiate glutamate-generated synchronizations, dynorphin-kappa opioid tone within the network served as a gate for synchronization initiation. These observations force a departure from the existing "KNDy hypothesis" for ARNKISS neuron synchronization. A "glutamate two-transition" mechanism is proposed to underlie synchronizations in this key hypothalamic central pattern generator driving mammalian fertility.

Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice.

The mechanisms underlying the homeostatic estrogen negative feedback pathway central to mammalian fertility have remained unresolved. Direct measurement of gonadotropin-releasing hormone (GnRH) pulse generator activity in freely behaving mice with GCaMP photometry demonstrated striking estradiol-dependent plasticity in the frequency, duration, amplitude, and profile of pulse generator synchronization events. Mice with Cre-dependent deletion of ESR1 from all kisspeptin neurons exhibited pulse generator activity identical to that of ovariectomized wild-type mice. An in vivo CRISPR-Cas9 approach was used to knockdown ESR1 expression selectively in arcuate nucleus (ARN) kisspeptin neurons. Mice with >80% deletion of ESR1 in ARN kisspeptin neurons exhibited the ovariectomized pattern of GnRH pulse generator activity and high frequency LH pulses but with very low amplitude due to reduced responsiveness of the pituitary. Together, these studies demonstrate that estrogen utilizes ESR1 in ARN kisspeptin neurons to achieve estrogen negative feedback of the GnRH pulse generator in mice.

Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron.

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic 'KNDy' neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

GnRH Pulse Generator Activity Across the Estrous Cycle of Female Mice.

A subpopulation of kisspeptin neurons located in the arcuate nucleus (ARN) operate as the GnRH pulse generator. The activity of this population of neurons can be monitored in real-time for long periods using kisspeptin neuron-selective GCaMP6 fiber photometry. Using this approach, we find that ARN kisspeptin neurons exhibit brief (∼50 seconds) periods of synchronized activity that precede pulses of LH in intact female mice. The dynamics and frequency of these synchronization episodes (SEs) are stable at approximately one event every 40 minutes throughout metestrus, diestrus, and proestrus, but slow considerably on estrus to occur approximately once every 10 hours. Evaluation of ARN kisspeptin neuron activity across the light-dark transition, including the time of onset of the proestrus LH surge, revealed no changes in SE frequency. Longer 24-hour recordings across proestrus into estrus demonstrated that an abrupt decrease in SEs occurred ∼4 to 5 hours after the onset of the LH surge to reach the low frequency of SEs observed on estrus. The frequency of SEs was stable across the 24-hour period from metestrus to diestrus. Administration of progesterone to diestrus mice resulted in the abrupt slowing of SEs. These observations show that the GnRH pulse generator exhibits an unvarying pattern of activity from metestrus through to the late evening of proestrus, at which time it slows dramatically, likely in response to postovulation progesterone secretion. The GnRH pulse generator maintains a constant frequency of activity across the time of the LH surge, demonstrating that it is not involved directly in surge generation.

Juvenile hormone enhances aversive learning performance in 2-day old worker honey bees while reducing their attraction to queen mandibular pheromone.

Previous studies have shown that exposing young worker bees (Apis mellifera) to queen mandibular pheromone (QMP) reduces their aversive learning performance, while enhancing their attraction to QMP. As QMP has been found to reduce the rate of juvenile hormone (JH) synthesis in worker bees, we examined whether aversive learning in 2-day old workers exposed to QMP from the time of adult emergence could be improved by injecting JH (10 µg in a 2 µl volume) into the haemolymph. We examined in addition, the effects of JH treatment on worker attraction to QMP, and on the levels of expression of amine receptor genes in the antennae, as well as in the mushroom bodies of the brain. We found that memory acquisition and 1-hour memory recall were enhanced by JH. In contrast, JH treatment reduced the bees' attraction towards a synthetic strip impregnated with QMP (Bee Boost). Levels of expression of the dopamine receptor gene Amdop1 were significantly lower in the mushroom bodies of JH-treated bees than in bees treated with vehicle alone (acetone diluted with bee ringer). Expression of the octopamine receptor gene, Amoa1, in this brain region was also affected by JH treatment, and in the antennae, Amoa1 transcript levels were significantly lower in JH-treated bees compared to controls. The results of this study suggest that QMP's effects on JH synthesis may contribute to reducing aversive learning performance and enhancing attraction to QMP in young worker bees.

Steroid hormone (20-hydroxyecdysone) modulates the acquisition of aversive olfactory memories in pollen forager honeybees.

Here, we examine effects of the steroid hormone, 20-hydroxyecdysone (20-E), on associative olfactory learning in the honeybee, Apis mellifera. 20-E impaired the bees' ability to associate odors with punishment during aversive conditioning, but did not interfere with their ability to associate odors with a food reward (appetitive learning). The steroid had a significant impact also on the expression of amine-receptor genes in centers of the brain involved in the formation and recall of associative olfactory memories (mushroom bodies). 20-E increased expression of the dopamine receptor gene, Amdop2, and reduced the expression of the putative dopamine/ecdysone receptor gene, Amgpcr19. Interestingly, Amgpcr19 tended to be highly expressed in the brains of foragers that exhibited strong aversive learning, but expressed at lower levels in bees that performed well in appetitive learning assays. In 2-d-old bees, transcript levels of the same gene could be reduced by queen mandibular pheromone, a pheromone that blocks aversive learning in young worker bees. As ecdysteroid levels rise to a peak ∼2 d after adult emergence and then fall to low levels in foragers, we examined aversive learning also in young worker bees. Aversive learning performance in 2-d-old bees was consistently poor. The results of this study indicate that learning in honeybees can be modulated by ecdysteroids. They highlight, in addition, a potential involvement of the putative dopamine/ecdysone receptor, AmGPCR19, in hormonal regulation of associative olfactory learning in the honeybee.

Mushroom bodies of the honeybee brain show cell population-specific plasticity in expression of amine-receptor genes.

Dopamine and octopamine released in the mushroom bodies of the insect brain play a critical role in the formation of aversive and appetitive memories, respectively. As recent evidence suggests a complex relationship between the effects of these two amines on the output of mushroom body circuits, we compared the expression of dopamine- and octopamine-receptor genes in three major subpopulations of mushroom body intrinsic neurons (Kenyon cells). Using the brain of the honeybee, Apis mellifera, we found that expression of amine-receptor genes differs markedly across Kenyon cell subpopulations. We found, in addition, that levels of expression of these genes change dramatically during the lifetime of the bee and that shifts in expression are cell population-specific. Differential expression of amine-receptor genes in mushroom body neurons and the plasticity that exists at this level are features largely ignored in current models of mushroom body function. However, our results are consistent with the growing body of evidence that short- and long-term olfactory memories form in different regions of the mushroom bodies of the brain and that there is functional compartmentalization of the modulatory inputs to this multifunctional brain center.

Effects of reproductive stage and 11-ketotestosterone on LPL mRNA levels in the ovary of the shortfinned eel.

To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT.

Peripheral modulation of worker bee responses to queen mandibular pheromone.

It is generally accepted that young worker bees (Apis mellifera L.) are highly attracted to queen mandibular pheromone (QMP). Our results challenge this widely held view. We have found that unless young workers are exposed to QMP early in adult life, they, like foragers, avoid contact with this pheromone. Our data indicate that responses to QMP are regulated peripherally, at the level of the antennal sensory neurons, and that a window of opportunity exists in which QMP can alter a young bee's response to this critically important pheromone. Exposing young bees to QMP from the time of adult emergence reduces expression in the antennae of the D1-like dopamine receptor gene, Amdop1. Levels of Amdop3 transcript, on the other hand, and of the octopamine receptor gene Amoa1, are significantly higher in the antennae of bees strongly attracted to QMP than in bees showing no attraction to this pheromone. A decline in QMP attraction with age is accompanied by a fall in expression in worker antennae of the D2-like dopamine receptor, AmDOP3, a receptor that is selectively activated by QMP. Taken together, our findings suggest that QMP's actions peripherally not only suppress avoidance behavior, but also enhance attraction to QMP, thereby facilitating attendance of the queen.

Expression of ovarian tumour suppressor OPCML in the female CD-1 mouse reproductive tract.

Opioid binding protein/cell adhesion molecule-like gene (OPCML) is frequently inactivated in epithelial ovarian cancer, but the role of this membrane protein in normal reproductive function is unclear. The ovarian surface epithelium (OSE) is thought to be the cell of origin of most epithelial ovarian cancers, some of which arise after transformation of OSE cells lining ovarian inclusion cysts, formed during ovulation. We used immunohistochemistry, immunoblotting and quantitative RT-PCR (qRT-PCR) to investigate OPCML expression in the uteri and ovaries of cycling 3-month CD-1 mice, as well as in ovaries from older mice containing inclusion cysts derived from rete ovarii tubules. Immunoblotting showed OPCML bands in uterine, but not whole ovarian or muscle extracts. Strong OPCML immunoreactivity was observed in oviduct, rete ovarii and uterus, whereas in ovary more immunoreactivity was seen in granulosa cells than OSE. No staining was observed in OSE around ovulation sites, where OSE cells divide to cover the site. OPCML immunoreactivity was also weaker in more dysplastic cells lining large ovarian inclusion cysts, compared with normal rete ovarii. No significant changes in Opcml mRNA expression were observed in whole ovarian and uterine extracts at different stages of the cycle. We conclude that murine OPCML is more consistently expressed in cells lining the uterus, oviduct and rete ovarii than in ovary and is not expressed in OSE associated with ovulation sites. This observation supports the hypothesis that a proportion of epithelial ovarian cancers arise from ductal cells and other epithelia of the secondary Mullerian system, rather than the OSE.

E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in age-matched breeder and incessantly ovulated CD-1 mice.

BACKGROUND: Female CD-1/Swiss Webster mice subjected to incessant ovulation for 8 months and 12-month breeder mice both developed ovarian inclusion cysts similar to serous cystadenomas. The majority of cysts appeared to be dilated rete ovarii tubules, but high ovulation number resulted in more cortical inclusion cysts. We hypothesized that comparison of inclusion cyst pathology in animals of the same age, but with differences in total lifetime ovulation number, might allow us to determine distinguishing characteristics of the two types of cyst. METHODS: Ovaries from breeder mice (BR) or females subjected to incessant ovulation (IO) were compared at 6-, 9- and 12-months of age. Ovaries were serially sectioned and cysts characterized with regard to location and histology, E-cadherin immunoreactivity and rates of BrdU incorporation. RESULTS: Inclusion cysts developed with age in BR and IO ovaries. The majority of cysts were connected to the ovarian hilus. Two cortical inclusion cysts were observed in ten IO ovaries and one in ten BR ovaries. Low or no E-cadherin immuno-staining was seen in the OSE of all mice studied. Conversely, strong membrane immuno-staining was observed in rete ovarii epithelial cells. Variable E-cadherin immunoreactivity was seen in cells of hilar inclusion cysts, with strong staining observed in cuboidal ciliated cells and little or no staining in flat epithelial cells. Two of the three cortical cysts contained papillae, which showed E-cadherin immuno-staining at the edge of cells. However hilar and cortical cysts were not distinguishable by morphology, cell type or E-cadherin immunoreactivity. BrdU incorporation in cyst cells (1.4% [95% CI: 1.0 to 2.1]) was greater than in OSE (0.7% [95% CI: 0.4 to 1.2]) and very few BrdU-labeled cells were observed in rete ovarii at any age. Incessant ovulation significantly increased BrdU incorporation in OSE of older animals. CONCLUSION: These experiments confirm ovarian inclusion cysts develop with age in the CD-1 mouse strain, irrespective of total ovulation burden. We conclude longer periods of incessant ovulation do not lead to significant changes in inclusion cyst formation or steroidogenesis in CD-1 mice and inclusion cyst type can not be distinguished by morphology, cell proliferation rate or E-cadherin immunoreactivity.

Effects of sex steroids, sex, and sexual maturity on cortisol production: an in vitro comparison of chinook salmon and rainbow trout interrenals.

Sex steroids appear to be responsible for hyperactivation of the hypothalamus-pituitary-interrenal (HPI) axis that occurs in mature semelparous Pacific salmon as a prelude to post-spawning (programmed) death. This study was undertaken to examine the direct effects of sex steroids on interrenal activity of semelparous (chinook salmon) and iteroparous (rainbow trout) salmonids using an in vitro incubation system. In addition, phenotypic sex differences in cortisol production by interrenals of sexually mature (spawning) rainbow trout and chinook salmon were investigated. Interrenal tissue from juvenile and sexually mature chinook salmon and rainbow trout was incubated for 48 h in culture medium containing either no steroid (controls), 1 microM estradiol (E2) or 1 microM 11-ketotestosterone (11-KT). This tissue was then challenged for 3h with either pregnenolone, dibutyryladenosine 3('):5(')-cyclic monophosphate (dbcAMP) or forskolin, or synthetic human adrenocorticotropic hormone (ACTH(1-24)). Sex differences in in vitro interrenal cortisol production were assessed using separate tissue pools challenged with the same agents. Cortisol in media was measured by radioimmunoassay. E2 suppressed the ability of juvenile chinook salmon interrenals to utilize pregnenolone as substrate for cortisol synthesis. In mature female chinook salmon the suppressive effect of E2 was less pronounced, but was observed as a reduced response of interrenals to both pregnenolone and dbcAMP. E2 did not affect ACTH(1-24) stimulated cortisol production. Immature and mature rainbow trout interrenals were both relatively insensitive to E2. 11-KT did not affect cortisol production by juvenile chinook salmon and juvenile or mature rainbow trout, and had only minor effects in male and female spawning chinook salmon. In mature chinook salmon and rainbow trout, the interrenals of females were more responsive to ACTH stimulation and showed a greater utilization of pregnenolone as a substrate than interrenals of males. Mature female rainbow trout were also more responsive to dbcAMP stimulation than males. The results of this study suggest that the onset of sexual maturation and gonadal steroid production may contribute to sexually dimorphic cortisol responses in vitro.

Characterization and expression of steroidogenic acute regulatory protein and MLN64 cDNAs in trout.

Complementary DNA-encoding proteins with high homology to steroidogenic acute regulatory proteins (StAR) of mammals were cloned from rainbow trout head kidney and a mixture of several brook trout tissues. A cDNA encoding an MLN64 homolog was also cloned from brook trout. The C-terminal domains of rainbow trout StAR and brook trout StAR were very highly conserved compared with StAR of mammals. In rainbow trout, Northern and RT-PCR analyses showed abundant StAR transcripts in head kidney, ovary, and testis, and weaker signals were found in intestine, pyloric caeca, spleen, and kidney. Brief acute stress resulted in elevated plasma cortisol levels and a 2-fold increase in rainbow trout StAR transcripts in head kidneys sampled 3 h after exposure to the stressor. In brook trout, StAR transcripts were detected only in known steroidogenic tissues. Ovarian brook trout StAR mRNA was not seen until the onset of final maturation. Its abundance increased during germinal vesicle breakdown, peaked during and just following ovulation, and decreased by 2 wk post ovulation. Brook trout MLN64 transcripts were found in all tissues tested, and transcript abundance in ovarian samples did not vary during final oocyte maturation and ovulation. Both StAR structure and function appear to be highly conserved throughout the vertebrates.