Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS.

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17ß-estradiol (17ß-E2) promotes aggressive behavior, including during the nonbreeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17ß-E2 and disregard other estrogens. Here, we developed and validated a method to measure four estrogens [estrone (E1), 17ß-E2, 17α-estradiol (17α-E2), estriol (E3)] simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest E1 and 17ß-E2 levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the nonbreeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.

Steroid profiling of glucocorticoids in microdissected mouse brain across development.

Corticosterone is produced by the adrenal glands and also produced locally by other organs, such as the brain. Local levels of corticosterone in specific brain regions during development are not known. Here, we microdissected brain tissue and developed a novel liquid chromatography tandem mass spectrometry method (LC-MS/MS) to measure a panel of seven steroids (including 11-deoxycorticosterone (DOC), corticosterone, and 11-dehydrocorticosterone (DHC) in the blood, hippocampus (HPC), cerebral cortex (CC), and hypothalamus (HYP) of mice at postnatal day (PND) 5, 21, and 90. In a second cohort of mice, we measured the expression of three genes that code for steroidogenic enzymes that regulate corticosterone levels (Cyp11b1, Hsd11b1, and Hsd11b2) in the HPC, CC, and HYP. There were region-specific patterns of steroid levels across development, including higher corticosterone levels in the HPC and HYP than in the blood at PND5. In contrast, corticosterone levels were higher in the blood than in all brain regions at PND21 and PND90. Brain corticosterone levels were not positively correlated with blood corticosterone levels, and correlations across brain regions increased with age. Local corticosterone levels were best predicted by local DOC levels at PND5, but by local DHC levels at PND21 and PND90. Transcripts for the three enzymes were detectable in all samples (with highest expression of Hsd11b1) and showed region-specific changes with age. These data demonstrate that individual brain regions fine-tune local levels of corticosterone during early development and that coupling of glucocorticoid levels across regions increases with age.

Profiling of systemic and brain steroids in male songbirds: Seasonal changes in neurosteroids.

Steroids are secreted by the gonads and adrenal glands into the blood to modulate neurophysiology and behaviour. In addition, the brain can metabolise circulating steroids and synthesise steroids de novo. Songbirds show high levels of neurosteroid synthesis. In the present study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the measurement of 10 steroids in whole blood, plasma and microdissected brain tissue (1-2 mg) of song sparrows. Our assay is highly accurate, precise, specific and sensitive. Moreover, the liquid-liquid extraction is fast, simple and effective. We quantified steroids in the blood and brain of wild male song sparrows in both breeding and non-breeding seasons. As expected, systemic androgen levels were higher in the breeding season than in the non-breeding season. Brain androgens were detectable only in the breeding season; androstenedione and 5α-dihydrotestosterone levels were up to 20-fold higher in specific brain regions than in blood. Oestrogens were not detectable in blood in both seasons. Oestrone and 17ß-oestradiol were detectable in brain in the breeding season only (up to 1.4 ng g-1 combined). Progesterone levels in several regions were higher in the non-breeding season than the breeding season, despite the lack of seasonal changes in systemic progesterone. Corticosterone levels in the blood were higher in the breeding season than in the non-breeding season but showed few seasonal differences in the brain. In general, the steroid levels presented here are lower than those in previous reports using immunoassays, because of the higher specificity of mass spectrometry. We conclude that (i) brain steroid levels can differ greatly from circulating steroid levels and (ii) brain steroid levels show region-specific seasonal patterns that are not a simple reflection of circulating steroid levels. This approach using ultrasensitive LC-MS/MS is broadly applicable to other species and allows steroid profiling in microdissected brain regions.

Effects of aging on testosterone and androgen receptors in the mesocorticolimbic system of male rats.

As males age, systemic testosterone (T) levels decline. T regulates executive function, a collection of cognitive processes that are mediated by the mesocorticolimbic system. Here, we examined young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats, and measured systemic T levels in serum and local T levels in microdissected nodes of the mesocorticolimbic system (ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC)). We also measured androgen receptor (AR) immunoreactivity (-ir) in the mesocorticolimbic system. As expected, systemic T levels decreased with age. Local T levels in mesocorticolimbic regions - except the VTA - also decreased with age. Mesocorticolimbic T levels were higher than serum T levels at both ages. AR-ir was present in the VTA, NAc, mPFC, and OFC and decreased with age in the mPFC. Taken together with previous results, the data suggest that changes in androgen signaling may contribute to changes in executive function during aging.

Neuropeptide Y and orexin immunoreactivity in the sparrow brain coincide with seasonal changes in energy balance and steroids.

The transition between the breeding and nonbreeding states is often marked by a shift in energy balance. Despite this well-known shift in energy balance, little work has explored seasonal differences in the orexigenic neuropeptides that regulate food intake in wild animals. Here we tested the hypothesis that free-living male song sparrows (Melospiza melodia) show seasonal changes in energetic state, circulating steroids, and both neuropeptide Y (NPY) and orexin (OX) immunoreactivity. Nonbreeding song sparrows had more fat and muscle, as well as a ketone and triglyceride profile suggesting a greater reliance on lipid reserves. Breeding birds had higher plasma androgens; however, nonbreeding birds did maintain androgen precursors in circulation. Nonbreeding birds had more NPY immunoreactivity, specifically in three brain regions: lateral septum, bed nucleus of the stria terminalis, and ventral tegmental area. Furthermore, nonbreeding birds had more OX immunoreactivity in multiple brain regions. Taken together, the data indicate that a natural shift in energy balance is associated with changes in NPY and OX in a region-specific manner.

Preparing to migrate: expression of androgen signaling molecules and insulin-like growth factor-1 in skeletal muscles of Gambel's white-crowned sparrows.

Migratory birds, including Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), exhibit profound modifications of skeletal muscles prior to migration, notably hypertrophy of the pectoralis muscle required for powered flight. Muscle growth may be influenced by anabolic effects of androgens; however, prior to spring departure, circulating androgens are low in sparrows. A seasonal increase in local androgen signaling may occur within muscle to promote remodeling. We measured morphological parameters, plasma and tissue levels of testosterone, as well as mRNA expression levels of androgen receptor, 5α-reductase (converts testosterone to 5α-dihydrotestosterone), and the androgen-dependent myotrophic factor insulin-like growth factor-1. We studied the pectoralis muscle as well as the gastrocnemius (leg) muscle of male sparrows across three stages on the wintering grounds: winter (February), pre-nuptial molt (March), and pre-departure (April). Testosterone levels were low, but detectable, in plasma and muscles at all three stages. Androgen receptor mRNA and 5α-reductase Type 1 mRNA increased at pre-departure, but did so in both muscles. Notably, mRNA levels of insulin-like growth factor-1, an androgen-dependent gene critical for muscle remodeling, increased at pre-departure in the pectoralis but decreased in the gastrocnemius. Taken together, these data suggest a site-specific molecular basis for muscle remodeling that may serve to enable long-distance flight.

Rapid effects of 17ß-estradiol on aggressive behavior in songbirds: Environmental and genetic influences.

Contribution to Special Issue on Fast effects of steroids. 17ß-estradiol (E2) has numerous rapid effects on the brain and behavior. This review focuses on the rapid effects of E2 on aggression, an important social behavior, in songbirds. First, we highlight the contributions of studies on song sparrows, which reveal that seasonal changes in the environment profoundly influence the capacity of E2 to rapidly alter aggressive behavior. E2 administration to male song sparrows increases aggression within 20 min in the non-breeding season, but not in the breeding season. Furthermore, E2 rapidly modulates several phosphoproteins in the song sparrow brain. In particular, E2 rapidly affects pCREB in the medial preoptic nucleus, in the non-breeding season only. Second, we describe studies of the white-throated sparrow, which reveal how a genetic polymorphism may influence the rapid effects of E2 on aggression. In this species, a chromosomal rearrangement that includes ESR1, which encodes estrogen receptor α (ERα), affects ERα expression in the brain and the ability of E2 to rapidly promote aggression. Third, we summarize studies showing that aggressive interactions rapidly affect levels of E2 and other steroids, both in the blood and in specific brain regions, and the emerging potential for steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS). Such studies of songbirds demonstrate the value of an ethologically informed approach, in order to reveal how steroids act rapidly on the brain to alter naturally-occurring behavior.

Testosterone and Corticosterone in the Mesocorticolimbic System of Male Rats: Effects of Gonadectomy and Caloric Restriction.

Steroid hormones can modulate motivated behaviors through the mesocorticolimbic system. Gonadectomy (GDX) is a common method to determine how steroids influence the mesocorticolimbic system, and caloric restriction (CR) is often used to invigorate motivated behaviors. A common assumption is that the effects of these manipulations on brain steroid levels reflects circulating steroid levels. We now know that the brain regulates local steroid levels in a region-specific manner; however, previous studies have low spatial resolution. Using ultrasensitive liquid chromatography tandem mass spectrometry, we examined steroids in microdissected regions of the mesocorticolimbic system (ventral tegmental area, nucleus accumbens, medial prefrontal cortex). We examined whether GDX or CR influences systemic and local steroids, particularly testosterone (T) and steroidogenic enzyme transcripts. Adult male rats underwent a GDX surgery and/or CR for either 2 or 6 weeks. Levels of T, the primary steroid of interest, were higher in all brain regions than in the blood, whereas corticosterone (CORT) was lower in the brain than in the blood. Importantly, GDX completely eliminated T in the blood and lowered T in the brain. Yet, T remained present in the brain, even 6 weeks after GDX. CR decreased both T and CORT in the blood and brain. Steroidogenic enzyme (Cyp17a1, 3ß-hydroxysteroid dehydrogenase, aromatase) transcripts and androgen receptor transcripts were expressed in the mesocorticolimbic system and differentially affected by GDX and CR. Together, these results suggest that T is synthesized within the mesocorticolimbic system. These results provide a foundation for future studies examining how neurosteroids influence behaviors mediated by the mesocorticolimbic system.

Tyramide Signal Amplification Permits Immunohistochemical Analyses of Androgen Receptors in the Rat Prefrontal Cortex.

Research on neural androgen receptors (ARs) has traditionally focused on brain regions that regulate reproductive and aggressive behaviors, such as the hypothalamus and amygdala. Although many cells in the prefrontal cortex (PFC) also express ARs, the number of ARs per cell appears to be much lower, and thus, AR immunostaining is often hard to detect and quantify in the PFC. Here, we demonstrate that biotin tyramide signal amplification (TSA) dramatically increases AR immunoreactivity in the rat brain, including critical regions of the PFC such as the medial PFC (mPFC) and orbitofrontal cortex (OFC). We show that TSA is useful for AR detection with both chromogenic and immunofluorescent immunohistochemistry. Double-labeling studies reveal that AR+ cells in the PFC and hippocampus are NeuN+ but not GFAP+ and thus primarily neuronal. Finally, in gonadally intact rats, more AR+ cells are present in the mPFC and OFC of males than of females. Future studies can use TSA to further examine AR immunoreactivity across ages, sexes, strains, and different procedures (e.g., fixation methods). In light of emerging evidence for the androgen regulation of executive function and working memory, these results may help understand the distribution and roles of ARs in the PFC.

Sex steroid profiles in zebra finches: Effects of reproductive state and domestication.

The zebra finch is a common model organism in neuroscience, endocrinology, and ethology. Zebra finches are generally considered opportunistic breeders, but the extent of their opportunism depends on the predictability of their habitat. This plasticity in the timing of breeding raises the question of how domestication, a process that increases environmental predictability, has affected their reproductive physiology. Here, we compared circulating steroid levels in various "strains" of zebra finches. In Study 1, using radioimmunoassay, we examined circulating testosterone levels in several strains of zebra finches (males and females). Subjects were wild or captive (Captive Wild-Caught, Wild-Derived, or Domesticated). In Study 2, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined circulating sex steroid profiles in wild and domesticated zebra finches (males and females). In Study 1, circulating testosterone levels in males differed across strains. In Study 2, six steroids were detectable in plasma from wild zebra finches (pregnenolone, progesterone, dehydroepiandrosterone (DHEA), testosterone, androsterone, and 5α-dihydrotestosterone (5α-DHT)). Only pregnenolone and progesterone levels changed across reproductive states in wild finches. Compared to wild zebra finches, domesticated zebra finches had elevated levels of circulating pregnenolone, progesterone, DHEA, testosterone, androstenedione, and androsterone. These data suggest that domestication has profoundly altered the endocrinology of this common model organism. These results have implications for interpreting studies of domesticated zebra finches, as well as studies of other domesticated species.

Locally elevated cortisol in lymphoid organs of the developing zebra finch but not Japanese quail or chicken.

Glucocorticoids are important for production of functional lymphocytes and immunity. In altricial neonates, adrenal glands are unresponsive and local glucocorticoid synthesis in lymphoid organs may be necessary to support lymphocyte development. Precocial neonates, in contrast, have fully responsive adrenal glucocorticoid production, and lymphoid glucocorticoid synthesis may not be necessary. Here, we found that in altricial zebra finch hatchlings, lymphoid organs had dramatically elevated endogenous glucocorticoid (and precursor) levels compared to levels in circulating blood. Furthermore, while avian adrenals produce corticosterone, finch lymphoid organs had much higher levels of cortisol, an unexpected glucocorticoid in birds. In contrast, precocial Japanese quail and chicken offspring did not have locally elevated lymphoid glucocorticoid levels, nor did their lymphoid organs contain high proportions of cortisol. These results show that lymphoid glucocorticoids differ in identity, concentration, and possibly source, in hatchlings of three different bird species. Locally-regulated glucocorticoids might have species-specific roles in immune development.

Steroid profiling reveals widespread local regulation of glucocorticoid levels during mouse development.

Glucocorticoids (GCs) are produced by the adrenal glands and circulate in the blood to coordinate organismal physiology. In addition, different tissues may independently regulate their local GC levels via local GC synthesis. Here, we find that in the mouse, endogenous GCs show tissue-specific developmental patterns, rather than mirroring GCs in the blood. Using solid-phase extraction, HPLC, and specific immunoassays, we quantified endogenous steroids and found that in tissues of female and male mice, (1) local GC levels can be much higher than systemic GC levels, (2) local GCs follow age-related patterns different from those of systemic GCs, and (3) local GCs have identities different from those of systemic GCs. For example, whereas corticosterone is the predominant circulating adrenal GC in mice, high concentrations of cortisol were measured in neonatal thymus, bone marrow, and heart. The presence of cortisol was confirmed with liquid chromatography-tandem mass spectrometry. In addition, gene expression of steroidogenic enzymes was detected across multiple tissues, consistent with local GC production. Our results demonstrate that local GCs can differ from GCs in circulating blood. This finding suggests that steroids are widely used as local (paracrine or autocrine) signals, in addition to their classic role as systemic (endocrine) signals. Local GC regulation may even be the norm, rather than the exception, especially during development.

Inhibition of hippocampal aromatization impairs spatial memory performance in a male songbird.

Recent studies have revealed the presence and regulation of aromatase at the vertebrate synapse, and identified a critical role played by presynaptic estradiol synthesis in the electrophysiological response to auditory and other social cues. However, if and how synaptic aromatization affects behavior remains to be directly tested. We have exploited 3 characteristics of the zebra finch hippocampus (HP) to test the role of synaptocrine estradiol provision on spatial memory function. Although the zebra finch HP contains abundant aromatase transcripts and enzyme activity, immunocytochemical studies reveal widespread pre- and postsynaptic, but sparse to undetectable somal, localization of this enzyme. Further, the superficial location of the avian HP makes possible the more exclusive manipulation of its neurochemical characteristics without perturbation of the neuropil and the resultant induction of astroglial aromatase. Last, as in other vertebrates, the HP is critical for spatial memory performance in this species. Here we report that local inhibition of hippocampal aromatization impairs spatial memory performance in an ecologically valid food-finding task. Local aromatase inhibition also resulted in lower levels of estradiol in the HP, but not in adjacent brain areas, and was achieved without the induction of astroglial aromatase. The observed decrement in acquisition and subsequent memory performance as a consequence of lowered aromatization was similar to that achieved by lesioning this locus. Thus, hippocampal aromatization, much of which is achieved at the synapse in this species, is critical for spatial memory performance.

Xenopus oocyte meiosis lacks spindle assembly checkpoint control.

The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC.

Sex steroid levels and AD-like pathology in 3xTgAD mice.

Decreases in testosterone and 17ß-oestradiol (E(2)) are associated with an increased risk for Alzheimer's disease (AD), which has been attributed to an increase in ß-amyloid and tau pathological lesions. Although recent studies have used transgenic animal models to test the effects of sex steroid manipulations on AD-like pathology, almost none have systematically characterised the associations between AD lesions and sex steroid levels in the blood or brain in any mutant model. The present study evaluated age-related changes in testosterone and E(2) concentrations, as well as androgen receptor (AR) and oestrogen receptor (ER) α and ß expression, in brain regions displaying AD pathology in intact male and female 3xTgAD and nontransgenic (ntg) mice. We report for the first time that circulating and brain testosterone levels significantly increase in male 3xTgAD mice with age, but without changes in AR-immunoreactive (IR) cell number in the hippocampal CA1 or medial amygdala. The age-related increase in hippocampal testosterone levels correlated positively with increases in the conformational tau isoform, Alz50. These data suggest that the over-expression of human tau up-regulate the hypothalamic-pituitary-gonadal axis in these mice. Although circulating and brain E(2) levels remained stable with age in both male and female 3xTgAD and ntg mice, ER-IR cell number in the hippocampus and medial amygdala decreased with age in female transgenic mice. Furthermore, E(2) levels were significantly higher in the hippocampus than in serum, suggesting local production of E(2). Although triple transgenic mice mimic AD-like pathology, they do not fully replicate changes in human sex steroid levels, and may not be the best model for studying the effects of sex steroids on AD lesions.

Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes.

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

Measurement of steroid concentrations in brain tissue: methodological considerations.

It is well recognized that steroids are synthesized de novo in the brain (neurosteroids). In addition, steroids circulating in the blood enter the brain. Steroids play numerous roles in the brain, such as influencing neural development, adult neuroplasticity, behavior, neuroinflammation, and neurodegenerative diseases such as Alzheimer's disease. In order to understand the regulation and functions of steroids in the brain, it is important to directly measure steroid concentrations in brain tissue. In this brief review, we discuss methods for the detection and quantification of steroids in the brain. We concisely present the major advantages and disadvantages of different technical approaches at various experimental stages: euthanasia, tissue collection, steroid extraction, steroid separation, and steroid measurement. We discuss, among other topics, the potential effects of anesthesia and saline perfusion prior to tissue collection; microdissection via Palkovits punch; solid phase extraction; chromatographic separation of steroids; and immunoassays and mass spectrometry for steroid quantification, particularly the use of mass spectrometry for "steroid profiling." Finally, we discuss the interpretation of local steroid concentrations, such as comparing steroid levels in brain tissue with those in the circulation (plasma vs. whole blood samples; total vs. free steroid levels). We also present reference values for a variety of steroids in different brain regions of adult rats. This brief review highlights some of the major methodological considerations at multiple experimental stages and provides a broad framework for designing studies that examine local steroid levels in the brain as well as other steroidogenic tissues, such as thymus, breast, and prostate.

Antiapoptotic role for ornithine decarboxylase during oocyte maturation.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, is a nonredundant and essential gene in all eukaryotes. During the mitotic cell cycle, ODC exhibits two activity peaks: one at the G(1)/S transition and one during the G(2)/M transition. The physiological role of this cell cycle-dependent ODC activity dynamic is not clear. Previous studies have reported a significant elevation of ODC activity during Xenopus oocyte maturation, which resembles mitotic G(2)/M transition. In order to study the roles of ODC activity in the oocytes, we utilized antisense morpholino (xODC mo) oligonucleotides to inhibit ODC translation. We report here that xODC mo abolished ODC activity increase during oocyte maturation. xODC mo-injected oocytes underwent germinal vesicle breakdown, emitted the first polar body, and reached metaphase II, thus completing nuclear maturation. However, the metaphase II oocytes exhibited high levels of reactive oxygen species and became apoptotic. When transferred to host frogs and subsequently ovulated, these eggs were fertilized but exhibited embryo fragmentation. Translation of ODC is therefore integral to cytoplasmic maturation, protecting metaphase II oocytes from reactive oxygen species-induced apoptosis.

Polar body emission requires a RhoA contractile ring and Cdc42-mediated membrane protrusion.

Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. We report here that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion.

Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation.

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.