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Journal of Steroid Biochemistry & Molecular Biology 139 (2014) 154– 158

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry and Molecular Biology

journa l ho me p age: www.elsev ier .com/ locate / j sbmb

eview

ex hormones and expression pattern of cytoskeletal proteins in the rat brainhroughout pregnancy

liesha González-Arenasa, Ana Gabriela Pina-Medinaa, Oscar González-Floresb,gustín Galván-Rosasb, Porfirio Gómora-Arratib, Ignacio Camacho-Arroyoa,∗

Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, México, D.F., MéxicoCentro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-CINVESTAV, México

r t i c l e i n f o

rticle history:eceived 31 August 2012eceived in revised form0 December 2012ccepted 4 January 2013

eywords:rain

a b s t r a c t

Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronalcytoskeleton. In this physiological state, the brain is in contact with several hormones that it has neverbeen exposed, as well as with very high levels of hormones that the brain has been in touch throughoutlife. Among the latter hormones are progesterone and estradiol which regulate several brain functions,including learning, memory, neuroprotection, and the display of sexual and maternal behavior. Thesefunctions involve changes in the structure and organization of neurons and glial cells that require theparticipation of cytoskeletal proteins whose expression and activity is regulated by estradiol and proges-

ytoskeletonstradiolFAPAP2

regnancyrogesterone

terone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and GlialFibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestationand the start of lactation, suggesting that these proteins participate in the plastic changes observed inthe brain during pregnancy.

This article is part of a Special Issue entitled ‘Pregnancy and Steroids’.© 2013 Elsevier Ltd. All rights reserved.

au

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1542. Sex steroids during pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1553. MAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1554. Tau. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1555. GFAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1566. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

. Introduction

In mammals the pregnant female provides an intrauterine envi-onment suitable for fetus development. In the maternal brain,

large number of the physiological and behavioral changes that

In pregnancy, the brain responds to hormones it has not beenin contact with such as chorionic gonadotropin and placental lac-togen [5,6], and to a very high levels of several hormones such assex steroids. For example, in some strains, female rats are exposedto 45 pg/ml of estradiol between days 2 and 20 of pregnancy while

ccur along pregnancy and in the postpartum period such as thencrease in food intake and nesting construction depend on hor-

onal changes [1–4].

∗ Corresponding author. Facultad de Química, U.N.A.M., Ciudad Universitaria,oyoacán 04510, México, D. F. México, Tel.: +52 55 5622 3732;ax: +52 55 5616 2010.

E-mail address: camachoarroyo@gmail.com (I. Camacho-Arroyo).

960-0760/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.jsbmb.2013.01.005

they are exposed to 12 pg/ml on proestrus day [7]. Progesteroneand its reduced metabolites can be synthesized in the brain inlarge amounts during pregnancy [8,9]. As a result of the increaseand uninterrupted exposure to sex steroid hormones, the neuroen-docrine regulation of many functions is significantly modified. The

brain not only presents a different expression pattern of sex steroidreceptors during pregnancy, but the metabolism of steroids is alsomodified [10]. The content of other hormones such as prolactin andoxytocin also changes during pregnancy, and they play a key role

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role of Tau in microtubules polymerization is driven by phos-phorylation/dephosphorylation cycles. Thus, Tau phosphorylationat specific sites allows microtubules depolymerization, while Taudephosphorylation promotes their stabilization [54,55]. Estradiol

Table 1Changes in MAP2 content in the hippocampus and the preoptic area of the rat duringpregnancy and on day 2 of lactation.

Days G2 G14 G18 G21 L2

Hippocampus 100 ± 2 116 ± 4* 82 ± 9* 69 ± 3* 65 ± 11*

Preoptic area 100 ± 2 96 ± 8 71 ± 16 89 ± 16 54 ± 14*

A. González-Arenas et al. / Journal of Steroid Bio

n the preparation of the neural substrates involved in the displayf maternal behavior [11].

The brain presents a variety of morphophysiological changesuring pregnancy including cell plasticity [7,12,13]. Initial exper-

ments made by Diamond et al. showed a considerable increasen the thickness of the cortex during pregnancy, demonstratinghat hormones–brain interaction during a long period of time suchs pregnancy affects neuronal morphology [14]. Similar resultsave been found in late pregnant rats whose neurons soma fromhe medial preoptic area are bigger as compared with those fromvariectomized animals [15]. These data suggest that estradiol androgesterone play an important role in brain function during preg-ancy that is associated to the expression of several behaviors,

ncluding the onset of maternal behavior [16].In spatial memory tests, the performance of pregnant rats is bet-

er than that of non-pregnant ones [13,17]. Moreover, anxiety andear are diminished during pregnancy. The levels of estradiol, pro-esterone and their metabolites are markedly higher in pregnantats [7,8,18]. These high hormonal levels have been associated tohe change in behavior that occurs along pregnancy such as thoseelated with maternal behavior [19,20].

It has been demonstrated that in the CA1 field of the rodentippocampus there is an increase in neurogenesis, cell prolifer-tion, and the density of dendritic spines throughout pregnancy15,17,21–23]. These plastic changes involve cytoskeletal reorga-ization. This review is focused on the expression pattern of threeroteins involved in cytoskeletal organization of neurons and glialells: Microtubule associated protein 2 (MAP2), Tau and Glial fibril-ary acidic protein (GFAP) during pregnancy, and its relation withex steroid hormones profile.

. Sex steroids during pregnancy

Pregnancy is characterized by a progressive incrementing estra-iol and progesterone levels, both prepare the neural circuit relatedo the display of maternal behavior [24]. In the rat, estradiol lev-ls progressively increase during pregnancy reaching a peak beforearturition, while those of progesterone rapidly increase after mat-

ng, reaching a first peak on day 6 of gestation, which is maintainedntil day 12; after then there is maximum on day 14, continuingntil day 20 of pregnancy. Progesterone levels suddenly diminishear term, let parturition to occur and maternal behavior to bexpressed [8].

Changes in estradiol and progesterone intracellular receptorsER and PR, respectively) have also been reported in the brain dur-ng pregnancy. Two main subtypes of ER: ER� and ER� as well aswo PR isoforms, PR-A and PR-B, that exhibited different functionnd regulation have been reported [25–31].

ER binding changes in several brain regions during rat pregnancy32]. For example, in the preoptic area of the rat, estrogen bindingncreases between days 8 and 16 of pregnancy, staying high on day2 of parturition [27,33,34]. In the ventromedial nucleus, estrogeninding increases in early pregnancy but it is reduced on day 16,nd rises on day 22 [34]. In the amygdala, estrogen binding dra-atically increases during pregnancy (over 500%) [34]. On days 16

nd 22 of pregnancy, preoptic area expressed a higher number ofR-positive cells as compared with day 8 or postpartum day 1. Inhe ventromedial hypothalamus, ER immunoreactivity per cell wasigher on day 22 of pregnancy compared with day 16 and the firstostpartum day [34]. Changes in ER expression at critical times inhe preoptic area and the ventromedial nucleus suggest a key rolef this receptor in maternal behavior.

PR has been detected in the forebrain of the rat during preg-ancy using immunocytochemical procedures. It was found thathe number of PR-positive cells was low in several hypothalamicuclei on day 3, increasing later on days 15 and 21 of gestation [35].

istry & Molecular Biology 139 (2014) 154– 158 155

Progesterone is rapidly metabolized in the brain to ringA-reduced progestin such as allopregnanolone (5�-pregnan-3�-ol-20-one) by the actions of 5�-reductase and 3�-hydroxysteroiddehydrogenase. During late pregnancy, the expression and activ-ity of these enzymes are increased in several brain regions [10].All these changes in sex steroids levels and the expression oftheir receptors during pregnancy have been related to cytoskeletalremodelation involved in brain plasticity in this physiological state.

3. MAP

Microtubule-associated proteins (MAPs) play a key role inthe extension of neurites, axonal transport, neuronal plasticityand dendrite stability [36–39]. MAPs regulate the formation ofmicrotubules, determining neuronal shape and size of neuronalprocesses. MAP2 can also bind to actin and modify microfilamentstability in dendritic spines [40–42].

In the adult brain two proteins of high molecular weight(280 kDa), MAP2a and MAP2b, have been reported in dendritesand neuronal soma where they are mainly associated with micro-tubules [43,44] as well as with actin fibers in dendritic spinesand postsynaptic terminals [41,42]. MAP2 has an important rolein the regulation of dendritic outgrowth and synaptogenesis[45,46]. Reyna-Neyra et al., have demonstrated that estradiol andprogesterone augment MAP2 expression in the hippocampus ofovariectomized rats [47,48]. Furthermore, in the rat, estradiolincreases dendritic growth in mediobasal hypothalamic neurons,and the number of spines as well as that of dendritic synapses in theventromedial nucleus [49,50]. This may contribute to the increasein dendritic spine density observed both on proestrus day and inhippocampal cell cultures after steroids treatment [51,52].

We have evaluated the expression of MAP2 in the hippocampusand the preoptic area throughout rat gestation and the beginning oflactation. In the hippocampus of pregnant rats the content of MAP2decreased during pregnancy and in day 2 of lactation (Table 1). Onthe other hand, in preoptic area MAP2 content did not significantlychange during pregnancy, however, it decreased on the beginningof lactation (Table 1). The differences in MAP2 protein contentbetween hippocampus and preoptic area during pregnancy suggestthat tissue-specific factors are involved in the regulation of MAP2expression. Changes in MAP2 expression could be associated to theparticipation of different brain regions in the behavioral patternsobserved throughout pregnancy.

4. Tau

Tau is another protein associated to microtubules that belongsto a family of proteins (45–65 kDa) mainly located in axons whosehyperphosphorylated forms have been related with several neu-rodegenerative disorders such as Alzheimer’s disease [53]. The

Days 2, 14, 18, and 21 of gestation (G2, G14, G18, G21, respectively), and day 2of lactation (L2). Data show the percentage change in protein content of MAP2 ascompared with day 2 of gestation. Results are expressed as mean ± S.E.M. n = 4.

* P < 0.05 vs G2.

156 A. González-Arenas et al. / Journal of Steroid Biochemistry & Molecular Biology 139 (2014) 154– 158

Table 2Changes in Tau and PhosphoTau content in the hypothalamus, preoptic area, hippocampus, frontal cortex and cerebellum of the rat during pregnancy and on day 2 of lactation.

Days G2 G14 G18 G21 L2

Hypothalamus 100 ± 9 65 ± 19* 97 ± 15 45 ± 24* 55* ± 11*

100 ± 15 162 ± 19 149 ± 29 191 ± 12* 176 ± 21Preoptic Area 100 ± 6 86 ± 25 105 ± 15 102 ± 18 113 ± 10

100 ± 12 104 ± 10 86 ± 25 22 ± 13* 88 ± 15Hippocampus 100 ± 10 52 ± 10* 49 ± 29* 62 ± 40 79 ± 7

100 ± 52 218 ± 48 290 ± 23 359 ± 21* 327 ± 25Frontal Cortex 100 ± 15 91 ± 13 111 ± 9 79 ± 6 183 ± 12*

100 ± 10 78 ± 11 77 ± 14 113 ± 14 100 ± 4Cerebellum 100 ± 2 35 ± 15* 41 ± 11* 29 ± 26* 57 ± 14*

100 ± 22 188 ± 15 329 ± 11* 415 ± 32* 497 ± 17*

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ays 2, 14, 18, and 21 of gestation (G2, G14, G18, G21, respectively), and day 2 of lacau, PhosphoTau, (highlighted data) as compared with day 2 of gestation. Results a

* P < 0.05 vs G2.

ncreases Tau content in rat hypothalamic neurons in culture,hereas progesterone and its ring A-reduced metabolites reduce

t in the cerebellum of the rat [49,56]. In ovariectomized rats,e have reported that Tau expression was not modified by the

cute treatment with estradiol and progesterone in the hippocam-us and prefrontal cortex [57], but the chronic treatment withrogesterone increases Tau content in the prefrontal cortex [58].

nterestingly, estradiol and progesterone modify Tau phosphoryla-ion status in the brain. In the cerebellum of ovariectomized rats,rogesterone increases Tau phosphorylation, [56] while in humaneuroblastoma cells, estradiol prevents Tau hyperphosphorylation59].

It is clear that the combined action of estradiol and progesterones crucial for an adequate progress of pregnancy and other repro-uctive functions. The beneficial effects of estrogens in the brainave been well established, however, the role of progesterone aloner in combination with estradiol is still unclear. In a transgenicouse model of Alzheimer’s disease, the ovariectomy exacerbated

he symptoms of this pathology, but the administration of estradiolrecludes these symptoms and in combination with progesteroneeduced Tau hyperphosporylation and improved the behavioralmpairments in these animals. Thus, during pregnancy the continuexposition of the brain to sex steroids hormones could protect it foreurodegenerative damage [60].

Our lab has reported a tissue-specific expression pattern ofau and its phosphorylated forms in several regions of the brainhroughout rat gestation and on day 2 of lactation [61]. In theypothalamus, the hippocampus and the cerebellum, Tau con-ent diminished on day 14 of pregnancy and only hippocampusnd cerebellum maintained this decrease until day 18. All studiedegions except hypothalamus presented a high content of Tau onay 2 of lactation (Table 2).

Changes in Tau content in the hypothalamus, the hippocampus,nd the cerebellum may be associated with the changing levelsf estradiol, progesterone, oxytocin and cortisol exhibited duringregnancy [7,18]. Variations in Tau content might be associated tohe plastic changes of the brain during pregnancy, including spatial

emory and preparing the nursing [19].In addition, progesterone reduces Tau content in the rat cere-

ellum, but increased its phosphorylation [56]. In line with theseesults, we have demonstrated that the phosphorylation of TauPhosphoTau) progressively augmented in the hippocampus, theypothalamus and the cerebellum throughout pregnancy (Table 2)61]. In contrast, in the preoptic area the content of Phospho Tauecreased on day 21 of gestation, whereas in the frontal cortex, itid not change during pregnancy (Table 2) [61].

Differences in Tau protein content and in its phosphorylationattern in the different brain regions of the pregnant rat may beelated with its key role in the dynamic remodelation of neuronalytoskeleton during pregnancy.

(L2). Data show the percentage change in protein content of Tau or phosphorylatedressed as mean ± S.E.M. n = 4.

5. GFAP

GFAP is the main intermediate filament of astrocytes. Itregulates astrocyte motility and shape by stabilizing astrocyticprocesses. We have reported marked morphological variations inastrocytes and an increase in the number of GFAP immunoreactivecells in the hippocampus of the female rat during proestrus (whenestradiol and progesterone levels are high) compared with a femalerat during diestrus (when estradiol and progesterone levels arelow) [62]. Luquin et al. showed that in the hilus of the dentate gyrusthe surface density of GFAP-immunoreactive cells reached maximalvalues 24 h after estradiol and/or progesterone administration [63].

In the medial preoptic area, Featherstone et al. found an increasein the number of astrocytes in multiparous rats recently exposed topups than in non-pup exposed multiparous females [64]. Salmasoet al. found an increase in GFAP protein expression in the cingulatecortex within 3 h of parturition in rats [65]. All these data may indi-cate that hormonal repertoire and sensory signals that the femalereceives during pregnancy and lactation (i.e., suckling, tactile andolfactory stimulation) induce an up-regulation of GFAP in severalbrain areas involved in the display of behaviors essential for thesurvival of pups.

It has been reported that estradiol increases GFAP content inneurospheres from the subventricular zone of mice that is blockedby ICI, an antagonist of intracellular ER [66]. An increment in GFAPimmunoreactivity has been observed in the hippocampus and thepreoptic area of pregnant rodents compared with virgin ones [19].

We have observed that GFAP expression changes in the rat brainthroughout gestation and the start of lactation. GFAP expressionaugmented in the hippocampus along gestation, while a diminutionwas found in the cerebellum. In the frontal cortex, GFAP increasedon day 14 of gestation followed by a diminution on subsequentpregnancy days. The increase in GFAP on day 14 may be associatedwith an increment in anxiety related to mother’s perceptual fac-ulties. In contrast, a diminution in GFAP expression was found inthe preoptic area and hypothalamus on day 14 of gestation with anincrease in last days of pregnancy (Table 3) [67]. It has been shownan up-regulation of GFAP in the preoptic area and hypothalamus ofpostpartum multiparous rats that are in contact with pups, whencompared with pup-exposed primiparous rats [64].

Although the significance of the tissue-specific expressionpattern of GFAP in the pregnant brain is not known, it may beassociated with particular modifications of each brain area inresponse to changes in sex steroid hormone levels, as well as totheir specific participation in several behaviors along pregnancy[19,20]. For example, the increment in GFAP expression in the

hippocampus could be related to the better performance in spatialmemory tasks reported during pregnancy [68].

In relation to the role of estradiol and progesterone in GFAPexpression, it has been found that in the hippocampus of female

A. González-Arenas et al. / Journal of Steroid Biochemistry & Molecular Biology 139 (2014) 154– 158 157

Table 3Changes in GFAP content in the hypothalamus, preoptic area, hippocampus, frontal cortex and cerebellum of the rat during pregnancy and on day 2 of lactation.

Days G2 G14 G18 G21 L2

Hypothalamus 100 ± 2 89 ± 19 134 ± 9* 69 ± 10* 69 ± 12*

Preoptic Area 100 ± 11 27 ± 17* 84 ± 16 78 ± 13 62 ± 11Hippocampus 100 ± 13 139 ± 5* 172 ± 10* 142 ± 13 162 ± 15*

Frontal Cortex 100 ± 10 150 ± 11* 99 ± 12 83 ± 14 144 ± 13*

Cerebellum 100 ± 5 65 ± 9* 69 ± 11* 64 ± 29 111 ± 13

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ays 2, 14, 18, and 21 of gestation (G2, G14, G18, G21, respectively), and day 2 of laay 2 of gestation. Results are expressed as mean ± S.E.M. n = 4.

* P < 0.05 vs G2.

odents, GFAP immunoreactivity increased on proestrus day com-ared with diestrus day [62,69]. It has been proposed that gliallasticity modulated by sex steroid hormones could be involved

n the mechanisms of neuronal and synaptic plasticity regulation57,70].

. Conclusions

During pregnancy several morphophysiological modificationsccur in the brain. These changes implicate a re-organization ineuronal and glial cytoskeleton. Two major steroids hormones,stradiol and progesterone increase during pregnancy, and bothlay a key role in several neuronal functions that involve changes

n synaptic plasticity, and therefore in cell structure.Some of these changes are mediated by cytoskeletal proteins

uch as MAP2, Tau and GFAP. In five different brain areas (hypothal-mus, preoptic area, hippocampus, frontal cortex and cerebellum)he expression of these proteins changes during pregnancy in aissue specific manner, suggesting different roles in cytoskeletoneorganization. There are several promising research areas that willive us a better understanding about the participation of cytoskele-al proteins during pregnancy as well as the molecular mechanismsmplicated in the control of the expression and activity of these pro-eins, and consequently in the plastic changes of the brain duringregnancy.

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