Corticogenesis and folding process of the neopallium in the South American plains vizcacha, Lagostomus maximus.

The plains vizcacha, Lagostomus maximus, is a precocial hystricomorph rodent with a gyrencephalic brain. This work aimed to perform a time-lapse analysis of the embryonic brain cortical development in the plains vizcacha to establish a species-specific temporal window for corticogenesis and the gyrencephaly onset. Additionally, a comparative examination with evolutionarily related rodents was conducted. Embryos from 40 embryonic days (ED) until the end of pregnancy ( ∼ $\sim $ 154 ED) were evaluated. The neuroanatomical examination determined transverse sulci at 80 ED and rostral lateral and caudal intraparietal sulci around 95 ED. Histological examination of corticogenesis showed emergence of the subplate at 43 ED and expansion of the subventricular zone (SVZ) and its division into inner and outer SVZs around 54 ED. The neocortical layers formation followed an inside-to-outside spatiotemporal gradient beginning with the emergence of layers VI and V at 68 ED and establishing the final six neocortical layers around 100 ED. A progressive increment of gyrencephalization index (GI) from 1.005 ± 0.003 around 70 ED, which reflects a smooth cortex, up to 1.07 ± 0.009 at the end of gestation, reflecting a gyrencephalic neuroanatomy, was determined. Contrarily, the minimum cortical thickness (MCT) progressively decreased from 61 ED up to the end of gestation. These results show that the decrease in the cortical thickness, which enables the onset of neocortical invaginations, occurs together with the expansion and subdivision of the SVZ. The temporal comparison of corticogenesis in plains vizcacha with that in relative species reflects a prenatal long process compared with other rodents that may give an evolutionary advantage to L. maximus as a precocial species.

Neurovascular description in the South American plains vizcacha, Lagostomus maximus (Chinchilloidea, Caviomorpha). A study involving evolutionarily related species of Caviomorpha and Muroidea.

Oxygenated blood is required for the adequate metabolic activity of the brain. This is supplied by the circle of Willis (CoW) and the vertebrobasilar and carotid systems. The CoW ensures blood flow in case of arterial stenosis or occlusion. Different animal models have been explored for the CoW morphological and functional study. This work aims to characterize the vascular architecture of the CoW of the plains vizcacha, Lagostomus maximus (Suborder: Hystricomorpha), and to compare it with evolutionarily related species of Caviomorpha and Muroidea. The blood supply in adult plains vizcachas was studied using latex cerebrovascular casts and angiography. A caudo-rostral flow direction was determined, beginning in the spinal and vertebral arteries and converging in the basilar artery which bifurcates in the carotid-basilar communication in the caudal communicating arteries. In the first third of its course, the caudal cerebral arteries project laterally, and the middle and rostral cerebral arteries bifurcate from their rostral terminal segment, supplying the temporo-parietal and frontal cortex. The CoW architecture is mainly conserved between rodent species. Likewise, the small neurovascular variations observed could be considered phylogenetic morphological variations more than evolutionary adaptations. The absence of the rostral communicating artery that generates the rostral open architecture of the CoW in the vizcacha as in the other analyzed species, supports the need for a revision of the CoW classical function as a security system. Finally, this work supports the importance of expanding our understanding of brain anatomy among species, which may contribute to a better understanding of functional neuroanatomy.

Hypothalamic GnRH expression and pulsatility depends on a balance of prolactin receptors in the plains vizcacha, Lagostomus maximus.

In mammals, gestation is considered a physiological hyperprolactinemia status. Prolactin (PRL) is one of the modulators of gonadotropin-releasing hormone (GnRH) neurons function. The South American plains vizcacha (Lagostomus maximus) is a unique model to study the regulation of hypothalamic GnRH neurons by direct and indirect steroid-dependent pathways. The aim was to characterize the hypothalamic expression of endocrine markers in vizcacha during gestation as well as their response to experimental induced hyperprolactinemia. The possible involvement of PRL regulatory pathways on GnRH in the context of hypothalamic and pituitary reactivation in mid-gestating vizcachas was discussed. Using two in vivo approaches, we determined changes in the hypothalamic expression and distribution of prolactin receptor (PRLR), tyrosine hydroxylase (TH), and dopamine type 2 receptor. A significant increment in the number of tuberoinfundibular dopaminergic (TIDA) neurons was determined in the arcuate nucleus from early to term pregnancy. On the other hand, at preoptic area, the number of both TH+PRLR+ and GnRH+PRLR+ double-labeled neurons significantly decreased at mid-pregnancy probably allowing the recovery of GnRH expression indicating that both types of neurons may represent the key points of PRL indirect and direct pathways modulating GnRH. Moreover, in a model of induced hyperprolactinemic vizcachas, the inhibitory effect of PRL on GnRH at both expression and delivery levels were confirmed. These results suggest the concomitant participation of both PRL regulatory pathways on GnRH modulation and pinpoint the key role of PRL on GnRH expression enabling the recovery of the hypothalamic activity during the gestation in this species.

Achieving full-term pregnancy in the vizcacha relies on a reboot of luteal steroidogenesis in mid-gestation (Lagostomus maximus, Rodentia).

Reactivation of the hypothalamic-pituitary-ovarian (HPO) axis triggered by the decline in serum progesterone in mid-gestation is an uncommon trait that distinguishes the vizcacha from most mammals. Accessory corpora lutea (aCL) developed upon this event have been proposed as guarantors of the restoration of the progesterone levels necessary to mantain gestation. Therefore, the steroidogenic input of primary CL (pCL) vs aCL was evaluated before and after HPO axis-reactivation (BP and AP respectively) and in term pregnancy (TP). Nonpregnant-ovulated females (NP) were considered as the pCL-starting point group. In BP, the ovaries mainly showed pCL, whose LH receptor (LHR), StAR, 3ß-HSD, 20α-HSD, and VEGF immunoexpressions were similar or lower than those of NP. In AP, luteal reactivity increased significantly compared to the previous stages, and the pool of aCL developed in this stage represented 20% of the ovarian structures, equaling the percentage of pCL. Both pCL and aCL luteal cells shared similar histological features consistent with secretory activity. Although pCL and aCL showed equivalent labeling intensity for the luteotropic markers, pCL were significantly larger than aCL. Towards TP, both showed structural disorganization and loss of secretory characteristics. No significant DNA fragmentation was detected in luteal cells throughout gestation. Our findings indicate that the LH surge derived from HPO axis-reactivation targets the pCL and boost luteal steroidogenesis and thus progesterone production. Because there are many LHR-expressing antral follicles in BP, they also respond to the LH stimuli and luteinize without extruding the oocyte. These aCL certainly contribute but it is the steroidogenic restart of the pCL that is the main force that restores progesterone levels, ensuring that gestation is carried to term. Most importantly, the results of this work propose luteal steroidogenesis reboot as a key event in the modulation of vizcacha pregnancy and depict yet another distinctive aspect of its reproductive endocrinology.

Melatonin is involved in the modulation of the hypothalamic and pituitary activity in the South American plains vizcacha, Lagostomus maximus.

Melatonin, the key messenger of photoperiodic information, is synthesized in the pineal gland by arylalkylamine N-acetyltransferase enzyme (AANAT). It binds to specific receptors MT1 and MT2 located in the hypothalamus and pituitary gland. Melatonin can modulate the reproductive axis affecting the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). The South American plains vizcacha, Lagostomus maximus, shows natural poliovulation of up to 800 oocytes per estrous cycle, a 154-day long pregnancy, and reactivation of the reproductive axis at mid-gestation with pre-ovulatory follicular recruitment, presence of active corpora lutea, and variations of the endocrine status. Here we analyzed the involvement of melatonin in the modulation of the hypothalamic and pituitary gland physiology of vizcacha thorough several approaches, including histological localization of melatoninergic system components, assessment of melatoninergic components expression throughout the reproductive cycle, and evaluation of the effect of melatonin on hypothalamic and pituitary activities during the follicular and luteal phases of the estrous cycle. AANAT and melatonin receptors were localized in the pineal gland and preoptic area of the hypothalamus. Increase in pineal AANAT and serum melatonin expression was observed as pregnancy progressed, with the lowest hypothalamic MT1 and MT2 levels at mid-pregnancy. Pulsatility assays demonstrated that melatonin induces GnRH and LH secretion at luteal phase. The melatoninergic system effects on hypothalamic and pituitary gland hormones secretion during pregnancy pinpoint to melatonin as a potential key factor underlying the reactivation of the reproductive axis activity at mid-gestation.

Mammary gland-specific regulation of GNRH and GNRH-receptor gene expression is likely part of a local autoregulatory system in female vizcachas (Rodentia: Chinchillidae).

In addition to key mammotrophic hormones such as the pituitary prolactin (PRL) and the ovarian steroids progesterone and estradiol, there are local factors that modulate the tissue dynamics of the mammary glands during pregnancy and lactation. By immunohistochemistry and RT-PCR, we found local transcription and translation of gonadotropin-releasing hormone (GNRH), GNRH receptor (GNRHR), PRL and PRL receptor (PRLR) in mammary glands of adult vizcachas during pregnancy and lactation. Both GNRH and GNRHR showed a lag between protein expression and gene transcription throughout the gestational period: while the highest transcription levels of these genes were recorded at early-pregnancy, the epithelial immunoexpressions of both showed their maximum during lactation. RIA results corroborated the presence of GNRH in mammary glands at all the analyzed stages and confirmed the maximum amount of this peptide in the lactating group. Significant amounts of GNRH were detected in milk samples as well. Conversely, PRL and PRLR shared similar protein and gene expression profiles, all exhibiting maximum values during lactation. GNRH peptide content in mammary glands of females with sulpiride-induced hyperprolactinemia (HP) was significantly lower than that of control females (CT). Although PRL mRNA levels remained unchanged, there was a marked increase in theα-lactalbumin (LALBA) transcription in mammary glands of HP- vs CT-females. These results suggest that after targeting mammary glands, PRL stimulates the expression of milk protein genes, but also, tempers the local expression of GNRH. Mammary gland-explantssupplemented with a GNRH analogue (GN-explants) had no differences in terms of PRLR orLALBA transcription levels compared to CT-explants, so the mammary PRLR signaling would not appear to be modulated by GNRH. Yet, mRNA expression levels of both GNRH and the GNRHR-downstream factor, EGR1, were significantly higher in GN-explants compared to that of CT which would point to a GNRH-positive feedback mechanism. In summary, the local coupled expression of GNRH, GNRHR and EGR1 in the mammary gland throughout pregnancy of vizcachas, the PRL-dependent mammary GNRH secretion as well as the GNRH positive feedback on its own transcription suggest an autocrine-paracrine regulatory mechanism and propose an active role for GNRH in mammary gland tissue remodeling.

Neocortical Anatomy in the South American Plains Vizcacha, Lagostomus maximus, Reveals Different Strategies in Encephalic Development among Hystricomorpha and Myomorpha Rodents.

Depending on the presence or absence of sulci and convolutions, the brains of mammals are classified as gyrencephalic or lissencephalic. We analyzed the encephalic anatomy of the hystricomorph rodent Lagostomus maximus in comparison with other evolutionarily related species. The encephalization quotient (EQ), gyrencephaly index (GI), and minimum cortical thickness (MCT) were calculated for the plains vizcacha as well as for other myomorph and hystricomorph rodents. The vizcacha showed a gyrencephalic brain with a sagittal longitudinal fissure that divides both hemispheres, and 3 pairs of sulci with bilateral symmetry; that is, lateral-rostral, intraparietal, and transverse sulci. The EQ had one of the lowest values among Hystricomorpha, while GI was one of the highest. Besides, the MCT was close to the mean value for the suborder. The comparison of EQ, GI, and MCT values between hystricomorph and myomorph species allowed the detection of significant variations. Both EQ and GI showed a significant increase in Hystricomorpha compared to Myomorpha, whereas a Pearson's analysis between EQ and GI depicted an inverse correlation pattern for Hystricomorpha. Furthermore, the ratio between MCT and GI also showed a negative correlation for Hystricomorpha and Myomorpha. Our phylogenetic analyses showed that Hystricomorpha and Myomorpha do not differ in their allometric patterning between the brain and body mass, GI and brain mass, and MCT and GI. In conclusion, gyrencephalic neuroanatomy in the vizcacha could have developed from the balance between the brain size, the presence of invaginations, and the cortical thickness, which resulted in a mixed encephalization strategy for the species. Gyrencephaly in the vizcacha, as well as in other Hystricomorpha, advocates in favor of the proposal that in the more recently evolved Myomorpha lissencephaly would have arisen from a phenotype reversal process.

Structural organization, GABAergic and tyrosine hydroxylase expression in the striatum and globus pallidus of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The striatum is an essential component of the basal ganglia that regulatessensory processing, motor, cognition, and behavior. Depending on the species, the striatum shows a unique structure called caudate-putamen as in mice, or its separation into two regions called caudate and lenticular nuclei, the latter formed by putamen and globus pallidus areas, as in primates. These structures have two compartments, striosome and matrix. We investigated the structural organization, GABAergic and tyrosine hydroxylase (TH) expression in the striatum and globus pallidus of the South American plains vizcacha, Lagostomus maximus. Its striatum showed regionalization arising from the presence of an internal capsule, and a similar organization to a striosome-matrix compartmentalization. GABAergic neurons in the matrix of caudate exhibited parvalbumin, calretinin, calbindin, GAD65, and NADPH-d-immunoreactivity. These were also expressed in cells of the putamen with the exception of calretinin showing neurofibers localization. Globus pallidus showed parvalbumin- and GAD65-immunoreactive cells, and calretinin- and calbindin-immunoreactive neuropil, plus GABA-A-immunoreactive neurofibers. NADPH-d-, GAD65- and GABA-A-immunoreactive neurons were larger than parvalbumin-, calretinin-, and calbindin-immunoreactive cells, whereas calbindin-immunoreactive cells were the most abundant. In addition, TH-immunoreactive neuropil was observed in the matrix of the striatum. A significant larger TH-immunoreactive area and neuron number was found in females compared to males. The presence of an internal capsule suggests an adaptive advantage concerning motor and cognitive abilities favoring reaction time in response to predators. In an anatomy-evolutive perspective, the striatum of vizcacha seems to be closer to that of humans than to that of laboratory traditional models such as mouse.

Pituitary estrogen receptor alpha is involved in luteinizing hormone pulsatility at mid-gestation in the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

The South American plains vizcacha, Lagostomus maximus, is a caviomorph rodent native from Argentina, Bolivia and Paraguay. It shows peculiar reproductive features like pre-ovulatory follicle recruitment during pregnancy with an ovulatory process at around mid-gestation. We have described the activation of the hypothalamic - pituitary - ovarian (HPO) axis during pregnancy. A progressive decrease of progesterone (P4) at mid-pregnancy elicits the delivery of gonadotropin-releasing hormone (GnRH) with the consequent secretion of follicle stimulating hormone (FSH) and estradiol (E2) followed by luteinizing hormone (LH) release resulting in follicular luteinization and the P4 concentration recover. Pituitary gland is the central regulator of the HPO axis being E2 a key hormone involved in the regulation of its activity. In this work we analyzed the action of E2 on the pituitary response to the GnRH wave as well as its involvement on LH secretion at mid-gestation in L. maximus. The expression of GnRHR at the pituitary pars distalis showed a significant decrease at mid-pregnancy compared to early- and term-gestating females. ERα showed a significant increment from mid-gestation whereas ERß did not show variations throughout pregnancy; whereas the LH expression in the pituitary pars distalis showed a significant increase at mid-gestation, concordantly with serum LH, which was followed by a decrease at term-gestation with similar values than at early-pregnancy. The number of cells with co-localization of ERα and GnRHR showed a decline at mid-pregnancy related to early- and term-gestation, whereas the cells with co-localization of ERα and LH increased at mid- and term-pregnancy. On the other hand, ex vivo measuring of LH pulsatility showed a significant increment in the total mass of LH delivered at mid-pregnancy followed by a decrease at term-gestation. The stimulation of ERα with the PPT specific agonist induced a significant increment in the total mass of LH released, whereas no changes were determined when ERß was stimulated with its specific agonist MPP. These results suggest that LH pulsatility rise at mid-pregnancy would be enabled by the increase of E2 acting through ERα.

Hormonal behavior correlates with follicular recruitment at mid-gestation in the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

In mammals, hormonal regulation during gestation is crucial for embryo implantation and pregnancy success. This regulation is controlled through the level of progesterone (P4) that blocks the activity of the hypothalamic-hypophyseal-gonadal (HHG) axis. Previous studies in the pregnant South American plains vizcacha, Lagostomus maximus, have shown that the HHG axis activates around mid-gestation, promoting pre-ovulatory follicle formation. However, the characterization of the hormonal dynamics throughout gestation and its ovarian correlation has not been studied in depth. We studied the ovarian dynamics of L. maximus and its correlation with the hormonal profile during gestation, analyzing serum levels of P4, 17ß-estradiol (E2), 4Δ-androstenedione (A4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) as well as the ovarian distribution and expression of their receptors. Additionally, we have analyzed the folliculogenesis and accessory corpora lutea (ACL) formation. P4 showed two concentration peaks reaching its highest level at mid-gestation decreasing at 91-100days post-coitum. P4 decrease is followed by an increase of circulating levels of A4, E2, FSH and LH and with an elevated number of antral/pre-ovulatory follicles which express PGR, ESR1, ESR2, AR, LHR and FSHR. In addition, ACL with oocyte retention and cytoplasmic lipid droplets in luteal cells were detected at this time point. These results show that in L. maximus the decrease of P4 level from mid-gestation enables follicular recruitment until pre-ovulatory stage and the development of functional ACL.

ERα and GnRH co-localize in the hypothalamic neurons of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

Gonadotropin-releasing hormone (GnRH) is the key regulator of the hypothalamic-pituitary-gonadal axis. Estradiol (E2) affects GnRH synthesis and delivery. Hypothalamic estrogen receptors (ER) modulate GnRH expression acting as transcription factors. The South American plains vizcacha, Lagostomus maximus, is able to ovulate up to 800 oocytes per reproductive cycle, and shows continuous folliculogenesis with pre-ovulatory follicle formation and an ovulatory event at mid-gestation. The aim of this work was to analyze the hypothalamic expression of ER in the vizcacha at different gestational time-points, and its relationship with GnRH expression, serum luteinizing hormone (LH) and E2. The hormonal pattern of mid-gestating vizcachas was comparable to ovulating-females with significant increases in GnRH, LH and E2. Hypothalamic protein and mRNA expression of ERα varied during pregnancy with a significant increase at mid-gestation whereas ERß mRNA expression did not show significant variations. Hypothalamic immunolocalization of ERα was observed in neurons of the diagonal band of Brocca, medial preoptic area (mPOA), periventricular, suprachiasmatic, supraoptic (SON), ventromedial, and arcuate nuclei, and medial eminence, with a similar distribution throughout gestation. In addition, all GnRH neurons of the mPOA and SON showed ERα expression with no differences across the reproductive status. The correlation between GnRH and ERα at mid-gestation, and their co-localization in the hypothalamic neurons of the vizcacha, provides novel information compared with other mammals suggesting a direct action of estrogen as part of a differential reproductive strategy to assure GnRH synthesis during pregnancy.

Sequence analysis, tissue distribution and molecular physiology of the GnRH preprogonadotrophin in the South American plains vizcacha (Lagostomus maximus).

Gonadotropin-releasing hormone (GnRH) is the regulator of the hypothalamic-hypophyseal-gonadal (HHG) axis. GnRH and GAP (GnRH-associated protein) are both encoded by a single preprohormone. Different variants of GnRH have been described. In most mammals, GnRH is secreted in a pulsatile manner that stimulates the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The South-American plains vizcacha, Lagostomus maximus, is a rodent with peculiar reproductive features including natural poly-ovulation up to 800 oocytes per estrous cycle, pre-ovulatory follicle formation throughout pregnancy and an ovulatory process which takes place at mid-gestation and adds a considerable number of secondary corpora lutea. Such features should occur under a special modulation of the HHG axis, guided by GnRH. The aim of this study was to sequence hypothalamic GnRH preprogonadotrophin mRNA in the vizcacha, to compare it with evolutionarily related species and to identify its expression, distribution and pulsatile pattern of secretion. The GnRH1variant was detected and showed the highest homology with that of chinchilla, its closest evolutionarily related species. Two isoforms of transcripts were identified, carrying the same coding sequence, but different 5' untranslated regions. This suggests a sensitive equilibrium between RNA stability and translational efficiency. A predominant hypothalamic localization and a pulsatile secretion pattern of one pulse of GnRH every hour were found. The lower homology found for GAP, also among evolutionarily related species, depicts a potentially different bioactivity.

Variation in progesterone receptors and GnRH expression in the hypothalamus of the pregnant South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia).

In mammals, elevated levels of progesterone (P4) throughout gestation maintain a negative feedback over the hypothalamic-hypophyseal-gonadal (H-H-G) axis, avoiding preovulatory follicular growth and preventing ovulation. Recent studies showed that in the South American plains vizcacha (Lagostomus maximus) folliculogenesis progresses to preovulatory stages during gestation, and an ovulatory process seems to occur at midgestation. The aim of this work was to analyze hypothalamic gonadotropin-releasing hormone (GnRH) and P4 receptors (PR) expression and luteinizing hormone (LH) secretion and correlate these with the functional state of the ovary in nonovulating and ovulating females and gestating females with special emphasis in the supposedly ovulating females at midgestation. We investigated P4 and LH serum levels as well as the distribution, localization, and expression of PR and GnRH in the hypothalamus of L. maximus at different time points during gestation and in nongestating, ovulating and nonovulating, females. A significant increment in GnRH, P4, and LH was detected in midpregnant vizcachas with respect to early-pregnant and to ovulating females. PR was also significantly increased in midpregnant animals. PR was detected in neurons of the preoptic and hypothalamic areas. Coexistence of both PR and GnRH in neurons of medial preoptic area and supraoptic nucleus was detected. Midpregnant animals showed increased number of PR immunoreactive cells at median eminence, localized adjacently to GnRH immunoreactive fibers. High expression of hypothalamic GnRH and PR, despite an increased level of P4, was correlated with the presence of antral, preovulatory follicles, and luteinized unruptured follicles at midgestation that suggest a possible role of the H-H-G axis in the modulation of ovulation during gestation in L. maximus.

Hypothermia prevents gliosis and angiogenesis development in an experimental model of ischemic proliferative retinopathy.

PURPOSE: To develop a time course study of vascularization and glial response to perinatal asphyxia in hypoxic-ischemic animals, and to evaluate hypothermia as possible protective treatment. METHODS: We used retinas of 7-, 15-, 21-, and 30-day-old male Sprague-Dawley rats that were exposed to perinatal asphyxia at either 37°C (PA) or 15°C (HYP). Born to term animals were used as controls (CTL). We evaluated the thickness of the most inner layers of the retina (IR), including internal limiting membrane, the retinal nerve fiber layer, and the ganglion cell layer; and studied glial development, neovascularization, adrenomedullin (AM), and VEGF by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: A significant increment in IR thickness was observed in the PA group from postnatal day (PND) 15 on. This alteration was concordant with an increased number of new vessels and increased GFAP expression. The immunolocalization of GFAP in the internal limiting membrane and perivascular glia of the IR and in the inner processes of Müller cells was coexpressed with AM, which was also significantly increased from PND7 in PA animals. In addition, VEGF expression was immunolocalized in cells of the ganglion cell layer of the IR and this expression significantly increased in the PA group from PND15 on. The retinas of the HYP group did not show differences when compared with CTL at any age. CONCLUSIONS: This work demonstrates that aberrant angiogenesis and exacerbated gliosis seem to be responsible for the increased thickness of the inner retina as a consequence of perinatal asphyxia, and that hypothermia is able to prevent these alterations.

Expression of androgen receptor, estrogen receptors alpha and beta and aromatase in the fetal, perinatal, prepubertal and adult testes of the South American plains Vizcacha, Lagostomus maximus (Mammalia, Rodentia).

Androgens and androgen receptor play a critical role in spermatogenesis and fertility in mammals, and estrogens and their receptors contribute to regulation of testicular function through initiation and maintenance of spermatogenesis and germ cell division and survival. However, results from different species are still far from establishing a clear understanding of these receptors in the different cell types from the testis. We analyzed the expression of androgen receptor, estrogen receptors α and ß and aromatase protein by immunohistochemistry and real-time PCR, in relation to proliferation followed by the expression of proliferation cell nuclear antigen (PCNA) and germinal identity by VASA protein, in fetal, perinatal, prepubertal and adult testes of Lagostomus maximus, a rodent with sustained germ cell proliferation and an increasing number of OCT-4-expressing gonocytes in the developing ovary. AR expression was restricted to Leydig cells and peritubular cells before sexual maturity, at which point it also became expressed in Sertoli cells. ERα and ERß were expressed in seminiferous tubules and the interstitium, respectively, in both fetal and prepubertal testes. In adult testes, both ERα and ERß co-localized in Leydig and peritubular cells. The aromatase enzyme, which converts androgenic precursors into estrogens, was detectable in all developmental stages analyzed and was restricted to Leydig cells. PCNA remained high until sexual maturity. ERα nuclear detection in germ cells and AR in Leydig cells in PCNA-positive cells suggest the possibility of a stimulatory effect of estrogens on spermatogonia proliferation. This effect might explain the increase found in VASA-expressing cells in the adult testis.

Histological characterization of gonadotropin-releasing hormone (GnRH) in the hypothalamus of the South American plains vizcacha (Lagostomus maximus).

In contrast to most mammalian species, females of the South American plains vizcacha, Lagostomus maximus, show an extensive suppression of apoptosis-dependent follicular atresia, continuous folliculogenesis, and massive polyovulation. These unusual reproductive features pinpoint to an eventual peculiar modulation of the hypothalamo-hypophyseal-gonadal axis through its main regulator, the gonadotropin-releasing hormone (GnRH). We explored the hypothalamic histological landscape and cellular and subcellular localization of GnRH in adult non-pregnant L. maximus females. Comparison to brain atlases from mouse, rat, guinea pig and chinchilla enabled us to histologically define and locate the preoptic area (POA), the ventromedial nucleus, the median eminence (ME), and the arcuate nucleus (Arc) of the hypothalamus in vizcacha's brain. Specific immunolocalization of GnRH was detected in soma of neurons at medial POA (MPA), ventrolateral preoptic nucleus, septohypothalamic nucleus (SHy) and Arc, and in beaded fibers of MPA, SHy, ventromedial hypothalamic nucleus, anterior hypothalamic area and ME. Electron microscopy examination revealed GnRH associated to cytoplasmic vesicles of the ME and POA neurons, organized both in core and non-core vesicles within varicosities, and in neurosecretory vesicles within the myelinated axons of the MPA. Besides the peculiar and unusual features of folliculogenesis and ovulation in the vizcacha, these results show that hypothalamus histology and GnRH immune-detection and localization are comparable to those found in other mammals. This fact leads to the possibility that specific regulatory mechanisms should be in action to maintain continuous folliculogenesis and massive polyovulation.

Hypothermia prevents nitric oxide system changes in retina induced by severe perinatal asphyxia.

One-third of asphyctic neonates develop long-term neurological injuries, including several degrees of ischemic proliferative retinopathy (IPR) such as retinopathy of prematurity (ROP). Given that the retina is altered by perinatal asphyxia, our aim was to study the effects of nitric oxide (NO) in the retina in order to analyze its impact on the retinal injury. Application of hypothermia was evaluated as preventive treatment. Sprague-Dawley rats were subjected to perinatal asphyxia [either at 37°C (PA group) or at 15°C (HYP group)]. Full-term rats were used as controls (CTL). A significantly increased activity of both constitutive NO synthase (nNOS, Ca(2+)-dependent) and inducible NO synthase (iNOS, Ca(2+)-independent) was observed in PA retinas from 21 days old up to 60 days old with respect to age-matched CTL, with a significant increase along the time course in the PA. nNOS was immunolocalized at amacrine, horizontal, and ganglion cells of the PA group, with a significant increase in relative optical density (R.O.D.), cellular area, and number of cells. iNOS immunoreactivity was observed in the inner nuclear layer and in the internal Müller cell processes of PA, with a significant increase in R.O.D. and colocalizing with GFAP in the 60-day-old PA group. Six nitrated protein species were increased in retinas from PA rats. Nitrotyrosine immunoreactivity showed a localization similar to that of iNOS, with increased R.O.D. in the PA group and colocalization with GFAP in 60-day-old animals. HYP prevented all the changes observed in PA rats. Although the NO system displays changes induced by hypoxia-ischemia, hypothermia application shows a strong protective effect.

Hypothermia prevents the development of ischemic proliferative retinopathy induced by severe perinatal asphyxia.

Obstetric complications, such as perinatal asphyxia, may cause retinal injuries as retinopathy of prematurity (ROP), a type of ischemic proliferative retinopathy. Up to date there are no appropriate experimental models for studying the long-term sequels of this disease. In the present work, we present an experimental model of perinatal asphyxia which shows structural and ultrastructural retinal alterations at the most inner layers of the retina, such as neurodegeneration, development of neoformed vessels and glial reaction, which are compatible with the histopathological description of ROP. Besides, the application of hypothermia during perinatal asphyxia showed effective results preventing cellular and morphological alterations. This study may contribute to the development of therapies in order to either ameliorate or prevent retinal damage. In this manner, hypothermia may improve life quality and decrease medical, family and social costs of these avoidable causes of blindness.

Differential cerebral deposition of IDE and NEP in sporadic and familial Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid beta (A beta) accumulation in the brain and is classified as familial early-onset (FAD) or sporadic late-onset (SAD). Evidences suggest that deficits in the brain expression of insulin degrading enzyme (IDE) and neprilysin (NEP), both proteases involved in amyloid degradation, may promote A beta deposition in SAD. We studied by immunohistochemistry IDE and NEP cortical expression in SAD and FAD samples carrying the E280A presenilin-1 missense mutation. We showed that IDE, a soluble peptidase, is linked with aggregated A beta 40 isoform while NEP, a membrane-bound protease, negatively correlates with amyloid angiopathy and its expression in the senile plaques is independent of aggregated amyloid and restricted to SAD cases. NEP, but not IDE, is over-expressed in dystrophic neurites, both proteases are immunoreactive in activated astrocytes but not in microglia and IDE was the only one detected in astrocytes of white matter from FAD cases. Collectively, our results support the notion that gross conformational changes involved in the modification from "natively folded-active" to "aggregated-inactive" IDE and NEP may be a relevant pathogenic mechanism in SAD.

Nitric oxide system alteration at spinal cord as a result of perinatal asphyxia is involved in behavioral disabilities: hypothermia as preventive treatment.

Perinatal asphyxia (PA) is able to induce sequelae such as spinal spasticity. Previously, we demonstrated hypothermia as a neuroprotective treatment against cell degeneration triggered by increased nitric oxide (NO) release. Because spinal motoneurons are implicated in spasticity, our aim was to analyze the involvement of NO system at cervical and lumbar motoneurons after PA as well as the application of hypothermia as treatment. PA was performed by immersion of both uterine horns containing full-term fetuses in a water bath at 37 degrees C for 19 or 20 min (PA19 or PA20) or at 15 degrees C for 20 min (hypothermia during PA-HYP). Some randomly chosen PA20 rats were immediately exposed for 5 min over grain ice (hypothermia after PA-HPA). Full-term vaginally delivered rats were used as control (CTL). We analyzed NO synthase (NOS) activity, expression and localization by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity, inducible and neuronal NOS (iNOS and nNOS) by immunohistochemistry, and protein nitrotyrosilation state. We observed an increased NOS activity at cervical spinal cord of 60-day-old PA20 rats, with increased NADPH-d, iNOS, and nitrotyrosine expression in cervical motoneurons and increased NADPH-d in neurons of layer X. Lumbar neurons were not altered. Hypothermia was able to maintain CTL values. Also, we observed decreased forelimb motor potency in the PA20 group, which could be attributed to changes at cervical motoneurons. This study shows that PA can induce spasticity produced by alterations in the NO system of the cervical spinal cord. Moreover, this situation can be prevented by perinatal hypothermia.