Local effect of allopregnanolone in rat ovarian steroidogenesis, follicular and corpora lutea development.

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3ß-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABAA receptor increased after ALLO treatment. To evaluate if the ovarian GABAA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABAA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.

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.

Distribution of kisspeptin system and its relation with gonadotropin-releasing hormone in the hypothalamus of the South American plains vizcacha, Lagostomus maximus.

Kisspeptin (KISS), a key hormone involved in the regulation of the hypothalamic-pituitary-ovarian (HPO) axis, has been localized in the anteroventral periventricular (AVPV) nucleus and the neighboring rostral periventricular nucleus (PeVN), and in the arcuate (ARC) nucleus of the mammalian hypothalamus. In the ARC, the KISS neurons that co-express neurokinin B (NKB) and dynorphin A (Dyn) are named KNDy cells. The South American plains vizcacha is a rodent with peculiar reproductive traits. Around mid-pregnancy, vizcacha shows the reactivation of its HPO axis with the pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), an essential event for the success of gestation. Considering the role of KISS system in GnRH modulation, the aim of this work was to study their neuroanatomical distribution in adult vizcachas. AVPV showed sexual dimorphism with a significant smaller area in males (t-Test, p < 0.05), and KISS immunoreactivity was detected in somas and varicosities homogenously distributed in the AVPV with a concordant sex-related expression pattern. NKB and Dyn expression was also observed in cytoplasm of neurons scattered in the AVPV. Three subpopulations of neurons were detected in the AVPV: neurons expressing Dyn and NKB (DyNK cells), neurons expressing KISS and NKB (KiNK cells), and single NKB expressing neurons. Strikingly, KISS and Dyn were always expressed in different cells. In addition, in the ARC nucleus, KNDy cells were detected. On the other hand, KISS and GnRH expression was detected in different subpopulations of neurons, GnRH cells showed KISS receptor (KISSR or GPR-54) expression, and KISS immunoreactive afferent contacts were detected making close appositions onto somas and dendrites of GnRH cells. These results show similarities and differences between the KISS system in the hypothalamus of the vizcacha and other mammals, and constitute crucial observations about KISS and GnRH relation. Considering the peculiarity of HPO axis regulation in this species, the present work provides a neuroanatomical framework for the further elucidation of molecular mechanisms underlying GnRH expression and secretion.

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.

First record of an infection by tissue cyst-forming coccidia in wild vizcachas (Lagostomus maximus, Rodentia) of Argentina.

Endoparasites of the Sarcocystidae family share the ability to form tissue cysts in their intermediate hosts, ultimately leading to pathogenesis in the definitive hosts that include various mammals, reptiles and birds. In our research on the endocrinology of the female vizcachas (Lagostomus maximus), we have found abnormal cystic structures in the ovaries of some individuals. So far, no cases of infection by tissue cyst-forming parasites have been reported in this species. To evaluate whether this autochthonous wild rodent is an intermediate host of an undescribed endoparasite, histological sections from various organs were examined. Pinhead-sized tissue cysts were found in the ovaries, mammary glands, uterus, pituitary, brain, adrenals and spleen, of both pregnant and non-pregnant females. The presence of cysts in the adult brain and embryonic tissue is indicative of the ability of the parasite to cross both the blood-brain and placental barriers. The infected brains exhibited a lower cyst density than that seen in other organs. Regardless of their location in superficial or deep tissue, the cysts were surrounded by a layer of connective tissue. Histologically, the cyst wall consisted of an outer layer of fibroblasts and collagen fibers, and an inner, granular-looking layer composed of host nucleated cells surrounding thousands of spindle-shaped bradyzoites. Outside the cysts, the host cellular structures showed normal appearance. The remarkable morphological similarities between the cysts studied here with those reported in naturally infected rabbits from an area neighboring the one inhabited by the vizcachas point to Besnoitia sp. as a plausible candidate. More studies will be necessary to confirm the identity of the parasite. Nevertheless, this is the first report of L. maximus as an intermediate host for a tissue cyst-forming coccidia.

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.

PTEN and FOXO3 expression in the prenatal and postnatal human ovary.

PURPOSE: The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. METHODS: Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. RESULTS: PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. CONCLUSION: Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

The key action of estradiol and progesterone enables GnRH delivery during gestation in the South American plains vizcacha, Lagostomus maximus.

The South American plains vizcacha, Lagostomus maximus, is the only mammal described so far that shows expression of estrogen receptors (ERs) and progesterone receptors (PRs) in gonadotropin-releasing hormone (GnRH) neurons. This animal therefore constitutes an exceptional model for the study of the effect of steroid hormones on the modulation of the hypothalamic-pituitary-ovarian (HPO) axis. By using both in vivo and ex vivo approaches, we have found that pharmacological doses of progesterone (P4) and estradiol (E2) produced an inhibition in the expression of hypothalamic GnRH, while physiological doses produced a differential effect on the pulsatile release frequency or genomic expression of GnRH. Our ex vivo experiment indicates that a short-term effect of E2 modulates the frequency of GnRH release pattern that would be associated with membrane ERs. On the other hand, our in vivo approach suggests that a long-term effect of E2, acting through the classical nuclear ERs-PRs pathway, would produce the modification of GnRH mRNA expression during the GnRH pre-ovulatory surge. Particularly, P4 induced a rise in GnRH mRNA expression and protein release with a decrease in its release frequency. These results suggest different levels of action of steroid hormones on GnRH modulation. We conclude that the fine action of E2 and P4 constitute the key factor to enable the hypothalamic activity during the pregnancy of this mammal.

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.

Local production of neurostradiol affects gonadotropin-releasing hormone (GnRH) secretion at mid-gestation in Lagostomus maximus (Rodentia, Caviomorpha).

Females of the South American plains vizcacha, Lagostomus maximus, show peculiar reproductive features such as massive polyovulation up to 800 oocytes per estrous cycle and an ovulatory process around mid-gestation arising from the reactivation of the hypothalamic-hypophyseal-ovary (H.H.O.) axis. Estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) expression. Biosynthesis of estrogens results from the aromatization of androgens by aromatase, which mainly occurs in the gonads, but has also been described in the hypothalamus. The recently described correlation between GnRH and ERα expression patterns in the hypothalamus of the vizcacha during pregnancy, with coexpression in the same neurons of the medial preoptic area, suggests that hypothalamic synthesis of E2 may affect GnRH neurons and contribute with systemic E2 to modulate GnRH delivery during the gestation. To elucidate this hypothesis, hypothalamic expression and the action of aromatase on GnRH release were evaluated in female vizcachas throughout pregnancy. Aromatase and GnRH expression was increased significantly in mid-pregnant and term-pregnant vizcachas compared to early-pregnant and nonpregnant females. In addition, aromatase and GnRH were colocalized in neurons of the medial preoptic area of the hypothalamus throughout gestation. The blockage of the negative feedback of E2 induced by the inhibition of aromatase resulted in a significant increment of GnRH-secreted mass by hypothalamic explants. E2 produced in the same neurons as GnRH may drive intracellular E2 to higher levels than those obtained from systemic circulation alone. This may trigger for a prompt GnRH availability enabling H.H.O. activity at mid-gestation with ovulation and formation of accessory corpora lutea with steroidogenic activity that produce the necessary progesterone to maintain gestation to term and guarantee the reproductive success.

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.

Reproductive actions of prolactin mediated through short and long receptor isoforms.

Prolactin (PRL) is a polypeptide hormone with a wide range of physiological functions, and is critical for female reproduction. PRL exerts its action by binding to membrane bound receptor isoforms broadly classified as the long form and the short form receptors. Both receptor isoforms are highly expressed in the ovary as well as in the uterus. Although signaling through the long form is believed to be more predominant, it remains unclear whether activation of this isoform alone is sufficient to support reproductive functions or whether both types of receptor are required. The generation of transgenic mice selectively expressing either the short or the long form of PRL receptor has provided insight into the differential signaling mechanisms and physiological functions of these receptors. This review describes the essential finding that both long and short receptor isoforms are crucial for ovarian functions and female fertility, and highlights novel mechanisms of action for these receptors.

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.

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus).

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERß. Yet throughout pregnancy, ERα and ERß levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function.

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

The stimulation of HSD17B7 expression by estradiol provides a powerful feed-forward mechanism for estradiol biosynthesis in breast cancer cells.

Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17ß-hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)α, whereas ERß prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiol-induced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a -185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers.