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.

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 ovarian germinal reserve and apoptosis-related proteins in the infant and adolescent human ovary.

BACKGROUND: Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program. RESULTS: Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani's body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy. CONCLUSIONS: These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program.

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.

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.

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.