Alfredo Daniel Vitullo

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 ERβ. Yet throughout pregnancy, ERα and ERβ 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.

Early embryonic development and spatiotemporal localization of mammalian primordial germ cell-associated proteins in the basal rodent Lagostomus maximus

The gene network controlling primordial germ cell (PGC) specification in eutherian mammals has been exhaustively investigated in mice. The egg-cylinder morphology of the mouse embryo is the key event enabling inductive signals from the extra-embryonic ectoderm (ExE) to specify epiblast cells as PGCs early on. We investigated the embryonic development and the spatiotemporal localization of PGC-associated proteins in the basal Hystricognathi rodent Lagostomus maximus. L. maximus develops through a flat-disc epiblast far apart from the ExE. In the primitive streak stage, OCT4-positive cells are detected in the posterior pole of the embryo disc in the mesoderm of the proximal epiblast. In the neural plate stage, a reduced 8 to 12 OCT4-positive cell population transiently expresses FRAGILIS, STELLA and SOX17 in the posterior streak. Soon after translocation to the hindgut, pluripotent OCT4 cells start expressing VASA, and then, STELLA and FRAGILIS are turned on during migration toward the genital ridge. L. maximus shows a spatiotemporal pattern of PGC-associated markers divergent from the early PGC restriction model seen in mice. This pattern conforms to alternative models that are based on a pluripotent population in the embryonic axis, where PGCs are specified later during development.

Population Genetics of Franciscana Dolphins (Pontoporia blainvillei): Introducing a New Population from the Southern Edge of Their Distribution

Due to anthropogenic factors, the franciscana dolphin, Pontoporia blainvillei, is the most threatened small cetacean on the Atlantic coast of South America. Four Franciscana Management Areas have been proposed: Espiritu Santo to Rio de Janeiro (FMA I), São Paulo to Santa Catarina (FMA II), Rio Grande do Sul to Uruguay (FMA III), and Argentina (FMA IV). Further genetic studies distinguished additional populations within these FMAs. We analyzed the population structure, phylogeography, and demographic history in the southernmost portion of the species range. From the analysis of mitochondrial DNA control region sequences, 5 novel haplotypes were found, totalizing 60 haplotypes for the entire distribution range. The haplotype network did not show an apparent phylogeographical signal for the southern FMAs. Two populations were identified: Monte Hermoso (MH) and Necochea (NC)+Claromecó (CL)+Río Negro (RN). The low levels of genetic variability, the relative constant size over time, and the low levels of gene flow may indicate that MH has been colonized by a few maternal lineages and became isolated from geographically close populations. The apparent increase in NC+CL+RN size would be consistent with the higher genetic variability found, since genetic diversity is generally higher in older and expanding populations. Additionally, RN may have experienced a recent split from CL and NC; current high levels of gene flow may be occurring between the latter ones. FMA IV would comprise four franciscana dolphin populations: Samborombón West+Samborombón South, Cabo San Antonio+Buenos Aires East, NC+CL+Buenos Aires Southwest+RN and MH. Results achieved in this study need to be taken into account in order to ensure the long-term survival of the species.

Patterns of population structure at microsatellite and mitochondrial DNA markers in the franciscana dolphin (Pontoporia blainvillei)

Abstract The franciscana dolphin, Pontorporia blainvillei, is an endemic cetacean of the Atlantic coast of South America. Its coastal distribution and restricted movement patterns make this species vulnerable to anthropogenic factors, particularly to incidental bycatch. We used mitochondrial DNA control region sequences, 10 microsatellites, and sex data to investigate the population structure of the franciscana dolphin from a previously established management area, which includes the southern edge of its geographic range. F‐statistics and Bayesian cluster analyses revealed the existence of three genetically distinct populations. Based on the microsatellite loci, similar levels of genetic variability were found in the area; 13 private alleles were found in Monte Hermoso, but none in Claromecó. When considering the mitochondrial DNA control region sequences, lower levels of genetic diversity were found in Monte Hermoso, when compared to the other localities. Low levels of gene flow were found between most localities. Additionally, no evidence of isolation by distance nor sex‐biased dispersal was detected in the study area. In view of these results showing that populations from Necochea/Claromecó, Monte Hermoso, and Río Negro were found to be genetically distinct and the available genetic information for the species previously published, Argentina would comprise five distinct populations: Samborombón West/Samborombón South, Cabo San Antonio/Buenos Aires East, Necochea/Claromecó/Buenos Aires Southwest, Monte Hermoso, and Río Negro. In order to ensure the long‐term survival of the franciscana dolphin, management and conservation strategies should be developed considering each of these populations as different management units.

Psychostimulant-Induced Testicular Toxicity in Mice: Evidence of Cocaine and Caffeine Effects on the Local Dopaminergic System

Several organ systems can be affected by psychostimulant toxicity. However, there is not sufficient evidence about the impact of psychostimulant intake on testicular physiology and catecholaminergic systems. The aim of the present study was to further explore potential toxic consequences of chronic exposure to cocaine, caffeine, and their combination on testicular physiology. Mice were injected with a 13-day chronic binge regimen of caffeine (3x5mg/kg), cocaine (3×10mg/kg), or combined administration. Mice treated with cocaine alone or combined with caffeine showed reduced volume of the seminiferous tubule associated to a reduction in the number of spermatogonia. Cocaine-only and combined treatments induced increased lipid peroxidation evaluated by TBARS assay and decreased glutathione peroxidase mRNA expression. Importantly, caffeine-cocaine combination potentiated the cocaine-induced germ cell loss, and induced pro-apoptotic BAX protein expression and diminished adenosine receptor A1 mRNA levels. We analyzed markers of dopaminergic function in the testis and detected the presence of tyrosine hydroxylase (TH) in the cytoplasm of androgen-producing Leydig cells, but also in meiotic germs cells within seminiferous tubules. Moreover, using transgenic BAC-Drd1a-tdTomato and D2R-eGFP mice, we report for the first time the presence of dopamine receptors (DRs) D1 and D2 in testicular mouse Leydig cells. Interestingly, the presence of DRD1 was also detected in the spermatogonia nearest the basal lamina of the seminiferous tubules, which did not show TH staining. We observed that psychostimulants induced downregulation of DRs mRNA expression and upregulation of TH protein expression in the testis. These findings suggest a potential role of the local dopaminergic system in psychostimulant-induced testicular pathology.

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 ERβ 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.

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, 17β-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.

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.

Endoscopy, histology and electron microscopy analysis of foetal membranes in pregnant South American plains vizcacha reveal unusual excrescences on the yolk sac

The South American hystricognathe Lagostomus maximus is a fossorial rodent whose females show unique reproductive characteristics. They have a 155-day long gestation, show massive polyovulation and a selective process of embryonic resorption in the first half of gestation. In order to explore and perform an in-situ characterization of the reproductive tract, we visualized internal structures through ultrasonography and video-endoscopy in pregnant and non-pregnant females. We describe the finding of protruding structures that lie on the yolk sac and their histological and ultrastructural characterization. The placenta was covered with whitish, small pearl-shaped structures. These structures were also seen on the extra-embryonic space, being the amnion and the umbilical cord free of them. Pearl-shaped structures were composed with loose connective tissue, lacked blood vessels, and showed collagen fibers organized in a spiral form. They were anchored by pedicles to the villous surface of the extraembryonic membrane. We discuss the biological and evolutionary meaning of the pearl-shaped structures that relate L. maximus to the African origin of the South American hystricognathe fauna.

Prolactin Is a Strong Candidate for the Regulation of Luteal Steroidogenesis in Vizcachas (Lagostomus maximus)

Prolactin (PRL) is essential for the maintenance of the corpora lutea and the production of progesterone (P4) during gestation of mice and rats, which makes it a key factor for their successful reproduction. Unlike these rodents and the vast majority of mammals, female vizcachas (Lagostomus maximus) have a peculiar reproductive biology characterized by an ovulatory event during pregnancy that generates secondary corpora lutea with a consequent increment of the circulating P4. We found that, although the expression of pituitary PRL increased steadily during pregnancy, its ovarian receptor (PRLR) reached its maximum in midpregnancy and drastically decreased at term pregnancy. The luteinizing hormone receptor (LHR) exhibited a similar profile than PRLR. Maximum P4 and LH blood levels were recorded at midpregnancy as well. Remarkably, the P4-sinthesizing enzyme 3β-HSD accompanied the expression pattern of PRLR/LHR throughout gestation. Instead, the luteolytic enzyme 20α-HSD showed low expression at early and midpregnancy, but reached its maximum at the end of gestation, when PRLR/LHR/3ß-HSD expressions and circulating P4 were minimal. In conclusion, both the PRLR and LHR expressions in the ovary would define the success of gestation in vizcachas by modulating the levels of 20α-HSD and 3ß-HSD, which ultimately determine the level of serum P4 throughout gestation.