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The corpus luteum (CL) is a transient endocrine gland that plays a crucial role in establishing and maintaining pregnancy. Although autophagy and apoptosis have been suggested as cooperative mechanisms, their interaction within the CL of pregnant mammals has not been thoroughly investigated. To understand the collaborative function of autophagy and apoptosis in the CL, we analyzed both mechanisms during pregnancy in the South American plains vizcacha, Lagostomus maximus. This rodent undergoes a decline in progesterone levels during mid-gestation, a reactivation of the hypothalamus-hypophysis-gonadal axis, and the incorporation of new functional secondary CL. Our analysis of autophagy markers BECLIN 1 (BECN1), SEQUESTOSOME1 (SQSTM1), Microtubule-associated protein light chain 3 (LC3B), and lysosomal-associated membrane protein 1 (LAMP1) and anti- and pro-apoptotic markers BCL2 and ACTIVE CASPASE 3 (A-C3) revealed interactive behaviors between both processes. Healthy primary and secondary CL exhibited positive expression of BECN1, SQSTM1, LC3B, and LAMP1, while regressed CL displayed enhanced expression of these autophagy markers along with nuclear A-C3. Transmission electron microscopy revealed a significant formation of autophagic vesicles in regressed CL during full-term pregnancy, whereas healthy CL exhibited a low number of autophagy vesicles. The co-localization between LC3B and SQSTM1 and LC3B with LAMP1 was observed in both healthy and regressed CL during pregnancy, while co-localization of BECN1 and BCL2 was only detected in healthy CL. LC3B and ACTIVE CASPASE 3 co-localization were detected in a subset of luteal cells within the regressing CL. We propose that autophagy could act as a survival mechanism in the CL, allowing the pregnancy to progress until full-term, while also serving as a mechanism to eliminate remnants of regressed CL, thereby providing the necessary space for subsequent follicular maturation., (© 2024. The Author(s).)

Over the past two decades, the importance of fertility preservation has grown not only in the realm of medical and clinical patient care, but also in the field of basic and applied research in human reproduction. With advancements in cancer treatments resulting in higher rates of patient survival, it is crucial to consider the quality of life post-cure. Therefore, fertility preservation must be taken into account prior to antitumor treatments, as it can significantly impact a patient's future fertility. For postpubertal patients, gamete cryopreservation is the most commonly employed preservation strategy. However, for prepubertal patients, the situation is more intricate. Presently, ovarian tissue cryopreservation is the standard practice for prepubertal girls, but further scientific evidence is required in several aspects. Testicular tissue cryopreservation, on the other hand, is still experimental for prepubertal boys. The primary aim of this review is to address the strategies available for possible fertility preservation in prepubertal girls and boys, such as ovarian cryopreservation/transplantation, in vitro follicle culture and meiotic maturation, artificial ovary, transplantation of cryopreserved spermatogonia, and cryopreservation/grafting of immature testicular tissue and testicular organoids., (© 2023. The Author(s).)

Background: FOXO3/pFOXO3 and PTEN expression is known to regulate the dormancy/activation of ovarian primordial follicles. How chemotherapy could influence the expression of FOXO3 and PTEN in pre- and post-menarcheal girls with extra-gonadal cancer remains unexplored., Methods: Ovarian samples were collected from 27 girls suffering from extra-gonadal cancer. Of these, 8 patients had received chemotherapy before the time of sample collection. Ovarian tissue collected at the time of surgery was fixed in 10% formaldehyde for FOXO3/pFOXO3 and PTEN immunohistochemistry or immunofluorescence, or stored at -80 °C for Western blot, or preserved in RNA later for RT-PCR., Results: PTEN was detected in a limited number of primordial follicle-enclosed oocytes in approximately fifty percent of the patients, regardless of whether they had received anti-cancer treatment or not. However, there was a significant decrease in PTEN detection in patients who underwent chemotherapy treatment prior to the retrieval of the sample. Both primordial follicle-enclosed oocytes that expressed FOXO3 and those that did not were identified in patients who were treated with chemotherapy and those who were not. FOXO3-positive primordial follicles exhibited either nuclear FOXO3 localization or cytoplasmic pFOXO3 localization. Furthermore, transitional primordial follicles that expressed nuclear FOXO3 and cytoplasmic pFOXO3 were also observed. Primary follicle-enclosed oocytes displayed cytoplasmic pFOXO3 localization, whereas in more advanced stages of folliculogenesis, the expression moved to the somatic stratum. No significant statistical differences were identified in the detection of FOXO3 and pFOXO3 in patients who had or had not received chemotherapy prior to sample collection., Conclusion: Primordial follicles expressing and not expressing FOXO3 were equally present in both the ovaries of patients who underwent chemotherapy and those who did not. The expression of FOXO3 remained unaltered in response to chemotherapy treatment. Notably, the detection of PTEN was significantly reduced in the treated patients, thereby warranting in-depth investigation, given the limited sample size examined in the present study., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Accumulating evidence points out that sperm carry epigenetic instructions to embryo in the form of retained histones marks and RNA cargo that can transmit metabolic and behavioral traits to offspring. However, the mechanisms behind epigenetic inheritance of paternal environment are still poorly understood. Here, we curated male germ cells RNA-seq data and analyzed the expression profile of all known histone lysine writers and erasers enzymes across spermatogenesis, unraveling the developmental windows at which they are upregulated, and the specific activity related to canonical and non-canonical histone marks deposition and removal. We also characterized the epigenetic enzymes signature in the mature sperm RNA cargo, showing most of them positive translation at pre-cleavage zygote, suggesting that paternally-derived enzymes mRNA cooperate with maternal factors to embryo chromatin assembly. Our study shows several histone modifying enzymes not described yet in spermatogenesis and even more, important mechanistic aspects behind transgenerational epigenetics. Epigenetic enzymes not only can respond to environmental stressors, but could function as vectors of epigenetic information and participate in chromatin organization during maternal-to-zygote transition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Barbero, de Sousa Serro, Perez Lujan, Vitullo, González and González.)

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., Competing Interests: The authors of this publication have nothing to disclose., (© 2021 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.)

Plains vizcacha females are able to ovulate up to 800 oocytes per estrus cycle. However, just 10-12 embryos are implanted and only two of them, those located nearest the cervix, are gestated to term. Between 26 and 70 days post-coitum, a constitutive resorption occurs from the embryos located proximal to the ovary, extending progressively toward those distally implanted. Our previous studies on the dynamics of gestation in L. maximus, led us to hypothesize some kind of placental and nutritional insufficiency as the basis for the resorption process. We analyzed histology and arterial architecture of the reproductive tract in pregnant and non-pregnant females. Uterine horns are irrigated through the uterine artery, a branch of the internal iliac artery, in an ascending way from the cervix; segmental arteries irrigating the embryo vesicles become thinner as they approach the ovary. Contrast solution administered during angiographies accumulated in the placenta of embryos closest to cervix. Thus, blood stream favors the embryos nearest the cervix, indicating a gradual nutritional deficiency of those closest to the ovary. Besides, placenta becomes calcified early, at mid-gestation, during the resorption process. Finally, the detection of specialized endothelial venules and inflammatory cells suggest the concurrent participation of immunological processes in embryo vesicles undergoing resorption., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Follicular atresia is a cell death event that occurs in the great majority of follicles before ovulation in the mature mammalian ovary. Germ cell loss has been mainly associated to apoptosis although autophagy also seems to be at play. Aimed to increase our understanding on the possible cooperating role of autophagy and apoptosis in follicular atresia and/or follicular survival, we analyzed both programmed cell death mechanisms in a rodent model, the South American plains vizcacha, Lagostomus maximus. Female vizcacha shows highly suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation. This strategy of massive ovulation requires a permanent remodeling of the ovarian architecture to maintain the availability of quiescent primordial follicles throughout the individual's reproductive lifespan. We report here our analysis of autophagy (BECN1, LAMP1 and LC3B-I/II) and apoptosis (BCL2 and ACTIVE CASPASE-3) markers which revealed interactive behaviors between both processes, with autophagy promoting survival or cell death depending on the ovarian structure. Strong BECN1, LC3B-II and LAMP1 staining was observed in atretic follicles and degenerating corpora lutea that also expressed nuclear ACTIVE CASPASE-3. Healthy follicles showed a slight expression of autophagy proteins but a strong expression of BCL2 and no detectable ACTIVE CASPASE-3. Transmission electron microscopy revealed a high formation of autophagosomes, autolysosomes and lysosomes in atretic follicles and degenerating corpora lutea and a low number of autophagic vesicles in normal follicles. The co-expression of LC3B-BECN1, LC3B-LAMP1 and LC3B-ACTIVE CASPASE-3 was only detected in atretic follicles and degenerating corpora lutea, while co-expression of BCL2-BECN1 was only observed in normal follicles. We propose that autophagy could act as a mechanism to eliminate altered follicles and remnant corpora lutea providing the necessary space for maturation of primordial follicles that continuously enter the growing follicular pool to sustain massive ovulation., Competing Interests: The authors have declared that no competing interests exist.

Paternal environmental perturbations, including cocaine intake, can affect the development and behavior of the offspring through epigenetic inheritance. However, the mechanism by which cocaine alters the male germ cells epigenome is almost unexplored. Here, we report that cocaine-treated male mice showed alterations on specific histone post-translational modifications (PTMs) including increased silent chromatin marks H3K9me3 and H3K27me3 and decreased active enhancer and promoter marks H3K27ac and H3K4me3 in isolated germ cells. Also, cocaine increased H3K9ac and H4K16ac levels, involved in the replacement of histones by protamines that take place at round spermatid stage. Cocaine also altered histones H3/H4 epigenetic enzymes by increasing acetyltransferase KAT8/MOF, deacetylase SIRT1 and methyltransferase KMT1C/G9A, and decreasing deacetylases HDAC1/2 and demethylase KDM1A/LSD1 protein levels. Moreover, a pre-treatment with dopamine receptor 1 (DRD1) antagonist SCH23390 (SCH) blocked cocaine effects on H3K4me3, H3K27me3, and H4K16ac epigenetic marks. Interestingly, treatment with SCH-only was able to modify most of the histone marks tested here, pointing to a dopamine role in controlling histone PTMs in germ cells. Taken together, our data suggest a key role for DRD1 in mediating cocaine-triggered epigenetic modifications related to the silencing of gene transcription and the histone-to-protamine replacement that controls chromatin architecture of maturing sperm cells, and pinpoints a novel role of the dopaminergic system in the regulation of male germ cells reprogramming., (Copyright © 2020 González, Gancedo, Janeir Garazatua, Roldán, Vitullo and González.)

Research Question: Recent evidence suggests that cocaine administration in animal models can trigger non-genetic inheritance of addiction traits from father to offspring, affecting development and behaviour. Is chronic cocaine intake involved in alterations of epigenetic homeostasis in the testis?, Design: Epigenetic marks and mediators in testis and isolated germ cells of adult mice treated with cocaine (10 mg/kg) or vehicle (sterile saline solution) were evaluated in an intermittent binge protocol: three intraperitoneal injections, 1 h apart, one day on/off for 13 days, collecting tissue 24 h after the last binge administration (day 14)., Results: It was shown that chronic cocaine intake in mice disrupts testicular epigenetic homeostasis, increasing global methylated cytosine levels in DNA from germ cells and sperm. Cocaine also increased testicular and germ cell acetylated histone 3 and 4 and decreased expression of histone deacetylases HDAC1/2. Immunolocalization studies showed that HDAC1/2 and acetylated histone 3 and 4 proteins localize to meiotic germ cells. Analysis of mRNA expression in isolated germ cells shows decreased levels of Hdac1/2/8, Dnmt3b and Tet1 and increased levels of Dnmt3a gene expression after cocaine treatment., Conclusions: Cocaine intake is associated with testicular toxicity and significant reproductive function impairment. The results presented here broaden the basic knowledge of the impact of addictive stimulants on testicular pathophysiology, fertility and male reproductive health and imply that altered epigenetic homeostasis by cocaine may have potential consequences on future generations., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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.

Mammalian testis undergoes deep changes in their architecture and function during photoregression conditions in seasonal breeders. Particularly, the testicular mechanisms that regulate the transition between the active (functional) and inactive (regression) stage vary between species. The aim of the present study was to analyze the incidence of proliferation, apoptosis and autophagy in the testicular seminiferous ephitelium of a seasonal breeder, Lagostomus maximus, during the annual reproductive cycle. We observed that proliferating spermatogonia increased from the active testis until reaching the maximum peak in the activating testis. During the annual reproductive cycle, the quantity of apoptotic-TUNEL positive spermatogonia and meiotic germ cells was constant and this might be regulated by the members of the BCL2 family. Only in the activating testis, apoptosis of germ cells was almost undetectable. The analysis of the autophagic-related proteins BECN1 and LC3 showed their localization in Leydig cells and the germ cells in the active and activating testis. In the inactive testis, BECN1 and LC3 ceased to be immunolocalized within the seminiferous tubules and the mRNA expression of both regulators decreased. Moreover, the expression of BECN1 and LC3 and also the apoptotic index were up regulated in testicular cultures subjected to nutritional stress. These results suggest a possible interaction between apoptosis and autophagy in the active and activating testis (characterized by high metabolic requirement and nutrient), where autophagy could promote survival over cell death. In the inactive testis, the absence of autophagic-related proteins might explain the massive loss of germ cells, suggesting that autophagy plays new and key role in the alterations of the seminiferous epithelium during photoregression.

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.

Saliva is the first barrier to entry of bacteria and viruses into the body. The submandibular glands (SMG) contribute to the maintenance of oral health and regulation of immune/ inflam matory responses. Previous studies suggest that transforming growth factor beta 1 (TGFB1) may contribute to salivary gland fibrosis but the expression of the TGFB1 system in the SMG has not been elucidated. Thus, the aim of this study was to analyze in rat SMG the immunolocalization of TGFB1 and its specific receptors ALK5 (profibrotic) and ALK1 (proproliferative) and the coreceptor endoglin (EDG) in a bilateral experimental periodontitis (EP) model (cotton thread ligature around the neck of the first lower molars) for 1 and 6 weeks. Fixed SMG were embedded in paraffin and serially cut for routine hematoxylin-eosin staining for histological analysis or immunohistochemical techniques by diaminobenzidine detection. SMG histology from animals with EP showed timedependent structural changes involving marked reduction in the height of the contoured ducts, cell destruction, loss of secretory granules, periductal congestion and excess connective tissue surrounding these ducts indicative of a fibrotic process, compared to control SMG. TGFB1, ALK5 and ALK1 receptors and the coreceptor EDG were mainly immunolocalized in ductal cells and in the fibrotic areas in EP groups. The expression of the profibrotic ALK5 receptor was increased in areas of fibrosis in SMG of animals with EP. In SMG of rats with EP, the localization of the TGFB1 specific receptors in the ducts and cells from fibrotic areas, due to the expression of TGFB1 in the surrounding areas, might indicate paracrine and autocrine actions exerted by TGFB1 via its specific receptors. The results of this study suggest that TGFB1 promotes fibrosis, inducing cell proliferation via ALK1 and EDG receptors and stimulates fibrosis relatedprocesses via ALK5 receptor, which could lead to abnormal secretor activity of the SMG during periodontal disease., (Sociedad Argentina de Investigación Odontológica.)

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.

Background: Endometriosis is a gynaecological disorder that affects 6-10 % of female population. It is characterized by the presence of endometrial tissue outside the uterus, most often in the pelvic peritoneum or ovaries. Recent studies have indicated that mesenchymal endometrial stem cells might get involved in endometriosis progression. Although germ line stem cells have been proved to exist in the ovary, their involvement in ovarian endometriosis has not been investigated. In this preliminary report we aimed to identify germinal stem cell markers in ovarian endometriosis., Findings: Ten paraffin-embedded ovarian endometriosis samples were screened for germ cell-specific proteins DDX4 (VASA) and IFITM3, and its relation with stem cell marker OCT4, proliferation marker PCNA and estrogen receptor alpha (ESR1), by immunohistochemistry, immunofluorescence and PCR. DDX4 and IFITM3 proteins were expressed in isolated cells and clusters of cells in the cortical region of ovarian endometriotic cysts. DDX4 and IFITM3 co-localized in cells from endometriotic stroma, and DDX4/IFITM3-expressing cells were positive for ESR1, OCT4 and PCNA. No cells expressing neither DDX4 nor IFITM3 were detected in normal endometrial tissue., Conclusion: The identification of germ cell-specific proteins DDX4 and IFITM3 provides the first evidence of ovarian-sourced cells in ovarian endometriotic lesions and opens up new directions towards understanding the still confusing pathogenesis of endometriosis.

The Deleted in AZoospermia (DAZ) gene family plays an essential role in spermatogenesis and fertility in mammals. This gene family contains two autosomal genes, BOULE and DAZL (DAZ-Like), and the DAZ gene cluster in the Y chromosome. CDC25A (a cell cycle regulator) has been proposed as a putative substrate for the RNA-binding proteins of DAZ family. However, mechanisms regulating DAZ gene expression have been poorly investigated. We analyzed immunohistochemical localization of DAZL, BOULE and CDC25A, as well as the involvement of testosterone (T) and insulin-like growth factor 1 (IGF1) in the modulation of mRNA expression for DAZL, BOULE and CDC25A in the adult mouse testes. It was found that DAZL was mostly immunolocalized in spermatogonia, while BOULE and CDC25A were detected in spermatocytes and round spermatids. Three-color immunofluorescence showed that DAZL-positive cells also expressed proliferating cell nuclear antigen (PCNA). In vitro incubation of the testes showed that neither T nor IGF1 affected DAZL mRNA expression. However, either T or IGF1 increased BOULE mRNA expression. Antiandrogen flutamide abolished the T-induced increase in BOULE mRNA, but had no effect on the IGF1 induced increase in the mouse testes. Extracellular-signal-regulated kinase 1/2 (ERK1/2) inhibitor, U0126, prevented IGF1-induction of BOULE mRNA. It was found that IGF1 increased CDC25A mRNA expression and that U0126 - but not flutamide - abolished the IGF1-induced CDC25A mRNA expression. These results showed that IGF1 regulated the expression of BOULE and CDC25A mRNAs via ERK1/2 signaling and in T-independent pathway during spermatogenesis in the adult mouse testes., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

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.

Study Question: How do apoptosis-related BCL2 and BAX genes, known to regulate death or survival of germ cells in fetal and adult life, and germ-cell-specific VASA protein behave from birth to puberty in the human ovary?, Summary Answer: In resting primordial follicles in both infant and pubertal ovaries, BCL2 family members and germ-cell-specific VASA behave as in fetal life. After birth, once follicles leave the resting reserve to enter the growing follicular pool, detection of apoptosis-related genes moves from the germ cell to granulosa cells and VASA expression is lost., What Is Known Already: In the human ovary, around 85% of the 7 × 10⁶ potential oocytes produced at mid-gestation are lost before birth, an extra 10% before puberty, and loss continues throughout reproductive life until germinal exhaustion of the ovary. Oocyte loss is mainly driven through a balanced expression of BCL2 gene family members. Apoptosis-inducing BAX gene shows a sustained expression throughout fetal and adult life, whereas apoptosis-inhibiting BCL2 is detectable during the proliferative stage of primordial germ cells and oogonia in the fetal ovary and proliferation of granulosa cells in growing follicles in the adult ovary. The germ-cell marker VASA is detectable in the fetal ovary from early oogenesis and is conspicuously expressed in primordial follicles, where in late pregnancy it is associated with the Balbiani's vitelline space. VASA expression is not detectable in the adult ovary., Study Design, Size, Duration: This is a qualitative analysis involving infant/pubertal paraffin-embedded human ovaries screened for apoptosis-related proteins, DNA fragmentation and germ-cell identity., Participants/materials, Setting, Methods: Ovaries from 13 patients ranging in age from 4 to 16 years, undergoing gynaecological surgical procedures due to benign pathology, were studied. Tissues were fixed in 10% formalin, paraffin-embedded and processed for immunohistochemistry to screen the temporal and cellular localization of germ-cell-specific VASA protein and BCL2 and BAX apoptosis-related proteins. In addition, a terminal deoxynucleotidyl transferase-mediated deoxiuridinetriphosphate nick-end labelling (TUNEL) assay was performed to detect DNA fragmentation. General histology and tissue integrity were assessed by haematoxylin-eosin staining., Main Results and the Role of Chance: VASA showed a differential pattern of expression; in the resting primordial follicle reserve in infant and pubertal ovaries, it was associated with the Balbiani's body space in the germ cell. VASA remained detectable in primary follicles leaving the resting reserve, but once follicles entered the growing pool it became undetectable. This pattern of VASA expression is the same as in the fetal ovary. BAX was expressed both in the resting primordial reserve and in the pool of growing follicles, whereas BCL2 was detected only in granulosa cells in antral follicles in the growing pool. Apoptosis-related protein expression moved from the germ cell to the somatic stratum when primordial follicles left the resting reserve to enter the pool of growing follicles, irrespective of female age. Most TUNEL-positive cells were detected in the granulosa cells of antral follicles. No TUNEL-positive cells were found in resting primordial follicles., Limitations, Reasons for Caution: The study was limited by the qualitative nature of the immuno-histochemical analysis and the TUNEL assay. The results neither quantify the levels of germ-cell death nor exclude other concurrent cell death mechanisms that could act in the regulation of female germ-cell number., Wider Implications of the Finding: This study provides missing knowledge about apoptosis and germ-cell-specific VASA expression in the human ovary between birth and puberty and the participation of BCL2 and BAX genes in the balance between death and survival throughout female germ-line development. Intracellular localization of VASA in resting follicles emerges as a possible marker with prognostic value that needs further investigation, especially in infant patients entering ovarian cryo-preservation programmes. This knowledge will be valuable in optimizing the rescue and clinical use of germ cells to restore fertility in women., Study Funding/competing Interest(s): No external funding was obtained for this study. The authors have no conflicts of interest to declare.

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species.

The South American plains vizcacha, Lagostomus maximus, displays an exceptional ovulation rate of up to 800 eggs per cycle, the highest rate recorded for a mammal. Massive polyovulation arises from the overexpression of the apoptosis-inhibiting BCL2 gene leading to a suppression of apoptotic pathways responsible for follicular atresia in mammals. We analyzed the ovarian histology, ovarian apoptosis, and apoptosis-related protein expression with special emphasis in corpora lutea throughout the 5-mo-long gestation period, at parturition day and early postpartum, in L. maximus. Corpora lutea were abundant throughout gestation with no sign of structural regression even at the end of gestation. Both immunohistochemistry and Western blot analysis showed strong signals for apoptosis-inhibiting BCL2 protein, whereas the proapoptotic BAX protein was just detected in isolated luteal cells in gestating females and postpartum females. Apoptosis-associated DNA fragmentation detected by TUNEL was very scarce and occasional and correlated with BAX detection in luteal cells. Marked expression of progesterone and alpha-estrogen receptors in luteal cells was found at early, mid-, and late gestation as well as at parturition day and early postpartum samples. Additionally, serum level of progesterone increased markedly to reach maximal values at late gestation and decreasing at parturition to levels found at early gestation, suggesting that corpora lutea remained functional throughout gestation. These results point out that the unusual ovarian environment of L. maximus in which germ cell demise is abolished through antiapoptotic BCL2 gene overexpression also preserves structural integrity and functionality of corpora lutea during the whole gestation. Overexpression of antiapoptotic BCL2 gene may represent a strategy for an essential need of ovary and corpora lutea in order to maintain pregnancy until term.

Background: Germ cell number during ovarian organogenesis is regulated through programmed cell death. We investigated the expression of germ-cell-specific VASA protein, apoptosis-related proteins BAX and BCL-2 and DNA fragmentation in developing human ovaries from gestation week 12 to term., Methods: Human fetal ovaries from 13 women undergoing spontaneous abortion were fixed, paraffin-embedded and processed for immunohistochemistry to analyse temporal and cellular localization of VASA, BCL-2 and BAX, and to detect apoptosis by TUNEL assay., Results: VASA showed a differential pattern of expression throughout the differentiation and proliferative phase and prophase I to finally associate with Balbiani's body in primordial and primary follicles. BCL-2 was detected from week 12 to 17 and became undetectable thereafter. Strong BAX signal was detected in oogonia and oocytes from week 12 to term. Low levels (

It has been widely accepted that mammalian females are born with a non-renewing, finite pool of oocytes that will be continuously cleared by atresia, with only a small proportion of them reaching ovulation. Apoptosis regulates this mass germ cell death, especially through the balance between pro- and anti-apoptotic proteins encoded by the BCL-2 gene family. The caviomorph rodent Lagostomus maximus, the South American plains viscacha, displays the highest ovulation rate known for a mammal releasing 400-800 eggs per cycle. We tested the hypothesis that in L. maximus massive polyovulation is a consequence of reduced apoptosis resulting in suppressed follicular atresia. We found that anti-apoptotic BCL-2 gene is markedly expressed in all kind of follicles from primordial to fully mature antral stages in the adult ovary of L. maximus. On the other hand, pro-apoptotic BAX gene showed weak signals or was undetectable by immunohistochemical examination. Western blot against both proteins confirmed immunohistochemical results. Screening for DNA fragmentation by TUNEL assay was conspicuously negative in ovaries from both pregnant and non-pregnant females. In addition, alpha-oestrogen receptor also showed an enhanced expression from primordial stage to fully mature antral follicles. Our results show that natural preferential expression of BCL-2 and restricted BAX expression greatly suppresses apoptosis in the ovary of L. maximus. This prevents the decline of the oocyte reserve by abolishing follicular atresia and enables the highest ovulation rate known for a mammal, 400-800 or more eggs per cycle.