Naked mole rat ovaries allow investigation of ovarian reserve, in vitro germ cell expansion, and oocyte in vitro maturation within a single sample.

Recently, we described that in the naked mole rat ovary it is possible to study the ovarian reserve and the mitotic expansion of the germ cell postnatally. Herein, we show oocyte in vitro maturation and in vitro germ cell expansion using the same ovary.

Subcutaneous Application of a Gelatin/Hyaluronic Acid Hydrogel Induces the Production of Skin Extracellular Matrix.

The development of injectable hydrogels with natural biopolymers such as gelatin (Ge) and hyaluronic acid (Ha) is widely performed due to their biocompatibility and biodegradability. The combination of both polymers crosslinked with N-Ethyl-N'-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) can be used as an innovative dermal filler that stimulates fibroblast activity and increases skin elasticity and tightness. Thus, crosslinked Ge/Ha hydrogels with different concentrations of EDC were administered subcutaneously to test their efficacy in young and old rats. At higher EDC concentrations, the viscosity decreases while the particle size of the hydrogels increases. At all concentrations of EDC, amino and carboxyl groups are present. The histological analysis shows an acute inflammatory response, which disappears seven days after application. At one and three months post-treatment, no remains of the hydrogels are found, and the number of fibroblasts increases in all groups in comparison with the control. In addition, the elastic modulus of the skin increases after three months of treatment. Because EDC-crosslinked Ge/Ha hydrogels are biocompatible and induce increased skin tension, fibroblast proliferation, and de novo extracellular matrix production, we propose their use as a treatment to attenuate wrinkles and expression lines.

Characterization of the Postnatal Naked Mole-Rat Ovary: From Primordial Germ Cells to Meiotic Prophase I Oocytes.

The mammalian reproductive cycle, including those of humans and mice, begins very early in development. In utero, the ovaries become populated with primordial germ cells (PGCs) that will generate the oogonia. First, these cells proliferate mitotically, and then they trigger the meiotic program and initiate meiotic prophase I. Since these processes happen during gestation, their study had been very limited and challenging. Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the PGCs, and the initiation of the meiotic program occurs postnatally. In this chapter, we present a comprehensive collection of protocols that permit the analysis of naked mole-rat germ cells, from PGCs to oocytes, in meiotic prophase I, using in vivo and in vitro approaches.

Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Gelatin/Hyaluronic Acid Scaffold Coupled to CpG and MAGE-A5 as a Treatment against Murine Melanoma.

The half-time of cells and molecules used in immunotherapy is limited. Scaffolds-based immunotherapy against cancer may increase the half-life of the molecules and also support the migration and activation of leukocytes in situ. For this purpose, the use of gelatin (Ge)/hyaluronic acid (HA) scaffolds coupled to CpG and the tumor antigen MAGE-A5 is proposed. Ge and HA are components of the extracellular matrix that stimulate cell adhesion and activation of leucocytes; CpG can promote dendritic cell maturation, and MAGE-A5 a specific antitumor response. C57BL/6 mice were treated with Ge/HA/scaffolds coupled to MAGE-A5 and/or CpG and then challenged with the B16-F10 melanoma cell line. Survival, tumor growth rate and the immune response induced by the scaffolds were analyzed. Ge/HA/CpG and Ge/HA/MAGE-A5 mediated dendritic cell maturation and macrophage activation, increased survival, and decreased the tumor growth rate and a tumor parenchyma with abundant cell death areas and abundant tumor cells with melanin granules. Only the scaffolds coupled to MAGE-A5 induced the activation of CD8 T cells. In conclusion, Ge/HA scaffolds coupled to CpG or MAGE-A5, but not the mixture, can induce a successful immune response capable of promoting tumor cell clearance and increased survival.

Unraveling mitochondrial piRNAs in mouse embryonic gonadal cells.

Although mitochondria are widely studied organelles, the recent interest in the role of mitochondrial small noncoding RNAs (sncRNAs), miRNAs, and more recently, piRNAs, is providing new functional perspectives in germ cell development and differentiation. piRNAs (PIWI-interacting RNAs) are single-stranded sncRNAs of mostly about 20-35 nucleotides, generated from the processing of pre-piRNAs. We leverage next-generation sequencing data obtained from mouse primordial germ cells and somatic cells purified from early-differentiating embryonic ovaries and testis from 11.5 to 13.5 days postcoitum. Using bioinformatic tools, we elucidate (i) the origins of piRNAs as transcribed from mitochondrial DNA fragments inserted in the nucleus or from the mitochondrial genome; (ii) their levels of expression; and (iii) their potential roles, as well as their association with genomic regions encoding other sncRNAs (such as tRNAs and rRNAs) and the mitochondrial regulatory region (D-loop). Finally, our results suggest how nucleo-mitochondrial communication, both anterograde and retrograde signaling, may be mediated by mitochondria-associated piRNAs.

Immunomodulatory Properties of Masticadienonic Acid and 3α-Hydroxy Masticadienoic Acid in Dendritic Cells.

Dendritic cells are antigen-presenting cells, which identify and process pathogens to subsequently activate specific T lymphocytes. To regulate the immune responses, DCs have to mature by the recognition of TLR ligands, TNFα or IFNγ. These ligands have been used as adjuvants to activate DCs in situ or in vitro, with toxic effects. It has been shown that some molecules affect the immune system, e.g., Masticadienonic acid (MDA) and 3α-hydroxy masticadienoic acid (3α-OH MDA) triterpenes naturally occurring in several medicinal plants, since they activate the nitric oxide synthase in macrophages and induce T lymphocyte proliferation. The DCs maturation induced by MDA or 3a-OH MDA was determined by incubating these cells with MDA or 3α-OH MDA, and their phenotype was afterwards analyzed. The results showed that only 3α-OH MDA was able to induce DCs maturation. When mice with melanoma were inoculated with DCs/3α-OH MDA, a decreased tumor growth rate was observed along with an extended cell death area within tumors compared to mice treated with DCs incubated with MDA. In conclusion, it is proposed that 3α-OH MDA may be an immunostimulant molecule. Conversely, it is proposed that MDA may be a molecule with anti-inflammatory properties.

Epicutaneous Administration of 17ß-Estradiol Induces Langerhans Cells Depletion.

BACKGROUND: Langerhans cells (LC) number and function in mouse vaginal mucosa are affected by 17ß-estradiol (E2) application; nonetheless, its effect on epidermal LC has not been studied. The purpose of this study was to evaluate the effect of topical administration of E2 on the number, phenotype, and migratory ability of LC in mouse skin. METHODS: Ears of adult CD1 male mice were topically treated once with several doses. Immunohistochemical staining for CD207 and TUNEL staining were performed. LC migration to lymph nodes and the effect on the expression of costimulatory molecules on cultured dendritic cells (DC) were also evaluated. RESULTS: E2 decreased the number of CD207+ LC in a dose-dependent manner. One hour after treatment, 1 and 10 µg/mL E2 significantly reduced the LC number by 21% and 26%, respectively, after two hours, the reduction was 23% and 41%, respectively. After 48 hours, LC recovered, and after 96 hours of treatment, the CD207+/MHCII+ DC numbers were increased in regional lymph nodes. However, CD86 and CD40 molecules were expressed at lower levels than in positive control. The TUNEL assay did not show apoptotic cells. Furthermore, in cultured DC, E2 promoted a decrease in CD40 and CD86 expression and an increase in CD273, CD274, MHCII, and CCR7. CONCLUSIONS: The topical administration of E2 induced a transitory local diminution of LC population and a tolerogenic phenotype. This decrease in epidermal LC suggests that E2 may affect skin immune responses, inducing an inhibitory response, which should be considered when prescribing topical E2 medications.

Meiotic Cohesin and Variants Associated With Human Reproductive Aging and Disease.

Successful human reproduction relies on the well-orchestrated development of competent gametes through the process of meiosis. The loading of cohesin, a multi-protein complex, is a key event in the initiation of mammalian meiosis. Establishment of sister chromatid cohesion via cohesin rings is essential for ensuring homologous recombination-mediated DNA repair and future proper chromosome segregation. Cohesin proteins loaded during female fetal life are not replenished over time, and therefore are a potential etiology of age-related aneuploidy in oocytes resulting in decreased fecundity and increased infertility and miscarriage rates with advancing maternal age. Herein, we provide a brief overview of meiotic cohesin and summarize the human genetic studies which have identified genetic variants of cohesin proteins and the associated reproductive phenotypes including primary ovarian insufficiency, trisomy in offspring, and non-obstructive azoospermia. The association of cohesion defects with cancer predisposition and potential impact on aging are also described. Expansion of genetic testing within clinical medicine, with a focus on cohesin protein-related genes, may provide additional insight to previously unknown etiologies of disorders contributing to gamete exhaustion in females, and infertility and reproductive aging in both men and women.

Germ cell nests in adult ovaries and an unusually large ovarian reserve in the naked mole-rat.

The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35-40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen's suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species' protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR's long reproductive lifespan remains to be determined.

Sex- and Age-Specific Impact of ERK Loss Within the Pituitary Gonadotrope in Mice.

Extracellular signal-regulated kinase (ERK) signaling regulates hormone action in the reproductive axis, but specific mechanisms have yet to be completely elucidated. In the current study, ERK1 null and ERK2 floxed mice were combined with a gonadotropin-releasing hormone receptor (GnRHR)-internal ribosomal entry site-Cre (GRIC) driver. Female ERK double-knockout (ERKdko) animals were hypogonadotropic, resulting in anovulation and complete infertility. Transcript levels of four gonadotrope-specific genes (GnRHR and the three gonadotropin subunits) were reduced in pituitaries at estrus in ERKdko females, and the postcastration response to endogenous GnRH hyperstimulation was blunted. As females aged, they exhibited abnormal ovarian histology, as well as increased body weight. ERKdko males were initially less affected, showing moderate subfertility, up to 6 months of age. Male ERKdko mice also displayed a blunted response to endogenous GnRH following castration. By 12 months of age, ERKdko males had reduced testicular weights and sperm production. By 18 months of age, the ERKdko males displayed reduced testis and seminal vesicle weights, marked seminiferous tubule degeneration, and a 77% reduction in sperm production relative to controls. As the GRIC is also active in the male germ line, we examined the specific role of ERK loss in the testes using the stimulated by retinoic acid 8 (Stra8)-Cre driver. Whereas ERK loss in GRIC and Stra8 males resulted in comparable losses in sperm production, seminiferous tubule histological degeneration was only observed in the GRIC-ERKdko animals. Our data suggest that loss of ERK signaling and hypogonadotropism within the reproductive axis impacts fertility and gonadal aging.

Embarazo ectópico intersticial asociado a técnicas de reproducción asistida y ligadura tubaria bilateral. Reporte de caso / Interstitial Pregnancy associated to assisted reproductive techniques. Case report

Antecedentes: Sólo 2,4% de los embarazos ectópicos se desarrollan en la región intersticial de la trompa, la cual tiene un diámetro de 0,7 mm y 1-2 cm de largo, su ubicación anatómica causa que el embarazo ectópico se diagnostique tardíamente, y como se encuentra en la rama ascendente de la arteria uterina, predispone un riesgo de hemorragia, asociado a una tasa de mortalidad de 5,2%, en contraste con otras variedades de embarazo ectópico tubario. Descripción del caso: Presentamos el caso clínico de una paciente de 32 años con infertilidad primaria de 5 años de evolución, con antecedentes de endometriosis severa. La paciente fue tratada con fertilización in vitro después de la supresión hipofisaria con acetato de leuprolide, hiperestimulación ovárica controlada con FSH recombinante, captura de 8 ovocitos con adecuada fertilización, transferencia embrionaria (2 embriones); treinta y dos días después de la transferencia de embriones, en el examen ecográfico se reporta: saco gestacional de 18 mm, embrión de 8,2 mm y masa heterogénea anexial izquierda de 16x15 mm. Se le realiza salpingectomia en porción cornual por laparoscopia y estudio histopatológico que reporta embarazo ectópico. La paciente cursó postoperatorio normoevolutivo y sin complicaciones. Conclusiones: el embarazo ectópico es una complicación de las técnicas de reproducción asistida y el embarazo intersticial es una forma rara de embarazo tubario que implica un reto diagnóstico y es potencialmente mortal por el riesgo de hemorragia masiva, la evidencia de un embarazo intersticial merece una intervención rápida para evitar situaciones que amenacen la vida (AU)

2.4 % of ectopic pregnancies develop in the interstitial region of the tube. It has a diameter of 0.7 mm and 1.2 cm length, anatomic location causes a late diagnosed, it’s in the ascending branch of the uterine artery predisponding a risk of hemorrhage, has a mortality rate of 5.2%, in contrast to traditional tubal pregnancy. Case Report: Patient is 32 years with primary infertility of 5 years of evolution, history of severe endometriosis she was treated in vitro fertilization after pituitary suppression with leuprolide acetate. For ovarian hyperstimulation was used recombinant FSH. Were captured 8 eggs, which were suitable for fertilization All fertilized eggs. Pre-embryos were transferred at stage 2 11 C 2 + and blastocyst. Thirty-two days after embryo transfer, ultrasonographic examination is performed where 18-mm gestational sac, embryo of 8.2 mm and heterogeneous left adnexal mass of 16x15 mm. Were admitted for diagnostic and operative laparoscopy, the laparoscopic procedure was performed with 30 degree angle with the tripolar energy use for coagulation and exeresis of the uterine horn. The histopathologic result was left cornual pregnancy. The patient developed normally in early postoperative period, and leaving the next day in proper clinical status. Conclusion: ectopic pregnancy is a complication of assisted reproduction techniques and interstitial pregnancy is a rare form of tubal pregnancy involving a diagnostic challenge and is life-threatening because of the risk of massive bleeding , evidence of an interstitial pregnancy deserves a quick intervention avoid life-threatening situations (AU)

Endocrine disrupters, microRNAs, and primordial germ cells: a dangerous cocktail.

Endocrine-disrupting chemicals (EDCs) are environmental pollutants that may change the homeostasis of the endocrine system, altering the differentiation of germ cells with consequences for reproduction. In mammals, germ cell differentiation begins with primordial germ cells (PGCs) during embryogenesis. Primordial germ cell development and gametogenesis are genetically regulated processes, in which the posttranscriptional gene regulation could be mediated by small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Here, we review the deleterious effects of exposure during fetal life to EDCs mediated by deregulation of ncRNAs, and specifically miRNAs on PGC differentiation. Moreover, the environmental stress induced by exposure to some EDCs during the embryonic window of development could trigger reproductive dysfunctions transgenerationally transmitted by epigenetic mechanisms with the involvement of miRNAs expressed in germ line cells.

GK-1 improves the immune response induced by bone marrow dendritic cells loaded with MAGE-AX in mice with melanoma.

The aim of dendritic cell (DC) vaccination in cancer is to induce tumor-specific effector T cells that may reduce and control tumor mass. Immunostimulants that could drive a desired immune response are necessary to be found in order to generate a long lasting tumor immune response. GK-1 peptide, derived from Taenia crassiceps, induces not only increase in TNFα, IFNγ, and MCP-1 production in cocultures of DCs and T lymphocytes but also immunological protection against influenza virus. Moreover, the aim of this investigation is the use of GK-1 as a bone marrow DCs (BMDCs) immunostimulant targeted with MAGE antigen; thus, BMDC may be used as immunotherapy against murine melanoma. GK-1 induced in BMDCs a meaningful increment of CD86 and IL-12. In addition, the use of BMDCs TNFα/GK-1/MAGE-AX induced the highest survival and the smallest tumors in mice. Besides, the treatment helped to increase CD8 lymphocytes levels and to produce IFNγ in lymph nodes. Moreover, the histopathological analysis showed that BMDCs treated with GK-1/TNFα and loaded with MAGE-AX induced the apparition of more apoptotic and necrotic areas in tumors than in mice without treatment. These results highlight the properties of GK-1 as an immunostimulant of DCs and suggest as a potential candidate the use of this immunotherapy against cancer disease.

Telomeric repeat-containing RNA and telomerase in human fetal oocytes.

STUDY QUESTION: What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes? SUMMARY ANSWER: TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes. WHAT IS KNOWN ALREADY: TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown. STUDY DESIGN, SIZE, DURATION: For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT). LIMITATIONS, REASONS FOR CAUTION: We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.

Meiosis in a bottle: new approaches to overcome Mammalian meiocyte study limitations.

The study of meiosis is limited because of the intrinsic nature of gametogenesis in mammals. One way to overcome these limitations would be the use of culture systems that would allow meiotic progression in vitro. There have been some attempts to culture mammalian meiocytes in recent years. In this review we will summarize all the efforts to-date in order to culture mammalian sperm and oocyte precursor cells.

Inhaled vanadium pentoxide decrease gamma-tubulin of mouse testes at different exposure times.

Vanadium is an important environmental and industrial pollutant whose concentrations have increased in the last decades. Due to its status as reproductive toxicant and a microtubule damaging agent, the present study investigated by immunohistochemistry the effect of the inhalation of vanadium pentoxide on gamma-tubulin within somatic and testicular germ cells. Male mice inhaled vanadium pentoxide (V2O5) (0.02 M) 1 h/twice a week for 12 weeks. Our results demonstrated that vanadium accumulates in the testes starting with the initial inhalation (24 h), and this pattern remained until the last week of treatment. In general, vanadium was capable of significantly decreasing the percentage of gamma-tubulin in all analyzed testicular cells (Sertoli, Leydig and germ cells) starting with the first week of treatment. For all cell types studied, regression analysis revealed a negative and significant relationship between the percentage of immunopositive cells to gamma-tubulin and exposure time, showing a time dependent response in all cases. Our findings suggest that alterations on this protein might imply changes in microtubule-involved function such as cell division, which in the testes might lead to damage in the spermatogenesis, leading probably to infertility.

Eritroenzimopatías hereditarias en neonatos con hiperbilirrubinemia / Hereditary erythroenzymopathies in neonates with hyperbilirubinemia

Se presentan los resultados del tamizaje para eritroenzimopatías hereditarias en 707 recién nacidos a término con hiperbilirrubinemia (305 niñas y 402 varones), cuyo objetivo fue determinar su frecuencia relativa. Se tamizó por métodos fluorescentes para las eritroenzimopatías más frecuentes a saber: deficiencias de glucosa-6-fosfato deshidrogenasa (G-6-PD), piruvato cinasa (PC) y glucosa fosfatoisomerasa (GPI): estos son tres de los 14 errores congénitos del metabolismo del eritrocito claramente asociados con hemólisis. Se identificaron cuatro varones con deficiencia de G-6-PD y no se encontraron individuos con deficiencia de PC o GPI. Los resultados globales de éste y de estudios previos de nuestro grupo en una población de 2218 individuos sugieren que la frecuencia de la deficiencia de G-6-PD en varones recién nacidos con ictericia y/o hiperbilirrubinemia es de 1.08%. Se concluye que la deficiencia de G-6-PD como causa de hiperbilirrubinemia no es un problema de salud pública en la población estudiada; sin embargo, ya que 1% de los varones con ictericia y/o hiperbilirrubinemia neonatal tienen deficiencia de G-6-PD, se recomienda tomar en consideración este diagnóstico al evaluar a un neonato que presente anemia y/o hiperbilirrubinemia