Environmentally-relevant exposure to diethylhexyl phthalate (DEHP) alters regulation of double-strand break formation and crossover designation leading to germline dysfunction in Caenorhabditis elegans.

Exposure to diethylhexyl phthalate (DEHP), the most abundant plasticizer used in the production of polyvinyl-containing plastics, has been associated to adverse reproductive health outcomes in both males and females. While the effects of DEHP on reproductive health have been widely investigated, the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP and its metabolites impact the female germline in the context of a multicellular organism have remained elusive. Using the Caenorhabditis elegans germline as a model for studying reprotoxicity, we show that exposure to environmentally-relevant levels of DEHP and its metabolites results in increased meiotic double-strand breaks (DSBs), altered DSB repair progression, activation of p53/CEP-1-dependent germ cell apoptosis, defects in chromosome remodeling at late prophase I, aberrant chromosome morphology in diakinesis oocytes, increased chromosome non-disjunction and defects during early embryogenesis. Exposure to DEHP results in a subset of nuclei held in a DSB permissive state in mid to late pachytene that exhibit defects in crossover (CO) designation/formation. In addition, these nuclei show reduced Polo-like kinase-1/2 (PLK-1/2)-dependent phosphorylation of SYP-4, a synaptonemal complex (SC) protein. Moreover, DEHP exposure leads to germline-specific change in the expression of prmt-5, which encodes for an arginine methyltransferase, and both increased SC length and altered CO designation levels on the X chromosome. Taken together, our data suggest a model by which impairment of a PLK-1/2-dependent negative feedback loop set in place to shut down meiotic DSBs, together with alterations in chromosome structure, contribute to the formation of an excess number of DSBs and altered CO designation levels, leading to genomic instability.

Preservation of female genetic resources of common carp through oogonial stem cell manipulation.

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

Ovarian structure and oogenesis of the extremophile viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico.

The structure of the ovary and oogenesis of Poecilia mexicana from an active sulfur spring cave is documented. Poecilia mexicana is the only poeciliid adapted to a subterranean environment with high hydrogen sulfide levels and extreme hypoxic conditions. Twenty females were captured throughout one year at Cueva del Azufre, located in the State of Tabasco in Southern Mexico. Ovaries were processed with histological techniques. P. mexicana has a single, ovoid ovary with ovigerous lamella that project to the ovarian lumen. The ovarian wall presents abundant loose connective tissue, numerous melanomacrophage centers and large blood vessels, possibly associated with hypoxic conditions. The germinal epithelium bordering the ovarian lumen contains somatic and germ cells forming cell nests projecting into the stroma. P. mexicana stores sperm in ovarian folds associated with follicles at different developmental phases. Oogenesis in P. mexicana consisted of the following stages: (i) oogonial proliferation, (ii) chromatin nucleolus, (iii) primary growth, subdivided into: (a) one nucleolus, (b) multiple nucleoli, (c) droplet oils-cortical alveoli steps; (iv) secondary growth, subdivided in: (a) early secondary growth, (b) late secondary growth, and (c) full grown. Follicular atresia was present in all stages of follicular development; it was characterized by oocyte degeneration, where follicle cells hypertrophy and differentiate in phagocytes. The ovary and oogenesis are similar to these seen in other poeciliids, but we found frequent atretic follicles, melanomacrophage centers, reduced fecundity and increased of offspring size.

Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam.

Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

c-Fos Repression by Piwi Regulates Drosophila Ovarian Germline Formation and Tissue Morphogenesis.

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. We uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.

Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries.

Accruing evidence indicates that production of new oocytes (oogenesis) and their enclosure by somatic cells (folliculogenesis) are processes not limited to the perinatal period in mammals. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life. The recent purification of mitotically active oocyte progenitor cells, termed female germline stem cells (fGSCs) or oogonial stem cells (OSCs), from mouse and human ovaries opens up new avenues for research into the biology and clinical utility of these cells. Here we detail methods for the isolation of mouse and human OSCs from adult ovarian tissue, cultivation of the cells after purification, and characterization of the cells before and after ex vivo expansion. The latter methods include analysis of germ cell-specific markers and in vitro oogenesis, as well as the use of intraovarian transplantation to test the oocyte-forming potential of OSCs in vivo.

Dexamethasone induces germ cell apoptosis in the human fetal ovary.

CONTEXT: The 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. Pregnant women presenting a risk of genetic transmission may be treated with synthetic glucocorticoids such as dexamethasone (DEX) to prevent female fetus virilization. OBJECTIVE: The aim of this study was to assess the potential deleterious effects of DEX exposure on fetal ovarian development. SETTINGS: Human fetal ovaries, ranging from 8-11 weeks after fertilization, were harvested from material available after legally induced abortions. They were cultured in the absence or presence of DEX (2, 10, or 50 µm) over 14 d, and histological analyses were performed. RESULTS: The glucocorticoid receptor NR3C1 was present and the signaling pathway active in the fetal ovary as demonstrated by the expression of NR3C1 target genes, such as PLZF and FKBP5, in response to DEX exposure. DEX decreased germ cell density at the 10 and 50 µm doses. Exposure to DEX, even at the highest dose, did not change oogonial proliferation as monitored by 5-bromo-2'-deoxyuridine incorporation and significantly increased the apoptotic rate, detected with cleaved caspase 3 staining. Interestingly, the expression of the prosurvival gene KIT was significantly decreased in the presence of DEX during the course of the culture. CONCLUSION: We have demonstrated for the first time that in vitro exposure to high doses of DEX impairs human fetal oogenesis through an increase in apoptosis. These data are of high importance, and additional epidemiological studies are required to investigate the female fertility of those women who have been exposed to DEX during fetal life.

Ovarian folliculogenesis in the smallnose fanskate sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae) / Foliculogénesis ovárica en la raya marmorada, sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae)

Chondrichthyes have become an important economic resource in the last decades, with Argentina as one of the countries that exploits more sharks and skates, even at levels that exceed de limits of many species. However, there is a scarce knowledge of the reproductive biology of this group, particularly from species inhabiting the Southern hemisphere. This work shows the most relevant facts during folliculogenesis in Sympterygia bonapartii. Results show that germinal cells are present in immature and maturing females. The most important facts that vary along de follicular development are the number of types and layers of follicular cells, the establishment of thin projections from the follicular cells and the degree of development of the thecae. Follicular cells are, at least, of two different types and both of them emit projections that break through the zona pellucida. The outer theca shows signs of synthetic activity. Atretic follicles of different sizes are present in exemplars of all the reproductive stages. These results are discussed in a physiological and adaptive context.

Los Condrictios se han convertido en un recurso económico importante en las últimas décadas, siendo Argentina uno de los países que más explota tiburones y rayas, incluso a niveles que exceden los límites de varias especies. A pesar de esto, es poco lo que se conoce sobre la biología reproductiva de este grupo, particularmente en especies del Hemisferio Sur. En este trabajo se estudian los estadios más relevantes de la foliculogénesis en Sympterygia bonapartii. Los resultados muestran que las ovogonias están presentes tanto en ejemplares inmaduros como subadultos. Las características más importantes que varían a lo largo del desarrollo folicular son el número de capas y tipos celulares que constituyen el epitelio folicular, el desarrollo de proyecciones de las células de la granulosa y el grado de desarrollo de las tecas. Las células foliculares son, al menos, de dos tipos y ambos emiten proyecciones que atraviesan la zona pelúcida. La teca externa presenta características compatibles con la actividad sintética. Folículos atrésicos de distintos tamaños están presentes en ejemplares de todos los estadios de madurez sexual. Estos resultados se discuten en un marco fisiológico y adaptativo.

Identification of germline stem cells in the ovary of the teleost medaka.

Germline stem cells continually produce sperm in vertebrate testes, whereas there is no direct evidence showing that germline stem cells are present in adult vertebrate ovaries. By using transgenic methods and clonal analysis, we identified germline stem cells that supported oogenesis and the production of offspring in the ovaries of adult medaka fish. Early-stage germ cells were localized in clusters along interwoven threadlike cords of sox9b-expressing somatic cells (termed germinal cradles) where the germ cells developed. Germline stem cells gave rise to germ cells that divided to produce cysts, which then underwent cell death or separated to form follicles. Our results provide insight into the germline stem cell biology of medaka and provide a model system for studying vertebrate stem cell niches.

Pattern of expression of immune-relevant genes in the gonad of a teleost, the gilthead seabream (Sparus aurata L.).

Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b (il1b), il6, tumor necrosis factor-a (tnfa), cyclooxygenase-2 (cox2) and the NADPH oxidase subunit p40(phox) (ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 (tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 (tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor (mcsfr); (iv) lymphocyte receptors, such as the beta subunit of T-cell receptor (Tcrb) and the heavy chain of immunoglobulin M (ighm); (v) the anti-bacterial molecules lysozyme (lyz), hepcidin (hamp) and complement component 3 (c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein (mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (Sele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish.

Oogenesis in the swamp eel Synbranchus marmoratus (Bloch, 1795) (Teleostei; Synbranchidae). Ovarian anatomy, stages of oocyte development and micropyle structure

Synbranchus marmoratus (Synbranchidae), commonly known as the swamp eel, is a protogynous diandric teleost fish widely distributed throughout South America. The purpose of this work was to study the ovarian anatomy and to describe oocyte developmental stages in the swamp eel, Synbranchus marmoratus. S. marmoratus has a unique sacular ovary. It is covered by a conspicuous muscular wall, probably involved in an egg-releasing system acting as a peristaltic-like mechanism. The internal ovarian anatomy shows a U-shaped ovarian lamella delimiting a dorsal ovarian lumen. The microscopic study shows evidence of the existence of a germinal epithelium in the inner surface of the lamella, which contains germinal cells, pre-follicular cells and epithelial cells. The complete oogenesis process is divided into four stages: oogonia, primary growth, cortical alveoli and vitellogenesis. Besides, the ovulated oocytes, and atretic structures were described. The structure of the micropyle was studied by scanning electron microscopy (MEB). Near the animal pole the vitelline envelope forms crests that fuse together becoming furrow-like structures with a slightly spiraled direction that converge into the micropyle pit where is located the micropylar canal. Although the sex reversal process of Synbranchids has been subject of many studies, this is the first complete description of the ovarian anatomy and oogenesis.

Fine structure of human oogonia in the foetal ovary.

Foetal ovarian tissue is now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. This study examines the ultrastructure of oogonia at 13-15 weeks of gestation, which could be used as a control for culture and freezing of foetal ovaries. Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to dense follicle cells, which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts - beginning of folliculogenesis. Nuclei of oogonia contain one to three highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum (RER) form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristate presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The most remarkable feature of oogonia is that they have typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material, which nucleate microtubules, characteristic of functional centrosomes organizing the cytoskeleton. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced, while the paternal is dominant. Centrioles are most likely involved in mitosis of oogonia.

Effect of follicle-stimulating hormone on different cell sub-populations in the ovary of newly hatched chicks treated during embryonic development.

1. Cell sub-populations of the ovary of newly-hatched chicks were assessed following follicle stimulating hormone (FSH) treatment during embryonic development. Changes in cell number and the amount of oestradiol in serum were determined. 2. White Leghorn chick embryos received 1 microgram FSH applied to the chorioallantoic membrane at 13, 15, and 17 d of incubation. Within 24 h after hatching, animals were killed and blood was collected. The left ovary was immediately removed then weighed and processed by an enzymatic-mechanical dissociation method for total cell count. An air-drying method was also used for meiotic preparations to study the germinal cells. 3. The pre-follicular ovary is able to respond to FSH by inducing an increase both in the serum oestradiol concentration and in the number of steroidogenic cells and of poorly differentiated cells of the ovarian medulla. 4. FSH increases the number of oogonia, which are responsible for a sharp increase in the total population of germ cells in the FSH-treated ovary. 5. It is possible that FSH acts to increase the proliferation of oogonia and a delay in the meiotic prophase through a change in the microenvironment rather than by a direct effect on germ cells.

Karyological analysis of Cylindera trisignata (Latreille & Dejean, 1822) from Portugal (Coleoptera, Cicindelidae).

Cellular suspensions of germinal tissues of Cylindera trisignata provided the definition of its karyotype: 2n = 23 and 2n = 26 for the males and females respectively. This Palearctic species has a sex chromosome system of the X1X2X3X4Y/X1X1X2X2X3X3X4X4 type, only found until now in Cicindela maroccana pseudomaroccana. The heterosomes are not well-differentiated from the general morphological standpoint. To explain the origin of the 4X condition, a mechanism of dissociation of the X chromosomes rather than an incorporation of autosomal segments is proposed. However, based on the occurrence of distinct ploidy levels, both in male and female cells, with a relatively high incidence for the tetraploid condition with regular autosomal bivalents, the hypothesis of a possible evolutionary role of polyploidy is suggested.

The role of the rete ovarii in meiosis and follicle formation in the cat, mink and ferret.

Ovarian morphology was studied from the inception of meiosis in the cat, mink and ferret. It was shown that "open connections", allowing cellular contact, existed between the intra-ovarian rete cords and the groups of germ cells as well as between the surface epithelium and the germ cells. The germ cells in the innermost part of the cortex and lying in contact with the rete cells were those which were the first to enter meiotic prophase. Later, the more peripheral oogonia transformed to oocytes. The first follicular formations occurred at the innermost part of the cortex. The granulosa cell layers were in open connection with the intra-ovarian rete cords. In the mink and ferret, a certain part of the rete system at the hilus differentiated into the hilar rete body. In all animals, the extra-ovarian rete cells were actively secreting. It is proposed that the rete system interacts with the cortex, initiating the start of meiosis and that the rete cells as well as cells of the surface epithelium contribute to the granulosa cell layer.