Mature Gametes Research Articles

Oxidative Stress in the Male Germline: A Review of Novel Strategies to Reduce 4-Hydroxynonenal Production.

Germline oxidative stress is intimately linked to several reproductive pathologies including a failure of sperm-egg recognition. The lipid aldehyde 4-hydroxynonenal (4HNE) is particularly damaging to the process of sperm-egg recognition as it compromises the function and the stability of several germline proteins. Considering mature spermatozoa do not have the capacity for de novo protein translation, 4HNE modification of proteins in the mature gametes has uniquely severe consequences for protein homeostasis, cell function and cell survival. In somatic cells, 4HNE overproduction has been attributed to the action of lipoxygenase enzymes that facilitate the oxygenation and degradation of ω-6 polyunsaturated fatty acids (PUFAs). Accordingly, the arachidonate 15-lipoxygenase (ALOX15) enzyme has been intrinsically linked with 4HNE production, and resultant pathophysiology in various complex conditions such as coronary artery disease and multiple sclerosis. While ALOX15 has not been well characterized in germ cells, we postulate that ALOX15 inhibition may pose a new strategy to prevent 4HNE-induced protein modifications in the male germline. In this light, this review focuses on (i) 4HNE-induced protein damage in the male germline and its implications for fertility; and (ii) new methods for the prevention of lipid peroxidation in germ cells.

Generation of human oogonia from induced pluripotent stem cells in vitro.

Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell-like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming-genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs-and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.

Fertility preservation options in transgender people: A review.

Gender affirming procedures adversely affect the reproductive potential of transgender people. Thus, fertility preservation options should be discussed with all transpeople before medical and surgical transition. In transwomen, semen cryopreservation is typically straightforward and widely available at fertility centers. The optimal number of vials frozen depends on their reproductive goals and treatment options, therefore a consultation with a fertility specialist is optimal. Experimental techniques including spermatogonium stem cells (SSC) and testicular tissue preservation are technologies currently under development in prepubertal individuals but are not yet clinically available. In transmen, embryo and/or oocyte cryopreservation is currently the best option for fertility preservation. Embryo cryopreservation requires fertilization of the transman's oocytes with a donor or partner's sperm prior to cryopreservation, but this limits his future options for fertilizing the eggs with another partner or donor. Oocyte cryopreservation offers transmen the opportunity to preserve their fertility without committing to a male partner or sperm donor at the time of cryopreservation. Both techniques however require at least a two-week treatment course, egg retrieval under sedation and considerable cost. Ovarian tissue cryopreservation is a promising experimental method that may be performed at the same time as gender affirming surgery but is offered in only a limited amount of centers worldwide. In select places, this method may be considered for prepubertal children, adolescents, and adults when ovarian stimulation is not possible. Novel methods such as in-vitro activation of primordial follicles, in vitro maturation of immature oocytes and artificial gametes are under development and may hold promise for the future.

Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from onenonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and hasimplications for deciphering male-related reproductive disorders.

Transchem project – Part I: Impact of long-term exposure to pendimethalin on the health status of rainbow trout (Oncorhynchus mykiss L.) genitors

Pendimethalin is a herbicide active substance commonly used in terrestrial agricultural systems and is thus detected at high concentrations in the surface water of several European countries. Previous studies reported several histopathological changes, enzymatic antioxidant modulation and immunity disturbance in fish exposed to this pesticide. The objective of this work was to investigate the direct effects of long-term exposure to environmental concentrations of pendimethalin over a period of 18 months in rainbow trout (Oncorhynchus mykiss) genitors. To do so, an experimental system consisting of eight similar 400 L tanks with a flow-through of fresh river water was used to perform daily chemical contamination. Fish were exposed to 850 ng/L for one hour and the pendimethalin concentration was then gradually diluted during the day to maintain optimal conditions for the fish throughout the experiment and to achieve a mean theoretical exposure level of around 100 ng L−1 per day. Every November, males and females were stripped to collect eggs and sperm and two new first generations of offspring were obtained. Kinetic sampling revealed differences in immune system parameters and antioxidative defences in the contaminated trout compared to the controls, due to pesticide exposure combined with seasonal changes related to gamete maturation. Moreover, reproductive capacity was significantly affected by exposure to the herbicide; a time lag of more than five weeks was observed for egg maturation in contaminated females and high bioconcentrations of pendimethalin were measured in eggs and sperm. Chemical transfer from genitors to offspring via gametes may affect embryo development and negatively impact the early stages of development.

Gametogenesis and reproductive pattern of the reef-building coral Acropora millepora in northwestern Philippines

ABSTRACTCoral populations continue to decline in many regions because of natural and anthropogenic disturbances; and the persistence of coral populations mainly relies on natural recruitment through coral reproduction. However, studies on the reproduction of many important reef-building coral species in one of the most biodiverse region, the Coral Triangle, are very limited. Here, we investigated the reproductive biology of the coral Acropora millepora in Bolinao-Anda Reef Complex, northwestern Philippines from February 2014 to March 2015, through in situ assessment of gamete maturity, histological examination of gamete development and ex situ spawning observations. Results showed that A. millepora exhibited an annual gametogenic cycle. Formation of oocytes took place within 8 months, whereas spermatocytes developed in 3 months before the gamete release. Abundance and size of eggs in A. millepora were greatest during the months nearest to the spawning period, which coincides with the rapid increase in sea surface temperature. Ex situ broadcast spawning occurred around the months of February–March, which suggests an extended spawning pattern. Results provide additional information on coral reproduction in the region and reveal the potential of A. millepora in providing coral propagules for the replenishment and recovery of degraded coral populations.

Ion Channel Function During Oocyte Maturation and Fertilization.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca2+), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

Coral reproduction at Hall Bank, a high latitude coral assemblage in Western Australia

Research on coral reproduction has increased dramatically in recent times; however, there remain significant regions, in particular high latitude reefs, where research is limited. For example, the reproductive biology of species in the coral assemblage at Hall Bank, a high latitude site (32 degrees S) in southern Western Australia, remain unknown. Here, reproductive traits and the likely time of spawning for 12 of the approximately 16 species that occur at Hall Bank were established using histology between March 2009 and March 2011 at 7 discrete time points. Peak reproductive activity most likely occurs in February, as 7 of the 10 species sampled in this month had colonies with mature gametes. The sexuality, mode of larval development and transmission of symbionts were, as expected, consistent with previous work. The reproductive biology of the corals at Hall Bank is consistent with other regions of the Indo-Pacific, supporting the hypo thesis that reproductive traits such as sexuality and mode of larval development are evolutionarily conserved and do not vary biogeographically.

CRYOPRESERVATION OF OOCYTE-CUMULUS COMPLEXES OF COWS WITH DIFFERENT BIOACTIVE SUBSTANCES

Implementation of biotechnological process in livestock should be considered not only in terms of selection process intensification (obtain of embryos in vitro, their transplantation) and to a greater extent as development of effective methods of freezing and long storage of mammalian cells, including ova and embryos. Application of biotechnology in livestock breeding increases rate of genetic progress, preservation of gene pool of breeds via banks of sperm, embryos and cryobank of oocytes, obtaining and regulation of progeny of the desired sex, providing genetic evaluation of gametes and embryos, and it will enable to use genetic potential of animals after culling by age, replicate and create new genotypes with desired properties repeatedly. Solution to this problem is to improve medium and conditions of gametes and embryos freezing. Although the overall development of cryopreservation method is through simplification of the equilibration and vitrification solutions which would be able to ensure the full development frozen-thawed gametes. Addition of biologically active substances to the solution for cryopreservation contributes to protection of gametes during freezing and thawing, and determination of the consistent patterns of these substances will improve procedures of frozen-thawed oocyte cultivation outside the body. So it is necessary to deepen the fundamental research on the mechanisms of formation of a mature ovum of cows obtained from frozen-thawed oocytes to obtain embryos. The aim of the research is to conduct comparative analysis of different biologically active substances in equilibration and vitrification solutions at cryopreservation of oocyte-cumulus complexes of cows. Material and methods of the research. The objects of experimental studies were oocyte-cumulus complexes of black-and-white cows. The oocytes with homogeneous fine-grained ooplasm, undamaged pellucid zone, thick or partially loosened cumulus were used for freezing. The gametes of cows were treated by equilibration solution before freezing and then were transferred into vitrification solution. All the equilibration (10% glycerol + 20% propanediol) and vitrification (25% glycerol + 25% propanediol) solutions for cryopreservation of cows’ oocyte-cumulus complexes were prepared in Dulbecco phosphatebuffered saline with addition of 20% fetal serum of cows, 1х10-4 M unithiol, 1х10-6 M acetylcholine and without addition of bioactive substances. The research on adding some biologically active substances (fetal serum of cows – version A, unithiol – version B, acetylcholine – version C, without addition of biologically active substances – version D and not frozen cells of the control group (K)) in equilibration and vitrification solutions at freezing the oocyte-cumulus complexes of cows was carried out. It was found by the results of experimental studies that introduction of the above-mentioned components into the equilibration solution for freezing cows’ oocyte-cumulus complexes increased their cryoresistance, as evidenced by increasing indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis after 27-hour cultivation by 5,4-23,0% and decreasing indicator of number of oocytes with chromosomal abnormalities by 2,9-15,3%. The introduction of biologically active substances into the vitrification solution and subsequent cultivation during 27 hours after freezing and thawing cows’ oocyte-cumulus complexes showed that the indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis increased by 3,9-16,4% and the indicator of number of oocytes with chromosomal abnormalities decreased by 2,9-8,4%. Comparative analysis of the results of in vitro fertilization of cows’ frozen-thawed ova which were frozen using fetal serum of cows (version A) and without it (version - B) has shown a positive effect of adding it to equilibration and vitrification medium at freezing gametes of cows; it led to increase of obtaining bovine embryos in vitro by 11,5%. Thus, the analysis of experimental results showed different efficiency of using fetal serum of cows, unithiol, acetylcholine in the equilibration solution for freezing cows’ oocyte-cumulus complexes. The advantage of using these biologically active substances in the vitrification solution for cryopreservation of cow’s gametes by the indicator of maturation of frozen-thawed oocyte-cumulus complexes outside the body to metaphase-2 of meiosis wasn’t established. Conclusions. Introduction of fetal serum into cryopreservation solution increases cryoresistance of cow’s oocytes to cooling leading to increasing the indicator of the matured frozen-thawed gametes outside the body to metaphase-2 of meiosis by 23,0% and the obtained bovine embryos in vitro by 11,5%.

Split spawning realigns coral reproduction with optimal environmental windows

Split spawning in coral populations occurs when gamete maturation and mass spawning are split over two consecutive months. While split spawning has been observed at many reefs, little is known about the frequency and significance of these events. Here we show that split spawning occurred frequently and predictably over a decade at Scott Reef. Split spawning overlays the biannual spawning pattern in the region and occurs when the full moon falls in the first week of the usual spawning month, or the last week of the previous month. Additionally, in split years most species have their main spawning event after a 13-month lunar cycle, in the month following the usual spawning month. Without split spawning, spawn dates would shift by ~10 days each year to occur outside of optimal environmental windows. Our results suggest that split spawning is driven by a disconnect between lunar and seasonal cues, and is analogous with a ‘leap year’ in coral reproduction, realigning spawning dates with favourable conditions for reproduction.

Acquisition of oocyte competence to develop as an embryo: integrated nuclear and cytoplasmic events.

Infertility affects ~7% of couples of reproductive age with little change in incidence in the last two decades. ART, as well as other interventions, have made major strides in correcting this condition. However, and in spite of advancements in the field, the age of the female partner remains a main factor for a successful outcome. A better understanding of the final stages of gamete maturation yielding an egg that can sustain embryo development and a pregnancy to term remains a major area for improvement in the field. This review will summarize the major cellular and molecular events unfolding at the oocyte-to-embryo transition. We will provide an update on the most important processes/pathways currently understood as the basis of developmental competence, including the molecular processes involved in mRNA storage, its recruitment to the translational machinery, and its degradation. We will discuss the hypothesis that the translational programme of maternal mRNAs plays a key role in establishing developmental competence. These regulations are essential to assemble the machinery that is used to establish a totipotent zygote. This hypothesis further supports the view that embryogenesis begins during oogenesis. A better understanding of the events required for developmental competence will guide the development of novel strategies to monitor and improve the success rate of IVF. Using this information, it will be possible to develop new biomarkers that may be used to better predict oocyte quality and in selection of the best egg for IVF.

Stage-specific approaches promote in vitro induction for spermatogenesis.

Spermatogenesis in vitro has been demonstrated using spermatogonial stem cells (SSCs) in monolayer culture or testis tissue fragments in agarose-constructed three-dimensional (3-D) conditions. However, the low efficiency of gamete maturation and the lack of a novel induction platform have limited the progress of its use in further research and clinical applications. Here, we provide modified stage-specific induction approaches for spermatogenesis in in vitro culture with cells possessing a totipotent status. With this stage-specific propagation in a monolayer condition and a changing cytokine combination, we obtained spermatogenic cells in the forward to late meiosis stages with haploid features. Based on this technical platform, we refined a novel serum-free culture system with various cytokines in 3-D Matrigel for spermatogenesis that promote totipotent embryonic stem cells to meiosis stage with distinct SCP3 expression. And we also explored the effects of coculture with fibroblasts, the mutual interactions in the induction conditions promote the mouse embryonic fibroblasts underwent stromal cells differentiation. In further overexpression of spermatogenic gene Dazl in mouse embryonic fibroblasts, we found early stage initiation for spermatogenesis, and that will enhanced if cocultured with embryonic stem cells in the induction condition. Our results provide alternative approaches for effective spermatogenesis and support the development of promising avenues for infertility therapies.

Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish.

N6-methyladenosine (m6A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic messenger RNA (mRNA). Previous studies have implicated m6A modification in multiple biological processes, but the in vivo function of m6A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Zmettl3m/m , in zebrafish. We find that the oocytes in Zmettl3m/m adult females are stalled in early development and the ratio of full-grown stage (FG) follicles is significantly lower than that of wild type. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo In Zmettl3m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Zmettl3m/m , and defective gamete maturation is accompanied by decreased overall m6A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Zmettl3m/m Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m6A modifications are essential for optimal reproduction in vertebrates.

The Germline-Specific Factor OEF-1 Facilitates Coordinated Progression Through Germ Cell Development in Caenorhabditis elegans.

The purpose of germ cells is to ensure the faithful transmission of genetic material to the next generation. To develop into mature gametes, germ cells must pass through cell cycle checkpoints while maintaining totipotency and genomic integrity. How germ cells coordinate developmental events while simultaneously protecting their unique fate is not well understood. Here, we characterize a novel nuclear protein, Oocyte-Excluded Factor-1 (OEF-1), with highly specific germline expression in Caenorhabditis elegans OEF-1 is initially detected early in embryogenesis and is expressed in the nuclei of all germ cells during larval stages. In adults, OEF-1 expression abruptly decreases just prior to oocyte differentiation. In oef-1 mutants, the developmental progression of germ cells is accelerated, resulting in subtle defects at multiple stages of germ cell development. Lastly, OEF-1 is primarily associated with the bodies of germline-expressed genes, and as such is excluded from the X chromosome. We hypothesize that OEF-1 may regulate the rate of progression through germ cell development, providing insight into how these critical maturation events are coordinated.

A gonad-expressed opsin mediates light-induced spawning in the jellyfish Clytia.

Across the animal kingdom, environmental light cues are widely involved in regulating gamete release, but the molecular and cellular bases of the photoresponsive mechanisms are poorly understood. In hydrozoan jellyfish, spawning is triggered by dark-light or light-dark transitions acting on the gonad, and is mediated by oocyte maturation-inducing neuropeptide hormones (MIHs) released from the ectoderm. We determined in Clytia hemisphaerica that blue-cyan light triggers spawning in isolated gonads. A candidate opsin (Opsin9) was found co-expressed with MIH within specialised ectodermal cells. Opsin9 knockout jellyfish generated by CRISPR/Cas9 failed to undergo oocyte maturation and spawning, a phenotype reversible by synthetic MIH. Gamete maturation and release in Clytia is thus regulated by gonadal photosensory-neurosecretory cells that secrete MIH in response to light via Opsin9. Similar cells in ancestral eumetazoans may have allowed tissue-level photo-regulation of diverse behaviours, a feature elaborated in cnidarians in parallel with expansion of the opsin gene family.

The early development of germ cells in chicken.

Primordial germ cells (PGCs) are the founder cells for mature gametes, the vehicles by which individuals transmit genetic and epigenetic information to later generations. Since the 19th century, avian species (chickens in particular) have been widely used for germ cell research. Previous studies have used chicken PGCs for a variety of research applications, including as a model for studies focusing on germline development. Other applications of chicken PGCs, including conservation efforts for avian species and methods of producing transgenic birds, have further reinforced the importance of these cells. However, much remains to be revealed about the origin and role of PGCs during their development in the chicken. Here, we provide a comprehensive review of chicken PGCs, focusing in particular upon their initial profiles and physiological changes during development as regulated by environmental factors and/or intrinsic mechanisms. We also emphasise sex-dependent differences in PGC development after settlement within the gonads, as well as future applications for avian PGCs.

Metabolomic profiling of bovine oviductal fluid across the oestrous cycle using proton nuclear magnetic resonance spectroscopy.

In the present study we tested whether regulation of the metabolome in bovine oviductal fluid depended on the stage of the oestrous cycle, the side relative to ovulation and local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected in the preovulatory, postovulatory, mid- and late luteal phases, from cyclic cows at a local abattoir (18-27 cows per stage and side). The metabolomes were assessed by proton nuclear magnetic resonance spectroscopy (H-NMR). In all, 39 metabolites were identified, among which the amino acid glycine and the energy substrates lactate and myoinositol were the most abundant at all stages. The concentrations of 14 metabolites varied according to the stage of the oestrous cycle in at least one side relative to ovulation, of which four (choline, glucose-1-phosphate, glycine and pyruvate) were correlated with intraoviductal progesterone or oestradiol concentrations. Glucose-1-phosphate was most affected by the stage of the cycle, with four- to sixfold higher levels in luteal than periovulatory stages. These results provide new knowledge on the regulation of secretory activity in the oviduct and may help optimise culture media for gamete maturation, IVF and embryo production.

182 In Vitro Derivation of Male Germ Cells from Bovine Bone Marrow-Derived Mesenchymal Stem Cells

During the last few years, the in vitro derivation of germ cell lineages from stem cells has emerged as an exciting new strategy for obtaining mature gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Germ cell differentiation and gametogenesis is a complex process and potential of different stem cell donors (i.e. SSC, ESC, iPSC) for in vitro male germ cell derivation has been inconsistent. Mesenchymal stem cells (MSC) may be suitable candidates for in vitro gamete derivation considering their (1) plasticity that is not limited to mesodermal derivatives, (2) availability of abundant tissues sources for isolation, (3) high proliferative potential, (4) simple and inexpensive isolation, and (5) high potential for cell therapy, including autologous or allogenic transplantation. The present study aimed to induce differentiation of MSC isolated from bone marrow derived from bovine male fetuses (bfMSC) into the germ cell lineage using an in vitro approach based on the exogenous effect of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4). Differentiation media consisted in control media (DMEM with high glucose plus 10% fetal bovine serum, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1amphotericin B) supplemented with RA (0.01, 0.1, or 1 µM) or BMP4 (10, 50, or 100 ng mL−1). Cell samples were obtained from differentiating and control bfMSC cultures and analysed for expression of housekeeping genes β-ACTIN and GAPDH, pluripotent genes OCT4 and NANOG, germ cell genes FRAGILLIS, STELLA, and VASA, male germ cell genes DAZL, PIWIl2, and STRA8, and meiotic biomarker SCP3 by quantitative-PCR (Q-PCR). OCT4, NANOG, and DAZL were immunodetected in undifferentiated and differentiated bfMSC using flow-cytometry analysis. The mRNA expression of DAZL was activated by RA or BMP4 supplementation, although no differences (P > 0.05) were detected among different concentrations. DAZL and NANOG mRNA levels increased (P < 0.05) from Day 7 to Day 21 during supplementation of RA (0.1 μM). In comparison, DAZL mRNA levels increased (P < 0.05) at Day 14 during supplementation of BMP4 (100 ng). OCT4 and SCP3 mRNA levels were not affected by RA or BMP4 treatments. Transcripts of FRAGILLIS, STELLA, VASA, PIWIl2, and STRA8 were not detected in control or differentiated bfMSC. Higher (P < 0.05) percentages of undifferentiated bfMSC were positive for NANOG (80.6%) and OCT4 (83.4%). DAZL- and NANOG-positive cells were 2.1% and 2.9%, and 95.9% and 97.8% at Days 0 and 21 of RA treatment, respectively. Data indicated that expression of germ cell biomarker DAZL in bfMSC is activated and increased after in vitro supplementation of RA and BMP4. Moreover, NANOG mRNA levels were regulated by RA treatment. Similar levels of SCP3 mRNA expression suggest that differentiated bfMSC were not induced into meiosis. Thus, exposure of bfMSC to RA or BMP4 under in vitro conditions might induce an early stage of premeiotic germinal differentiation.

Changes in mercury concentration in muscles, ovaries and eggs of European eel during maturation under controlled conditions

The degradation of the environment, namely due to pollution causes increasing impact on the organisms living on it, especially those at the top of the trophic chain. The accumulation of heavy metals such as mercury in live organisms may have a negative effect on the efficiency of reproduction. The aim of this study was to determine the mercury (Hg) content in muscles, ovaries and eggs during final maturation of gametes and after ovulation of European eel females. To this end, 65 (53 as treated and 12 as control) female European eels collected in Poland were analysed for mercury content. Eels were caught during commercial fishing activities in fresh water in the Warmia and Mazury region. Females were stimulated with carp pituitary homogenate (CPH) at 18mgkg−1, at 5-day intervals, for 20 weeks. The hormones were not given to the fish in the control group. The mercury content in tissues was determined by atomic absorption using a Milestone DMA-80 and analysis showed statistically significant differences (P < 0.05) between the content of mercury in muscle, ovary and eggs. The total mercury concentration in females following spawning increased in muscles and ovaries. Female eel eggs (0.0031 ± 0.0003mgkg−1 w/w) were found to contain eight times less mercury than their ovaries (0.026 ± 0.002mgkg−1 w/w) and 78 times less than their muscles (0.24 ± 0.03mgkg−1 w/w). The concentration of mercury in both muscles and ovaries in fish increases after spawning, which can additionally confirm that mercury is transmitted in small amounts from the mother's body to eggs.

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.