Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence†.

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A two-dimensional attachment mouse secondary follicle culture system was used to assess the oocyte's capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral (PA) and early antral (EA) follicles was performed. Established endpoints were the oocyte's potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from PA and EA cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections with no significant differences in meiotic and developmental competence compared with those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as nonhuman chorionic gonadotropin triggered in vitro maturation.

The endocannabinoid system modulates the ovarian physiology and its activation can improve in vitro oocyte maturation.

The cannabinoid (CB) system has been involved in many aspects of reproduction and it is known that the systemic chronic use of exogenous CBs are deleterious to reproductive processes. Even so, it is not known what happens in relation to the physiology of the ovary when CB receptors are absent. The present study investigated the effect of the lack of CB1 and CB2 receptors in mice ovarian morphology, folliculogenesis, oocyte retrieval, and oocyte maturation and evaluated the use of Δ9-tetrahydrocannabinol (THC) on oocyte in vitro maturation (IVM) by comparing classical IVM and two-step IVM by analyzing the meiotic competence of the oocytes and their evolution toward embryos. Thus, when CB1 and CB2 receptors were missed, the ovary area and volume was significantly less and the action of the equine chorionic gonadotropin (eCG) hormone was diminished. In addition, the mutant genotypes had fewer ovarian follicles and they were less competent after eCG administration compared with wild-type mice, and this lack of CB receptors showed a mismatch of oocyte maturation. However, the in vitro use of THC showed improvements in oocytes IVM after a Pre-IVM step for 48 hr, as those oocytes reached a significantly higher polar body rate, a larger diameter and the best result on blastocysts rate was achieved when THC was used during the IVM step.

Evaluation of the association between exosomal levels and female reproductive system and fertility outcome during aging: a systematic review protocol.

BACKGROUND: Exosomes may have critical roles in the maternal-embryo cross-talk for the recognition and maintenance of pregnancy during maternal aging. Exosomes have the capability to carry developmental signaling molecules with the ability to modulate gene expressions and affect growth and regulation of embryo during pregnancy. Systematic review aims to evaluate age-related alterations in the exosomal content and functions that can influence the reproductive performance in human and animal models as conveyors of senescence signals. METHODS: A literature search of electronic databases including MEDLINE (PubMed), Embase, ProQuest, Scopus, Google Scholar, WHO, SID, MAGIRAN, and Barakat will be conducted. Following the online search, articles will be screened by two independent reviewers according to inclusion and exclusion criteria. Eligible studies will be critically appraised by reviewers at the study level for methodological quality using Joanna Briggs Institute's standardized critical appraisal tools. The extracted data from selected studies will cover the study populations, methods, current evidence about the physiological role of extracellular vesicles and exosomes in reproductive system, relevant outcomes, and possible conclusions about the effectiveness of exposure. DISCUSSION: Regarding the role of exosomes and their cargoes in the function of reproductive tract, the possible beneficial or adverse effects following exosomal administration from younger women to older women will be evaluated in the systematic review. As a result, exosome therapy could be suggested as a novel therapeutic agent if the favorable results are identified.

Physiological impact of extracellular vesicles on female reproductive system; highlights to possible restorative effects on female age-related fertility.

An alternative mechanism of cell-to-cell communication via extracellular vesicles (EVs) has recently raised increasing attention. EVs are spherical structures comprising exosomes and microvesicles, capable of transferring regulatory molecules and genetic information from one cell to another. EVs act as modulators which can alter a wide spectrum of functions at the cellular level in the recipient cells, taking part in a variety of biological processes in both physiological and pathological conditions. Alteration in EVs content, notably exosomes, was reported during cellular senescence and in patients with age-related diseases. Most studies reported regulating the impacts of exosomes on fertility and pregnancy outcomes via their capability in carrying developmental signaling molecules like proteins, RNA cargos, influencing gene expressions, affecting growth, and development of embryos during aging. Alterations in the exosomal content and functions can influence the reproductive performance in human and animals as conveyors of senescence signals from outside of the cells. This review aimed to summarize evidence on the role of EVs on modulating fertility, early embryonic development, maternal-embryo crosstalk for the recognition, and maintenance of pregnancy during maternal aging. Advanced clinical studies are required to strengthen the findings that the benefit of exosomes can be extended to subjects undergoing reproductive aging. © 2019 BioFactors, 45(3):293-303, 2019.

In vitro follicle culture in the context of IVF.

The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

Immature Oocytes from Unprimed Juvenile Mice Become a Valuable Source for Embryo Production When Using C-Type Natriuretic Peptide as Essential Component of Culture Medium.

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of prepubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25 nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in the presence of estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominantly dispersed to a condensed configuration. Following in vitro maturation, oocytes cultured under CNP were capable of extruding the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, with CNP as the pivotal factor, that can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.

The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

Removal of GPI-anchored membrane proteins causes clustering of lipid microdomains in the apical head area of porcine sperm.

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.

Bovine cumulus cells protect maturing oocytes from increased fatty acid levels by massive intracellular lipid storage.

Metabolic conditions characterized by elevated free fatty acid concentrations in blood and follicular fluid are often associated with impaired female fertility. Especially elevated saturated fatty acid levels can be lipotoxic for several somatic cell types. The aim of this study was to determine the impact of elevated free fatty acid concentrations in follicular fluid on neutral lipids (fatty acids stored in lipid droplets) inside cumulus cells and oocytes and their developmental competence. To this end, cows were exposed to a short-term fasting period during final oocyte maturation. This resulted in elevated, but distinct, free fatty acid concentrations in blood and follicular fluid and a rise in the concentrations of in particular fatty acids with a chain length of 14-18 carbon atoms. Interestingly, elevated free fatty acid concentrations in follicular fluid resulted in a massive increase in the level of neutral lipids in cumulus cells, whereas the level of neutral lipid in oocytes was hardly affected. Furthermore, competence of oocytes to develop to the blastocyst stage after fertilization and culture of cumulus-oocyte-complexes of the experimental and control group was not different. In conclusion these data suggest that short-term elevated free fatty acid concentrations in follicular fluid do not harm oocyte developmental competence. We propose that the involvement of high levels of mobilized oleic acid in follicular fluid in combination with the induced lipid storage in cumulus cells serves to prevent harmful saturated fatty acid exposure to the oocyte.

Oleic acid prevents detrimental effects of saturated fatty acids on bovine oocyte developmental competence.

Mobilization of fatty acids from adipose tissue during metabolic stress will increase the amount of free fatty acids in blood and follicular fluid and, thus, may affect oocyte quality. In this in vitro study, the three predominant fatty acids in follicular fluid (saturated palmitic and stearic acid and unsaturated oleic acid) were presented to maturing oocytes to test whether fatty acids can affect lipid storage of the oocyte and developmental competence postfertilization. Palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence. Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence. Remarkably, the adverse effects of palmitic and stearic acid could be counteracted by oleic acid. These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.

The endocannabinoid system and pivotal role of the CB2 receptor in mouse spermatogenesis.

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(2) receptors. In fact, we found that the selective CB(2) receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

ATRA and KL promote differentiation toward the meiotic program of male germ cells.

While it is known that retinoic acid (RA) induces meiosis in mouse female fetal gonads, the mechanisms which regulate this process during spermatogenesis are poorly understood. We show that the All trans RA derivative (ATRA) and Kit Ligand (KL) increase meiotic entry of postnatal mouse spermatogonia in vitro without synergism. Competence to enter meiosis is reached by spermatogonia only at the stage in which they undergo Kit-dependent divisions. Besides increasing Kit expression in spermatogonia, ATRA also upregulates KL expression in Sertoli cells. Both ATRA and KL increase the expression of Stimulated by Retinoic Acid Gene 8 and Dmc1, an early meiotic marker. A specific Kit tyrosine kinase inhibitor prevents the increase in the number of meiotic cells induced by both the two factors, suggesting that they converge on common Kit-dependent signalling pathways. Meiotic entry induced by ATRA and KL is independent from their ability to affect germ cell viability, and is mediated by the activation of PI3K and MAPK pathways through Kit autophosphorylation. ATRA-induced phosphorylation of the two downstream kinases is mediated by a non-genomic mechanism. These data suggest that RA may control the timing of meiosis by influencing both the somatic and the germ cell compartment of the postnatal testis through the activation of the KL/Kit system.

Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population.

Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.

Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand.

Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation.