Gene expression programs in mammalian spermatogenesis.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies.

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Unfolding the ventral nerve center of chaetognaths.

BACKGROUND: Chaetognaths are a clade of marine worm-like invertebrates with a heavily debated phylogenetic position. Their nervous system superficially resembles the protostome type, however, knowledge regarding the molecular processes involved in neurogenesis is lacking. To better understand these processes, we examined the expression profiles of marker genes involved in bilaterian neurogenesis during post-embryonic stages of Spadella cephaloptera. We also investigated whether the transcription factor encoding genes involved in neural patterning are regionally expressed in a staggered fashion along the mediolateral axis of the nerve cord as it has been previously demonstrated in selected vertebrate, insect, and annelid models. METHODS: The expression patterns of genes involved in neural differentiation (elav), neural patterning (foxA, nkx2.2, pax6, pax3/7, and msx), and neuronal function (ChAT and VAChT) were examined in S. cephaloptera hatchlings and early juveniles using whole-mount fluorescent in situ hybridization and confocal microscopy. RESULTS: The Sce-elav + profile of S. cephaloptera hatchlings reveals that, within 24 h of post-embryonic development, the developing neural territories are not limited to the regions previously ascribed to the cerebral ganglion, the ventral nerve center (VNC), and the sensory organs, but also extend to previously unreported CNS domains that likely contribute to the ventral cephalic ganglia. In general, the neural patterning genes are expressed in distinct neural subpopulations of the cerebral ganglion and the VNC in hatchlings, eventually becoming broadly expressed with reduced intensity throughout the CNS in early juveniles. Neural patterning gene expression domains are also present outside the CNS, including the digestive tract and sensory organs. ChAT and VAChT domains within the CNS are predominantly observed in specific subpopulations of the VNC territory adjacent to the ventral longitudinal muscles in hatchlings. CONCLUSIONS: The observed spatial expression domains of bilaterian neural marker gene homologs in S. cephaloptera suggest evolutionarily conserved roles in neurogenesis for these genes among bilaterians. Patterning genes expressed in distinct regions of the VNC do not show a staggered medial-to-lateral expression profile directly superimposable to other bilaterian models. Only when the VNC is conceptually laterally unfolded from the longitudinal muscle into a flat structure, an expression pattern bearing resemblance to the proposed conserved bilaterian mediolateral regionalization becomes noticeable. This finding supports the idea of an ancestral mediolateral patterning of the trunk nervous system in bilaterians.

From signalling to form: the coordination of neural tube patterning.

The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.

Neuromesodermal specification during head-to-tail body axis formation.

The anterior-to-posterior (head-to-tail) body axis is extraordinarily diverse among vertebrates but conserved within species. Body axis development requires a population of axial progenitors that resides at the posterior of the embryo to sustain elongation and is then eliminated once axis extension is complete. These progenitors occupy distinct domains in the posterior (tail-end) of the embryo and contribute to various lineages along the body axis. The subset of axial progenitors with neuromesodermal competency will generate both the neural tube (the precursor of the spinal cord), and the trunk and tail somites (producing the musculoskeleton) during embryo development. These axial progenitors are called Neuromesodermal Competent cells (NMCs) and Neuromesodermal Progenitors (NMPs). NMCs/NMPs have recently attracted interest beyond the field of developmental biology due to their clinical potential. In the mouse, the maintenance of neuromesodermal competency relies on a fine balance between a trio of known signals: Wnt/ß-catenin, FGF signalling activity and suppression of retinoic acid signalling. These signals regulate the relative expression levels of the mesodermal transcription factor Brachyury and the neural transcription factor Sox2, permitting the maintenance of progenitor identity when co-expressed, and either mesoderm or neural lineage commitment when the balance is tilted towards either Brachyury or Sox2, respectively. Despite important advances in understanding key genes and cellular behaviours involved in these fate decisions, how the balance between mesodermal and neural fates is achieved remains largely unknown. In this chapter, we provide an overview of signalling and gene regulatory networks in NMCs/NMPs. We discuss mutant phenotypes associated with axial defects, hinting at the potential significant role of lesser studied proteins in the maintenance and differentiation of the progenitors that fuel axial elongation.

Hox genes and patterning the vertebrate body.

The diversity of vertebrate body plans is dizzying, yet stunning for the many things they have in common. Vertebrates have inhabited virtually every part of the earth from its coldest to warmest climates. They locomote by swimming, flying, walking, slithering, or climbing, or combinations of these behaviors. And they exist in many different sizes, from the smallest of frogs, fish and lizards to giraffes, elephants, and blue whales. Despite these differences, vertebrates follow a remarkably similar blueprint for the establishment of their body plan. Within the relatively small amount of time required to complete gastrulation, the process through which the three germ layers, ectoderm, mesoderm, and endoderm are created, the embryo also generates its body axis and is simultaneously patterned. For the length of this axis, the genes that distinguish the neck from the rib cage or the trunk from the sacrum are the Hox genes. In vertebrates, there was evolutionary pressure to maintain this set of genes in the organism. Over the past decades, much has been learned regarding the regulatory mechanisms that ensure the appropriate expression of these genes along the main body axes. Genetic functions continue to be explored though much has been learned. Much less has been discerned on the identity of co-factors used by Hox proteins for the specificity of transcriptional regulation or what downstream targets and pathways are critical for patterning events, though there are notable exceptions. Current work in the field is demonstrating that Hox genes continue to function in many organs long after directing early patterning events. It is hopeful continued research will shed light on remaining questions regarding mechanisms used by this important and conserved set of transcriptional regulators.

Generation of patterns in the paraxial mesoderm.

The Segmentation Clock is a tissue-level patterning system that enables the segmentation of the vertebral column precursors into transient multicellular blocks called somites. This patterning system comprises a set of elements that are essential for correct segmentation. Under the so-called "Clock and Wavefront" model, the system consists of two elements, a genetic oscillator that manifests itself as traveling waves of gene expression, and a regressing wavefront that transforms the temporally periodic signal encoded in the oscillations into a permanent spatially periodic pattern of somite boundaries. Over the last twenty years, every new discovery about the Segmentation Clock has been tightly linked to the nomenclature of the "Clock and Wavefront" model. This constrained allocation of discoveries into these two elements has generated long-standing debates in the field as what defines molecularly the wavefront and how and where the interaction between the two elements establishes the future somite boundaries. In this review, we propose an expansion of the "Clock and Wavefront" model into three elements, "Clock", "Wavefront" and signaling gradients. We first provide a detailed description of the components and regulatory mechanisms of each element, and we then examine how the spatiotemporal integration of the three elements leads to the establishment of the presumptive somite boundaries. To be as exhaustive as possible, we focus on the Segmentation Clock in zebrafish. Furthermore, we show how this three-element expansion of the model provides a better understanding of the somite formation process and we emphasize where our current understanding of this patterning system remains obscure.

Shaping gene expression and its evolution by chromatin architecture and enhancer activity.

Transcriptional regulation plays a pivotal role in orchestrating the intricate genetic programs governing embryonic development. The expression of developmental genes relies on the combined activity of several cis-regulatory elements (CREs), such as enhancers and silencers, which can be located at long linear distances from the genes that they regulate and that interact with them through establishment of chromatin loops. Mutations affecting their activity or interaction with their target genes can lead to developmental disorders and are thought to have importantly contributed to the evolution of the animal body plan. The income of next-generation-sequencing approaches has allowed identifying over a million of sequences with putative regulatory potential in the human genome. Characterizing their function and establishing gene-CREs maps is essential to decode the logic governing developmental gene expression and is one of the major challenges of the post-genomic era. Chromatin 3D organization plays an essential role in determining how CREs specifically contact their target genes while avoiding deleterious off-target interactions. Our understanding of these aspects has greatly advanced with the income of chromatin conformation capture techniques and fluorescence microscopy approaches to visualize the organization of DNA elements in the nucleus. Here we will summarize relevant aspects of how the interplay between CRE activity and chromatin 3D organization regulates developmental gene expression and how it relates to pathological conditions and the evolution of animal body plan.

The control of transitions along the main body axis.

Although vertebrates display a large variety of forms and sizes, the mechanisms controlling the layout of the basic body plan are substantially conserved throughout the clade. Following gastrulation, head, trunk, and tail are sequentially generated through the continuous addition of tissue at the caudal embryonic end. Development of each of these major embryonic regions is regulated by a distinct genetic network. The transitions from head-to-trunk and from trunk-to-tail development thus involve major changes in regulatory mechanisms, requiring proper coordination to guarantee smooth progression of embryonic development. In this review, we will discuss the key cellular and embryological events associated with those transitions giving particular attention to their regulation, aiming to provide a cohesive outlook of this important component of vertebrate development.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.

Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization.

The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.

Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Maternal Diet High in Linoleic Acid Alters Renal Branching Morphogenesis and mTOR/AKT Signalling Genes in Rat Fetal Kidneys.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is obtained from the maternal diet during pregnancy, and is essential for normal fetal growth and development. A maternal high-LA (HLA) diet alters maternal and offspring fatty acids, maternal leptin and male/female ratio at embryonic (E) day 20 (E20). We investigated the effects of an HLA diet on embryonic offspring renal branching morphogenesis, leptin signalling, megalin signalling and angiogenesis gene expression. Female Wistar Kyoto rats were fed low-LA (LLA; 1.44% energy from LA) or high-LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring were sacrificed and mRNA from kidneys was analysed by real-time PCR. Maternal HLA decreased the targets involved in branching morphogenesis Ret and Gdnf in offspring, independent of sex. Furthermore, downstream targets of megalin, namely mTOR, Akt3 and Prkab2, were reduced in offspring from mothers consuming an HLA diet, independent of sex. There was a trend of an increase in the branching morphogenesis target Gfra1 in females (p = 0.0517). These findings suggest that an HLA diet during pregnancy may lead to altered renal function in offspring. Future research should investigate the effects an HLA diet has on offspring kidney function in adolescence and adulthood.

Fetal Hypoglycemia Induced by Placental SLC2A3-RNA Interference Alters Fetal Pancreas Development and Transcriptome at Mid-Gestation.

Glucose, the primary energy substrate for fetal oxidative processes and growth, is transferred from maternal to fetal circulation down a concentration gradient by placental facilitative glucose transporters. In sheep, SLC2A1 and SLC2A3 are the primary transporters available in the placental epithelium, with SLC2A3 located on the maternal-facing apical trophoblast membrane and SLC2A1 located on the fetal-facing basolateral trophoblast membrane. We have previously reported that impaired placental SLC2A3 glucose transport resulted in smaller, hypoglycemic fetuses with reduced umbilical artery insulin and glucagon concentrations, in addition to diminished pancreas weights. These findings led us to subject RNA derived from SLC2A3-RNAi (RNA interference) and NTS-RNAi (non-targeting sequence) fetal pancreases to qPCR followed by transcriptomic analysis. We identified a total of 771 differentially expressed genes (DEGs). Upregulated pathways were associated with fat digestion and absorption, particularly fatty acid transport, lipid metabolism, and cholesterol biosynthesis, suggesting a potential switch in energetic substrates due to hypoglycemia. Pathways related to molecular transport and cell signaling in addition to pathways influencing growth and metabolism of the developing pancreas were also impacted. A few genes directly related to gluconeogenesis were also differentially expressed. Our results suggest that fetal hypoglycemia during the first half of gestation impacts fetal pancreas development and function that is not limited to ß cell activity.

Somatostatin Receptor Gene Functions in Growth Regulation in Bivalve Scallop and Clam.

Bivalves hold an important role in marine aquaculture and the identification of growth-related genes in bivalves could contribute to a better understanding of the mechanism governing their growth, which may benefit high-yielding bivalve breeding. Somatostatin receptor (SSTR) is a conserved negative regulator of growth in vertebrates. Although SSTR genes have been identified in invertebrates, their involvement in growth regulation remains unclear. Here, we identified seven SSTRs (PySSTRs) in the Yesso scallop, Patinopecten yessoensis, which is an economically important bivalve cultured in East Asia. Among the three PySSTRs (PySSTR-1, -2, and -3) expressed in adult tissues, PySSTR-1 showed significantly lower expression in fast-growing scallops than in slow-growing scallops. Then, the function of this gene in growth regulation was evaluated in dwarf surf clams (Mulinia lateralis), a potential model bivalve cultured in the lab, via RNA interference (RNAi) through feeding the clams Escherichia coli containing plasmids expressing double-stranded RNAs (dsRNAs) targeting MlSSTR-1. Suppressing the expression of MlSSTR-1, the homolog of PySSTR-1 in M. lateralis, resulted in a significant increase in shell length, shell width, shell height, soft tissue weight, and muscle weight by 20%, 22%, 20%, 79%, and 92%, respectively. A transcriptome analysis indicated that the up-regulated genes after MlSSTR-1 expression inhibition were significantly enriched in the fat digestion and absorption pathway and the insulin pathway. In summary, we systemically identified the SSTR genes in P. yessoensis and revealed the growth-inhibitory role of SSTR-1 in bivalves. This study indicates the conserved function of somatostatin signaling in growth regulation, and ingesting dsRNA-expressing bacteria is a useful way to verify gene function in bivalves. SSTR-1 is a candidate target for gene editing in bivalves to promote growth and could be used in the breeding of fast-growing bivalves.

Histone Lactylation Is Involved in Mouse Oocyte Maturation and Embryo Development.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.