ResearchPad - sperm Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Deletion of inositol polyphosphate 4-phosphatase type-II B affects spermatogenesis in mice]]> Inositol polyphosphate-4-phosphatase type II (INPP4B) is a dual-specificity phosphatase that acts as a tumor suppressor in multiple cancers. INPP4B dephosphorylates phospholipids at the 4th position of the inositol ring and inhibits AKT and PKC signaling by hydrolyzing of PI(3,4)P2 and PI(4,5)P2, respectively. INPP4B protein phosphatase targets include phospho-tyrosines on Akt and phospho-serine and phospho-threonine on PTEN. INPP4B is highly expressed in testes, suggesting its role in testes development and physiology. The objective of this study was to determine whether Inpp4b deletion impacts testicular function in mice. In testis, Inpp4b expression was the highest in postmeiotic germ cells in both mice and men. The testes of Inpp4b knockout male mice were significantly smaller compared to the testes of wild-type (WT) males. Inpp4b-/- males produced fewer mature sperm cells compared to WT, and this difference increased with age and high fat diet (HFD). Reduction in early steroidogenic enzymes and luteinizing hormone (LH) receptor gene expression was detected, although androgen receptor (AR) protein level was similar in WT and Inpp4b-/- testes. Germ cell apoptosis was significantly increased in the knockout mice, while expression of meiotic marker γH2A.X was decreased. Our data demonstrate that INPP4B plays a role in maintenance of male germ cell differentiation and protects testis functions against deleterious effects of aging and high fat diet.

<![CDATA[Drosophila melanogaster tPlus3a and tPlus3b ensure full male fertility by regulating transcription of Y-chromosomal, seminal fluid, and heat shock genes]]>

Spermatogenesis in Drosophila melanogaster is characterized by a specific transcriptional program during the spermatocyte stage. Transcription of thousands of genes is regulated by the interaction of several proteins or complexes, including a tTAF-containing TFIID variant, tMAC, Mediator, and chromatin interactors, e.g., bromodomain proteins. We addressed how distinct subsets of target genes are selected. We characterized the highly similar proteins tPlus3a and tPlus3b, which contain a Plus3 domain and are enriched in the testis, mainly in spermatocytes. In tPlus3a and tplus3b deletion mutants generated using the CRISPR/Cas9 system, fertility was severely reduced and sperm showed defects during individualization. tPlus3a and tPlus3b heterodimerized with the bromodomain protein tBRD-1. To elucidate the role of the tPlus3a and tPlus3b proteins in transcriptional regulation, we determined the transcriptomes of tplus3a-tplus3b and tbrd-1 deletion mutants using next-generation sequencing (RNA-seq) and compared them to that of the wild-type. tPlus3a and tPlus3b positively or negatively regulated the expression of nearly 400 genes; tBRD-1 regulated 1,500 genes. Nearly 200 genes were regulated by both tPlus3a and tPlus3b and tBRD-1. tPlus3a and tPlus3b activated the Y-chromosomal genes kl-3 and kl-5, which indicates that tPlus3a and tPlus3b proteins are required for the function of distinct classes of genes. tPlus3a and tPlus3b and tBRD-1 repress genes relevant for seminal fluid and heat shock. We hypothesize that tPlus3a and tPlus3b proteins are required to specify the general transcriptional program in spermatocytes.

<![CDATA[Effects of highly active antiretroviral therapy on semen parameters of a cohort of 770 HIV-1 infected men]]>


HIV-1 infected patients show impaired semen parameters. Currently, it is not clear whether HIV-1 infection itself or antiretroviral therapy have an effect on semen parameters. We aim evaluate semen quality in a large cohort of fertile HIV-1 infected men under stable highly active antiretroviral therapy (HAART) and to assess the effect of HAART type and duration on semen parameters.

Materials and methods

Between January 2010 and June 2014, we enrolled in a retrospective case-control study 770 HIV-1 patients under stable HAART asking a reproductive counselling with their HIV negative partner. Co-infections with HBV or HCV, genital tract infections and known causes of infertility represented exclusion criteria. Semen samples were analysed and compared with the WHO reference values. A multivariate analysis including HAART type and duration, age, viral load and CD4 count, was performed on 600 patients out of 770.


The median values of all semen parameters were significantly lower among HIV-1 infected patients compared to the WHO reference group, with a significant proportion of patients having values below the 5th percentile of the WHO reference value. In a multivariate analysis, only age and viral load negatively impacted progressive motility (β -0.3 (95% CI: -0.5; -0.0) %, p<0.05) and semen morphology (β -0.00 (95% CI: -0.00; -0.00) %, p≤0.01), while no associations were detected as regards HAART type and duration.


HIV-1 infected patients showed a significant impairment of semen parameters compared to the reference values. HAART type and duration showed no associations with semen quality. Further research is needed to investigate implications for clinical care of HIV infected men desiring a child.

<![CDATA[Dual role of DMXL2 in olfactory information transmission and the first wave of spermatogenesis]]>

Gonad differentiation is a crucial step conditioning the future fertility of individuals and most of the master genes involved in this process have been investigated in detail. However, transcriptomic analyses of developing gonads from different animal models have revealed that hundreds of genes present sexually dimorphic expression patterns. DMXL2 was one of these genes and its function in mammalian gonads was unknown. We therefore investigated the phenotypes of total and gonad-specific Dmxl2 knockout mouse lines. The total loss-of-function of Dmxl2 was lethal in neonates, with death occurring within 12 hours of birth. Dmxl2-knockout neonates were weak and did not feed. They also presented defects of olfactory information transmission and severe hypoglycemia, suggesting that their premature death might be due to global neuronal and/or metabolic deficiencies. Dmxl2 expression in the gonads increased after birth, during follicle formation in females and spermatogenesis in males. DMXL2 was detected in both the supporting and germinal cells of both sexes. As Dmxl2 loss-of-function was lethal, only limited investigations of the gonads of Dmxl2 KO pups were possible. They revealed no major defects at birth. The gonadal function of Dmxl2 was then assessed by conditional deletions of the gene in gonadal supporting cells, germinal cells, or both. Conditional Dmxl2 ablation in the gonads did not impair fertility in males or females. By contrast, male mice with Dmxl2 deletions, either throughout the testes or exclusively in germ cells, presented a subtle testicular phenotype during the first wave of spermatogenesis that was clearly detectable at puberty. Indeed, Dmxl2 loss-of-function throughout the testes or in germ cells only, led to sperm counts more than 60% lower than normal and defective seminiferous tubule architecture. Transcriptomic and immunohistochemichal analyses on these abnormal testes revealed a deregulation of Sertoli cell phagocytic activity related to germ cell apoptosis augmentation. In conclusion, we show that Dmxl2 exerts its principal function in the testes at the onset of puberty, although its absence does not compromise male fertility in mice.

<![CDATA[Epigenome-wide analysis of sperm cells identifies IL22 as a possible germ line risk locus for psoriatic arthritis]]>

Psoriasis and its associated inflammatory arthritis, psoriatic arthritis (PsA), have a clear heritable component, but a large proportion of the heritable risk remains unexplained by gene sequence variation. This study aimed to determine if epigenetic factors contribute to the missing heritability in psoriatic disease. DNA methylation profiling was performed on sperm cells from 23 probands with psoriasis without PsA (PsC), 13 PsA probands, and 18 unaffected controls. Differentially methylated CpGs and regions (DMRs) were identified and validated by pyrosequencing. Underlying AluY and copy number variation (CNV) in the HCG26 and IL22 genes, respectively, were assessed by genotyping. Array, subject’s age, age of psoriasis onset, psoriasis severity, and medication usage were found to influence methylation at many genes and were included as covariates in the analysis. Between PsC probands vs. controls, 169 DMRs were found; 754 DMRs were found between PsA probands vs. controls, and 86 between PsA and PsC probands (adjusted p<0.05). Differences in methylation across DMRs were generally subtle (<10%) but correlated well with pyrosequencing. Biological inference prioritized notable DMRs associated with skin disease (SIGLEC14, JAM3, PCOLCE, RXRB), skin and/or joint disease (MBP, OSBPL5, SNORD115, HCG26), and joint disease (IL22, ELF5, PPP2R2D, PTPRN2, HCG26). Hypermethylation of the DMR within the first exon of arthritis-associated IL22 showed significant correlation (rho = 0.34, 95% CI 0.06–0.57, p = 0.01) between paired sperm and blood samples, independent of a CNV within the same region. Further studies are needed to rule out underlying genetic causes and determine if these represent heritable, constitutional epimutations, or are the result of exposure of germ cells to endogenous or exogenous environmental factors.

<![CDATA[Dual functions for the ssDNA-binding protein RPA in meiotic recombination]]>

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.

<![CDATA[Epigenetic inheritance of telomere length in wild birds]]>

Telomere length (TL) predicts health and survival across taxa. Variation in TL between individuals is thought to be largely of genetic origin, but telomere inheritance is unusual, because zygotes already express a TL phenotype, the TL of the parental gametes. Offspring TL changes with paternal age in many species including humans, presumably through age-related TL changes in sperm, suggesting an epigenetic inheritance mechanism. However, present evidence is based on cross-sectional analyses, and age at reproduction is confounded with between-father variation in TL. Furthermore, the quantitative importance of epigenetic TL inheritance is unknown. Using longitudinal data of free-living jackdaws Corvus monedula, we show that erythrocyte TL of subsequent offspring decreases with parental age within individual fathers, but not mothers. By cross-fostering eggs, we confirmed the paternal age effect to be independent of paternal age dependent care. Epigenetic inheritance accounted for a minimum of 34% of the variance in offspring TL that was explained by paternal TL. This is a minimum estimate, because it ignores the epigenetic component in paternal TL variation and sperm TL heterogeneity within ejaculates. Our results indicate an important epigenetic component in the heritability of TL with potential consequences for offspring fitness prospects.

<![CDATA[Metabolomic profiling reveals correlations between spermiogram parameters and the metabolites present in human spermatozoa and seminal plasma]]>

In 50% of all infertility cases, the male is subfertile or infertile, however, the underlying mechanisms are often unknown. Even when assisted reproductive procedures such as in vitro fertilization and intracytoplasmic sperm injection are performed, the causes of male factor infertility frequently remain elusive. Since the overall activity of cells is closely linked to their metabolic capacity, we analyzed a panel of 180 metabolites in human sperm and seminal plasma and elucidated their associations with spermiogram parameters. Therefore, metabolites from a group of 20 healthy donors were investigated using a targeted LC-MS/MS approach. The correlation analyses of the amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and sugars from sperm and seminal plasma with standard spermiogram parameters revealed that metabolites in sperm are closely related to sperm motility, whereas those in seminal plasma are closely related to sperm concentration and morphology. This study provides essential insights into the metabolome of human sperm and seminal plasma and its associations with sperm functions. This metabolomics technique could be a promising screening tool to detect the factors of male infertility in cases where the cause of infertility is unclear.

<![CDATA[Adverse effects of paternal obesity on the motile spermatozoa quality]]>

Growing evidence suggests that paternal obesity may decrease male fertility potential. During infertility treatment with intra-cytoplasmic sperm injection (ICSI), a morphologically normal motile spermatozoon is injected into a mature egg, when possible. However, sperm motility and morphology per se do not reflect the sperm molecular composition. In this study, we aimed to assess the quality of motile spermatozoa in the context of obesity by analysing their conventional and molecular characteristics as well as their ability to promote early embryonic development. A prospective study was conducted on 128 infertile men divided into three groups: 40 lean, 42 overweight, and 46 obese men. Conventional sperm parameters (concentration, motility and morphology) and sperm molecular status (chromatin composition and integrity, 5-methycytosine (5-mC) and 5-hydroxycytosine (5-hmC) contents and oxidative stress level) were analysed on raw semen and/or on motile spermatozoa selected by density gradient or swim-up techniques. Morphokinetic analysis of the embryos derived from ICSI was performed using the Embryoviewer software. Our results showed that the motile sperm-enriched fraction from obese men exhibited higher levels of retained histones (p<0.001), elevated percentage of altered chromatin integrity (p<0.001), and decreased contents of 5-hmC (p<0.001), and 5-mC (p<0.05) levels as compared to that from lean men. Importantly, there were no statistically significant correlations between these molecular parameters and the percentages of morphologically normal motile spermatozoa. Regarding embryo morphokinetics, the CC1 (p<0.05) and CC3 (p<0.05) embryonic cell cycles were significantly delayed in the cleavage embryos of the obese group as compared to the embryos of the lean group. Our data is of particular interest because, besides demonstrating the negative impacts of obesity on motile spermatozoa molecular composition, it also highlights the possible risk of disturbing early embryonic cell cycles kinetics in the context of paternal obesity.

<![CDATA[OpenCASA: A new open-source and scalable tool for sperm quality analysis]]>

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson’s correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

<![CDATA[Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions]]>

MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3′-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.

<![CDATA[Beliefs, attitudes and funding of assisted reproductive technology: Public perception of over 6,000 respondents from 6 European countries]]>


Fertility rates in Europe are among the lowest in the world, which may be attributed to both biological and lifestyle factors. Cost and reimbursement of fertility treatments vary across Europe, although its citizens enjoy wide access to fertility care. Since few regional studies evaluating public support for fertility treatment exist, we conducted the Listening IVF and Fertility in Europe (LIFE) survey to ascertain public perception of in vitro fertilization (IVF) and gamete donation as a treatment for infertility among European men and women.

Methods and findings

This survey was distributed via an online questionnaire to 8,682 individuals who were voluntary participants in an online research panel residing in France, Germany, Italy, Spain, Sweden, or the UK. The survey covered items to determine respondents’ beliefs regarding IVF and its success, the need for public funding, the use of IVF among modern families with different lifestyles, and the support for gamete donation. Results were analyzed by age, country of origin, sex, and sexual orientation. A total of 6,110 (70% of total) men and women responded. Among all respondents, 10% had undergone IVF treatment and 48% had considered or would consider IVF in case of infertility. Respondents estimated IVF mean success rate to be 47% and over half of respondents believed that availability of IVF would encourage people to delay conception. Although 93% of respondents believed that IVF treatment should be publicly funded to some extent, a majority believed that secondary infertility or use of fertility treatments allowing to delay parenthood should be financed privately. Survey respondents believed that the mean number of stimulated IVF cycles funded publicly should be limited 2 to 3 (average 2.4). 79% of respondents were willing to pay for IVF if needed with a mean amount of 5,400 € for a child brought to life through IVF. According to respondents, mean minimum and maximum ages for IVF should be 29 and 42 years old, respectively. The current survey showed support for egg and sperm donation (78%), for IVF in single women (61%) and for same-sex female couples (64%). When analyzing the results per group (i.e., sex, age, sexual orientation, and countries), youngest age groups, homosexuals, bisexuals, German respondents, and men had similar overall positive attitudes and beliefs toward IVF and opinions on public funding. Perceived limits to availability were stronger in women.


Overall, the survey results demonstrate a positive attitude among respondents in an online panel toward IVF, gamete donation, and support for public funding for fertility treatment. These findings could potentially drive discussions between patients and prescribers to explore IVF treatment and among legislators and payers to support public funding for these procedures.

<![CDATA[Transition from a meiotic to a somatic-like DNA damage response during the pachytene stage in mouse meiosis]]>

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early “meiotic” repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a “somatic-like” repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.

<![CDATA[Genetic and genomic analyses of testicular hypoplasia in Nellore cattle]]>

Reproductive performance is a key indicator of the long-term sustainability of any livestock production system. Testicular hypoplasia (TH) is a morphological and functional reproductive disorder that affects bulls around the world and consequently causes major economic losses due to reduced fertility rates. Despite the improvements in management practices to enhance performance of affected animals, the use of hypoplastic animals for reproduction might contribute to expand the prevalence of this disorder. The aim of this study was to identify genomic regions that are associated with TH in Nellore cattle by performing a genome-wide association study (GWAS) and functional analyses. Phenotypic and pedigree data from 47,563 animals and genotypes (500,689 Single Nucleotide Polymorphism, SNPs) from 265 sires were used in this study. TH was evaluated as a binary trait measured at 18 months of age. The estimated breeding values (EBVs) were calculated by fitting a single-trait threshold animal model using a Bayesian approach. The SNP effects were estimated using the Bayes C method and de-regressed EBVs for TH as the response variable (pseudo-phenotype). The top-15 ranking windows (5-adjacent SNPs) that explained the highest proportion of variance were identified for further functional and biological network analyses. The posterior mean (95% highest posterior density) of the heritability for TH was 0.16 (0.08; 0.23). The most important genomic windows were located on BTA1, BTA3, BTA4, BTA5, BTA9, BTA22, BTA23, and BTA25. These windows explained together 22.69% of the total additive genetic variance for TH. Strong candidate genes associated with metabolism and synthesis of steroids, cell survival, spermatogenesis process and sperm motility were identified, which might play an important role in the expression of TH. Our findings contribute to a better biological understanding of TH and future characterization of causal variants might enable improved genomic prediction of this trait in beef cattle.

<![CDATA[The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish]]>

The mechanisms that ensure fertilization of egg by a sperm are not fully understood. In all teleosts, a channel called the ‘micropyle’ is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the vitelline envelope by a single specialized follicle cell, the micropylar cell. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish (Danio rerio). Genome editing mutants affecting taz can grow to adults. However, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. We find that taz mutant eggs have no micropyle. Taz protein is specifically enriched in mid-oogenesis in the micropylar cell located at the animal pole of wild type oocyte, where it might regulate the cytoskeleton. Taz protein and micropylar cells are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

<![CDATA[<i>PLoS Genetics</i> Issue Image | Vol. 15(1) January 2019]]>

DNA repair during meiosis and chromosomal bridges.

Meiotic cells respond to DNA damage triggering diverse repair mechanisms in a cell cycle-dependent manner. Sequential activation of these mechanisms contribute to accurately maintain genome integrity. However, when spermatocytes are exposed to exogenous DNA damaging agents, like gamma radiation, repair homeostasis may be stressed and chromosomes sometimes engage in aberrant connections with non-homologous chromosomes. Super-resolution fluorescence image (STED) of two meiotic bivalents labelled with an antibody against the SYCP3 protein of the synaptonemal complex. Parallel lines represent the trajectory of homologous chromosomes within each bivalent. A protein filament bridges from one bivalent to the other, connecting two non-homologous chromosomes. See Enguita-Marruedo et al.

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Image Credit: Marta Martín-Ruiz

<![CDATA[Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time]]>

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.

<![CDATA[Revisiting aneuploidy profile of surgically retrieved spermatozoa by whole exome sequencing molecular karyotype]]>

Previous studies, including our own, have reported that spermatozoa isolated from the testis have remarkably higher occurrence of aneuploidy once isolated from azoospermic men. This notion, however, did not translate into a lower pregnancy rate nor a greater proportion of miscarriages. Indeed, ICSI offspring generated from surgically retrieved gametes did not suffer from increased karyotypic aneuploidy than children generated from ejaculated specimens. In recent years, aneuploidy assessments on a larger number of cells and utilizing more chromosome probes have reported a progressive decrease in chromosomal aberrations in spermatozoa directly retrieved from the seminiferous tubules. In light of the availability of more accurate molecular genetic techniques, we have decided to challenge the notion that sampling epididymal and testicular tissues yields spermatozoa with higher incidence of aneuploidy than those retrieved in the ejaculate. In a retrospective manner, we have carried out an analysis by FISH with 9 chromosome probes on at least 1000 cells from the ejaculates of 87 consenting men and the specimens of 6 azoospermic men, while spermatozoa of fertile donors were used as control. Aneuploidy by FISH yielded 0.9% for the donor control but rose in the study group to 3.6% in the ejaculated, 1.2% for the epididymal, and 1.1% for testicular spermatozoa. There were no differences in autosomal or gonosomal disomies, nor nullisomies. In this group, once the specimens of these men were used for ICSI, ejaculated spermatozoa yielded a 22% clinical pregnancy rate that resulted in 62.5% pregnancy loss. The surgically retrieved specimens yielded a 50% clinical pregnancy rate that progressed to term. To confirm our findings, in a prospective analysis, DNA sequencing was carried out on the ejaculates and surgical samples of 22 men with various spermatogenic characteristics. In this comparison, the findings were similar with actually a higher incidence of aneuploidy in the ejaculated spermatozoa (n = 16) compared to those surgically retrieved (n = 6) (P<0.0001). For this group, the clinical pregnancy rate for the ejaculated specimens was 47.2% with 29.4% pregnancy loss, while the surgically retrieved yielded a 50% clinical pregnancy rate, all progressing to term. A subsequent prospective combined assessment on ejaculated and surgically retrieved spermatozoa by FISH and NGS was performed on non-azoospermic men with high DNA fragmentation in their ejaculate. The assessment by FISH evidenced 2.8% chromosomal defects in the ejaculated and 1.2% in testicular biopsies while by NGS became 8.4% and 1.3% (P = 0.02), respectively. Interestingly, we evidenced a pregnancy rate of 0% with ejaculated while 100% with the testicular spermatozoa in this latter group. This indicates that improved techniques for assessing sperm aneuploidy on a wider number of cells disproves earlier reports and corroborates the safe utilization of testicular spermatozoa with a positive impact on chances of pregnancy.

<![CDATA[Seminal plasma amino acid profile in different breeds of chicken: Role of seminal plasma on sperm cryoresistance]]>

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

<![CDATA[Reproduction and population structure of the sea urchin Heliocidaris crassispina in its newly extended range: The Oga Peninsula in the Sea of Japan, northeastern Japan]]>

Ocean warming has facilitated the range expansion of commercially important sea urchin species to higher latitudes. Heliocidaris crassispina was recorded to extend northward to Toga Bay along the Oga Peninsula, Japan following an increase in seawater temperatures, and replacement of local sea urchin species Mesocentrotus nudus. In order to identify evidence of adaptation occurring in response to a range extension of H. crassispina to the newly extended environments, we randomly collected 106 H. crassispina in August 2014 in Toga Bay, determined the growth and age composition and examined gonad traits (size, color and development). To confirm the gonad development, 30 H. crassispina with > 30 mm diameter were collected in July, August and September 2017. We found slower growth in the extended range than the central range. More delayed gonad development of males than those of females and a large variety of developmental stages in the acini of testis indicated that the spawning of both sexes of the sea urchins were asynchronous. In terms of gonad color, L* (lightness) values increased with increasing GI, while b* (yellowness) values decreased with increasing age. The population consisted of seven year-classes from 2006 to 2012, suggesting persistent juvenile recruitment. Long-term water temperature data indicated that the range extension of H. crassispina was due to ocean warming, in particular during the summer spawning season.