The Association of HLA-G Gene Polymorphism and Its Soluble Form With Male Infertility

Successful reproduction depends on many factors. Male factors contribute to infertility in approximately 50% of couples who fail to conceive. Seminal plasma consists of secretions from different accessory glands containing a mixture of various cytokines, chemokines, and growth factors, which together can induce a local immune response that might impact on a male’s as well as a female’s fertility. Human leukocyte antigen (HLA)-G expression has been suggested as an immunomodulatory molecule that influences pregnancy outcome. The HLA-G gene encodes either membrane-bound or/and soluble proteins. The aim of this study was the evaluation of HLA-G polymorphisms and their impact on soluble HLA-G (sHLA-G) production. We tested the HLA-G polymorphism in three positions: rs1632947: c.-964G>A; rs1233334: c.-725G>C/T in the promoter region; rs371194629: c.∗65_∗66insATTTGTTCATGCCT in the 3′ untranslated region. We tested two cohorts of men: 663 who participated in in vitro fertilization (test material was blood or sperm), and 320 fertile controls who possessed children born after natural conception (test material was blood). Since 50% of men visiting assisted reproductive clinics have abnormal semen parameters, we wondered if men with normal sperm parameters differ from those with abnormal parameters in terms of HLA-G polymorphism and secretion of sHLA-G into semen. We found that certain rs1632947-rs1233334-rs371194629 HLA-G haplotypes and diplotypes were associated with male infertility, while others were protective. Normozoospermic men with the A-C-del haplotype and A-C-del/A-C-del diplotype secreted the most sHLA-G into semen (574.1 IU/mL and 1047.0 IU/mL, respectively), while those with the G-C-ins haplotype and G-C-ins/G-C-ins diplotype – the least (80.8 IU/mL and 75.7 IU/mL, respectively). Men with the remaining haplotypes/diplotypes secreted sHLA-G at an intermediate level. However, only in one haplotype, namely G-C-ins, did we observe strong significant differences in the concentration of sHLA-G in the semen of men with teratozoospermia compared to men with normal sperm parameters (p = 0.009). In conclusion, fertile men differ in the profile of HLA-G polymorphism from men participating in IVF. Among all HLA-G haplotypes, the most unfavorable for male fertility is the G-C-ins haplotype, which determines the secretion of the lowest concentration of the soluble HLA-G molecule. This haplotype may reduce sperm parameters.

Ovary Structure and Oogenesis of Trypophloeus klimeschi (Coleoptera: Curculionidae: Scolytinae)

The female reproductive system, ovary structure and ultrastructure of Trypophloeus klimeschi (Coleoptera: Curculionidae: Scolytinae) were investigated using light microscopy, scanning electron microscopy, and transmission electron microscopy. Its female reproductive system is comprised of two ovaries (each ovary has two ovarioles), lateral oviducts, common oviduct, spermathecal sac, spermathecal pump, two accessory glands and bursa copulatrix. Well-developed endoplasmic reticulum can be clearly seen in the secretory cells of spermathecal sac. This species has telotrophic meroistic ovarioles that are comprised of terminal filament, tropharium, vitellarium and pedicel. The terminal filaments are simple; each is comprised of cellular peritoneal sheath. The presence of several clusters of nurse cells in the tropharium is indicative that its ovarioles conform to the transition stage. This indicates that there are at least two different types (transition stage and secondary stage) of ovarioles in Curculionidae.

Seminal fluid proteins induce transcriptome changes in the Aedes aegypti female lower reproductive tract

Background Mating induces behavioral and physiological changes in the arbovirus vector Aedes aegypti, including stimulation of egg development and oviposition, increased survival, and reluctance to re-mate with subsequent males. Transferred seminal fluid proteins and peptides derived from the male accessory glands induce these changes, though the mechanism by which they do this is not known. Results To determine transcriptome changes induced by seminal proteins, we injected extract from male accessory glands and seminal vesicles (MAG extract) into females and examined female lower reproductive tract (LRT) transcriptomes 24 h later, relative to non-injected controls. MAG extract induced 87 transcript-level changes, 31 of which were also seen in a previous study of the LRT 24 h after a natural mating, including 15 genes with transcript-level changes similarly observed in the spermathecae of mated females. The differentially-regulated genes are involved in diverse molecular processes, including immunity, proteolysis, neuronal function, transcription control, or contain predicted small-molecule binding and transport domains. Conclusions Our results reveal that seminal fluid proteins, specifically, can induce gene expression responses after mating and identify gene targets to further investigate for roles in post-mating responses and potential use in vector control.

Discovering Novel Genes in Non-Model Fly Accessory Glands Using De Novo Nanopore Transcriptomics

BackgroundOxford Nanopore Technologies (ONT) long-read transcriptomes offer many advantages including long reads (>10kbp), end-to-end transcripts, structural variants, isoform-level resolution of genes and expression. However, uptake of ONT transcriptomics is still low, largely due to high error rates (2 to 13%) and reliance on reference databases that are unavailable for many non-model species. Additionally, bioinformatics tools and pipelines for de novo ONT transcriptomics are still in early stages of development. ResultsHere, we use de novo ONT GridION transcriptomics to discover novel genes from the male accessory glands (AG) of a widespread, non-model dung fly, Sepsis punctum. Insect AGs are of particular interest for this as they are hotspots for rapid evolution of novel reproductive genes, and they synthesize seminal fluid proteins that lack homology to any other known proteins. We implement a completely de novo ONT GridION transcriptome pipeline, incorporating quality-filtering and rigorous error-correction procedures, to characterize this novel gene set and to quantify their expression. Specifically, we compare these ONT genes and their expression against de novo lllumina HiSeq transcriptome data. We find 40 high-quality and high-confidence ONT genes that cross-verify against Illumina genes; twenty-six of which are novel and specific to S. punctum. Read count based expression quantification in ONT samples is highly congruent with Illumina’s Transcript per Million (TPM), both in overall pattern and within functional categories. Novel genes account for an average of 81% of total gene expression underscoring their functional importance in S. punctum AGs. Eighty percentage of these genes are secretory in nature, responsible for 74% total gene expression. Notably, median sequence similarities of ONT nucleotide and protein sequences match within-Illumina sequence similarities indicating that our de novo ONT transcriptome pipeline successfully mitigated sequencing errors. ConclusionsThis is the first study to adapt ONT transcriptomics for completely de novo characterization of novel genes in animals. Our study demonstrates that ONT long-reads, constituting a quarter of the number of bases sequenced at less than a third the cost of Illumina reads, can be a resource-friendly and cost-effective solution for end-to-end sequencing of unknown genes even in the absence of a reference database.

Unraveling the role of male reproductive tract and haemolymph in cantharidin-exuding Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae): a comparative transcriptomics approach

Background Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. Results Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. Conclusions The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Extreme climate event promotes phenological mismatch between sexes in hibernating ground squirrels

Hibernating ground squirrels rely on a short active period for breeding and mass accrual, and are thus vulnerable to extreme climate events that affect key periods in their annual cycle. Here, we document how a heatwave in March 2012 led to a phenological mismatch between sexes in Richardson’s ground squirrels (Urocitellus richardsonii). Females emerged from hibernation and commenced breeding earlier in 2012 relative to average female emergence. Although males had descended testes and pigmented scrota, it appeared that not all males were physiologically prepared to breed since 58.6% of males had non-motile sperm when breeding commenced. Body condition, relative testes size, and the relative size of accessory glands were significant predictors of sperm motility. Males with non-motile sperm had smaller accessory glands than males with motile sperm. There was no decrease in the number of juveniles that emerged in 2012 or female yearlings recruited in 2013, nor did juveniles emerge later than other years. The impact of this heatwave on male ground squirrels emphasizes the importance of assessing the consequences of climate change on the breeding success of hibernating species in both sexes, since the different sensitivity to external cues for emergence led to a mismatch in timing under this event.

Natural Products as Protective Agents for Male Fertility

The male reproductive system is highly susceptible to noxious influences, such as oxidative stress, inflammation, drugs, and even diseases that can induce germ cell damage and alterations in spermatogenesis. All of these factors, which are caused by actions at the testicular level and/or at the excurrent ducts and accessory glands, significantly affect sperm parameters and male fertility. For this reason, it is of major importance to investigate possible ways to protect the male reproductive system since males are exposed to these toxic factors constantly. For centuries, natural products have been used by humans in folk medicine as therapeutic agents, and because of their beneficial properties for human health, plenty of them have been introduced to the pharmaceutical market as supplementary therapies. The present review aims to compile available information regarding different natural exogenous factors that demonstrate potential useful activity in the male reproductive system. The studies presented here reopen the perspective of using natural products as protective agents and eventually as new supplementary therapeutic options for the recovery of hampered spermatogenesis and/or male infertility.