Ccdc38 is required for sperm flagellum biogenesis and male fertility in mouse

Sperm flagellum is essential for male fertility, defects in flagellum biogenesis are associated with male infertility. Deficiency of CCDC42 is associated with malformation of the mouse sperm flagella. Here, we find that the testis-specific expressed protein CCDC38 (coiled coil domain containing 38) interacts with CCDC42 and localizes on manchette and sperm tail during spermiogenesis. Inactivation of CCDC38 in male mice results in distorted manchette, multiple morphological abnormalities of the flagella (MMAF) of spermatozoa, and eventually male sterility. Furthermore, we find that CCDC38 interacts with intra-flagellar transport protein 88 (IFT88) as well as the outer dense fibrous 2 (ODF2), and its depletion reduces the transportation of ODF2 to flagellum. Altogether, our results uncover the essential role of CCDC38 during sperm flagellum biogenesis, and suggesting the defects of these genes might be associated with male infertility in human being.

Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development

Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

Angiogenin mediates paternal inflammation-induced metabolic disorders in offspring through sperm tsRNAs

Paternal environmental inputs can influence various phenotypes in offspring, presenting tremendous implications for basic biology and public health and policy. However, which signals function as a nexus to transmit paternal environmental inputs to offspring remains unclear. Here we show that offspring of fathers with inflammation exhibit metabolic disorders including glucose intolerance and obesity. Deletion of a mouse tRNA RNase, Angiogenin (Ang), abolished paternal inflammation-induced metabolic disorders in offspring. Additionally, Ang deletion prevented the inflammation-induced alteration of 5′-tRNA-derived small RNAs (5′-tsRNAs) expression profile in sperm, which might be essential in composing a sperm RNA ‘coding signature’ that is needed for paternal epigenetic memory. Microinjection of sperm 30–40 nt RNA fractions (predominantly 5′-tsRNAs) from inflammatory Ang+/+ males but not Ang–/– males resulted in metabolic disorders in the resultant offspring. Moreover, zygotic injection with synthetic 5′-tsRNAs which increased in inflammatory mouse sperm and decreased by Ang deletion partially resembled paternal inflammation-induced metabolic disorders in offspring. Together, our findings demonstrate that Ang-mediated biogenesis of 5′-tsRNAs in sperm contributes to paternal inflammation-induced metabolic disorders in offspring.

Quercetin-treated rat sperm enables refrigerated transport with motility and fertility for five days

Shipment of laboratory rats between animal facilities is frequently performed using special containers. However, the shipment of live animals is associated with potential risks of infectious diseases, escape and death during shipment and animal welfare issues. The transport of cold-stored sperm avoids such risks; however, there have been no reports on cold storage of rat sperm. We previously reported that dimethyl sulfoxide (DMSO) and quercetin maintained the motility and fertilising abilities of cold-stored mouse sperm stored for 10 days. The present study investigated the efficacy of DMSO and quercetin in the cold storage of rat sperm. Quercetin maintained motility and fertility of cold-stored rat sperm stored for 5 days. After in vitro fertilisation using cold-stored sperm, pronuclear and two-cell embryos developed normally to pups following embryo transfer. Therefore, we demonstrated that live pups could be obtained from sperm transported using the cold-storage system. We conclude that cold storage of rat sperm may provide an efficient system for transporting rat resources as an alternative to shipping live animals.

Human Sperm Remain Motile After a Temporary Energy Restriction but do Not Undergo Capacitation-Related Events

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca2+ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca2+ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro.

Conserved Mechanism of Bicarbonate-Induced Sensitization of CatSper Channels in Human and Mouse Sperm

To fertilize an egg, mammalian sperm must undergo capacitation in the female genital tract. A key contributor to capacitation is the calcium (Ca2+) channel CatSper, which is activated by membrane depolarization and intracellular alkalinization. In mouse epididymal sperm, membrane depolarization by exposure to high KCl triggers Ca2+ entry through CatSper only in alkaline conditions (pH 8.6) or after in vitro incubation with bicarbonate (HCO3–) and bovine serum albumin (capacitating conditions). However, in ejaculated human sperm, membrane depolarization triggers Ca2+ entry through CatSper in non-capacitating conditions and at lower pH (< pH 7.4) than is required in mouse sperm. Here, we aimed to determine the mechanism(s) by which CatSper is activated in mouse and human sperm. We exposed ejaculated mouse and human sperm to high KCl to depolarize the membrane and found that intracellular Ca2+ concentration increased at pH 7.4 in sperm from both species. Conversely, intracellular Ca2+ concentration did not increase under these conditions in mouse epididymal or human epididymal sperm. Furthermore, pre-incubation with HCO3– triggered an intracellular Ca2+ concentration increase in response to KCl in human epididymal sperm. Treatment with protein kinase A (PKA) inhibitors during exposure to HCO3– inhibited Ca2+ concentration increases in mouse epididymal sperm and in both mouse and human ejaculated sperm. Finally, we show that soluble adenylyl cyclase and increased intracellular pH are required for the intracellular Ca2+ concentration increase in both human and mouse sperm. In summary, our results suggest that a conserved mechanism of activation of CatSper channels is present in both human and mouse sperm. In this mechanism, HCO3– in semen activates the soluble adenylyl cyclase/protein kinase A pathway, which leads to increased intracellular pH and sensitizes CatSper channels to respond to membrane depolarization to allow Ca2+ influx. This indirect mechanism of CatSper sensitization might be an early event capacitation that occurs as soon as the sperm contact the semen.

FAM209 associates with DPY19L2 and is required for sperm acrosome biogenesis and fertility in mice

Infertility afflicts up to 15% of couples globally each year with men a contributing factor in half of these cases. Globozoospermia is a rare condition found in infertile men that is characterized by defective acrosome biogenesis leading to the production of round shaped sperm. Here, we report a novel gene, Fam209 (Family with sequence similarity 209), that is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 result in fertility defects secondary to abnormalities in acrosome biogenesis during spermiogenesis reminiscent of globozoospermia. Proteomic analysis of the FAM209 proteome identified DPY19L2, a protein involved in the majority of globozoospermia cases. While mutations in human and mouse DPY19L2 have been shown to cause globozoospermia, no in vivo interacting partners of DPY19L2 have been identified until now. FAM209 colocalizes with DPY19L2 to the inner nuclear membrane to maintain the developing acrosome. This report identifies FAM209 as the first interacting partner of DPY19L2 and the second protein that is essential for acrosome biogenesis and that co-localizes with DPY19L2 to the inner nuclear membrane.

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency, and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.