Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Loss of the importin Kpna2 causes infertility in male mice by disrupting the translocation of testis-specific transcription factors.

Karyopherins mediate the movement between the nucleus and cytoplasm of specific proteins in diverse cellular processes. Through a loss-of-function approach, we here examine the role of Karyopherin Subunit Alpha 2 (Kpna2) in spermatogenesis. Knockout male mice exhibited reduced body size and sperm motility, increased sperm abnormalities, and led to the dysregulation of testis gene expression and ultimately to infertility. Impaired mRNA expression mainly affected clusters of genes expressed in spermatids and spermatocytes. Downregulated genes included a set of genes that participate in cell adhesion and extracellular matrix (ECM) organization. We detected both the enrichment of some transcription factors that bind to regions around transcription start sites of downregulated genes and the impaired transport of specific factors to the nucleus of spermatid cells. We propose that Kpna2 is essential in the seminiferous tubules for promoting the translocation of testis-specific transcription factors that control the expression of genes related to ECM organization.

Effect of fermented soy beverage in aged female mice model.

Soy beverage is a rich source of phytoestrogens isoflavones, with potential benefits on health. The effect of those compounds depends greatly on their bacterial metabolization into their aglycone forms. This study evaluated the health effects of two soy beverages, non-fermented (SB) and fermented with Bifidobacterium pseudocatenulatum INIA P815 (FSB), in acyclic and cyclic C57BL/6J aged female mice as a model of menopause and premenopause, respectively. SB and FSB treatments were administrated for 36 days and, subsequently, body weight, lipid and inflammatory profile and fertility were analyzed and compared. In addition, hepatic gene expression and faecal microbiota composition were also assessed. After fermentation, FSB presented a high content in the aglycones daidzein and genistein and a higher antioxidant activity. FSB treated cyclic mice showed a significant increase in the number of retrieved oocytes and zigotes. Differences in serum lipids were observed in triglycerides, which were lower in FSB than in SB groups. None of the treatments influenced the inflammatory profile or caused a dramatic change in the intestinal microbiota profile or hepatic gene expression in any of the groups. Our data showed that FSB provided greater health benefits than SB in lipid profile and fertility in cyclic mice. These beneficial effects could be attributed to the fermentation process, which produces more bioavailable and bioactive compounds, achieving a greater impact on health.

Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants.

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis.

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.

Acquisition of fertilization competence in guinea pig spermatozoa under different capacitation protocols.

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs.

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome.

BACKGROUND: Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. METHODS: We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. RESULTS: The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05). CONCLUSION: Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates.

Concurrent Zrsr2 mutation and Tet2 loss promote myelodysplastic neoplasm in mice.

RNA splicing and epigenetic gene mutations are the most frequent genetic lesions found in patients with myelodysplastic neoplasm (MDS). About 25% of patients present concomitant mutations in such pathways, suggesting a cooperative role in MDS pathogenesis. Importantly, mutations in the splicing factor ZRSR2 frequently associate with alterations in the epigenetic regulator TET2. However, the impact of these concurrent mutations in hematopoiesis and MDS remains unclear. Using CRISPR/Cas9 genetically engineered mice, we demonstrate that Zrsr2m/mTet2-/- promote MDS with reduced penetrance. Animals presented peripheral blood cytopenia, splenomegaly, extramedullary hematopoiesis, and multi-lineage dysplasia, signs consistent with MDS. We identified a myelo-erythroid differentiation block accompanied by an expansion of LT-HSC and MPP2 progenitors. Transplanted animals presented a similar phenotype, thus indicating that alterations were cell-autonomous. Whole-transcriptome analysis in HSPC revealed key alterations in ribosome, inflammation, and migration/motility processes. Moreover, we found the MAPK pathway as the most affected target by mRNA aberrant splicing. Collectively, this study shows that concomitant Zrsr2 mutation and Tet2 loss are sufficient to initiate MDS in mice. Understanding this mechanistic interplay will be crucial for the identification of novel therapeutic targets in the spliceosome/epigenetic MDS subgroup.

Zrsr2 and functional U12-dependent spliceosome are necessary for follicular development.

ZRSR2 is a splicing factor involved in recognition of 3'-intron splice sites that is frequently mutated in myeloid malignancies and several tumors; however, the role of mutations of Zrsr2 in other tissues has not been analyzed. To explore the biological role of ZRSR2, we generated three Zrsr2 mutant mouse lines. All Zrsr2 mutant lines exhibited blood cell anomalies, and in two lines, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2 mu secondary follicles showed aberrations in gene expression and showed altered alternative splicing (AS) events involving enrichment of U12-type intron retention (IR), supporting the functional Zrsr2 action in minor spliceosomes. IR events were preferentially associated with centriole replication, protein phosphorylation, and DNA damage checkpoint. Notably, we found alterations in AS events of 50 meiotic genes. These results indicate that ZRSR2 mutations alter splicing mainly in U12-type introns, which may affect peripheral blood cells, and impede oogenesis and female fertility.

The impact of CD160 deficiency on alloreactive CD8 T cell responses and allograft rejection.

CD160 is a member of the immunoglobulin superfamily with a pattern of expression mainly restricted to cytotoxic cells. To assess the functional relevance of the HVEM/CD160 signaling pathway in allogeneic cytotoxic responses, exon 2 of the CD160 gene was targeted by CRISPR/Cas9 to generate CD160 deficient mice. Next, we evaluated the impact of CD160 deficiency in the course of an alloreactive response. To that aim, parental donor WT (wild-type) or CD160 KO (knock-out) T cells were adoptively transferred into non-irradiated semiallogeneic F1 recipients, in which donor alloreactive CD160 KO CD4 T cells and CD8 T cells clonally expanded less vigorously than in WT T cell counterparts. This differential proliferative response rate at the early phase of T cell expansion influenced the course of CD8 T cell differentiation and the composition of the effector T cell pool that led to a significant decreased of the memory precursor effector cells (MPECs) / short-lived effector cells (SLECs) ratio in CD160 KO CD8 T cells compared to WT CD8 T cells. Despite these differences in T cell proliferation and differentiation, allogeneic MHC class I mismatched (bm1) skin allograft survival in CD160 KO recipients was comparable to that of WT recipients. However, the administration of CTLA-4.Ig showed an enhanced survival trend of bm1 skin allografts in CD160 KO with respect to WT recipients. Finally, CD160 deficient NK cells were as proficient as CD160 WT NK cells in rejecting allogeneic cellular allografts or MHC class I deficient tumor cells. CD160 may represent a CD28 alternative costimulatory molecule for the modulation of allogeneic CD8 T cell responses either in combination with costimulation blockade or by direct targeting of alloreactive CD8 T cells that upregulate CD160 expression in response to alloantigen stimulation.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Sperm selection by rheotaxis improves sperm quality and early embryo development.

The objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates.

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism.

BACKGROUND: Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. OBJECTIVE: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. STUDY DESIGN: Experiments comparing in vitro and in vivo produced embryos. METHODS: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10 mmol/L glucose respectively; n = 87); 5-17 (5 and 17.5 mmol/L; n = 66); and 10-17 (10 and 17.5 mmol/L; n = 117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. RESULTS: No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5 mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. MAIN LIMITATIONS: Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. CONCLUSIONS: Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

Embryo responses to stress induced by assisted reproductive technologies.

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health.

Successful ICSI in Mice Using Caput Epididymal Spermatozoa.

Spermatozoa undergo their last phase of spermiogenesis, known as maturation, as they pass through the epididymis. A recent report indicates that mouse immature spermatozoa retrieved from the caput epididymis for intracytoplasmic sperm injection (ICSI) give rise to embryos with multiple developmental defects. Further, these embryos were unable to develop to term after their transfer to surrogate mothers. Herein, we examined the potential of mouse caput spermatozoa to produce normal embryos by comparing the use of caput vs. cauda epididymal spermatozoa for in vitro fertilization (IVF) or ICSI. Two methods for the separation of sperm heads prior to ICSI were also compared: freezing/thawing or drawing through a syringe. We found that in contrast to caudal spermatozoa, caput spermatozoa failed to produce embryos via IVF, confirming their immature state. However, regardless of the method employed for the separation of sperm heads, similar efficiencies of blastocyst production in vitro and development to term after transfer to surrogate mothers were observed following ICSI using both caput and cauda epididymal spermatozoa. It therefore seems that mice spermatozoa recovered from the caput epididymis are as valid as caudal spermatozoa for the production of embryos and offspring by ICSI.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

Sperm selection by thermotaxis improves ICSI outcome in mice.

The ejaculate is a heterogeneous pool of spermatozoa containing only a small physiologically adequate subpopulation for fertilization. As there is no method to isolate this subpopulation, its specific characteristics are unknown. This is one of the main reasons why we lack effective tools to identify male infertility and for the low efficiency of assisted reproductive technologies. The aim of this study was to improve ICSI outcome by sperm selection through thermotaxis. Here we show that a specific subpopulation of mouse and human spermatozoa can be selected in vitro by thermotaxis and that this subpopulation is the one that enters the fallopian tube in mice. Further, we confirm that these selected spermatozoa in mice and humans show a much higher DNA integrity and lower chromatin compaction than unselected sperm, and in mice, they give rise to more and better embryos through intracytoplasmic sperm injection, doubling the number of successful pregnancies. Collectively, our results indicate that a high quality sperm subpopulation is selected in vitro by thermotaxis and that this subpopulation is also selected in vivo within the fallopian tube possibly by thermotaxis.