Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 ( Rnf133 ); RING finger protein 148 ( Rnf148 ); RING finger protein 151 ( Rnf151 ); and Zinc finger SWIM-type containing 2 ( Zswim2 )]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148 , Rnf151 , and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

Objective:To evaluate the feasibility of generating a center-specific embryo morphokinetic algorithm by time-lapse microscopy to predict clinical pregnancy rates. Design:A retrospective cohort analysis. Setting:Academic fertility clinic in a tertiary hospital setting. Patients:Patients who underwent in vitro fertilization with embryos that underwent EmbryoScope time-lapse microscopy and subsequent transfer between 2014 and 2018. Interventions:None. Main Outcome Measures:Clinical pregnancy. Results:A supervised, random forest learning algorithm from 367 embryos successfully predicted clinical pregnancy from a training set with overall 65% sensitivity and 74% positive predictive value, with an area under the curve of 0.7 for the test set. Similar results were achieved for live birth outcomes. For the secondary analysis, embryo growth morphokinetics were grouped into five clusters using unsupervised clustering. The clusters that had the fastest morphokinetics (time to blastocyst = 97 hours) had pregnancy rates of 54%, whereas a cluster that had the slowest morphokinetics (time to blastocyst = 122 hours) had a pregnancy rate of 71%, although the differences were not statistically significant (P=.356). Other clusters had pregnancy rates of 51%-60%. Conclusions:This study shows the feasibility of a clinic-specific, noninvasive embryo morphokinetic simple machine learning model to predict clinical pregnancy rates.

Additional file 6: Table S2. The single-guide RNAs (sgRNAs) to generate KO mice. The sequences of sgRNAs targeting mouse Zswim2, Rnf133, Rnf148, and Rnf151.

Additional file 1: Table S1. Primers for RT-PCR. Human and mouse RT-PCR primer sequences used for verification of expression in reproductive tract.

Additional file 17: Table S4. Primers for cloning. The sequences of primers for cloning human UBE2J1, human RNF133, human UBE2C, human RNF151, mouse Ube2J1, mouse Rnf133, and human RNF133-mutant.

Infertility afflicts up to 15% of couples globally each year with men a contributing factor in 50% of these cases. Globozoospermia is a rare condition found in infertile men, which is characterized by defective acrosome biogenesis leading to the production of round-headed sperm. Here, we report that family with sequence similarity 209 (Fam209) is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 results in fertility defects that are secondary to abnormalities in acrosome biogenesis during spermiogenesis, reminiscent of globozoospermia. Analysis of the FAM209 proteome identified DPY19L2, whose human orthologue is involved in the majority of globozoospermia cases. Although 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 at the inner nuclear membrane to maintain the developing acrosome. Here, we identified FAM209 as the first interacting partner of DPY19L2, and the second protein that is essential for acrosome biogenesis that localizes to the inner nuclear membrane.

Receptor accessory protein 6 (REEP6) is a member of the REEP/Ypt-interacting protein family that we recently identified as essential for normal endoplasmic reticulum homeostasis and protein trafficking in the retina of mice and humans. Interestingly, in addition to the loss of REEP6 in our knockout (KO) mouse model recapitulating the retinal degeneration of humans with REEP6 mutations causing retinitis pigmentosa (RP), we also found that male mice are sterile. Herein, we characterize the infertility caused by loss of Reep6. Expression of both Reep6 mRNA transcripts is present in the testis; however, isoform 1 becomes overexpressed during spermiogenesis. In vitro fertilization assays reveal that Reep6 KO spermatozoa are able to bind the zona pellucida but are only able to fertilize oocytes lacking the zona pellucida. Although spermatogenesis appears normal in KO mice, cauda epididymal spermatozoa have severe motility defects and variable morphological abnormalities, including bent or absent tails. Immunofluorescent staining reveals that REEP6 expression first appears in stage IV tubules within step 15 spermatids, and REEP6 localizes to the connecting piece, midpiece, and annulus of mature spermatozoa. These data reveal an important role for REEP6 in sperm motility and morphology and is the first reported function for a REEP protein in reproductive processes. Additionally, this work identifies a new gene potentially responsible for human infertility and has implications for patients with RP harboring mutations in REEP6.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

Families with sequence similarity 170 members A and B (FAM170A and FAM170B) are testis-specific, paralogous proteins that share 31% amino acid identity and are conserved throughout mammals. While previous in vitro experiments suggested that FAM170B, an acrosome-localized protein, plays a role in the mouse sperm acrosome reaction and fertilization, the role of FAM170A in the testis has not been explored. In this study, we used CRISPR/Cas9 to generate null alleles for each gene, and homozygous null (-/-) male mice were mated to wild-type females for 6 months to assess fertility. Fam170b-/- males were found to produce normal litter sizes and had normal sperm counts, motility, and sperm morphology. In contrast, mating experiments revealed significantly reduced litter sizes and a reduced pregnancy rate from Fam170a-/- males compared with controls. Fam170a-/-;Fam170b-/- double knockout males also produced markedly reduced litter sizes, although not significantly different from Fam170a-/- alone, suggesting that Fam170b does not compensate for the absence of Fam170a. Fam170a-/- males exhibited abnormal spermiation, abnormal head morphology, and reduced progressive sperm motility. Thus, FAM170A has an important role in male fertility, as the loss of the protein leads to subfertility, while FAM170B is expendable. The molecular functions of FAM170A in spermatogenesis are as yet unknown; however, the protein localizes to the nucleus of elongating spermatids and may mediate its effects on spermatid head shaping and spermiation by regulating the expression of other genes. This work provides the first described role of FAM170A in reproduction and has implications for improving human male infertility diagnoses.

Group A streptococcus (GAS) produces millions of infections worldwide, including mild mucosal infections, postinfection sequelae, and life-threatening invasive diseases. During infection, GAS readily acquires nutritional iron from host heme and hemoproteins. Here, we identified a new heme importer, named SiaFGH, and investigated its role in GAS pathophysiology. The SiaFGH proteins belong to a group of transporters with an unknown ligand from the recently described family of energy coupling factors (ECFs). A siaFGH deletion mutant exhibited high streptonigrin resistance compared to the parental strain, suggesting that iron ions or an iron complex is the likely ligand. Iron uptake and inductively coupled plasma mass spectrometry (ICP-MS) studies showed that the loss of siaFGH did not impact GAS import of ferric or ferrous iron, but the mutant was impaired in using hemoglobin iron for growth. Analysis of cells growing on hemoglobin iron revealed a substantial decrease in the cellular heme content in the mutant compared to the complemented strain. The induction of the siaFGH genes in trans resulted in the induction of heme uptake. The siaFGH mutant exhibited a significant impairment in murine models of mucosal colonization and systemic infection. Together, the data show that SiaFGH is a new type of heme importer that is key for GAS use of host hemoproteins and that this system is imperative for bacterial colonization and invasive infection. IMPORTANCE ECF systems are new transporters that take up various vitamins, cobalt, or nickel with a high affinity. Here, we establish the GAS SiaFGH proteins as a new ECF module that imports heme and demonstrate its importance in virulence. SiaFGH is the first heme ECF system described in bacteria. We identified homologous systems in the genomes of related pathogens from the Firmicutes phylum. Notably, GAS and other pathogens that use a SiaFGH-type importer rely on host hemoproteins for a source of iron during infection. Hence, recognizing the function of this noncanonical ABC transporter in heme acquisition and the critical role that it plays in disease has broad implications.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception.

Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components. Male fecundity was determined by consecutively pairing knockout males with wild-type females and comparing the fecundity of wild-type controls. Phenotypic analyses of testis appearance and weight, testis and epididymis histology, and sperm movement were further carried out to examine any potential spermatogenic or sperm maturation defect in mutant males. In this study, we uncovered 13 testis- or epididymis-enriched evolutionarily conserved genes that are individually dispensable for male fertility in mice. Owing to their dispensable nature, it is not feasible to use these targets for the development of a male contraceptive.

To develop and examine the feasibility of a time-lapse microscopy (TLM)- based assay to predict pregnancy outcomes in patient IVF cycles. Retrospective and prospective cohort analyses of clinical pregnancy data with embryo TLM data that were transferred at an academic fertility center. Embryos that underwent EmbryoScopeTM TLM and subsequent transfer with clinical pregnancy (defined by fetal heart beat after 6 weeks) or no clinical pregnancy were included from 2014 to 2018. Machine classifiers used were morphokinetic parameters from fertilization to blastocyst formation that were annotated manually. Data were analyzed by multivariate analysis of covariance, Fisher’s exact, Chi-square tests, and binomial logistic regression using R and Scikit learn, and Python software. Learning curves were applied to a training set of 180 embryos and a validation set of 80 embryos. Supervised algorithms tested included naïve bayes classifiers, support vector machines, logistic regression, and decision trees. Highest accuracy scores were achieved with logistic regression and decision tree models. Accuracy scores of 0.6 for the two sets converged for the logistic regression model at 100 training set numbers whereas Decision Tree algorithm reached a 0.7 accuracy score converging at 180 training set numbers. Furthermore, the logistic regression prediction model (Chi square=26.3, p=0.010), termed the Yang-Peavey Embryo Enhancement Algorithm, correctly predicted 70% of clinical pregnancies for patients under age 35. A receiver operating characteristic curve was developed and found to be significant with an area under the curve value of 0.757 (95% CI 0.667-0.848, p<0.001). In a separate prospective cohort study, the algorithm was applied to 140 embryos that were transferred, which were manually annotated and blinded from the pregnancy outcome. The YPEEA algorithm yield 68% sensitivity and 53% positive predictive value (PPV) when applied to the full dataset. This study demonstrates that novel Machine Learning algorithms can be used for embryo selection based on as few as 200 embryos, and be applicable to a prospective cohort of embryos. The predictive value of the algorithm is comparable or even superior in a subset of patients to that of preimplantation genetics screening and therefore is a valuable non-invasive technology to predict clinical pregnancy in IVF.

BACKGROUND:Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation.OBJECTIVES:Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis.MATERIALS AND METHODS:KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6 months to check fertility.RESULTS:We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males.DISCUSSION AND CONCLUSION:We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.

To develop and examine the feasibility of a clinic-specific embryo morphokinetic assay by time-lapse microscopy (TLM) to predict clinical pregnancy rate. Retrospective cohort analysis of TLM of embryos with clinical pregnancy data (Phase I), and a prospective, blinded cohort analysis of TLM of embryos that were transferred at an academic fertility center (Phase II). Embryos that underwent EmbryoScopeTMTLM and subsequent frozen transfer with or without clinical pregnancy (defined by fetal heart beat after 6 weeks) were included from 2014 to 2016. For phase II, embryos that were transferred in 2017-2018 were included and with blinded pregnancy outcomes from the annotator. All morphokinetic parameters from fertilization to blastocyst formation were manually annotated. Data were analyzed by multivariate analysis of covariance, Fisher's exact, Chi-square tests, and binomial logistic regression with SPSS. In the phase I study, embryos that resulted in clinical pregnancy (n=94) were faster to develop to 4-cell stage (37.8h vs 40.2h, p=0.057, OR 0.74) compared to embryos that did not result in clinical pregnancy (n=121). A logistic regression prediction model (Chi square=26.3, p=0.010) using morphokinetic parameters, termed the Yang-Peavey Embryo Enhancement Algorithm (YPEEA). A receiver operating characteristic curve was developed and found to be significant with area under the curve 0.757 (95% CI 0.667-0.848, p<0.001). In the phase II prospective cohort study, the algorithm was applied to 132 embryos that were transferred, which were manually annotated and blinded from the pregnancy outcome. When applied to a subset of patients under the age of 35 (n=63), the YPEEA algorithm yielded positive predictive value (PPV) of 70% vs 60% pregnancy rate of euploid embryos. When applied to the total cohort,the YPEEA algorithm yielded 68% sensitivity and 53% PPV. This study shows the feasibility and development of a clinic-specific, novel embryo morphokinetic predictive model with as few as 200 embryo outcomes, applicable to a prospective cohort of embryos. The predictive value of the algorithm is comparable or even superior in a subset of patients to that of preimplantation genetics testing and therefore is a valuable non-invasive technology to predict clinical pregnancy in IVF.

Introduction: Group B streptococcus (GBS), also known as S. agalactiae, is a beta-hemolytic opportunistic pathogen that is part of the normal flora in the gastrointestinal tract. Like many other streptococcal pathogens, it requires iron from the host for growth and to establish virulence. Several heme binding and acquisition proteins have been identified that have provided significant insight into the heme metabolism in other streptococcus spp. A previous microarray analysis Group A streptococcus (GAS) showed up-regulation of specific genes in the presence of iron. Further bioinformatic analysis suggested a homologue in GBS. In addition, sequence analysis showed that the gene of interest lies near an iron uptake system. In the present study, we aim to successfully clone the gene in an inducible vector system for recombinant protein expression. In the future, this recombinant protein will be evaluated for its iron and heme binding abilities.

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.