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Rationale: Nonbenzodiazepine benzodiazepine receptor agonists (NBZRA, e.g., zolpidem) are frequently used to treat insomnia among patients with chronic obstructive pulmonary disease (COPD). However, multiple observational studies find that patients with COPD who are prescribed NBZRAs have greater risks for mortality and respiratory complications than patients without such prescriptions. Without an active comparator, these studies are susceptible to confounding by indication. Objectives: Compare the risk of death or inpatient COPD exacerbation among patients receiving zolpidem relative to patients receiving other hypnotics. Methods: Using nationwide Veterans Health Administration (VA) data, we identified patients with clinically diagnosed COPD and new receipt of zolpidem or another hypnotic available on VA formulary without prior authorization (melatonin, trazodone, doxepin). We excluded those receiving traditional benzodiazepines or multiple concurrent hypnotics. We propensity-matched patients receiving zolpidem to other hypnotics on 32 variables, including demographics, comorbidities, and markers of COPD severity. We compared risk of the primary composite outcome of death or inpatient COPD exacerbation over 1 year. In secondary analyses, we propensity-matched patients receiving zolpidem to those without hypnotic receipt. Results: Among 283,740 patients meeting inclusion criteria, 1,126 (0.4%) received zolpidem and 3,057 (1.1%) received other hypnotics. We propensity-matched patients receiving zolpidem 1:1 to peers receiving other hypnotics. We did not find a difference in the primary composite outcome of death or inpatient exacerbation (hazard ratio, 0.97; 95% confidence interval [CI], 0.77-1.23). In secondary analyses comparing patients receiving zolpidem to matched peers without hypnotic receipt, we observed greater risk of death or inpatient exacerbation with zolpidem (hazard ratio, 1.40; 95% CI, 1.09-1.81). Conclusions: Among patients with COPD, we did not observe greater risks after new receipt of zolpidem relative to other hypnotics. However, we did observe greater risks relative to those without hypnotic receipt. This latter finding may reflect: 1 ) residual, unmeasured confounding related to insomnia; or 2 ) true adverse effects of hypnotics across classes. Future work is needed to better understand the risks of hypnotics in COPD.

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Spermatogenesis is a complex developmental process that involves the proliferation of diploid cells, meiotic division, and haploid differentiation. Many genes are shown to be essential for male fertility using knockout (KO) mice; however, there still remain genes to be analyzed to elucidate their molecular mechanism and their roles in spermatogenesis. Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitously expressed protein that possesses three paralogs: CIB2, CIB3, and CIB4. It is reported that Cib1 KO male mice are sterile due to impaired haploid differentiation. In this study, we discovered that Cib4 is expressed strongly in mouse and human testis and begins expression during the haploid phase of spermatogenesis in mice. To analyze the function of CIB4 in vivo, we generated Cib4 KO mice using the CRISPR/Cas9 system. Cib4 KO male mice are sterile due to impaired haploid differentiation, phenocopying Cib1 KO male mice. Spermatogenic cells isolated from seminiferous tubules demonstrate an essential function of CIB4 in the formation of the apical region of the sperm head. Further analysis of CIB4 function may shed light on the etiology of male infertility caused by spermatogenesis defects, and CIB4 could be a target for male contraceptives because of its dominant expression in the testis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The t-complex is defined as naturally occurring variants of the proximal third of mouse chromosome 17 and has been studied by mouse geneticists for decades. This region contains many genes involved in processes from embryogenesis to sperm function. One such gene, t-complex protein 11 (Tcp11), was identified as a testis-specific gene whose protein is present in elongating spermatids. Later work on Tcp11 localized TCP11 to the sperm surface and acrosome cap and implicated TCP11 as important for sperm capacitation through the cyclic AMP/Protein Kinase A pathway. Here, we show that TCP11 is cytoplasmically localized to elongating spermatids and absent from sperm. In the absence of Tcp11, male mice have severely reduced fertility due to a significant decrease in progressively motile sperm; however, Tcp11-null sperm continues to undergo tyrosine phosphorylation, a hallmark of capacitation. Interestingly, null sperm displays reduced PKA activity, consistent with previous reports. Our work demonstrates that TCP11 functions in elongated spermatids to confer proper motility in mature sperm., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex ( N -DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N -DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1 -null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1 -null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility., Competing Interests: The authors declare no conflict of interest.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

In this report, we describe four cases of granulomatous amebic encephalitis caused by Balamuthia (Leptomyxid ameba) in four previously healthy Mexican patients. All four cases were characterized by focal neurologic signs, increased intracranial pressure, and cerebral hyperdense lesions in computed tomography scans of the head. These patients underwent craniotomies for evaluation of mass lesions for possible brain tumors. Granulomatous chronic inflammatory reaction and amebic trophozoites were found in brain biopsies. At autopsy, areas of hemorrhagic encephalomalacia were located in both basal frontal lobes, right parieto-occipital lobes, and, less often, in the brainstem and cerebellum. Angiitis, necrotizing granulomatous encephalitis, and large numbers of amebic trophozoites in perivascular spaces were present. Amebic trophozoites were seen in the left adrenal gland in one of the cases. The amebas in all four cases were identified as Balamuthia mandrillaris (Leptomyxiidae) based on their reactivity with the anti-Balamuthia (Leptomyxiidae) serum in an immunofluorescence test.