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Preserving sperm fertility Reproducing the complex process of spermatogenesis in vitro might lead to the development of new diagnostic and therapeutic techniques for male infertility. Takehiko Ogawa and colleagues have now established in vitro organ culture conditions that can support the production of fertile sperm from spermatogonia of neonatal mice. Spermatids and sperm that were derived in vitro produced healthy and fertile mice. In addition, neonatal testis tissues that were cryopreserved for several months resumed complete spermatogenesis in vitro on thawing. The organ culture method is simple and, with further refinements, could be applicable to a variety of mammalian species. This work suggests that cryopreservation of the testis tissue of paediatric cancer patients could become a practical way of ensuring future fertility. Reproducing the complex process of spermatogenesis in vitro might lead to the development of new diagnostic and therapeutic techniques for male infertility. This study establishes in vitro organ culture conditions that can support complete spermatogenesis in mice. The in - vitro -derived spermatids and sperm produced healthy and fertile mice, and testis tissue fragments used as a starting material for in vitro spermatogenesis could be cryopreserved for months and then resumed full spermatogenesis in vitro . Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation 1 , 2 . The whole process, therefore, has never been reproduced in vitro in mammals 3 , 4 , 5 , nor in any other species with a very few exceptions in some particular types of fish 6 , 7 . Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas–liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro . Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans -Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells (SSCs) produce haploid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N -methyladenosine (m A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactivation of the m A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m A and depletion of SSCs. m A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Combined deletion of Mettl3 and Mettl14 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettl14 in advanced germ cells show normal spermatogenesis. The spermatids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermiogenesis. This study highlights crucial roles of mRNA m A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.

The RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs) and small interfering RNAs (siRNAs). The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis. Being interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids. We demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

Centrosomes, composed of centrioles and pericentriolar matrix proteins, are traditionally viewed as essential microtubule-organizing centers (MTOCs) that facilitate bipolar spindle formation and chromosome segregation during spermatogenesis. In this study, we investigated the role of centrioles in male germ cell development by using a murine conditional knockout (cKO) of Sas4 , a critical component of centriole biogenesis. We found that while centriole duplication was impaired in Sas4 cKO spermatocytes, these cells were still capable of progressing through meiosis I and II. Chromosome segregation was able to proceed through the formation of a non-centrosomal MTOC, indicating that centrioles are not required for meiotic divisions. However, spermatids that inherited fewer than two centrioles exhibited severe defects in spermiogenesis, including improper manchette formation, constricted perinuclear rings, disrupted acrosome morphology, and failure to form flagella. Consequently, Sas4 cKO males were infertile due to the absence of functional spermatozoa. Our findings demonstrate that while centrioles are dispensable for meiosis in male germ cells, they are essential for spermiogenesis and sperm maturation. This work provides key insights into the role of centrosomes in male fertility and may have implications for understanding certain conditions of male infertility associated with centriole defects.

Generation of functional spermatids from human spermatogonial stem cells (SSCs) in vitro is of utmost importance for uncovering mechanisms underlying human germ cell development and treating infertility. Here we report a three-dimensional-induced (3D-I) system by which human SSCs were efficiently differentiated into functional haploid spermatids. Human SSCs were isolated and identified phenotypically. Meiotic chromatin spreads and DNA content assays revealed that spermatocytes and haploid cells were effectively generated from human SSCs by 3D-I system. Haploid cells derived from human SSCs harbored normal chromosomes and excluded Y chromosome microdeletions. RNA sequencing and bisulfite sequencing analyses reflected similarities in global gene profiles and DNA methylation in human SSCs-derived spermatids and normal round spermatids. Significantly, haploid spermatids generated from human SSCs via 3D-I system were capable of fertilizing mouse oocytes, which subsequently enabled the development of hybrid embryos. This study thus provides invaluable human male gametes for treating male infertility.

Spermatid individualization is a common stage of spermiogenic failure suggesting that a male germline checkpoint may act at this stage during sperm development in Drosophila . However, the molecular identity of such a male germline checkpoint has remained elusive. Here, we show that Overdrive ( Ovd ), a gene dispensable for male fertility, is required for the selective elimination of post-meiotic spermatids targeted by selfish chromosomes. Gamete elimination occurs during the individualization stage of spermiogenesis, following histone-to-protamine transition failure of targeted spermatids. We show that Ovd is necessary for the selfish behavior of segregation distorters across distantly related Drosophila species, indicating that independent selfish chromosomes use common mechanisms. Our study suggests that the normal function of an Ovd- mediated germline checkpoint involves the elimination of abnormal gametes during male germline development, which is exploited by selfish chromosomes into eliminating competing sperm. A hidden quality-control system to filter out defective sperm during development is exploited by selfish genes to eliminate competing chromosomes.

In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

Many long noncoding RNA (lncRNA) species have been identified in gametes. However, the biogenesis and function of other categories of lncRNAs in gametes is poorly understood. Here, we profiled the expression of lncRNAs and mRNAs in spermatogonial stem cells (SSC), type A spermatogonia (A), pachytene spermatocytes (PS) and round spermatids (RS) by microarray analysis. We analyze the total expression of lncRNA/mRNA in these four germ cells and found that the maximum number of lncRNAs expression is in A (22127) and the minimum is in PS (14456). Also, the maximum number of mRNAs is in A (19923) and the minimum is in PS (13941). Furthermore, the trend in the number of specific lncRNAs was similar to the number of specific mRNAs in each type of germ cells (e.g., maximum in A and minimum in PS). The trend in the number of lncRNAs was similar to the number of mRNAs in two continued types of germ cells (e.g., maximum in SSC to A and minimum in PS to RS). The correlation analysis showed a high correlation coefficient of lncRNAs/mRNAs expression (R = 0.992). The results suggested that the sequential expression of long noncoding RNA as mRNA gene expression exhibits coordinated changes in male spermatogenesis.

Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.