Abstract The development of mouse spermatozoa is a continuous process from spermatogonia, spermatocytes, spermatids to mature sperm. Those developing germ cells (spermatogonia, spermatocyte, and spermatids) together with supporting sertoli cells are all enclosed inside seminiferous tubules of the testis, their identification is key to testis histology and pathology analysis. Automated segmentation of all these cells is a challenging task because of their dynamical changes in different stages. The accurate segmentation of testicular cells is critical in developing computerized spermatogenesis staging. In this paper, we present a novel segmentation model, SED‐Net, which incorporates a squeeze‐and‐excitation (SE) module and a dense unit. The SE module optimizes and obtains features from different channels, whereas the dense unit uses fewer parameters to enhance the use of features. A human‐in‐the‐loop strategy, named deep interactive learning, is developed to achieve better segmentation performance while reducing the workload of manual annotation and time consumption. Across a cohort of 274 seminiferous tubules from stages VI to VIII, the SED‐Net achieved a pixel accuracy of 0.930, a mean pixel accuracy of 0.866, a mean intersection over union of 0.710, and a frequency weighted intersection over union of 0.878, respectively, in terms of four types of testicular cell segmentation. There is no significant difference between manual annotated tubules and segmentation results by SED‐Net in cell composition analysis for tubules from stages VI to VIII. In addition, we performed cell composition analysis on 2346 segmented seminiferous tubule images from 12 segmented testicular section results. The results provided quantitation of cells of various testicular cell types across 12 stages. The rule reflects the cell variation tendency across 12 stages during development of mouse spermatozoa. The method could enable us to not only analyze cell morphology and staging during the development of mouse spermatozoa but also potentially could be applied to the study of reproductive diseases such as infertility.
Members of the Pumilio (also called PUF) gene family belong to a class of highly conserved developmental regulators that are present in both flies and humans. Much is known about the function of Pumilio genes in invertebrate development, in particular their role as stem cell factors required for maintenance and/or self-renewal of germline stem cells in Drosophila and Caenorhabditis elegans. It remains unknown whether Pumilio genes are also required for development in mammals; however, several lines of evidence suggest similar functions based on extensive sequence homology, similar RNA-binding properties to their invertebrate counterparts and well-documented interactions with germ cell factors required for fertility. Here we report characterization of a gene trap mutation that disrupts the mouse Pumilio-2 (Pum2) gene. Our data confirm that Pumilio-2 is expressed most abundantly in germ cells with the highest expression in undifferentiated gonocytes and spermatogonia. Furthermore, the mutation in Pum2 results in significantly smaller testes although the mutants are otherwise viable and fertile. In addition, we observed no stronger reproductive defects on a genetic background homozygous for a Pum2 null mutation and heterozygous for a Dazl mutation than Pum2 mutant alone. Thus, as in C. elegans where single members of the Pumilio gene family are dispensable for reproductive development and viability, this individual member of the Pumilio gene family in mice is also not essential for reproduction or viability.
Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden-Spatz syndrome) is a rare but devastating neurodegenerative disorder, resulting from an inherited defect in coenzyme A biosynthesis. As pathology in the human condition is limited to the central nervous system, specifically the retina and globus pallidus, we have generated a mouse knock-out of the orthologous murine gene (Pank2) to enhance our understanding of the mechanisms of disease and to serve as a testing ground for therapies. Over time, the homozygous null mice manifest retinal degeneration, as evidenced by electroretinography, light microscopy and pupillometry response. Specifically, Pank2 mice show progressive photoreceptor decline, with significantly lower scotopic a- and b-wave amplitudes, decreased cell number and disruption of the outer segment and reduced pupillary constriction response when compared with those of wild-type littermates. Additionally, the homozygous male mutants are infertile due to azoospermia, a condition that was not appreciated in the human. Arrest occurs in spermiogenesis, with complete absence of elongated and mature spermatids. In contrast to the human, however, no changes were observed in the basal ganglia by MRI or by histological exam, nor were there signs of dystonia, even after following the mice for one year. Pank2 mice are 20% decreased in weight when compared with their wild-type littermates; however, dysphagia was not apparent. Immunohistochemistry shows staining consistent with localization of Pank2 to the mitochondria in both the retina and the spermatozoa.
Circular RNAs (circRNAs) are a large family of newly identified transcripts, and their physiological roles and evolutionary significance require further characterization. Here, we identify circRNAs generated from a conserved reproductive gene, Boule, in species from Drosophila to humans. Flies missing circular Boule (circBoule) RNAs display decreased male fertility, and sperm of circBoule knockout mice exhibit decreased fertilization capacity, when under heat stress conditions. During spermatogenesis, fly circBoule RNAs interact with heat shock proteins (HSPs) Hsc4 and Hsp60C, and mouse circBoule RNAs in sperm interact with HSPA2. circBoule RNAs regulate levels of HSPs by promoting their ubiquitination. The interaction between HSPA2 and circBoule RNAs is conserved in human sperm, and lower levels of the human circBoule RNAs circEx3-6 and circEx2-7 are found in asthenozoospermic sperm. Our findings reveal conserved physiological functions of circBoule RNAs in metazoans and suggest that specific circRNAs may be critical modulators of male reproductive function against stresses in animals.
It is generally believed that Drosophila melanogaster has no closely related species with which it can produce the viable and fertile hybrids that are essential for the genetic analysis of speciation. Following the recent report of molecular differentiation between a Zimbabwe, Africa, population and two United States populations, we provide evidence that strong sexual isolation exists between the D. melanogaster population in Zimbabwe and populations of other continents. In the presence of males of their own kind, females from most isofemale lines of Zimbabwe would not mate with males from elsewhere; the reciprocal mating is also significantly reduced, but to a lesser degree. The genes for sexual behaviors are apparently polymorphic in Zimbabwe and postmating reproductive isolation between this and other populations has not yet evolved. Whole chromosome substitutions indicate significant genetic contributions to male mating success by both major autosomes, whereas the X chromosome effect is too weak to measure. In addition, the relative mating success between hybrid and pure line males supports the interpretation of strong female choice. These observations suggest that we are seeing the early stages of speciation in this group and that it is driven by sexual selection. The genetic and molecular tractability of D. melanogaster offers great promise for the detailed analysis of this apparent case of incipient speciation.
Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3'UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.
Abstract Motivation Differentiating 12 stages of the mouse seminiferous epithelial cycle is vital towards understanding the dynamic spermatogenesis process. However, it is challenging since two adjacent spermatogenic stages are morphologically similar. Distinguishing Stages I–III from Stages IV–V is important for histologists to understand sperm development in wildtype mice and spermatogenic defects in infertile mice. To achieve this, we propose a novel pipeline for computerized spermatogenesis staging (CSS). Results The CSS pipeline comprises four parts: (i) A seminiferous tubule segmentation model is developed to extract every single tubule; (ii) A multi-scale learning (MSL) model is developed to integrate local and global information of a seminiferous tubule to distinguish Stages I–V from Stages VI–XII; (iii) a multi-task learning (MTL) model is developed to segment the multiple testicular cells for Stages I–V without an exhaustive requirement for manual annotation; (iv) A set of 204D image-derived features is developed to discriminate Stages I–III from Stages IV–V by capturing cell-level and image-level representation. Experimental results suggest that the proposed MSL and MTL models outperform classic single-scale and single-task models when manual annotation is limited. In addition, the proposed image-derived features are discriminative between Stages I–III and Stages IV–V. In conclusion, the CSS pipeline can not only provide histologists with a solution to facilitate quantitative analysis for spermatogenesis stage identification but also help them to uncover novel computerized image-derived biomarkers. Availability and implementation https://github.com/jydada/CSS. Supplementary information Supplementary data are available at Bioinformatics online.
Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.
The Deleted in AZoospermia ( DAZ ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE . With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30–40 million years ago and whose deletion is linked to infertility in men; DAZL, the “father” of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the “grandfather” of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule , the proposed ortholog of DAZ , in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies ( BOULE and DAZL ) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.
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