Human circBOULE RNAs as potential biomarkers for sperm quality and male infertility.

Reliable molecular biomarkers to predict fertility remain scarce. The current study explored the potential of testis-specific circBOULE RNAs as biomarkers for male infertility and sperm quality. Using RT-PCR and RT-qPCR assays, we identified seven circular RNAs from the human BOULE gene in human sperm. We found that sperm circEx3-6 RNA exhibited a significantly decreased expression in asthenozoospermia while circEx2-6 and circEx2-7 expression decreased in teratozoospermia, compared with the controls. Furthermore, circEx2-6 expression exhibited a negative correlation with sperm DNA Fragmentation Index (DFI), and circEx2-7 levels were correlated with both fertilization and cleavage rates involving assisted reproductive technologies. Further functional analyses in a transgenic fly model lent support for the roles of circBOULE RNAs in sperm development and human fertility. Collectively, our findings support that sperm circBOULE RNAs may serve as diagnostic biomarkers for assessing sperm motility and DNA quality. Hence clinical application and significance of sperm circular RNAs in assisted reproductive technologies warrant further investigation.

A novel pipeline for computerized mouse spermatogenesis staging.

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.

RNA binding protein BOULE forms aggregates in mammalian testis.

Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration. New evidence however increasingly suggests that non-pathological amyloids are formed in animals during normal development. Amyloid-like aggregate formation was originally thought to be a conserved feature of animal gametogenesis. This hypothesis was based on findings which suggest that regulated amyloid formations govern yeast meiosis by way of meiosis-specific RNA binding proteins. Additional support came from studies which demonstrate that DAZL, a mammalian gametogenesis-specific RNA binding protein, also forms SDS-resistant aggregates in vivo. Here, we report evidence of aggregated BOULE formations, another DAZ family protein, during sperm development. Data suggest that in mouse testis, BOULE forms SDS-resistant amyloid-like aggregates. BOULE aggregate formation correlates with dynamic developmental expression during spermatogenesis but disappeared in Boule knockout testis. We also mapped essential small region in vitro BOULE aggregations, immediately downstream DAZ repeats, and found that aggregations positively correlated with temperature. We also performed enhanced UV cross-linking immunoprecipitation on BOULE aggregates from mouse testes and found that aggregates bind with a large number of spermatogenesis-related mRNAs. These findings provide insight into the amyloidogenic properties of gametogenesis-specific RNA binding proteins as a conserved feature in mammalian reproduction. Further investigation is warranted to understand the functional significance of BOULE amyloid-like formation during mouse spermatogenesis.

Sertoli cell PUMILIO proteins modulate mouse testis size through translational control of cell cycle regulators.

Testis size determination is an important question of reproductive biology. Sertoli cells are known to be a key determinant of mammalian testis size but the underlying molecular mechanisms remain incompletely understood. Previously we showed that highly conserved germ cell RNA-binding proteins, PUMILIO1(PUM1) and PUMILIO2 (PUM2), control mouse organ and body size through translational regulation, but how different cell types of the organs contribute to their organ size regulation has not been established. Here, we report a somatic role of PUM in gonad size determination. PUM1 is highly expressed in the Sertoli cells of the developing testis from embryonic and postnatal mice as well as in germ cells. Removal of Sertoli cell, but not germ cell, Pum1 gene, led to reduced testis size without significantly affecting sperm number or fertility. Knockout of PUM1 target, Cdkn1b, rescued the phenotype of reduced testis size, supporting a key role of Sertoli cell PUM1 mediated Cdkn1b repression in the testis size control. Furthermore, removal of Pum2 or both Pum1 and Pum2 in the Sertoli cells also only affected the testis size, not sperm development, with the biggest size reduction in Pum1/2 double knockout mice. We propose that PUM1 and PUM2 modulate the testis size through their synergistic translational regulation of cell cycle regulators in the Sertoli cell. Further investigation of the ovary or other organs could reveal if PUM-mediated translational control of cell proliferation of the supporting cell represents a general mechanism for organ size modulation.

PUMILIO-mediated translational control of somatic cell cycle program promotes folliculogenesis and contributes to ovarian cancer progression.

Translational control is a fundamental mechanism regulating animal germ cell development. Gonadal somatic cells provide support and microenvironment for germ cell development to ensure fertility, yet the roles of translational control in gonadal somatic compartment remain largely undefined. We found that mouse homolog of conserved fly germline stem cell factor Pumilio, PUM1, is absent in oocytes of all growing follicles after the primordial follicle stage, instead, it is highly expressed in somatic compartments of ovaries. Global loss of Pum1, not oocyte-specific loss of Pum1, led to a significant reduction in follicular number and size as well as fertility. Whole-genome identification of PUM1 targets in ovarian somatic cells revealed an enrichment of cell proliferation pathway, including 48 key regulators of cell phase transition. Consistently granulosa cells proliferation is reduced and the protein expression of the PUM-bound Cell Cycle Regulators (PCCR) were altered accordingly in mutant ovaries, and specifically in granulosa cells. Increase in negative regulator expression and decrease in positive regulators in the mutant ovaries support a coordinated translational control of somatic cell cycle program via PUM proteins. Furthermore, postnatal knockdown, but not postnatal oocyte-specific loss, of Pum1 in Pum2 knockout mice reduced follicular growth and led to similar expression alteration of PCCR genes, supporting a critical role of PUM-mediated translational control in ovarian somatic cells for mammalian female fertility. Finally, expression of human PUM protein and its regulated cell cycle targets exhibited significant correlation with ovarian cancer and prognosis for cancer survival. Hence, PUMILIO-mediated cell cycle regulation represents an important mechanism in mammalian female reproduction and human cancer biology.

Deep SED-Net with interactive learning for multiple testicular cell types segmentation and cell composition analysis in mouse seminiferous tubules.

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.

PUM1 represses CDKN1B translation and contributes to prostate cancer progression.

Posttranscriptional regulation of cancer gene expression programs plays a vital role in carcinogenesis; identifying the critical regulators of tumorigenesis and their molecular targets may provide novel strategies for cancer diagnosis and therapeutics. Highly conserved RNA-binding protein Pumilio-1 (PUM1) regulates mouse growth and cell proliferation, propelling us to examine its role in cancer. We found human PUM1 is highly expressed in a diverse group of cancer, including prostate cancer; enhanced PUM1 expression is also correlated with reduced survival among prostate cancer patients. Detailed expression analysis in twenty prostate cancer tissues showed enhanced expression of PUM1 at mRNA and protein levels. Knockdown of PUM1 reduced prostate cancer cell proliferation and colony formation, and subcutaneous injection of PUM1 knockdown cells led to reduced tumor size. Downregulation of PUM1 in prostate cancer cells consistently elevated cyclin-dependent kinase inhibitor 1B (CDKN1B) protein expression through increased translation but did not impact its mRNA level, while overexpression of PUM1 reduced CDKN1B protein level. Our finding established a critical role of PUM1 mediated translational control, particularly the PUM1-CDKN1B axis, in prostate cancer cell growth and tumorigenesis. We proposed that PUM1-CDKN1B regulatory axis may represent a novel mechanism for the loss of CDKN1B protein expression in diverse cancers and potential targets for therapeutics development.

Computerized spermatogenesis staging (CSS) of mouse testis sections via quantitative histomorphological analysis.

Spermatogenesis in mammals is a cyclic process of spermatogenic cell development in the seminiferous epithelium that can be subdivided into 12 subsequent stages. Histological staging analysis of testis sections, specifically of seminiferous tubule cross-sections, is the only effective method to evaluate the quality of the spermatogenic process and to determine developmental defects leading to infertility. Such staging analysis, however, is tedious and time-consuming, and it may take a long time to become proficient. We now have developed a Computerized Staging system of Spermatogenesis (CSS) for mouse testis sections through learning of an expert with decades of experience in mouse testis staging. The development of the CSS system comprised three major parts: 1) Developing computational image analysis models for mouse testis sections; 2) Automated classification of each seminiferous tubule cross-section into three stage groups: Early Stages (ES: stages I-V), Middle Stages (MS: stages VI-VIII), and Late Stages (LS: stages IV-XII); 3) Automated classification of MS into distinct stages VI, VII-mVIII, and late VIII based on newly developed histomorphological features. A cohort of 40 H&E stained normal mouse testis sections was built according to three modules where 28 cross-sections were leveraged for developing tubule region segmentation, spermatogenic cells types and multi-concentric-layers segmentation models. The rest of 12 testis cross-sections, approximately 2314 tubules whose stages were manually annotated by two expert testis histologists, served as the basis for developing the CSS system. The CSS system's accuracy of mean and standard deviation (MSD) in identifying ES, MS, and LS were 0.93 ± 0.03, 0.94 ± 0.11, and 0.89 ± 0.05 and 0.85 ± 0.12, 0.88 ± 0.07, and 0.96 ± 0.04 for one with 5 years of experience, respectively. The CSS system's accuracy of MSD in identifying stages VI, VII-mVIII, and late VIII are 0.74 ± 0.03, 0.85 ± 0.04, and 0.78 ± 0.06 and 0.34 ± 0.18, 0.78 ± 0.16, and 0.44 ± 0.25 for one with 5 years of experience, respectively. In terms of time it takes to collect these data, it takes on average 3 hours for a histologist and 1.87 hours for the CSS system to finish evaluating an entire testis section (computed with a PC (I7-6800k 4.0 GHzwith 32GB of RAM & 256G SSD) and a Titan 1080Ti GPU). Therefore, the CSS system is more accurate and faster compared to a human histologist in staging, and further optimization and development will not only lead to a complete staging of all 12 stages of mouse spermatogenesis but also could aid in the future diagnosis of human infertility. Moreover, the top-ranking histomorphological features identified by the CSS classifier are consistent with the primary features used by histologists in discriminating stages VI, VII-mVIII, and late VIII.

Circular RNAs from BOULE play conserved roles in protection against stress-induced fertility decline.

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.

LYPD4, mouse homolog of a human acrosome protein, is essential for sperm fertilizing ability and male fertility†.

Fertilization is one of the fundamental biological processes, but so far, we still do not have a full understanding of the underlying molecular mechanism. We have identified a human acrosome protein, LY6/PLAUR domain containing 4 (LYPD4), expressed specifically in human testes and sperm, and conserved within mammals. Mouse Lypd4, also specific to the testis and sperm, is essential for male fertility. LYPD4 protein first appeared in round spermatids during acrosome biogenesis and became part of acrosomes during spermatogenesis and in mature sperm. Lypd4 knockout mice are infertile with normal sperm number and motility. Mutant sperm, however, failed to reach oviduct during sperm migration inside the female reproductive tract, leading to fertilization failure and infertility. In addition, Lypd4 mutant sperms were unable to fertilize denuded egg via IVF (in vitro fertilization) but could fertilize eggs within intact Cumulus-Oocyte Complex, supporting an additional role in sperm-zona interaction. Out of more than five thousand spermatozoa proteins identified by mass spectrometry analysis, only a small subset of proteins (26 proteins) was changed in the absence of LYPD4, revealing a whole proteome picture of mutant sperm defective in sperm migration and sperm-zona binding. ADAM3, a key component of fertilization complex, as well as other sperm ADAM proteins are significantly reduced. We hence propose that LYPD4 plays an essential role in mammalian fertilization, and further investigation of its function and its interaction with other sperm membrane complexes may yield insights into human fertilization and novel strategy to improve IVF success.

DAZL is a master translational regulator of murine spermatogenesis.

Expression of DAZ-like (DAZL) is a hallmark of vertebrate germ cells, and is essential for embryonic germ cell development and differentiation, yet the gametogenic function of DAZL has not been fully characterized and most of its in vivo direct targets remain unknown. We showed that postnatal stage-specific deletion of Dazl in mouse germ cells did not affect female fertility, but caused complete male sterility with gradual loss of spermatogonial stem cells, meiotic arrest and spermatid arrest. Using the genome-wide high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation and mass spectrometry approach, we found that DAZL bound to a large number of testicular mRNA transcripts (at least 3008) at the 3'-untranslated region and interacted with translation proteins including poly(A) binding protein. In the absence of DAZL, polysome-associated target transcripts, but not their total transcripts, were significantly decreased, resulting in a drastic reduction of an array of spermatogenic proteins and thus developmental arrest. Thus, DAZL is a master translational regulator essential for spermatogenesis.

Mammalian Pum1 and Pum2 Control Body Size via Translational Regulation of the Cell Cycle Inhibitor Cdkn1b.

Body and organ size regulation in mammals involves multiple signaling pathways and remains largely enigmatic. Here, we report that Pum1 and Pum2, which encode highly conserved PUF RNA-binding proteins, regulate mouse body and organ size by post-transcriptional repression of the cell cycle inhibitor Cdkn1b. Binding of PUM1 or PUM2 to Pumilio binding elements (PBEs) in the 3' UTR of Cdkn1b inhibits translation, promoting G1-S transition and cell proliferation. Mice with null mutations in Pum1 and Pum2 exhibit gene dosage-dependent reductions in body and organ size, and deficiency for Cdkn1b partially rescues postnatal growth defects in Pum1-/- mice. We propose that coordinated tissue-specific expression of Pum1 and Pum2, which involves auto-regulatory and reciprocal post-transcriptional repression, contributes to the precise regulation of body and organ size. Hence PUM-mediated post-transcriptional control of cell cycle regulators represents an additional layer of control in the genetic regulation of organ and body size.

Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice.

PUMILIO 2 (PUM2) is a member of Pumilio and FBF (PUF) family, an RNA binding protein family with phylogenetically conserved roles in germ cell development. The Drosophila Pumilio homolog is also required for dendrite morphogenesis and synaptic function via translational control of synaptic proteins, such as glutamate receptors, and recent mammalian studies demonstrated a similar role in neuronal culture with associated motor and memory abnormalities in vivo. Importantly, transgenic mice with PUM2 knockout show prominent epileptiform activity, and patients with intractable temporal lobe epilepsy and mice with pilocarpine-induced seizures have decreased neuronal PUM2, possibly leading to further seizure susceptibility. However, how PUM2 influences synaptic function in vivo and, subsequently, seizures is not known. We found that PUM2 is highly expressed in the brain, especially in the temporal lobe, and knockout of Pum2 (Pum2-/- ) resulted in significantly increased pyramidal cell dendrite spine and synapse density. In addition, multiple proteins associated with excitatory synaptic function, including glutamate receptor 2 (GLUR2), are up-regulated in Pum2-/- mice. The expression of GLUR2 protein but not mRNA is increased in the Pum2-/- mutant hippocampus, Glur2 transcripts are increased in mutant polysome fractions, and overexpression of PUM2 led to repression of reporter expression containing the 3'Untranslated Region (3'UTR) of Glur2, suggesting translation of GLUR2 was increased in the absence of Pum2. Overall, these studies provide a molecular mechanism for the increased temporal lobe excitability observed with PUM2 loss and suggest PUM2 might contribute to intractable temporal lobe epilepsy.

Essential requirement of mammalian Pumilio family in embryonic development.

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.

Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

Separation of germ cells from somatic cells is a widespread feature of animal sexual reproduction, with a core set of germ cell factors conserved among diverse animals. It is not known what controls their conserved gonad-specific expression. Core components of epigenetic machinery are ancient, but its role in conserved tissue expression regulation remains unexplored. We found that promoters of the reproductive genes BOULE and DAZL exhibit differential DNA methylation, consistent with their gonad-specific expression in humans and mice. Low or little promoter methylation from the testicular tissue is attributed to spermatogenic cells of various stages in the testis. Such differential DNA methylation is present in the orthologous promoters not only of other mammalian species, but also of chickens and fish, supporting a highly conserved epigenetic mechanism. Furthermore, hypermethylation of DAZL and BOULE promoters in human sperm is associated with human infertility. Our data strongly suggest that epigenetic regulation may underlie conserved germ-cell-specific expression, and such a mechanism may play an important role in human fertility.-Zhang, C., Xue, P., Gao, L., Chen, X., Lin, K., Yang, X., Dai, Y., Xu, E. Y. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

LncRNA, a new component of expanding RNA-protein regulatory network important for animal sperm development.

Spermatogenesis is one of the fundamental processes of sexual reproduction, present in almost all metazoan animals. Like many other reproductive traits, developmental features and traits of spermatogenesis are under strong selective pressure to change, both at morphological and underlying molecular levels. Yet evidence suggests that some fundamental features of spermatogenesis may be ancient and conserved among metazoan species. Identifying the underlying conserved molecular mechanisms could reveal core components of metazoan spermatogenic machinery and provide novel insight into causes of human infertility. Conserved RNA-binding proteins and their interacting RNA network emerge to be a common theme important for animal sperm development. We review research on the recent addition to the RNA family - Long non-coding RNA (lncRNA) and its roles in spermatogenesis in the context of the expanding RNA-protein network.

Loss of preimplantation embryo resulting from a Pum1 gene trap mutation.

Pumilio is a member of the highly conserved PUF family of RNA-binding proteins that function as a developmental regulator in diverse animal species. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals and are found to be involved in sperm development, neuron development as well as human diseases such as neurodegeneration. Generation of animal models disrupting different parts of Pum protein could help to further dissect their physiological function. Here we described characterization and analysis of a mouse line possessing a gene trap mutation of the Pumilio1 (Pum1) gene. Mice homozygous for the mutation (Pum1(XE002)) cannot be recovered in the adult offspring, at birth or at different time points of embryonic development (E18, E14, E12). Careful analysis of preimplantation embryos showed that no homozygous blastocysts could be detected on day 3.5 of gestation. 96-hr in vitro culture of 1-cell embryos either by natural mating or in vitro fertilization between heterozygotes failed to uncover any homozygous blastocysts, suggesting an early loss of homozygous preimplantation embryos. The lack of Pum1 gene trap homozygotes suggests a role of Pum1 in very early embryonic development or fertilization. This novel animal model affecting the beginning of embryonic development could help to understand not only the genetic mechanism underlying preimplantation embryonic development but also the translational regulation in development and diseases.

Widespread presence of human BOULE homologs among animals and conservation of their ancient reproductive function.

Sex-specific traits that lead to the production of dimorphic gametes, sperm in males and eggs in females, are fundamental for sexual reproduction and accordingly widespread among animals. Yet the sex-biased genes that underlie these sex-specific traits are under strong selective pressure, and as a result of adaptive evolution they often become divergent. Indeed out of hundreds of male or female fertility genes identified in diverse organisms, only a very small number of them are implicated specifically in reproduction in more than one lineage. Few genes have exhibited a sex-biased, reproductive-specific requirement beyond a given phylum, raising the question of whether any sex-specific gametogenesis factors could be conserved and whether gametogenesis might have evolved multiple times. Here we describe a metazoan origin of a conserved human reproductive protein, BOULE, and its prevalence from primitive basal metazoans to chordates. We found that BOULE homologs are present in the genomes of representative species of each of the major lineages of metazoans and exhibit reproductive-specific expression in all species examined, with a preponderance of male-biased expression. Examination of Boule evolution within insect and mammalian lineages revealed little evidence for accelerated evolution, unlike most reproductive genes. Instead, purifying selection was the major force behind Boule evolution. Furthermore, loss of function of mammalian Boule resulted in male-specific infertility and a global arrest of sperm development remarkably similar to the phenotype in an insect boule mutation. This work demonstrates the conservation of a reproductive protein throughout eumetazoa, its predominant testis-biased expression in diverse bilaterian species, and conservation of a male gametogenic requirement in mice. This shows an ancient gametogenesis requirement for Boule among Bilateria and supports a model of a common origin of spermatogenesis.

A novel requirement in mammalian spermatid differentiation for the DAZ-family protein Boule.

Reproduction is required for the survival of all animals, yet few reproductive genes have been shown to have a conserved requirement for fertility across the animal kingdom. Remarkably, the RNA binding protein BOULE, the oldest member of the DAZ (Deleted in AZoospermia) family of genes, appears to have maintained its conserved functional motif and spermatogenic expression from insects to humans. Boule mutations lead to a pachytene meiotic arrest before metaphase in Drosophila males and C. elegans females, and human BOULE can restore meiosis in the fly testis, suggesting a conserved meiotic function of human BOULE. However, the physiological function of BOULE in mammals is not yet known. We generated Boule knockout mice and found it to be required only for spermatogenesis, as in Drosophila. Interestingly, meiosis completed normally in the absence of Boule, and haploid round spermatids were readily detected. However, round spermatids did not progress beyond step 6, revealing a novel role for Boule in spermiogenesis, the differentiation of round spermatids into mature spermatozoa. Expression of key regulators of spermiogenesis was unaffected in Boule(-/-) mice, suggesting that Boule regulates germ-cell differentiation through a novel pathway.

Identification and characterization of novel mammalian spermatogenic genes conserved from fly to human.

Spermatogenesis is a complex and highly regulated developmental process by which round spermatogonial stem cells undergo mitotic proliferation and meiosis, followed by extraordinary differentiation into highly specialized elongated mature sperm. Extensive differences in terms of sperm production such as testicular structure and organization, hormonal regulation are reported between humans and insects, yet it is not known to what extent components of the process could be conserved and furthermore to what extent the underlying genetic regulators could be shared from insects to mammals. We hence take a genomic approach to identify genes which are expressed in the testes of both fly and mouse through in silico analysis and are phylogenetically conserved across metazoans. Fifty eight testis-enriched, phylogenetically conserved from fly to mouse genes were identified. Among them, 12 genes are novel. Detailed characterization of their murine and human homologs indicate most of them are testis-restricted or enriched and developmentally regulated, thus suggesting that they are important regulators of sperm development in mammals and potential human fertility factors. Our results reveal the existence of spermatogenic homologs with similar testicular expression across a large evolutionary distance, further functional study will be needed to explore the functional conservation among those spermatogenic orthologs.