Mouse Pramel1 regulates spermatogonial development by inhibiting retinoic acid signaling during spermatogenesis.

Spermatogenesis begins when cell fate-committed prospermatogonia migrate to the basement membrane and initiate spermatogenesis in response to retinoic acid (RA) in the neonatal testis. The underlying cellular and molecular mechanisms in this process are not fully understood. Here, we report findings on the involvement of a cancer/testis antigen, PRAMEL1, in the initiation and maintenance of spermatogenesis. By analyzing mouse models with either global or conditional Pramel1 inactivation, we found that PRAMEL1 regulates the RA responsiveness of the subtypes of prospermatogonia in the neonatal testis, and affects their homing process during the initiation of spermatogenesis. Pramel1 deficiency led to increased fecundity in juvenile males and decreased fecundity in mature males. In addition, Pramel1 deficiency resulted in a regional Sertoli cell-only phenotype during the first round of spermatogenesis, which was rescued by administration of the RA inhibitor WIN18,446, suggesting that PRAMEL1 functions as an inhibitor of RA signaling in germ cells. Overall, our findings suggest that PRAMEL1 fine-tunes RA signaling, playing a crucial role in the proper establishment of the first and subsequent rounds of spermatogenesis.

Reversible Phase Transformations in a Double-Walled Diamondoid Coordination Network with a Stepped Isotherm for Methane.

Flexible metal-organic materials (FMOMs) with stepped isotherms can offer enhanced working capacity in storage applications such as adsorbed natural gas (ANG) storage. Unfortunately, whereas >1000 FMOMs are known, only a handful exhibit methane uptake of >150 cm3/cm3 at 65 atm and 298 K, conditions relevant to ANG. Here, we report a double-walled 2-fold interpenetrated diamondoid (dia) network, X-dia-6-Ni, [Ni2L4(µ-H2O)]n, comprising a new azo linker ligand, L- (L- = (E)-3-(pyridin-4-yldiazenyl)benzoate) and 8-connected dinuclear molecular building blocks. X-dia-6-Ni exhibited gas (CO2, N2, CH4) and liquid (C8 hydrocarbons)-induced reversible transformations between its activated narrow-pore ß phase and γ, a large-pore phase with ca. 33% increase in unit cell volume. Single-crystal X-ray diffraction (SCXRD) studies of the as-synthesized phase α, ß, and γ revealed that structural transformations were enabled by twisting of the azo moiety and/or deformation of the MBB. Further insight into these transformations was gained from variable temperature powder XRD and in situ variable pressure powder XRD. Low-temperature N2 and CO2 sorption revealed stepped Type F-II isotherms with saturation uptakes of 422 and 401 cm3/g, respectively. X-dia-6-Ni exhibited uptake of 200 cm3/cm3 (65 atm, 298 K) and a high CH4 working capacity of 166 cm3/cm3 (5-65 bar, 298 K, 33 cycles), the third highest value yet reported for an FMOM and the highest value for an FMOM with a Type F-II isotherm.

Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45 h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

Highly Robust Microporous Metal-Organic Frameworks for Efficient Ethylene Purification under Dry and Humid Conditions.

Two C2 H6 -selective metal-organic framework (MOF) adsorbents with ultrahigh stability, high surface areas, and suitable pore size have been designed and synthesized for one-step separation of ethane/ethylene (C2 H6 /C2 H4 ) under humid conditions to produce polymer-grade pure C2 H4 . Experimental results reveal that these two MOFs not only adsorb a high amount of C2 H6 but also display good C2 H6 /C2 H4 selectivity verified by fixed bed column breakthrough experiments. Most importantly, the good water stability and hydrophobic pore environments make these two MOFs capable of efficiently separating C2 H6 /C2 H4 under humid conditions, exhibiting the benchmark performance among all reported adsorbents for separation of C2 H6 /C2 H4 under humid conditions. Moreover, the affinity sites and their static adsorption energies were successfully revealed by single crystal data and computation studies. Adsorbents described in this work can be used to address major chemical industrial challenges.

Bottom-Up Synthesis of 8-Connected Three-Dimensional Covalent Organic Frameworks for Highly Efficient Ethylene/Ethane Separation.

Developing cost-/energy-efficient separation techniques for purifying ethylene from an ethylene/ethane mixture is highly important but very challenging in the industrial process. Herein, using a bottom-up [8 + 2] construction approach, we rationally designed and synthesized three three-dimensional covalent organic frameworks (COFs) with 8-connected bcu networks, which can selectively remove ethane from an ethylene/ethane mixture with high efficiency. These COF materials, which are fabricated by the condensation reaction of a customer-designed octatopic aldehyde monomer with linear diamino linkers, possess high crystallinity, good structural robustness, and high porosity. Attributed to the well-organized micro-sized pores with a nonpolar/inert pore environment, these COFs display high ethane adsorption capacity and good selectivity over ethylene, making them among the best ethane-selective adsorbents for ethylene purification. Their excellent ethylene/ethane separation performance is validated by dynamic breakthrough experiments with high-purity ethylene (>99.99%) produced through a single adsorption process. The separation performance surpasses all reported C2H6-selective COFs and even some benchmark metal-organic frameworks. This work provides important guidance for the design of new adsorbents for value-added gas purification.

Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG-derived small RNAs present in mature spermatozoa.

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle-associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

Scalable Room-Temperature Synthesis of Highly Robust Ethane-Selective Metal-Organic Frameworks for Efficient Ethylene Purification.

The development of new techniques and materials that can separate ethylene from ethane is highly relevant in modern applications. Although adsorption-based separation techniques using metal-organic frameworks (MOFs) have gained increasing attention, the relatively low stability (especially water resistance) and unscalable synthesis of MOFs severely limit their application in real industrial scenarios. Addressing these challenges, we rationally designed and synthesized two new C2H6-selective MOF adsorbents (NKMOF-8-Br and -Me) with ultrahigh chemical and thermal stability, including water resistance. Attributed to the nonpolar/hydrophobic pore environments and appropriate pore apertures, the MOFs can capture C2 hydrocarbon gases at ambient conditions even in high humidity. The single-crystal structures of gas@NKMOF-8 realized the direct visualization of adsorption sites of the gases. Both the single-crystal data and simulated data elucidate the mechanism of selective adsorption. Moreover, the NKMOF-8 possesses high C2H6 adsorption capacity and high selectivity, allowing for efficient C2H6/C2H4 separation, as verified by experimental breakthrough tests. Most importantly, NKMOF-8-Br and -Me can be scalably synthesized through stirring at room temperature in minutes, which confers them with great potential for industrial application. This work offers new adsorbents that can address major chemical industrial challenges and provides an in-depth understanding of the gas binding sites in a visual manner.

The PRAME family of cancer testis antigens is essential for germline development and gametogenesis†.

Preferentially expressed antigen in melanoma (PRAME) belongs to a group of cancer/testis antigens that are predominately expressed in the testis and a variety of tumors, and are involved in immunity and reproduction. Much of the attention on PRAME has centered on cancer biology as PRAME is a prognostic biomarker for a wide range of cancers and a potential immunotherapeutic target. Less information is available about the PRAME family's function (s) during gametogenesis and in the overall reproduction process. Here, we review the current knowledge of the PRAME gene family and its function in germline development and gametogenesis. Members of the PRAME family are leucine rich repeat proteins, localized in nucleus and cytoplasm, with multifaceted roles in germ cells. As transcriptional regulators, the PRAME family proteins are involved in germline development, particularly in the maintenance of embryonic stem cell pluripotency, development of primordial germ cells, and differentiation/proliferation of spermatogenic and oogenic cells. The PRAME family proteins are also enriched in cytoplasmic organelles, such as rough endoplasmic reticulum, Golgi vesicle, germinal granules, centrioles, and play a role in the formation of the acrosome and sperm tail during spermiogenesis. The PRAME gene family remains transcriptionally active in the germline throughout the entire life cycle and is essential for gametogenesis, with some members specific to either male or female germ cells, while others are involved in both male and female gametogenesis. A potential molecular mechanism that underlies the function of PRAME, and is shared by gametogenesis and oncogenesis is also discussed.

321 W high-efficiency continuous-wave green laser produced by single-pass frequency doubling of narrow-linewidth fiber laser.

In this study, a high-power continuous-wave green laser for copper processing is investigated. The laser is produced by single-pass second-harmonic generation (SHG) of a narrow-linewidth fiber laser. Based on Sellmeier equations, the factors (e.g., fundamental wave linewidth, temperature, and angle) that decide LBO noncritical phase matching are theoretically analyzed for optimizing the SHG setup. A narrow-linewidth polarization-maintained fiber laser is used as the fundamental laser with a linewidth of 20 GHz and polarization extinction ratio of greater than 15 dB. Type I noncritical phase-matching LBO is used as the nonlinear crystal in the SHG. A green laser (up to 321 W) is obtained from a 784 W fundamental laser with a conversion efficiency of 40.9%. The green laser is near-diffraction-limited and has a $ M^{2} $ factor of 1.07.

Deletion of the mouse X-linked Prame gene causes germ cell reduction in spermatogenesis.

Preferentially expressed antigen in melanoma (PRAME) is cancer/testis antigen and a transcriptional repressor, inhibiting the signaling of retinoic acid through the retinoic acid receptor (RAR) for promoting cell proliferation and preventing cell apoptosis in cancer cells. The role of PRAME in testis and germline is unknown. We report here the generation and characterization of an X-linked Prame conditional knockout (cKO) mouse. Although fertile, the testis size (p < .01) and sperm count (p < .05) of the Prame cKO mice were significantly reduced by 12% at 4 months of age compared with the Prame floxed mice. Histological, immunofluorescence with germ cell-specific markers and terminal deoxynucleotidyl transferase dUTP nick end labeling analyses of testis cross-sections at postnatal day 7 (P7), P14, P21, P35, P120, and P365 indicated a significant increase in apoptotic germ cells at P7 and P14 and an increase in abnormal seminiferous tubules at P21 and P35. Germ cells were gradually lost resulting in two different phenotypes in the Prame cKO testes: Sertoli-cell-only for some of the affected tubules in young mice (at P35) and germ cell arrest at spermatogonia stage for other affected tubules in mature mice. Both phenotypes were a consequence of disruption in RAR signaling pathway by the depletion of Prame at a different time point during the first and subsequent rounds of spermatogenesis. The results suggest that Prame plays a minor, but important role in spermatogenesis and different paralogs in the Prame gene family may be functionally and partially redundant.

Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included T cell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

DNA methylation patterns in peripheral blood mononuclear cells from Holstein cattle with variable milk yield.

BACKGROUND: Milk yield for Holstein cows has doubled over five decades due to genetic selection and changes to management, but the molecular mechanisms that facilitated this increase are mostly unknown. Epigenetic modifications to the cattle genome are a plausible molecular mechanism to cause variation in milk yield and our objective was to describe genome-wide DNA methylation patterns in peripheral blood mononuclear cells (PBMC) from mature Holstein dairy cows with variable milk yield. RESULTS: Whole genome MeDIP-seq was performed following DNA extraction from PBMC of 6 lactating dairy cows from 4 different herds that varied in milk yield from 13,556 kg to 23,105 kg per 305 day lactation. We describe methylation across the genome and for 13,677 protein coding genes. Repetitive element reads were primarily mapped to satellite (36.4%), SINE (29.1%), and LINE (23.7%) regions and the majority (78.4%) of CpG sites were sequenced at least once. DNA methylation was generally low upstream of genes with the nadir occurring 95 bp prior to the transcription start site (TSS). Methylation was lower in the first exon than in later exons, was highest for introns near the intron-exon junctions, and declined downstream as the distance from the gene increased. We identified 72 differentially methylated regions (DMR) between high milk yield cows and their control, and 252 DMR across herd environments. CONCLUSIONS: This reference methylome for cattle with extreme variation in milk yield phenotype provides a resource to more fully evaluate relationships between DNA methylation and phenotype in populations subject to selection. The detection of DMR in cows of varying milk yield suggests potential to exploit epigenetic variation in cattle improvement programs.

Acquisition technology for optical ground stations in satellite-ground quantum experiments.

To establish optical links between an optical ground station (OGS) and a quantum experiment satellite, a method of acquisition for the Micius satellite is proposed in this paper and the acquisition technological specification of the OGS system is analyzed. An acquisition strategy for the OGS is designed to meet the requirements of the quantum experiments. A method is designed to point accurately at the quantum satellite and improve the absolute pointing precision. The results show that the correction accuracy is better than 5 µrad, the acquisition time is less than 5 s, and the acquisition probability is 100% so far.

A novel testis-specific protein, PRAMEY, is involved in spermatogenesis in cattle.

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is predominantly expressed in normal testicular tissues and a variety of tumors. The function of the PRAME family in spermatogenesis remains unknown. This study was designed to characterize the Y-linked PRAME (PRAMEY) protein during spermatogenesis in cattle. We found that PRAMEY is a novel male germ cell-specific, and a germinal granule-associated protein that is expressed in spermatogenic cells during spermatogenesis. The intact PRAMEY protein (58 kDa) was detected in different ages of testes but not in epididymal spermatozoa. A PRAMEY isoform (30 kDa) was highly expressed only in testes after puberty and in epididymal spermatozoa. This isoform interacts with PP1γ2 and is likely the mature protein present in the testes and sperm. Immunofluorescent staining demonstrated that PRAMEY was located predominantly in the acrosome granule of spermatids, and in acrosome and flagellum of spermatozoa. Immunogold electron microscopy further localized the PRAMEY protein complex to the nucleus and several cytoplasmic organelles, including the rough endoplasmic reticulum, some small vesicles, the intermitochondrial cement, the chromatoid body and the centrioles, in spermatogonia, spermatocytes, spermatids and/or spermatozoa. PRAMEY was highly enriched in and structurally associated with the matrix of the acrosomal granule (AG) in round spermatids, and migrated with the expansion of the AG during acrosomal biogenesis. While the function of PRAMEY remains unclear during spermatogenesis, our results suggest that PRAMEY may play an essential role in acrosome biogenesis and spermatogenesis.Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/153/6/847/suppl/DC1.FreeSpanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/153/6/847/suppl/DC2.

Worldwide patterns of ancestry, divergence, and admixture in domesticated cattle.

The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.

Male-specific region of the bovine Y chromosome is gene rich with a high transcriptomic activity in testis development.

The male-specific region of the mammalian Y chromosome (MSY) contains clusters of genes essential for male reproduction. The highly repetitive and degenerative nature of the Y chromosome impedes genomic and transcriptomic characterization. Although the Y chromosome sequence is available for the human, chimpanzee, and macaque, little is known about the annotation and transcriptome of nonprimate MSY. Here, we investigated the transcriptome of the MSY in cattle by direct testis cDNA selection and RNA-seq approaches. The bovine MSY differs radically from the primate Y chromosomes with respect to its structure, gene content, and density. Among the 28 protein-coding genes/families identified on the bovine MSY (12 single- and 16 multicopy genes), 16 are bovid specific. The 1,274 genes identified in this study made the bovine MSY gene density the highest in the genome; in comparison, primate MSYs have only 31-78 genes. Our results, along with the highly transcriptional activities observed from these Y-chromosome genes and 375 additional noncoding RNAs, challenge the widely accepted hypothesis that the MSY is gene poor and transcriptionally inert. The bovine MSY genes are predominantly expressed and are differentially regulated during the testicular development. Synonymous substitution rate analyses of the multicopy MSY genes indicated that two major periods of expansion occurred during the Miocene and Pliocene, contributing to the adaptive radiation of bovids. The massive amplification and vigorous transcription suggest that the MSY serves as a genomic niche regulating male reproduction during bovid expansion.

A limited number of Y chromosome lineages is present in North American Holsteins.

Holsteins are the most numerous dairy cattle breed in North America and the breed has undergone intensive selection for improving milk production and conformation. Theoretically, this intensive selection could lead to a reduction of the effective population size and reduced genetic diversity. The objective of this study was to investigate the effective population size of the Holstein Y chromosome and the effects of limited Y chromosome lineages on male reproduction and the future of the breed. Paternal pedigree information of 62,897 Holstein bulls born between 1950 and 2013 in North America and 220,872 bulls evaluated by multiple-trait across-country genetic evaluations of Interbull (Uppsala, Sweden) were collected and analyzed. The results indicated that the number of Y chromosome lineages in Holsteins has undergone a dramatic decrease during the past 50 years because of artificial selection and the application of artificial insemination (AI) technology. All current Holstein AI bulls in North America are the descendants of only 2 ancestors (Hulleman and Neptune H) born in 1880. These 2 ancestral Y-lineages are continued through 3 dominant pedigrees from the 1960s; namely, Pawnee Farm Arlinda Chief, Round Oak Rag Apple Elevation, and Penstate Ivanhoe Star, with a contribution of 48.78, 51.06, and 0.16% to the Holstein bull population in the 2010s, respectively. The Y-lineage of Penstate Ivanhoe Star is almost eliminated from the breed. The genetic variations in the 2 ancestral Y-lineages were evaluated among 257 bulls by determining the copy number variations (CNV) of 3 Y-linked gene families: PRAMEY, HSFY, and ZNF280BY, which are spread along the majority (95%) of the bovine Y chromosome male-specific region (MSY). No significant difference was found between the 2 ancestral Y-lineages, although large CNV were observed within each lineage. This study suggests minimal genetic diversity on the Y chromosome in Holsteins and provides a starting point for investigating the effect of the extremely limited number of Y-lineages on male reproduction and other traits important for the future of the Holstein breed.

Copy number variations of the extensively amplified Y-linked genes, HSFY and ZNF280BY, in cattle and their association with male reproductive traits in Holstein bulls.

BACKGROUND: Recent transcriptomic analysis of the bovine Y chromosome revealed at least six multi-copy protein coding gene families, including TSPY, HSFY and ZNF280BY, on the male-specific region (MSY). Previous studies indicated that the copy number variations (CNVs) of the human and bovine TSPY were associated with male fertility in men and cattle. However, the relationship between CNVs of the bovine Y-linked HSFY and ZNF280BY gene families and bull fertility has not been investigated. RESULTS: We investigated the copy number (CN) of the bovine HSFY and ZNF280BY in a total of 460 bulls from 15 breeds using a quantitative PCR approach. We observed CNVs for both gene families within and between cattle breeds. The median copy number (MCN) of HSFY among all bulls was 197, ranging from 21 to 308. The MCN of ZNF280BY was 236, varying from 28 to 380. Furthermore, bulls in the Bos taurus (BTA) lineage had a significantly higher MCN (202) of HSFY than bulls in the Bos indicus (BIN) lineage (178), while taurine bulls had a significantly lower MCN (231) of ZNF280BY than indicine bulls (284). In addition, the CN of ZNF280BY was positively correlated to that of HSFY on the BTAY. Association analysis revealed that the CNVs of both HSFY and ZNF280BY were correlated negatively with testis size, while positively with sire conception rate. CONCLUSION: The bovine HSFY and ZNF280BY gene families have extensively expanded on the Y chromosome during evolution. The CN of both gene families varies significantly among individuals and cattle breeds. These variations were associated with testis size and bull fertility in Holstein, suggesting that the CNVs of HSFY and ZNF280BY may serve as valuable makers for male fertility selection in cattle.

Synergistic enhancement of the mouse Pramex1 and Pramel1 in repressing retinoic acid (RA) signaling during gametogenesis.

BACKGROUND: PRAME constitutes one of the largest multi-copy gene families in Eutherians, encoding cancer-testis antigens (CTAs) with leucine-rich repeats (LRR) domains, highly expressed in cancer cells and gametogenic germ cells. This study aims to elucidate genetic interactions between two members, Pramex1 and Pramel1, in the mouse Prame family during gametogenesis using a gene knockout approach. RESULT: Single-gene knockout (sKO) of either Pramex1 or Pramel1 resulted in approximately 7% of abnormal seminiferous tubules, characterized by a Sertoli-cell only (SCO) phenotype, impacting sperm count and fecundity significantly. Remarkably, sKO female mice displayed normal reproductive functions. In contrast, Pramex1/Pramel1 double knockout (dKO) mice exhibited reduced fecundity in both sexes. In dKO females, ovarian primary follicle count decreased by 50% compared to sKO and WT mice, correlating with a 50% fecundity decrease. This suggested compensatory roles during oogenesis in Pramex1 or Pramel1 sKO females. Conversely, dKO males showed an 18% frequency of SCO tubules, increased apoptotic germ cells, and decreased undifferentiated spermatogonia compared to sKO and WT testes. Western blot analysis with PRAMEX1- or PRAMEL1-specific antibodies on sKO testes revealed compensatory upregulation of each protein (30-50%) in response to the other gene's deletion. Double KO males exhibited more severe defects in sperm count and litter size, surpassing Pramex1 and Pramel1 sKO accumulative effects, indicating a synergistic enhancement interaction during spermatogenesis. Additional experiments administering trans-retinoic acid (RA) and its inhibitor (WIN18,446) in sKO, dKO, and WT mice suggested that PRAMEX1 and PRAMEL1 synergistically repress the RA signaling pathway during spermatogenesis. CONCLUSION: Data from sKO and dKO mice unveil a synergistic interaction via the RA signaling pathway between Pramex1 and Pramel1 genes during gametogenesis. This discovery sets the stage for investigating interactions among other members within the Prame family, advancing our understanding of multi-copy gene families involved in germ cell formation and function.

Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.