Role In Spermatogenesis Research Articles

Reproductive Consequences of Electrolyte Disturbances in Domestic Animals.

Electrolyte balance is essential to maintain homeostasis in the body. The most crucial electrolytes are sodium (Na+), potassium (K+), magnesium (Mg2+), chloride (Cl-), and calcium (Ca2+). These ions maintain the volume of body fluids, and blood pressure, participate in muscle contractions, and nerve conduction, and are important in enzymatic reactions. The balance is mainly ensured by the kidneys, which are an important organ that regulates the volume and composition of urine, together with which excess electrolytes are excreted. They are also important in the reproductive system, where they play a key role. In the male reproductive system, electrolytes are important in acrosomal reaction and sperm motility. Sodium, calcium, magnesium, and chloride are related to sperm capacitation. Moreover, Mg2+, Ca2+, and Na+ play a key role in spermatogenesis and the maintenance of morphologically normal spermatozoa. Infertility problems are becoming more common. It is known that disturbances in the electrolyte balance lead to reproductive dysfunction. In men, there is a decrease in sperm motility, loss of sperm capacitation, and male infertility. In the female reproductive system, sodium is associated with estrogen synthesis. In the contraction and relaxation of the uterus, there is sodium, potassium, and calcium. Calcium is associated with oocyte activation. In turn, in women, changes in the composition of the follicular fluid are observed, leading to a restriction of follicular growth. Imbalance of oocyte electrolytes, resulting in a lack of oocyte activation and, consequently, infertility.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

ObjectivesBCL2‐associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24‐skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis.Materials and MethodsCRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high‐testosterone mice model. H&E staining, qRT‐PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis.Results Bag6 exon24 knockout mice show sub‐fertility along with partially impaired blood‐testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24‐deficient testes and sterol regulatory element‐binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up‐regulated, which causes a high serum testosterone level. Additionally, serine/arginine‐rich splicing factor 1 down‐regulates BAG6 exon24‐skipped transcripts in porcine Sertoli cells by binding to 35–51 nt on BAG6 exon24 via its N‐terminal RNA‐recognition domain.ConclusionsOur findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

Mixed model-based eQTL analysis reveals lncRNAs associated with regulation of genes involved in sex determination and spermatogenesis: The key to understanding human gender imbalance

BackgroundAn imbalance in the prenatal sex ratio in humans may be due to several factors affecting sperm physiology, including genetic features. In this study, we conducted a transcriptome-wide analysis of expression quantitative trait loci (eQTLs) to identify target genes associated with previously described QTLs associated with gender imbalance. MethodsA mixed model explaining polygenic effects by genomic covariance among individuals was used to identify the eQTLs using gene expression and genotype data from 462 European/African individuals. ResultsEight eGenes were associated with four QTLs (P < 4.00 × 10-5), with strong associations found (P < 4.00 × 10-8) between rs2485007 and eGenes ANKRD26P3 (P = 3.40 × 10-9) and LINC00421 (P = 1.35 × 10-9). ANKRD26P3 and LINC00421 are both lncRNAs associated with the control of testis-dominant genes PELP1, TAF15, NANOG, TEX14, TCF3, ZNF433, ZNF555, TEX37, FATE1, TCP11, and CYLC2 and Y-linked genes SRY and ZFY, as well as several genes with roles in spermatogenesis (ODF1, SPATC1, SPATA3, SPATA31E1, SPERT, SPATA16, MOSPD1, SPATA24, and SPO11) and sex determination (SOX family genes). ConclusionsThe above eGenes contribute directly or indirectly to gene regulation for sex determination and spermatogenesis, thereby serving as important functional clues for gender-biased selection.

Identification of the TSSK4 Alternative Spliceosomes and Analysis of the Function of the TSSK4 Protein in Yak (Bos grunniens).

Simple SummaryIn mammals, the testis-specific serine/threonine kinase (TSSK) is essential for spermatogenesis and male fertility. This study aimed to analyze the possible alternative spliceosomes of the TSSK4 gene in the yak testis tissue using PCR amplification and cloning techniques. A total of six alternative spliceosomes were obtained, of which there were only two alternative spliceosomes in the yak testis before sexual maturity and four alternative spliceosomes in the yak testis after sexual maturity.In mammals, the testis-specific serine/threonine kinase (TSSK) is essential for spermatogenesis and male fertility. TSSK4 belongs to the family of the testis-specific serine/threonine-protein kinase (TSSK), with a crucial role in spermatogenesis. This study aimed to analyze the variable spliceosome of the TSSK4 gene in the yak for understanding the regulatory function of the TSSK4 spliceosome in yak testis development using PCR amplification and cloning techniques. The GST pull-down was used for pulling down the protein interacting with TSSK4, and then the protein interacting with TSSK4 was identified using LC–MS/MS. The results of the PCR amplification demonstrated multiple bands of the TSSK4 gene in the yak. The cloning and sequencing yielded a total of six alternative spliceosomes, which included only two alternative spliceosomes before sexual maturity and four alternative spliceosomes after sexual maturity. The sub-cells of the alternative spliceosomes were found to localize in the nucleus before sexual maturity and in the cytoplasm after sexual maturity. The LC–MS/MS analysis of the alternative spliceosome with the highest expression after sexual maturity yielded a total of 223 interacting proteins. The enrichment analysis of the 223 interacting proteins revealed these proteins participate in biological processes, cell composition, and molecular functions. The KEGG analysis indicated that the TSSK4-interacting protein participates in the estrogen signaling pathways, tight junctions, endoplasmic reticulum protein processing, and other signaling pathways. This study cloned the six alternative spliceosomes of the TSSK4 gene laying the foundation for studying the function of each spliceosome in the future.

The microRNA-202 as a Diagnostic Biomarker and a Potential Tumor Suppressor.

MicroRNA-202 (miR-202) is a member of the highly conserved let-7 family that was discovered in Caenorhabditis elegans and recently reported to be involved in cell differentiation and tumor biology. In humans, miR-202 was initially identified in the testis where it was suggested to play a role in spermatogenesis. Subsequent research showed that miR-202 is one of the micro-RNAs that are dysregulated in different types of cancer. During the last decade, a large number of investigations has fortified a role for miR-202 in cancer. However, its functions can be double-edged, depending on context they may be tumor suppressive or oncogenic. In this review, we highlight miR-202 as a potential diagnostic biomarker and as a suppressor of tumorigenesis and metastasis in several types of tumors. We link miR-202 expression levels in tumor types to its involved upstream and downstream signaling molecules and highlight its potential roles in carcinogenesis. Three well-known upstream long non-coding-RNAs (lncRNAs); MALAT1, NORAD, and NEAT1 target miR-202 and inhibit its tumor suppressive function thus fueling cancer progression. Studies on the downstream targets of miR-202 revealed PTEN, AKT, and various oncogenes such as metadherin (MTDH), MYCN, Forkhead box protein R2 (FOXR2) and Kirsten rat sarcoma virus (KRAS). Interestingly, an upregulated level of miR-202 was shown by most of the studies that estimated its expression level in blood or serum of cancer patients, especially in breast cancer. Reduced expression levels of miR-202 in tumor tissues were found to be associated with progression of different types of cancer. It seems likely that miR-202 is embedded in a complex regulatory network related to the nature and the sensitivity of the tumor type and therapeutic (pre)treatments. Its variable roles in tumorigenesis are mediated in part thought its oncogene effectors. However, the currently available data suggest that the involved signaling pathways determine the anti- or pro-tumorigenic outcomes of miR-202’s dysregulation and its value as a diagnostic biomarker.

The Seasonal and Stage-Specific Expression Patterns of HMGB2 Suggest Its Key Role in Spermatogenesis in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

HMGB2, a member of the high-mobility group (HMG) proteins, was identified as a male-biased gene and plays a crucial role in the germ cells differentiation of mammals. However, its role in spermatogenesis of turtle is still poorly understood. Here, we cloned the Pelodiscus sinensis HMGB2 and analyzed its expression profile in different tissues and in testis at different developmental ages. P. sinensis HMGB2 mRNA was highly expressed in the testis of 3-year-old turtles (P < 0.01), but was hardly detected in ovaries and other somatic tissues. The results of chemical in situ hybridization (CISH) showed that HMGB2 mRNA was specifically expressed in germ cells, where it was mainly distributed in round spermatids and sperm, but not detected in somatic cells, spermatogonia, primary spermatocytes, or secondary spermatocyte. The relative expression of HMGB2 also responded to seasonal changes in testis development in P. sinensis. In different seasons of the year, the relative expression of HMGB2 transcripts in the testis of 1year and 2year olds showed an overall upward trend, whereas, in the testis of 3year old, it peaked in July and then declined in October. Moreover, in April and July, with an increase in ages, the expression of HMGB2 transcripts showed an upward trend. However, in January and October, there was a decline in expression in testis in 3-year-old turtles. These results showed that HMGB2 is closely related to spermatogenesis in P. sinensis.

Seminal plasma visfatin levels negatively correlate with sperm concentration.

Visfatin is involved in nicotinamide adenine dinucleotide biosynthesis, with a possible role in spermatogenesis. We investigated seminal plasma visfatin levels and its possible correlations with sperm parameters (concentration, motility, morphology) and BMI.

Androgens and spermatogenesis

Male infertility contributes to 50% of all cases of infertility. The main cause is low quality and quantity of sperm. In humans, spermatogenesis starts at the beginning of puberty and lasts lifelong. It is under the control of FSH and testicular androgens, and mainly testosterone (T), and therefore requires a normal gonadotroph axis, intratesticular T production by Leydig cells and functional androgen receptors (ARs) within testicular Sertoli cells. Various clinical cases illustrate the roles of T in human spermatogenesis. Men with complete congenital hypogonadotropic hypogonadism (HH) are usually azoospermic. Treatment by exogenous testosterone injection and FSH is not able to produce sperm. However, combined treatment with FSH and hCG is effective. This example shows that intratesticular T plays a major role in spermatogenesis. Furthermore, testicular histology of men with LH receptor mutations shows Leydig cell hypoplasia/agenesis/dysplasia with conserved Sertoli cell count. The sperm count is reduced, as in males with partial inactivating mutation of the androgen receptor. Some protocols of hormonal male contraception or exogenous androgen abuse induce negative feedback in the hypothalamic pituitary axis, decreasing FSH, LH and T levels and inducing sperm defects and testicular atrophy. The time to recovery after cessation of drug abuse is around 14 months for sperm output and 38 months for sperm motility. In summary, abnormal androgen production and/or AR signaling impairs spermatogenesis in humans. The minimal level of intratesticular T for normal sperm production is a matter of debate. Interestingly, some animal models showed that completely T-independent spermatogenesis is possible, potentially through strong FSH activation. Finally, recent data suggest important roles of prenatal life and minipuberty in adult spermatogenesis.

The function of nuclear hormone receptor 4A signaling in the human reproductive system: A review.

This review aims to summarize the research focused upon the functions of nuclear hormone receptor 4A (NR4A) in the human reproductive system. The research questions addressed are to decipher what role the NR4A subfamily plays in the regulation of the human reproductive system and effects upon fertility issues through regulation of the expression of the NR4A subfamily. The electronic database PubMed was searched for studies published before November 2021. Keywords included "NR4A," "trophoblast," "decidualization," "folliculogenesis," "estrogen," "pregnancy," "Leydig cells," "fertility," and "reproductive." Relevant references from retrieved manuscripts and review articles were also searched manually. NR4A subfamily are involved in trophoblast differentiation, endometrial decidualization, embryo adhesion, secretion of related hormones, and regulation of spontaneous term labor. Besides, many studies have provided strong evidence that they play critical roles in spermatogenesis. Furthermore, Multiple mechanisms can affect the expression of NR4As. Broadly, NR4A family receptors affect the human reproductive system in multiple ways. Further research is needed to specifically dissect the functions and regulatory mechanisms of these receptors and their pharmaceutical antagonists and agonists. The connection between the NR4A subfamily and a variety of reproductive disorders needs to be proven experimentally such that further examination of human tissue is required to assess the role of these receptors in human reproductive diseases.

Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster.

The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, we applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO2 ) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. We identified 182 phosphopeptides, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis.

The solute carrier family 7 member 11 (SLC7A11) is regulated by LH/androgen and required for cystine/glutathione homeostasis in mouse Sertoli cells

Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.

DNMT3A-dependent DNA methylation is required for spermatogonial stem cells to commit to spermatogenesis.

DNA methylation plays a critical role in spermatogenesis, as evidenced by the male sterility of DNA methyltransferase (DNMT) mutant mice. Here, we report a division of labor in the establishment of the methylation landscape of male germ cells and its functions in spermatogenesis. Although DNMT3C is essential for preventing retrotransposons from interfering with meiosis, DNMT3A broadly methylates the genome (with the exception of DNMT3C-dependent retrotransposons) and controls spermatogonial stem cell (SSC) plasticity. By reconstructing developmental trajectories through single-cell RNA sequencing and profiling chromatin states, we found that Dnmt3A mutant SSCs can only self-renew and no longer differentiate in association with spurious enhancer activation that enforces an irreversible stem cell gene program. Our findings therefore highlight a key function of DNA methylation in male fertility: the epigenetic programming of SSC commitment to differentiation and lifelong spermatogenesis supply.

The molecular characteristics in different procedures of spermatogenesis

Spermatogenesis is a multistep biological process. In addition to somatic cells, it involves the orderly differentiation of dozens of spermatogenic cells. In this process, the regulatory networks between different spermatogenic cell populations are significantly different. RNA m6A regulators and miRNAs have been found to be closely related to spermatogenesis in recent years, and they are an important part of the above regulatory networks. Understanding gene expression and its rules in different spermatogenic cell populations will help in the in-depth exploration of their detailed roles in spermatogenesis. This study collected a public dataset of nonobstructive azoospermia (NOA). Based on the Johnson score, the testicular samples of NOA were divided into three types, Sertoli-cell only syndrome, meiotic arrest and postmeiotic arrest, which represented the loss of three germ cell populations, including whole spermatogenic cells, postmeiotic spermatogenic cells, and a mixture of late spermatids and spermatozoa, respectively. The aforementioned three types of testis data were compared with normal testis data, and the molecular expression characteristics of the abovementioned three germ cell populations were obtained. Our study showed that different germ cell populations have different active molecules and their pathways. In addition, RNA m6A regulators, including METTL3, IGF2BP2 and PRRC2A, and miRNAs, including hsa-let-7a-2, hsa-let-7f-1, hsa-let-7g, hsa-miR-15a, hsa-miR-197, hsa-miR-21, hsa-miR-30e, hsa-miR-32, hsa-miR-503 and hsa-miR-99a, also presented regulatory roles in almost all germ cells.

Seminal plasma piRNA array analysis and identification of possible biomarker piRNAs for the diagnosis of asthenozoospermia.

Asthenozoospermia (AZS) is characterized by reduced sperm motility and its pathogenesis remains poorly understood. Piwi-interacting RNAs (piRNAs) have been indicated to serve important roles in spermatogenesis. However, little is known about the correlation of piRNA expression with AZS. In the present study, small RNA sequencing (small RNA-seq) was performed on sperm samples from AZS patients and fertile controls. Reverse transcription-quantitative (RT-q) PCR was used to validate the small RNA-seq results. Bioinformatics analyses were performed to predict the functions of differentially expressed piRNAs (DEpiRNAs). Logistic regression models were constructed and receiver operating characteristic curve (ROC) analysis was used to evaluate their diagnostic performance. A total of 114 upregulated and 169 downregulated piRNAs were detected in AZS patients. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the DEpiRNAs were mainly associated with transcription, signal transduction, cell differentiation, metal ion binding and focal adhesion. These results were verified by RT-qPCR analysis of eight selected piRNAs. The PCR results were consistent with the sequencing results in patients with AZS compared with controls in the first cohort. The expression of piR-hsa-32694, piR-hsa-26591, piR-hsa-18725 and piR-hsa-18586 was significantly upregulated in patients with AZS. The diagnostic power of the four piRNAs was further analyzed using ROC analysis; piR-hsa-26591 exhibited an area under the ROC curve (AUC) of 0.913 (95% CI: 0.795-0.994). Logistic regression modelling and subsequent ROC analysis indicated that the combination of the 4 piRNAs achieved good diagnostic efficacy (AUC: 0.935).

BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori.

Sperm deliver the male complement of DNA to the ovum, and thus play a key role in sexual reproduction. Accordingly, spermatogenesis has outstanding significance in fields as disparate as infertility treatments and pest-control, making it a broadly interesting and important focus for molecular genetics research in a wide range of species. Here we investigate spermatogenesis in the model lepidopteran insect Bombyx mori (silkworm moth), with particular focus on the gene PMFBP1 (polyamine modulated factor 1 binding protein 1). In humans and mouse, PMFBP1 is essential for spermatogenesis, and mutations of this gene are associated with acephalic spermatozoa, which cause infertility. We identified a B. mori gene labeled as “PMFBP1” in GenBank’s RefSeq database and sought to assess its role in spermatogenesis. Like in mammals, the silkworm version of this gene (BmPMFBP1) is specifically expressed in testes. We subsequently generated BmPMFBP1 mutants using a transgenic CRISPR/Cas9 system. Mutant males were sterile while the fertility of mutant females was comparable to wildtype females. In B. mori, spermatogenesis yields two types of sperm, the nucleated fertile eupyrene sperm, and anucleated unfertile apyrene sperm. Mutant males produced abnormal eupyrene sperm bundles but normal apyrene sperm bundles. For eupyrene sperm, nuclei were mislocated and disordered inside the bundles. We also found the BmPMFBP1 deficiency blocked the release of eupyrene sperm bundles from testes to ejaculatory seminalis. We found no obvious abnormalities in the production of apyrene sperm in mutant males, and double-matings with apyrene-deficient sex-lethal mutants rescued the ΔBmPMFBP1 infertility phenotype. These results indicate BmPMFBP1 functions only in eupyrene spermatogenesis, and highlight that distinct genes underlie the development of the two sperm morphs commonly found in Lepidoptera. Bioinformatic analyses suggest PMFBP1 may have evolved independently in lepidoptera and mammals, and that despite the shared name, are likely not homologous genes.

MEIG1 determines the manchette localization of IFT20 and IFT88, two intraflagellar transport components in male germ cells

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.

Cysteine is highly enriched in the canonical N-linked glycosylation motif of bovine spermatozoa N-Glycoproteome

Glycosylation, one of the most important post-translational modifications of proteins, plays an irreplaceable role in the whole process of spermatogenesis, sperm-egg recognition, and fertilization. Herein, we mapped the first bovine sperm N-linked glycoproteome and a total of 1188 N-glycosylated sites on 626 proteins were identified. Bioinformatics analysis revealed that bovine sperm N-glycosylated proteins were classified into “extracellular region” and “lysosome” groups based on cellular component annotation and enrichment of glycoproteins with proteolytic and reproductive functions. Notably, cysteines were highly enriched in the canonical N-glycosylation motifs N-!P-[S/T/C] and the conservative motifs N–C-[S/T] were also significantly enriched, indicating these modifications play extraordinary roles in bovine spermatogenesis and maturation. The percentage of cysteine at the second position relative to modified asparagine was 7.5%, much higher than that of the previously reported N-linked glycoproteome. A total of 120 cysteine enriched N-glycoproteins were identified, which had significantly upregulated metalloendopeptidase activity and metal ion binding compared with the whole bovine sperm glycoproteome. Strikingly, 15 of 58 N–C-[S/T] motif-containing glycoproteins had a disintegrin and metalloproteinase (ADAM) protein domain. Thus, we hypothesized that ADAM-containing conserved free cysteine residues in N-linked glycoprotein motifs may be key cysteine-switches and may have extraordinary roles in bovine spermatozoa. In conclusion, almost all bovine sperm glycoproteins have enzyme activity, participate in proteolysis, and play indispensable roles in spermatogenesis, sperm-egg recognition, and eventual fertilization. The mapping of N-glycosylation on bovine sperm may provide a new means to explore potential biomarkers for improving sperm quality and fertility.

COX4-like, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in Drosophila melanogaster.

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene—i.e., Cytochrome c oxidase 4-like (COX4L)—in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.