Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1−/+/Hdac2−/− or Hdac2−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2−/− oocytes. The infertility of mice harboring Hdac1−/+/Hdac2−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.
Oocyte development is becoming of increasing interest not only in the broad research community but also within the general public due, in part, to the ever increasing demand for and use of assisted reproductive technologies (ART) to treat human infertility, and because the oocyte-to-embryo transition encompasses a natural reprogramming of gene expression, a process central to forming iPS cells. Dramatic changes in chromatin structure and gene expression occur during oocyte development, but the role of such changes in generating oocytes that are capable of maturing, being fertilized, and giving rise to offspring is very poorly understood. Histone deacetylases (HDACs) are critically involved in modulating chromatin structure. Here, we describe the effect of specifically deleting the gene encoding Hdac2 in mouse oocytes and find the fertility of female mice harboring such oocytes is compromised. Although such mutant oocytes can grow they fail to mature properly to become an egg. The primary defect is that histone H4 acetylated on lysine 16 fails to become deacetylated as the oocyte matures to become an egg, with the consequence that the ability of chromosomes to interact with spindle microtubules is compromised, which in turn leads to improper chromosome segregation.
Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ2 = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.
male infertility; teratozoospermia; abnormal head morphology; ZPBP1 cDNA mutations; sp38
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.
► Bromodomain, testis-specific (BRDT) is a contraceptive target ► JQ1 is a BRDT inhibitor that causes a reversible contraceptive effect in male mice ► JQ1 alters spermatogenesis at the spermatocyte and round spermatid stages ► JQ1 treatment targets the male germline and reduces spermatozoa number and motility
Inhibition of the chromatin reader protein BRDT with the small molecule JQ1 provides an approach for reversible male contraception.
Infertility is one of the most prevalent public health problems facing young adult males in today’s society. A clear, treatable cause of infertility cannot be determined in a large number of these patients, and a growing body of evidence suggests that infertility in many of these men may be due to genetic causes. Studies utilizing animal models, and most importantly, mouse knockout technology, have been integral not only for the study of normal spermatogenesis but also for identifying proteins essential for this process, which in turn are candidate genes for causing human male infertility. Successful spermatogenesis depends on a delicate balance of local signaling factors, and this review focuses specifically on the genes that encode these factors. Normal functioning of all testicular cell types is not only essential for normal fertility but, as recently hypothesized, may also be crucial to prevent germ cell oncogenesis. Analysis of these processes using mouse models in vivo has provided investigators with an invaluable tool to effectively translate basic science research to the research of human disease and infertility.
male infertility; Sertoli cell; spermatogenesis; knockout mouse; testis signaling
Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.
Epigenetic modifications, and methylation of histones in particular, dynamically change during spermatogenesis. Among various methylations of histone H3, methylation of histone H3 lysine 9 (H3K9) and its regulation are essential for spermatogenesis. Trimethytransferases as well as dimethyltransferase are required for meiotic progression. In addition, didemethylase of H3K9 is also critical for spermatogenesis through transcriptional regulation of spermatid-specific genes. However, the requirement for demethylation of trimethylated H3K9 (H3K9me3) during spermatogenesis remains to be elucidated. Here, we report the targeted disruption of KDM4D, a testis-enriched tridemethylase of H3K9. Kdm4d-null mice are viable and fertile and do not show any obvious phenotype. However, H3K9me3 accumulates significantly in Kdm4d-null round spermatids, and the distribution of methylated H3K9 in germ cells is dramatically changed. Nevertheless, the progression of spermatogenesis and the number of spermatozoa are normal, likely secondary to the earlier nuclear localization of another H3K9 tridemethylase, KDM4B, in Kdm4d-null elongating spermatids. These results suggest that demethylation of H3K9me3 in round spermatids is dispensable for spermatogenesis but that possible defects in Kdm4d-null elongating spermatids could be rescued by functional redundancy of the KDM4B demethylase.
The testis-enriched histone demethylase, KDM4D, regulates global methylation of histone H3 lysine 9 during spermatogenesis but is not essential for completion of spermatogenesis and fertility.
epigenetics; gamete biology; histone demethylase; spermatogenesis; testis
Tektins are evolutionarily-conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial non-redundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for non-syndromic asthenozoospermia in humans.
coiled-coil; axoneme; knockout; motility
A conserved feature of germ cell cytokinesis is the formation of stable intercellular bridges between daughter cells. These intercellular bridges are seen in diverse species from Drosophila melanogaster to Homo sapiens and have been shown to have roles in communication of large numbers of germ cells. In testis expressed gene 14 (Tex14) knockout mice, intercellular bridges do not form during spermatogenesis, and male mice are sterile, demonstrating an essential role for intercellular bridges in postnatal spermatogenesis in mammals. Intercellular bridges also form between dividing germ cells in both male and female embryos. However, little is known about the formation or role of the embryonic intercellular bridges in mammals. In females, embryonic intercellular bridges have been proposed to have a role in development of the presumptive oocyte. Herein, we show that TEX14 is an essential component of male and female embryonic intercellular bridges. In addition, we demonstrate that mitotic kinesin-like protein 1 (MKLP1, official symbol KIF23), which we have discovered is a component of intercellular bridges during spermatogenesis, is also a component of male and female embryonic intercellular bridges. Germ cell intercellular bridges are readily identified by KIF23 immunofluorescence between the gonocytes and oogonia of control mice but are absent between germ cells of Tex14-null mice. Furthermore, by electron microscopy, intercellular bridges are present in all control newborn ovaries but are absent in the Tex14 knockout ovaries. Despite the absence of embryonic intercellular bridges in the Tex14-null mice, male mice initiate spermatogenesis, and female mice are fertile. Although fewer oocytes were present in Tex14-null neonatal ovaries, folliculogenesis was still active at 1 yr of age. Thus, while TEX14 and intercellular bridges have an essential role in postnatal spermatogenesis, they are not required in the embryo.
TEX14 is present in embryonic germ cells, but it is not required for female fertility.
follicle; gamete biology; gametogenesis; oocyte development; ovary
The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. We observed overlapping expression of these factors in uterine epithelial and stromal compartments of pregnant mice prior to embryo implantation. Conditional ablation of both Msx1 and Msx2 in the uterus resulted in female infertility due to a failure in implantation. In these mutant mice (Msx1/2d/d), the uterine epithelium exhibited persistent proliferative activity and failed to attach to the embryos. Gene expression profiling of uterine epithelium and stroma of Msx1/2d/d mice revealed an elevated expression of several members of the Wnt gene family in the preimplantation uterus. Increased canonical Wnt signaling in the stromal cells activated β-catenin, stimulating the production of a subset of fibroblast growth factors (FGFs) in these cells. The secreted FGFs acted in a paracrine manner via the FGF receptors in the epithelium to promote epithelial proliferation, thereby preventing differentiation of this tissue and creating a non-receptive uterus refractory to implantation. Collectively, these findings delineate a unique signaling network, involving Msx1/2, Wnts, and FGFs, which operate in the uterus at the time of implantation to control the mesenchymal-epithelial dialogue critical for successful establishment of pregnancy.
During implantation, various tissue compartments within the uterus, including epithelium and stroma, undergo sequential proliferation and differentiation as the embryo attaches to the uterus and invades into the maternal tissue. There is only limited understanding of the molecular signaling pathways that interconnect these tissue compartments to achieve a functional state of the uterus conducive to implantation. This study reveals that a unique signaling network regulated by the homeobox transcription factors MSX1 and MSX2 in the mouse uterus critically controls female fertility. Targeted mutation of Msx1 and Msx2 genes in female mice, which results in infertility, established that these factors suppress signaling by the morphogenic ligands, WNTS, in the uterus. In the absence of Msx1 and Msx2, the WNT signaling is elevated, leading to the production of a subset of fibroblast growth factors (FGFs) in uterine stroma. These FGFs act in a paracrine manner on the uterine epithelium to promote epithelial proliferation, which results in lack of uterine receptivity and implantation failure. This work, therefore, uncovers an important mechanism in mammalian reproduction and development by identifying key paracrine signals that arise from the uterine stroma to control epithelial function during implantation.
Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP), and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7, and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.
TEX14; intercellular bridge; cytoplasmic bridge; ring canal; knockout; centralspindlin; septins; midbody
The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.
Approximately 20% of infertile couples in the United States have unexplained causes. Many vital aspects of female fertility are regulated by a family of growth factors called the transforming growth factor β (TGFβ) superfamily. These factors exert their functions via specific receptors and downstream signal mediators. Perturbation of components in this pathway can lead to reproductive dysfunction. We identified a novel role for a TGFβ receptor (called TGFBR1) in female fertility. We demonstrated that female mice with disruption of Tgfbr1 in the reproductive tract are unable to successfully conceive, although they can ovulate and produce fertilizable oocytes. Most importantly, these mice have a striking deformity in the oviduct, marked by the formation of oviductal outpouchings (diverticula) that prevent embryos from reaching the uterus. Concomitant aberrations in the uterine smooth muscle layers are additional features of mice lacking TGFBR1. Therefore, TGFBR1 is critical for the structural integrity and function of the female reproductive tract. Our model can be further exploited to study the development of smooth muscle cells of the female reproductive tract. Genetic mutations in TGFBR1 or other TGFβ signaling machinery may lead to fertility defects in women.
Lipid storage diseases are debilitating inherited metabolic disorders that stem from the absence of specific lysosomal enzymes that degrade selected lipids. Most characteristically, these disorders affect the nervous and the reticulo-endothelial systems, with massive organomegaly resulting from the presence of engorged, lipid-laden macrophages. In this issue of the JCI, Yildiz et al. describe the role of the ER-resident enzyme β-glucosidase 2 (GBA2) in mice (see the related article beginning on page 2985). Surprisingly, GBA2 deficiency leaves bile acid and cholesterol metabolism intact, instead causing lipid accumulation in the ER of testicular Sertoli cells, round-headed sperm (globozoospermia), and impaired male fertility.
The transforming growth factor beta superfamily ligand activin A controls juvenile testis growth by stimulating Sertoli cell proliferation. Testicular levels are highest in the first postnatal week, when Sertoli cells are proliferating and spermatogonial stem cells first form. Levels decrease sharply as Sertoli cell proliferation ceases and spermatogenic differentiation begins. We hypothesized that changing activin levels also affect germ cell maturation. We detected an acute and developmentally regulated impact of activin on Kit mRNA in cocultures of Sertoli cells and germ cells from Day 8, but not Day 4, mice. Both stereological and flow cytometry analyses identified an elevated spermatogonium:Sertoli cell ratio in Day 7 testes from InhbaBK/BK mice, which have decreased bioactive activin, and the germ cell markers Sycp3, Dazl, and Ccnd3 were significantly elevated in InhbaBK/BK mice. The flow cytometry measurements demonstrated that surface KIT protein is significantly higher in Day 7 InhbaBK/BK germ cells than in wild-type littermates. By Day 14, the germ cell:Sertoli cell ratio did not differ between genotypes, but the transition of type A spermatogonia into spermatocytes was altered in InhbaBK/BK testes. We conclude that regulated activin signaling not only controls Sertoli cell proliferation, as previously described, but also influences the in vivo progression of germ cell maturation in the juvenile testis at the onset of spermatogenesis.
Activin modulates germ cell and Sertoli cell development in the mouse testis in vivo.
activin; KIT; KITL; Sertoli cells; spermatogenesis; spermatogonia; testis
MicroRNAs (miRNAs) regulate complex patterns of gene expression, and the relevance of altered miRNA expression to ovarian cancer remains to be elucidated. By comprehensively profiling expression of miRNAs and mRNAs in serous ovarian tumors and cell lines and normal ovarian surface epithelium, we identified hundreds of potential miRNA-mRNA targeting associations underlying cancer. Functional overexpression of miR-31, the most underexpressed miRNA in serous ovarian cancer, repressed predicted miR-31 gene targets including cell cycle regulator E2F2. MIR31 and CDKN2A, which encodes p14ARF and p16INK4A, are located at 9p21.3, a genomic region commonly deleted in ovarian and other cancers. p14ARF promotes p53 activity, and E2F2 overexpression in p53 wild-type cells normally leads via p14ARF to an induction of p53-dependent apoptosis. In a number of serous cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, and SKOV3), miR-31 overexpression inhibited proliferation and induced apoptosis; however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Additionally, the osteosarcoma cell line U2OS and the prostate cancer cell line PC3 (p14ARF-deficient and p53-deficient, respectively) were also sensitive to miR-31. Furthermore, miR-31 overexpression induced a global gene expression pattern in OVCAR8 associated with better prognosis in tumors from patients with advanced stage serous ovarian cancer, potentially impacting many genes underlying disease progression. Our findings reveal that loss of miR-31 is associated with defects in the p53 pathway and functions in serous ovarian cancer and other cancers, suggesting that patients with cancers deficient in p53 activity might benefit from therapeutic delivery of miR-31.
microRNA; serous ovarian carcinoma; cancer therapy; miR31; TP53
Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.
Testicular cell adhesion molecule 1 (Tcam1) is a testis-expressed gene that is evolutionarily conserved in most mammalian species. The putative location of TCAM1 on the cell surface makes it an attractive contraceptive target to study. We found that Tcam1 transcription is enriched in the adult testis, and in situ hybridization revealed that Tcam1 is expressed in pachytene to secondary spermatocytes. Immunofluorescence for TCAM1 protein showed strong expression along cell membranes of spermatocytes and weak localization to round spermatids. In light of this evidence, we hypothesized that TCAM1 interacts with an unknown receptor on the surface of Sertoli cells and that this interaction is important for germ cell-Sertoli cell interactions. However, Tcam1 knockout mice that we generated are fertile, and testis weights and sperm counts were not significantly altered. Therefore, we conclude that TCAM1 is not essential for male fertility or germ cell function in Mus musculus.
Spermatocyte; Male Fertility; Integral Membrane Protein; Transgenic mice
Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin βA (Inhba) and βB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.
BMP15; recombinant protein; oocyte; granulosa cell
The causes of male infertility are heterogeneous but more than 50% of cases have a genetic basis. Specific genetic defects have been identified in less than 20% of infertile males and, thus, most causes remain to be elucidated. The most common cytogenetic defects associated with nonobstructive azoospermia are numerical and structural chromosome abnormalities, including Klinefelter syndrome (47,XXY) and Y chromosome microdeletions. To refine the incidence and nature of chromosomal aberrations in males with infertility we reviewed cytogenetic results in 668 infertile men with oligozoospermia and azoospermia.
Materials and Methods
High resolution Giemsa banding chromosome analysis and/or fluorescence in situ hybridization were done in 668 infertile males referred for routine cytogenetic analysis between January 2004 and March 2009.
The overall incidence of chromosomal abnormalities was about 8.2%. Of the 55 patients with abnormal cytogenetic findings sex chromosome aneuploidies were observed in 29 (53%), including Klinefelter syndrome in 27 (49%). Structural chromosome abnormalities involving autosomes (29%) and sex chromosomes (18%) were detected in 26 infertile men. Abnormal cytogenetic findings were observed in 35 of 264 patients (13.3%) with azoospermia and 19 of 365 (5.2%) with oligozoospermia.
Structural chromosomal defects and low level sex chromosome mosaicism are common in oligozoospermia cases. Extensive cytogenetic assessment and fluorescence in situ hybridization may improve the detection rate in males with oligozoospermia. These findings highlight the need for efficient genetic testing in infertile men so that couples may make informed decisions on assisted reproductive technologies to achieve parenthood.
infertility; male; aneuploidy; azoospermia; oligospermia; sex chromosome aberrations
In somatic cells, abscission, the physical separation of daughter cells at the completion of cytokinesis, requires CEP55, ALIX, and TSG101. In contrast, cytokinesis is arrested prior to abscission in differentiating male germ cells that are interconnected by TEX14-positive intercellular bridges. We have previously shown that targeted deletion of TEX14 disrupts intercellular bridges in all germ cells and causes male sterility. Although these findings demonstrate that intercellular bridges are essential for spermatogenesis, it remains to be shown how TEX14 and other proteins come together to prevent abscission and form stable intercellular bridges. Using a biochemical enrichment of male germ cell intercellular bridges, we identified additional bridge proteins, including CEP55. Although CEP55 is highly expressed in testes at the RNA level, there is no report of the presence of CEP55 in germ cells. We show here that CEP55 becomes a stable component of the intercellular bridge and that an evolutionarily conserved GPPX3Y motif of TEX14 binds strongly to CEP55 to block similar GPPX3Y motifs of ALIX and TSG101 from interacting and localizing to the midbody. Thus, TEX14 prevents the completion of cytokinesis by altering the destiny of CEP55 from a nidus for abscission to an integral component of the intercellular bridge.
Mouse oocytes produce members of the transforming growth factor beta (TGFbeta) superfamily, including bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), as well as fibroblast growth factors (FGFs). These growth factors cooperate to regulate cumulus cell function. To identify potential mechanisms involved in these interactions, the ability of fully grown oocytes to regulate expression of BMP or FGF antagonists in cumulus cells was examined. Oocytes promoted cumulus cell expression of transcripts encoding antagonists to TGFbeta superfamily members, including Grem2, Htra1, Htra3, and Nog mRNAs. In contrast, oocytes suppressed cumulus cell expression of Spry2 mRNA, which encodes a regulator of receptor tyrosine kinase signals, such as FGF and epidermal growth factor (EGF) receptor signals. The regulation of Spry2 mRNA levels in cumulus cells was studied further as a model for analysis of potential mechanisms for cooperativity of FGF/EGF signaling with oocyte-derived members of the TGFbeta superfamily. Oocytes suppressed basal and FGF-stimulated Spry2 mRNA levels in cumulus cells but promoted EGF-stimulated levels. Furthermore, recombinant TGFbeta superfamily proteins, including BMP15 and GDF9, mimicked these effects of oocytes. Elevated expression of Spry2 mRNA in cumulus and mural granulosa cells correlated with human chorionic gonadotropin-induced expression of mRNAs encoding EGF-like peptides. Therefore, oocyte-derived members of the TGFbeta superfamily suppress FGF-stimulated Spry2 mRNA levels before the luteinizing hormone surge but promote Spry2 mRNA levels stimulated by EGF receptor-mediated signals after the surge.
Oocyte-derived TGFβ superfamily members suppress FGF-stimulated Spry2 mRNA levels before the LH surge but promote Spry2 mRNA levels stimulated by EGF receptor-mediated signals after the surge.
BMP15; EGF; FGF8; follicular development; GDF9; gene regulation; granulosa cells; oocytes; sprouty 2
Targeted disruption of the inhibin α gene (Inha−/−) in mice results in an ovarian phenotype of granulosa cell tumors that renders the animals infertile. Little is known about the reproductive defects prior to tumor development. Here, we report novel data on early follicle dynamics in Inha−/− mice, which demonstrate that inhibin α has important consequences upon follicle development. Morphological changes in both germ and somatic cells were evident in postnatal day 12 ovaries, with Inha−/− mice exhibiting numerous multilayered follicles that were far more advanced than those observed in age-matched controls. These changes were accompanied by alterations in follicle dynamics such that Inha−/− ovaries had fewer follicles in the resting pool and more committed in the growth phase. Absence of inhibin α resulted in advanced follicular maturation as marked by premature loss of anti-Müllerian hormone (AMH) in secondary follicles. Additionally, gene expression analysis revealed changes in factors known to be vital for oocyte and follicle development. Together, these data provide key evidence to suggest that regulation of the inhibin/activin system is essential for early folliculogenesis in the prepubertal mouse ovary.
Inhibin; activin; folliculogenesis; oocyte; follicle; prepubertal mouse
Conditional knockout mouse strategies identify the histone methyltranferase MLL2 as a key player in epigenetic reprogramming of female gametes.
During gametogenesis and pre-implantation development, the mammalian epigenome is reprogrammed to establish pluripotency in the epiblast. Here we show that the histone 3 lysine 4 (H3K4) methyltransferase, MLL2, controls most of the promoter-specific chromatin modification, H3K4me3, during oogenesis and early development. Using conditional knockout mutagenesis and a hypomorph model, we show that Mll2 deficiency in oocytes results in anovulation and oocyte death, with increased transcription of p53, apoptotic factors, and Iap elements. MLL2 is required for (1) bulk H3K4me3 but not H3K4me1, indicating that MLL2 controls most promoters but monomethylation is regulated by a different H3K4 methyltransferase; (2) the global transcriptional silencing that preceeds resumption of meiosis but not for the concomitant nuclear reorganization into the surrounded nucleolus (SN) chromatin configuration; (3) oocyte survival; and (4) normal zygotic genome activation. These results reveal that MLL2 is autonomously required in oocytes for fertility and imply that MLL2 contributes to the epigenetic reprogramming that takes place before fertilization. We propose that once this task has been accomplished, MLL2 is not required until gastrulation and that other methyltransferases are responsible for bulk H3K4me3, thereby revealing an unexpected epigenetic control switch amongst the H3K4 methyltransferases during development.
It is well established that gametes and early mammalian embryos undergo extensive epigenetic changes, which are changes in phenotype or gene expression that do not entail changes in DNA sequence. However, the machinery responsible for epigenetic modification in these situations is poorly understood. In mice, we conditionally deleted the histone 3 lysine 4 (H3K4) methyltransferase Mll2, an enzyme that alters DNA structure and packaging, either in gametes or in somatic cells of the ovary and also produced a mouse hypomorph expressing low levels of MLL2. We show that MLL2 is required in oocytes during gametogenesis and is also needed as a maternally derived factor during early development. Oocytes deficient in Mll2 display decreased methylation of H3K4 (H3K4me3) and show abnormal maturation and gene expression, in particular of pro-apoptotic factors. In addition, we demonstrate that embryonic genome activation is compromised in the absence of Mll2. Together our results identify MLL2 as one of the key players in the epigenetic reprogramming required for female fertility in the mouse.
The basic biology of the menstrual cycle is a complex, coordinated sequence of events involving the hypothalamus, anterior pituitary, ovary, and endometrium. The menstrual cycle with all its complexities can be easily perturbed by environmental factors such as stress, extreme exercise, eating disorders, and obesity. Furthermore, genetic influences such as fragile X premutations (Chapter X), X chromosome abnormalities (Chapter X), and galactose-1-phosphate uridyltransferase (GALT) point mutations (galactosemia) also contribute to perturbations of the menstrual cycle. Although not perfect, mouse model have helped to identify and confirm additional components and pathways in menstrual cycle function and dysfunction in humans.
Biology; menstrual cycle; mouse models; premature ovarian insufficiency (POI); genetics; endometriosis; folliculogenesis
Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice.
Activin is required for testis development. Activin signals via the phosphorylation and nuclear accumulation of SMAD2 and SMAD3. We present novel findings of developmentally regulated activin signalling leading to specific transcriptional outcomes in testicular Sertoli cells. In immature, proliferating, Sertoli cells, activin A induces nuclear accumulation of SMAD3, but not SMAD2, although both proteins become phosphorylated. In post-mitotic differentiating cells, both SMAD proteins accumulate in the nucleus. Furthermore, immature Sertoli cells are sensitive to activin dosage; higher concentrations induce maximal SMAD3 nuclear accumulation and a small increase in nuclear SMAD2. Microarray analysis identified distinct transcriptional outcomes correlating with differential SMAD utilization and new activin target genes, including Gja1 and Serpina5, which are essential for Sertoli cell development and male fertility. In transgenic mice with altered activin bioactivity that display fertility phenotypes, Gja1 and Serpina5 are significantly altered. Thus, differential SMAD utilization in response to activin features during Sertoli cell maturation.
morphogen gradient; development; TGFβ; Sertoli; testis; Rarres1; TIG-1; PDGF; Gja1; Serpina5