Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders.

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCß1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCß1 and PI-PLCÉ£1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCß1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCß1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.

Impaired fertility and motor function in a zebrafish model for classic galactosemia.

Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate galactose-1-phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder.

MicroRNA-200c impairs uterine receptivity formation by targeting FUT4 and α1,3-fucosylation.

Successful embryo implantation requires the establishment of a receptive endometrium. Poor endometrial receptivity has generally been considered as a major cause of infertility. Protein glycosylation is associated with many physiological and pathological processes. The fucosylation is catalyzed by the specific fucosyltransferases. Fucosyltransferase IV (FUT4) is the key enzyme for the biosynthesis of α1,3-fucosylated glycans carried by glycoproteins, and the previous studies showed FUT4 expression changed dynamically during perimplantation. MicroRNAs (miRNAs) are known to regulate specific gene expression. However, the relationship between specific miRNA and FUT4, as well as the role of miRNA/FUT4 in the establishment of uterine receptivity remains elusive. In the current study, we reported that the levels of miR-200 family members were significantly increased in serum from infertility and abortion patients relative to healthy non-pregnancy and early-pregnancy women. Among these, miR-200c was the most sensitive diagnostic criterion for infertility by receiver operating characteristic curve analysis. FUT4 was lower in the serum from infertility and abortion patients compared with the healthy non-pregnancy and early-pregnancy women. Using endometrial cell lines and a mouse model, we demonstrated that miR-200c targeted and inhibited FUT4 expression, leading to the dysfunction of uterine receptivity. Our results also revealed that miR-200c decreased α1.3-fucosylation on glycoprotein CD44, which further inactivated Wnt/ß-catenin signaling pathway. Taken together, miR-200c hampers uterine receptivity formation by targeting FUT4 and α1.3-fucosylation on CD44. miR-200c and FUT4 may be applied together as the potential markers for endometrial receptivity, and useful diagnostic and therapeutic targets for infertility.

Involvement of serum glucocorticoid-regulated kinase 1 in reproductive success.

Reproductive processes, in particular events that concern pregnancy, are fine-tuned to produce offspring. Reproductive success is of prime importance for the survival of every species. The highly conserved and ubiquitously expressed serum glucocorticoid-regulated kinase 1 (SGK1) was first implicated in infertility as a regulator of a Na+ channel. In this review, we emphasize the prominent role of SGK1 during early pregnancy: 1) balancing uterine luminal fluid secretion and reabsorption to aid blastocyst adhesion and to import nutrients and energy; 2) transducing signals from the blastocyst to the receptive endometrium; 3) inducing multiple genes that are involved in uterine receptivity and trophoblast invasion; 4) regulating cell differentiation and antioxidant defenses at the fetomaternal interface; and 5) contributing to the proliferation and survival of decidual stromal cells. Accordingly, SGK1 coordinates many cellular processes that are crucial to reproductive activities. Aberrant expression or function of SGK1 results in implantation failure and early pregnancy loss. Further investigation of the molecular mechanisms of the function of SGK1 might provide novel diagnostic tools and interventions for reproductive complications.-Lou, Y., Hu, M., Mao, L., Zheng, Y., Jin, F. Involvement of serum glucocorticoid-regulated kinase 1 in reproductive success.

Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models.

Oxidative stress is associated with some forms of both male and female infertility. However, there is insufficient knowledge of the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. There are a number of animal models for understanding age-dependent decrease of reproductive ability and diabetic embryopathy, especially abnormal spermatogenesis, oogenesis and embryogenesis with mitochondrial dysfunctions. Several important processes occur in mitochondria, including ATP synthesis, calcium ion storage, induction of apoptosis and production of reactive oxygen species (ROS). These events have different effects on the several aspects of reproductive function. Tet-mev-1 conditional transgenic mice, developed after studies with the mev-1 mutant of the nematode C. elegans, offer the ability to carefully regulate expression of doxycycline-induced mutated SDHC(V69E) levels and hence modulate endogenous oxidative stress. The mev-1 models have served to illuminate the effects of complex II deficiency-dependent mitochondrial ROS production, although interestingly they maintain normal mitochondrial and intracellular ATP levels. In this review, the reproductive dysfunctions are presented focusing on fertility potentials in each gamete, early embryogenesis, maternal conditions with placental function and neonatal development.

[Relationship of MTHFR gene polymorphisms with infertility].

The folate metabolic pathway plays important roles in cellular physiology by participating in nucleotide synthesis, DNA repair and methylation, and maintenance and stability of the genome. Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme involved in folate metabolism. Polymorphisms of MTHFR may change the level of homocysteine and affect DNA synthesis and methylation, leading to an increased oxidative stress and disturbed methylation reactions and consequently affecting reproductive function. This article presents an overview on MTHFR gene polymorphisms, proposing that multicentered, large-sample and long-term prospective studies are needed to reveal the relationship between MTHFR gene polymorphisms and infertility.

Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

PURPOSE: The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. METHODS: An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. RESULTS: Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. CONCLUSIONS: Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection.

OBJECTIVE: To study the relationship of total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta, with fertilization rates after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). DESIGN: Laboratory study; controls vs. patients after IVF (n = 27) or ICSI (n = 17) treatment. SETTING: Fertility center. PATIENT(S): A total of 44 semen samples, subjected to either IVF or ICSI treatment. Oocyte collection, ICSI or IVF, determination of sperm concentration and motility, and immunocytochemical analyses of phospholipase C zeta (PLCζ). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Percentages of sperm exhibiting PLCζ. RESULT(S): Significant positive correlation between ICSI fertilization rates and total levels, localization patterns, and the proportion (percentage) of sperm exhibiting PLCζ. Total levels, localization patterns, and the proportion of sperm exhibiting PLCζ are correlated with fertilization rates for ICSI, but not for IVF. CONCLUSION(S): Evaluating total levels, localization patterns, and proportions of PLCζ may represent a useful diagnostic tool for clinical purposes in men for whom IVF is not advised or has previously failed. This clinical study further supports the fundamental role of PLCζ in the oocyte activation process.

Do alterations in follicular fluid proteases contribute to human infertility?

PURPOSE: Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. METHODS: Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. RESULTS: The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. CONCLUSIONS: Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

Management considerations for the adult with congenital adrenal hyperplasia.

The congenital adrenal hyperplasias (CAH) are a group of genetic defects in cortisol biosynthesis, most commonly steroid 21-hydroxylase deficiency (21OHD). With the advent of cortisone therapy in the 1960s and newborn screening in the 1990s, most children with 21OHD now reach adulthood. The needs and concerns of adults with 21OHD overlap with those of children, but the focus and approach shift as these patients reach adulthood. Cohort studies suggest that adults with 21OHD experience significant health concerns such as infertility, obesity, short stature, neoplasia, and bone loss, as well as reduced quality of life. Nevertheless, the spectrum of health status and disease severity is broad, but only some of the reasons for these disparities are known. This review will summarize the current state of knowledge and suggested approaches to management adults with classic 21OHD, plus a few major considerations for adults with nonclassic 21OHD.

Sustained high protein-tyrosine phosphatase 1B activity in the sperm of obese males impairs the sperm acrosome reaction.

Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment.

Characterization of Prdm9 in equids and sterility in mules.

Prdm9 (Meisetz) is the first speciation gene discovered in vertebrates conferring reproductive isolation. This locus encodes a meiosis-specific histone H3 methyltransferase that specifies meiotic recombination hotspots during gametogenesis. Allelic differences in Prdm9, characterized for a variable number of zinc finger (ZF) domains, have been associated with hybrid sterility in male house mice via spermatogenic failure at the pachytene stage. The mule, a classic example of hybrid sterility in mammals also exhibits a similar spermatogenesis breakdown, making Prdm9 an interesting candidate to evaluate in equine hybrids. In this study, we characterized the Prdm9 gene in all species of equids by analyzing sequence variation of the ZF domains and estimating positive selection. We also evaluated the role of Prdm9 in hybrid sterility by assessing allelic differences of ZF domains in equine hybrids. We found remarkable variation in the sequence and number of ZF domains among equid species, ranging from five domains in the Tibetan kiang and Asiatic wild ass, to 14 in the Grevy's zebra. Positive selection was detected in all species at amino acid sites known to be associated with DNA-binding specificity of ZF domains in mice and humans. Equine hybrids, in particular a quartet pedigree composed of a fertile mule showed a mosaic of sequences and number of ZF domains suggesting that Prdm9 variation does not seem by itself to contribute to equine hybrid sterility.

The protein disulfide isomerase family: key players in health and disease.

SIGNIFICANCE: Protein disulfide isomerase (PDI) and its homologs have essential roles in the oxidative folding and chaperone-mediated quality control of proteins in the secretory pathway. In this review, the importance of PDI in health and disease will be examined, using examples from the fields of lipid homeostasis, hemostasis, infectious disease, cancer, neurodegeneration, and infertility. RECENT ADVANCES: Recent structural studies, coupled with cell biological, biochemical, and clinical approaches, have demonstrated that PDI family proteins are involved in a wide range of physiological and disease processes. CRITICAL ISSUES: Critical issues in the field include understanding how and why a PDI family member is involved in a given disease, and defining the physiological client specificity of the various PDI proteins when they are expressed in different tissues. FUTURE DIRECTIONS: Future directions are likely to include the development of new and more specific reagents to study and manipulate PDI family function. The development of conditional mouse models in concert with clinical data will help us to understand the in vivo function of the different PDIs at the organism level. Taken together with advances in structural biology and biochemical studies, this should help us to further understand and modify PDIs' functional interactions.

Galactosemia and amenorrhea in the adolescent.

Hereditary galactosemia is a biochemical genetic disease due to a deficiency of galactose-1-phosphate uridyltransferase (GALT) enzyme activity (OMIM 606999). Acute manifestations occur in the neonatal period and are, with rare exceptions, related to lactose ingestion. They include poor feeding and growth, emesis, jaundice, liver disease, bleeding diathesis, anemia, renal tubulopathy, cataracts, encephalopathy and death from E. coli sepsis. Chronic manifestations, which also develop in prospectively treated patients, involve (a) the brain, resulting in delayed language acquisition, speech defects, and learning problems, and (b) the ovary, in the majority of females, producing hypergonadotropic hypogonadism. The serum FSH level is elevated in infancy/early childhood in many, but not all patients with a severe phenotype. There are few reports of patients with classic galactosemia having undergone pregnancy, labor, and delivery. The pathologic findings in the ovary, including a persistence of primordial follicles and streak gonads, have been variable. The etiology of primary ovarian insufficiency (POI) in galactosemia is unknown. Clinical surveillance includes screening for abnormalities in ovarian function at an early age. Treatment consists of estrogen/progesterone supplementation at the appropriate age. Reduced BMD has been reported. Future research is needed (1) to delineate the mechanisms behind reduced ovarian function in these young women; (2) to determine the timing of the lesion: prenatal, postnatal, and both pre- and postnatal; (3) to determine whether elevated galactose-1-phosphate is both necessary and sufficient to induce primary ovarian insufficiency; and (4) to understand the mechanism(s) behind the reduced BMD seen in children and adolescents with galactosemia.

Delta6-desaturase (FADS2) deficiency unveils the role of omega3- and omega6-polyunsaturated fatty acids.

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and alpha-linolenic acid. EFAs are converted into omega3- and omega6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of delta6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2 -/- mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood-testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2 -/- mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.

Functional dynamics of H3K9 methylation during meiotic prophase progression.

Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ-lineage displayed sterility due to a drastic loss of mature gametes. G9a-deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono- and di-methylation of H3K9 (H3K9me1 and 2) in G9a-deficient germ cells were significantly reduced and G9a-regulated genes were overexpressed during meiosis, suggesting that G9a-mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex-differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis.

[Aromatase inhibitors--is it a new opportunity in the treatment of infertility?]. / Aromataza inkhibitorite--dali tova e nova vuzmozhnost za lechenie na infertiliteta?

Aromatase P-450 is a key enzyme in the production of estrogens, that is, the conversion of androstenedione and testosterone to estrone and estradiol. Aromatase is a good target for selective inhibition. New aromatase inhibitors provide a good opportunity for successful treatment during infertility management. They have a potential to replace clomiphene citrate (CC) as the first-line treatment for ovulation induction. Applying aromatase inhibitors during assisted reproduction followed: reducing the FSH dose needed to achieve optimum controlled ovarian hyperstimulation (COH); improving ovarian response to FSH in poor responders; terminating positive feedback loop and improving ovarian response to COH in infertile case with endometriosis; improving implantation rates in assisted reproduction technology (ART); reducing estrogen levels to reduce the risk of OHSS during COH.

Expression of cathepsin L in human testis under diverse infertility conditions.

The cathepsin family of proteolytic enzymes play an important role in the remodeling seminiferous epithelia in rodent testis. In an effort to uncover the cathepsin L expression in diverse pathological conditions in human testis, the immunohistochemical localization of cathepsin L was conducted in human testis under diverse male infertility condition including spermatogenic hypoplasia and testis cancer. In seminiferous tubule of normal, non-obstructive azoospermic, decreased spermatogenesis, and maturation arrest conditions, cathepsin L was found in both germ cells and Sertoli cells. In contrast, there was no visible expression of cathepsin L in seminiferous tubule tissues from Sertoli cell-only syndrome, spermatogenic hypoplasia, and testicular cancer. Our result suggests that the cross-talk between germ cells and Sertoli cells is crucial for the control of cathepsin L expression in human testis. The absence of expression of cathepsin L in germ cell cancer emphasizes that cathepsin L expression in Sertoli cells is regulated by functional germ cells in human testis.