Novel variants in UBE2B gene and idiopathic male infertility

The UBE2B gene encodes ubiquitin-conjugating enzyme, which is involved in DNA repair. Ube2b knockout mice were found to be infertile because of structural abnormality of sperm. However, there is no genetic study on the role of the UBE2B gene in human fertility; therefore, the present investigation was designed to study genetic variations in the UBE2B gene and its role in human male infertility. Sequence analyses of the UBE2B gene in 530 infertile (350 azoospermic, 105 oligoasthenoteratozoospermic, and 75 oligoasthenozoospermic) and 300 fertile control men revealed the presence of 5 substitution single-nucleotide polymorphisms (SNPs) in 221 individuals (199 infertile [37.5%] and 22 fertile [7.3%] men). Of these, 2 (g.5197:T>G; g.9157:A>G) of the 5 substitutions were novel and observed only in infertile men. Distribution of haplotypes TA, TG, GA, and GG are not uniform between the patient and the control group of this study. Interestingly, our study suggests that the haplotype TG conferred significantly increased risk for male infertility (odds ratio=5.07, 95% CI=1.29-23.29, p=.007). In silico analysis of SNPs that were specific to infertile men predicted that these SNPs lead to defective splicing by destroying or creating the potential binding site of splicing factors or causing alteration in predicted regulatory sequences. In the light of the above, our study suggests that the UBE2B gene is associated with male infertility in Indian men, hence, providing evidence for additional genetic factors for male infertility

<i>In vitro</i> fertilization results with control (A-D) and MT- spermatozoa (E-H).

<p>Oocytes fertilized using control spermatozoa showed proper fertilization (PF) as judged by the presence of both polar bodies (PBs) and both pronuclei [PN].(A-D). Oocytes fertilized with MT-spermatozoa showed defective fertilization. In these spermatozoa, only meiotic spindle reorganization was visible (asterisk, E-H) and the 2^nd polar body extrusion had failed (red arrow in E-H). Oocytes were stained with Hoechst 33342 to visualize the polar bodies and pronuclei and the images presented are a merge of both brightfield and fluorescence. Magnification used was 400 x. Scale bars indicate 5 µm.</p

Acrosome reaction studies after induction with calcium ionophore, A23187.

<p>Induction of acrosome reaction was seen in MT- spermatozoa with 0.2 µM A23187, when evaluated at 3, 4 and 5 h of capacitation. Values with same superscript indicate statistically significant changes at p<0.05. Values represent mean ± SD.</p

Fertilization with MT-spermatozoa pretreated with calcium ionophore, A23187.

<p>Control: Control spermatozoa in TALP-PVA medium; MT-: MICA treated spermatozoa; MT-PreCa: MT-spermatozoa pretreated with 0.2 µM calcium ionophore A23187; Post-fertilization: treatment of defective oocytes with 40 nM A23187, post-fertilization.</p><p>Values represent mean±SD.</p><p>Values with the same superscript differ significantly at p<0.05.</p

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

<div><p>Background/Aims</p><p>The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster <i>in vitro</i> fertilization (IVF) via <i>in vitro</i> sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.</p><p>Methodology and Principal Findings</p><p>Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes <i>in vitro</i> which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.</p><p>Conclusions</p><p>This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.</p></div

Graph showing the intracellular pH of spermatozoa (primary axis) under various sperm treatments and their corresponding fertilization outcomes (%) [secondary axis].

<p>Graph showing the intracellular pH of spermatozoa (primary axis) under various sperm treatments and their corresponding fertilization outcomes (%) [secondary axis].</p

The RNA Demethylase ALKBH5 Maintains Endoplasmic Reticulum Homeostasis by Regulating UPR, Autophagy, and Mitochondrial Function

Eukaryotic cells maintain cellular fitness by employing well-coordinated and evolutionarily conserved processes that negotiate stress induced by internal or external environments. These processes include the unfolded protein response, autophagy, endoplasmic reticulum-associated degradation (ERAD) of unfolded proteins and altered mitochondrial functions that together constitute the ER stress response. Here, we show that the RNA demethylase ALKBH5 regulates the crosstalk among these processes to maintain normal ER function. We demonstrate that ALKBH5 regulates ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain the calcium flux between the ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently, mitochondrial biogenesis. Thus, these findings reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness

Fertilization with MICA-treated spermatozoa capacitated in G medium (MICA-treated-G) with or without ammonium chloride [NH₄Cl].

<p>Control: Control spermatozoa in TALP-PVA medium; Control-G: control spermatozoa in G medium; MT-: MICA-treated spermatozoa; MT-G: MT spermatozoa in G medium; MT-15NH₄Cl: MT spermatozoa in TALP-PVA medium alkalinized with 15 mM ammonium chloride; MT-G-5NH₄Cl: MT-G spermatozoa alkalinized with 5 mM ammonium chloride; MT-5NH₄Cl: MT- spermatozoa alkalinized with 5 mM ammonium chloride.</p>#<p>Values represent mean±SD.</p><p>Values with the same superscript differ significantly at p<0.05.</p

Graph showing the sperm intracellular calcium levels (nM) under different sperm treatments and the fertilization outcomes (%) [secondary axis].

<p>Graph showing the sperm intracellular calcium levels (nM) under different sperm treatments and the fertilization outcomes (%) [secondary axis].</p