Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation.

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

Metabolic syndrome and male fertility disorders: Is there a causal link?

Infertility is a global health problem affecting 10-15% of couples in reproductive age. Recent studies have provided growing evidence supporting that lifestyle factors can affect male fertility through alterations in endocrine profiles, spermatogenesis and/or sperm function. One of these critical factors could be the change in the food intake behavior in modern societies that produces metabolic alterations. Regarding this, metabolic syndrome (MetS) prevalence has increased in epidemic in the last 40-50 years. Although MetS is associated with advanced age, changes in lifestyles have accelerated the appearance of symptoms in the reproductive age. We review herein the current understanding of the relationship between MetS and the male reproductive status. For this purpose, in this narrative review a comprehensive literature search was made in both animal models and men, allowing us to evaluate such relationship. This analysis showed a high variability in the reproductive phenotypes observed in patients and mice suffering MetS, including sperm parameters, fertility and offspring health. In view of this, we proposed that the reproductive effects, which are diverse and not robust, observed among MetS-affected males, might depend on additional factors not associated with the metabolic condition and contributed not only by the affected male but also by his partner. With this perspective, this review provides a more accurate insight of this syndrome critical for the identification of specific diagnostic indicators and treatment of MetS-induced fertility disorders.

Postnatal metformin treatment alters rat Sertoli cell proliferation and daily sperm production.

BACKGROUND: The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. OBJECTIVE: The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. MATERIALS AND METHODS: Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200 mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. RESULTS: MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. DISCUSSION AND CONCLUSION: These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation.

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4 Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.

PI3K/Akt Cooperates with Oncogenic Notch by Inducing Nitric Oxide-Dependent Inflammation.

The PI3K/Akt signaling pathway, Notch, and other oncogenes cooperate in the induction of aggressive cancers. Elucidating how the PI3K/Akt pathway facilitates tumorigenesis by other oncogenes may offer opportunities to develop drugs with fewer side effects than those currently available. Here, using an unbiased in vivo chemical genetic screen in Drosophila, we identified compounds that inhibit the activity of proinflammatory enzymes nitric oxide synthase (NOS) and lipoxygenase (LOX) as selective suppressors of Notch-PI3K/Akt cooperative oncogenesis. Tumor silencing of NOS and LOX signaling mirrored the antitumor effect of the hit compounds, demonstrating their participation in Notch-PI3K/Akt-induced tumorigenesis. Oncogenic PI3K/Akt signaling triggered inflammation and immunosuppression via aberrant NOS expression. Accordingly, activated Notch tumorigenesis was fueled by hampering the immune response or by NOS overexpression to mimic a protumorigenic environment. Our lead compound, the LOX inhibitor BW B70C, also selectively killed human leukemic cells by dampening the NOTCH1-PI3K/AKT-eNOS axis.

Functional and structural characterisation of AgMNPV ie1.

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the -492 and -357 versions contains sequence motifs important for the level of the lacZ reporter gene expression.