Localization and Distribution of Testicular Angiotensin I Converting Enzyme (ACE) in Neck and Mid-Piece of Spermatozoa from Infertile Men in Relation to Sperm Motility.

Testicular angiotensin converting enzyme (ACE) is known to play an essential role in the male reproduction and fertility. Data about tACE in cases of male infertility are quite scarce, and in this respect we aimed to study localization and distribution of tACE protein in the neck and mid-piece of spermatozoa from pathological samples in relation to sperm motility. The enzyme expression during capacitation and acrosome reaction was quantitatively assessed. In human ejaculated spermatozoa tACE is localized on sperm plasma membrane of the head, the neck and mid-piece of the tail. The immunoreactivity becomes stronger in capacitated spermatozoa followed by a decrease in acrosome reacted sperm. In different cases of semen pathology (oligozoospermia, asthenozoospermia and teratozoospermia) fluorescent signals in the neck and mid-piece are in punctate manner whereas in normozoospermia they were uniformly distributed. The expression area of tACE the neck and mid-piece was decreased in ejaculated and capacitated sperm from pathological semen samples compared to normospermia. Significant positive correlation was established between tACE area and progressive sperm motility, whereas with immotile sperm the correlation was negative. Our data suggest that proper distribution of tACE in the neck and mid-piece is required for normal sperm motility that could be used as a novel biomarker for male infertility.

A mouse testis serine protease, TESP1, as the potential SPINK3 receptor protein on mouse sperm acrosome.

Serine protease inhibitor Kazal type 3 (SPINK3) from mouse seminal vesicles is a Kazal-type trypsin inhibitor. It has been shown to bind to the sperm acrosome and modify sperm activity by influencing the sub-cellular Ca2+ influx. Previously, SPINK3 was reported to suppress in vitro sperm capacitation. However, under natural coitus, SPINK3 is removed from the mouse acrosome in the female reproductive tract, leading to successful fertilisation. Identification of the SPINK3 binding partner becomes essential to develop a contraceptive that works by prolonging the binding of SPINK3 to the sperm acrosome. We identified the SPINK3 receptor by using recombinant SPINK3 (rSPINK3). Testicular serine protease 1 (TESP1) was identified as the receptor for SPINK3 by 2D gel electrophoresis coupled with western blot analysis. To authenticate TESP1 as the receptor for SPINK3, sperm cells were incubated with TESP1 peptide antibody followed by determining the intracellular [Ca2+]i concentration by flow cytometry using Fluo-3 AM as a calcium probe. Furthermore, the 3D structures of SPINK3 and TESP1 were predicted by homology modelling (Schrodinger suite) using the crystal structure of pancreatic secretory trypsin inhibitor (PDB ID-1TGS) and human prostasin (PDB ID-3DFJ) as templates. The modelled protein structures were validated and subjected to molecular dynamics simulation (MDS) using GROMACS v5.0.5. Protein-protein docking was performed using HDOCK and the complex was validated by MDS. The results predicted that SPINK3 and TESP1 had strong binding affinity, with a dock score of -430.70 and 14 hydrogen bonds as key active site residues. If the binding affinity between SPINK3 and TESP1 could be increased, the SPINK3-TESP1 association will be prolonged, which will be helpful in the development of a male contraceptive.

The protein phosphatase with EF-hand domain 1 is a calmodulin-binding protein that interacts with proteins involved in sperm capacitation, binding to the zona pellucida, and motility.

Spermatozoa are highly specialized cells whose fertilizing and motility functions highly depend on intracellular Ca2+ -mediated events and protein posttranslational modifications like phosphorylation. Our group previously identified PPEF1, the Ser/Thr phosphatase with EF-hand domain 1, among calmodulin-affinity pulled down sperm proteins. As the mammalian ortholog of the Drosophila phosphatase rdgC that dephosphorylates rhodopsin, PPEF1 has been studied mostly in the retina. The presence and importance of this Ca2+ /calmodulin-binding protein phosphatase has not been studied in sperm or testicular functions despite its high expression level. In this study, we show that PPEF1 is present in testicular germ cells, and in mouse, human and bull spermatozoa where it is localized predominantly in the neck and acrosome areas. Different transcript variants encoding four predicted isoforms were detected by reverse transcription polymerase chain reaction in bull testis, spermatocytes and spermatids. Phosphatase activity of immunoprecipitated sperm PPEF1 was detected using the substrate pNPP and analysis of the coimmunoprecipitated proteins reveal an enrichment in the biological processes of sperm capacitation, binding to the zona pellucida and motility. Although this is the first demonstration of the presence of PPEF1 in sperm and testicular germ cells, its involvement in sperm fertilizing ability and motility, and the mechanisms regulating its activity remain to be further investigated.

Sialylation of Asparagine 612 Inhibits Aconitase Activity during Mouse Sperm Capacitation; a Possible Mechanism for the Switch from Oxidative Phosphorylation to Glycolysis.

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC-MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

Phospholipase C zeta profiles are indicative of optimal sperm parameters and fertilisation success in patients undergoing fertility treatment.

BACKGROUND: Oocyte activation is driven by intracellular calcium (Ca2+ ) oscillations induced by sperm-specific PLCζ, abrogation of which causes oocyte activation deficiency in humans. Clinical PLCζ investigations have been limited to severe male infertility conditions, while PLCζ levels and localisation patterns have yet to be associated with general sperm viability. MATERIALS AND METHODS: PLCζ profiles were examined within a general population of males attending a fertility clinic (65 patients; aged 29-53), examining PLCζ throughout various fractions of sperm viability. Male recruitment criteria required a minimum sperm count of 5 × 106 spermatozoa/mL, while all female patients included in this study yielded at least five oocytes for treatment. Sperm count, motility and semen volume were recorded according to standard WHO reference guidelines and correlated with PLCζ profiles examined via immunoblotting and immunofluorescence. Appropriate fertility treatments were performed following routine clinical standard operating protocols, and fertilisation success determined by successful observation of second polar body extrusion. RESULTS AND DISCUSSION: Four distinct PLCζ patterns were observed at the equatorial, acrosomal + equatorial regions of the sperm head, alongside a dispersed pattern, and a population of spermatozoa without any PLCζ. Acrosomal + equatorial PLCζ correlated most to sperm health, while dispersed PLCζ correlated to decreased sperm viability. Total levels of PLCζ exhibited significant correlations with sperm parameters. PLCζ variance corresponded to reduced sperm health, potentially underlying cases of male sub-fertility and increasing male age. Finally, significantly higher levels of PLCζ were exhibited by cases of fertilisation success, alongside higher proportions of Ac + Eq, and lower levels of dispersed PLCζ. CONCLUSIONS: PLCζ potentially represents a biomarker of sperm health, and fertilisation capacity in general cases of patients seeking fertility treatment, and not just cases of repeated fertilisation. Further focused investigations are required with larger cohorts to examine the full clinical potential of PLCζ.

[The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties].

OBJECTIVE: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS: LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.

Human sperm acrosome function assays are predictive of fertilization rate in vitro: a retrospective cohort study and meta-analysis.

OBJECTIVE: To determine whether acrosome function scoring-including acrosomal enzyme (AE) levels and acrosome reaction (AR) results-can predict fertilization rate in vitro. METHODS: We examined the predictive value of acrosomal enzymes (AE) determined by spectrophotometry/N-α-benzoyl-DL-arginine-p-nitroanilide for fertilization rate (FR) in vitro in a retrospective cohort study of 737 infertile couples undergoing IVF therapy. Additionally, a meta-analysis was done for prospective cohort or case-control studies; the following summary measures were reported to expand upon the findings: pooled spearman correlation coefficient (Rs), standardized mean difference (SMD), sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic score (DS), diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC). RESULTS: Lower AE levels determined by spectrophotometry with a cut-off value of <25µIU/106 spermatozoa were predictive of total fertilization failure (TFF) with moderate SEN (88.23%) and low SPE (16.50%). On meta-analysis, a total of 44 unique articles were selected, but given the multiple techniques described there was a total of 67 total datasets extracted from these 44 articles, comprising 5356 infertile couples undergoing IVF therapy. The AE levels or induced AR% was positively correlated with FR (Rs = 0.38, SMD = 0.79; Rs = 0.40, SMD = 0.86, respectively). Lower AE levels or induced AR% was predictive of lower fertilization rate with moderate accuracy (AUC = 0.78, AUC = 0.84, respectively); this was accompanied by low SEN/moderate SPE (0.57/0.85), moderate SEN/moderate SPE (0.79/0.87), respectively. For AE assay, the diagnostic performance in Asia (Rs = 0.24, SMD = 0.50) was inferior to that in North America (Rs = 0.54, SMD = 0.81) and Europe (Rs = 0.46, SMD = 0.92). Cryopreserved spermatozoa (SMD = 0.20, P = 0.204) were inferior to fresh spermatozoa (SMD = 0.89, P <  0.001). Sperm preparation yielded inferior results as compared to no preparation; spermatozoa after swim up were weak relevant (Rs = 0.27, P = 0.044); and there was no correlation for spermatozoa after a discontinuous gradient (SMD = 1.07, P >  0.05). Lower AE levels determined by fluorometry or substrate assay were used for predicting lower FR with low sensitivity and high specificity; the spectrophotometry assay had an uncertain predictive value. For induced AR assay, the diagnostic performance in the other areas was inferior to that in Africa (Rs = 0.65, SMD = 1.86). No preparation or double preparation yielded inferior results as compared to one preparation (Rs = 0.41); discontinuous gradient (Rs = 0.17, SMD = 0.47) was inferior to swim up (Rs =0.65, SMD = 1.51). Nonphysiological triggers (SMD = 0.81) did not differ from physiological triggers (SMD = 0.95) in general; ZP (Rs = 0.63) or mannose (Rs = 0.59) was superior to other physiological or nonphysiological triggers; and there was no correlation for human follicle fluid, progesterone, cyclic adenosine 3'-5'-phosphate analogue and phorbol ester-BSA-GlcNAc Neoglycoproteins with N-acetylglucosamine residues. Lower induced AR% determined by indirect immunofluorescence, direct immunofluorescence with lection, or triple stain was used for predicting lower FR, with moderate sensitivity/high specificity, moderate sensitivity/high specificity, or high sensitivity/low specificity. CONCLUSIONS: Although the correlation between acrosome function scoring and FR was significant, the assays were neither highly sensitive nor specific. Additionally, the diagnostic performance showed regional effects as well as an effect of the sperm preparation or assay method. More studies of multicenter, large-scale, careful design and synthesizing multiple sperm functional assays and oocyte quality assays are still needed in clinical settings to better predict fertilization outcome in IVF.

Elevated aminopeptidase N affects sperm motility and early embryo development.

Aminopeptidase N (APN) is a naturally occurring ectopeptidase present in mammalian semen. Previous studies have demonstrated that APN adversely affects male fertility through the alteration of sperm motility. This enzyme constitutes 0.5 to 1% of the seminal plasma proteins, which can be transferred from the prostasomes to sperms by a fusion process. In the present study, we investigated the molecular mechanism of action of APN and its role in regulating sperm functions and male fertility. In this in vitro study, epididymal mouse spermatozoa were incubated in a capacitating media (pH 7) containing 20 ng/mL of recombinant mouse APN for 90 min. Our results demonstrated that the supplementation of recombinant APN in sperm culture medium significantly increased APN activity, and subsequently altered motility, hyperactivated motility, rapid and medium swimming speeds, viability, and the acrosome reaction of mouse spermatozoa. These effects were potentially caused by increased toxicity in the spermatozoa. Further, altered APN activity in sperm culture medium affected early embryonic development. Interestingly, the effect of elevated APN activity in sperm culture medium was independent of protein tyrosine phosphorylation and protein kinase A activity. On the basis of these results, we concluded that APN plays a significant role in the regulation of several sperm functions and early embryonic development. In addition, increased APN activity could potentially lead to several adverse consequences related to male fertility.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Testis-specific isoform of angiotensin-converting enzyme (tACE) is involved in the regulation of bovine sperm capacitation.

We hypothesized that the testis-specific isoform of angiotensin-converting enzyme (tACE) is released during bovine sperm capacitation, and its peptidase activity is required for capacitation. Specific objectives of this study were to (i) develop an anti-tACE antibody; (ii) characterize expression of tACE in bovine testes and sperm; and (iii) determine the role of tACE in capacitation. A 110-kDa protein, consistent with the mass of tACE, was detected in sperm extract by our anti-tACE immunoserum. This immunotarget localized at the acrosomal region and principal piece, but was only expressed in testis of mature bulls. When bull sperm were incubated in Sp-TALP (0 and 4 hr) plus 10 µg/ml heparin (capacitation group) or 10 µg/ml heparin + 10 µM captopril (an ACE inhibitor) for 4 hr, the number of acrosome-reacted (40.1 vs. 24.0%, respectively) and hyperactivated (15.0 vs. 9.7%) sperm increased, and tyrosine phosphoprotein content were higher (p < 0.05) for sperm in heparin alone. tACE activity was also higher (0.04 U/ml; p < 0.01) in incubation medium of sperm exposed to heparin compared to 0- and 4-hr incubation controls or heparin + captopril conditions (0, 0.005, and 0.009 U/ml, respectively). Furthermore, capacitation-associated shedding of a portion of tACE into the medium decreased sperm content of the 110-kDa tACE, but concurrently increased the abundance of a 60-kDa tACE variant. Thus, a portion of the extracellular region of tACE (containing its catalytic site) is released from bovine sperm during capacitation, and tACE activity may be required for sperm capacitation.

Ubiquitin Carboxy-Terminal HydrolaseL3 Correlates with Human Sperm Count, Motility and Fertilization.

Ubiquitin C-terminal hydrolase L3 (UCHL3) belongs to the group of deubiquitinating enzymes and plays a part in apoptosis of germ cells and the differentiation of spermatocytes into spermatids. However, the exact role of UCHL3 in human spermatogenesis and sperm function remains unknown. Here we examined the level and activity of UCHL3 in spermatozoa from men with asthenozoospermia (A), oligoasthenozoospermia (OA) or normozoospermia (N). Immunofluorescence indicated that UCHL3 was mainly localized in the acrosome and throughout the flagella, and western blotting revealed a lower level in A or OA compared with N (p < 0.05). The catalytic activity of UCHL3 was decreased in spermatozoa from A or OA (p < 0.05, p < 0.001, respectively). The level and activity of UCHL3 were positively correlated with sperm count, concentration and motility. The UCHL3 level was positively correlated with the normal fertilization rate (FR) and percentage of embryos suitable for transfer/cryopreservation of in vitro fertilization (IVF). The UCHL3 activity was also positively correlated with FR, the percentage of embryos suitable for transfer/cryopreservation and high-quality embryos rate of IVF. Aforementioned correlations were not manifested in intra-cytoplasmic sperm injection (ICSI). These findings suggest that UCHL3 may play a role in male infertility.

CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

[Application of spontaneous acrosome reaction of sperm in prediction of outcome of in-vitro fertilization and embryo transfer].

OBJECTIVE: To investigate the clinical application of spontaneous acrosome reaction (AR) rate of sperm in predicting the outcome of in-vitro fertilization and embryo transfer (IVF-ET). METHODS: The spontaneous AR rate of the sperm of patients who underwent IVF-ET treatment in our center during the period from November to December 2014 were studied. The cut-off value from 6% to 12% were set and analyzed its association between the IVF-ET outcomes (including fertility rates, normal fertilization rates and high-quality embryo rates). For those who underwent fresh embryo transplantation, the rates of chemical pregnancy and clinical pregnancy were calculated, and compared the spontaneous AR rates and quantity of acrosomal enzyme according to the pregnancy outcome. RESULTS: There were 202 patients in this study and the mean spontaneous AR rate was 5.99%±5.18%. For patients with the spontaneous AR rate ≥9% versus <9%, the fertility rate, normal fertilization rate and high-quality embryo rate were 81.33% vs 83.85%, 60.53% vs 60.99%, and 51.10% vs 59.67%, respectively, with statistically significant difference in the high-quality embryo rate (P=0.02). For patients who underwent fresh embryo transplantation, when comparison was made between those with spontaneous AR rate ≥8% and those <8%, the rate of chemical pregnancy and clinical pregnancy were 48.57% (17/35) vs 69.64% (78/112) and 37.14% (13/35) vs 63.39% (71/112), respectively, both with statistically significant difference (P=0.02 and P<0.01). The patients with clinical pregnancy had lower spontaneous AR rate than those without clinical pregnancy (5.41%±3.87% vs 7.40%±6.79%, P=0.04), while the quantity of acrosomal enzyme showed no significant difference [(131.79±68.50) vs (153.62±59.59) µU/10(6,) P=0.06]. Logistic regression analysis demonstrated association between spontaneous AR rates and clinical pregnancy (OR=0.93, 95%CI: 0.87-0.99, P=0.03). CONCLUSIONS: The spontaneous AR rate of sperm may have clinical significance in predicting the outcome of IVF-ET, as it is reversely correlated with IVF high-quality embryo rate and pregnancy rate. The quantity of acrosomal enzyme may not have significant predictive value for the outcome of IVF.

Anti-Müllerian hormone in serum and seminal plasma in comparison with other male fertility parameters.

Anti-Müllerian hormone (AMH) is a factor most associated with female fertility and especially with the ovarian reserve. AMH is also used as a parameter of fertility in men as it arises from the epithelium of the seminiferous tubules that contain Sertoli cells which produce the AMH. To investigate the relationship between AMH production and sperm related parameters we compared the AMH levels in serum and seminal plasma between a group of healthy males (n=65) and male patients (n=68) of infertile couples with semen pathology. We assessed the following fertility parameters: sperm count (SC), presence of intra-acrosomal enzymes (IAE), and antispermatozoal antibodies (ASA). Infertile men were divided into four subgroups according to: SC less than 15 million, SC less than 15 million and lack of IAE, SC less than 15 million and presence of ASA, presence of all three pathological parameters. The mean AMH serum level in the healthy group was 6.95 ng/ml and no significant difference was observed in serum AMH levels. The mean AMH seminal plasma level in the healthy group was 14.21 ng/ml. We observed a statistically significant decrease in the group with a SC with less than 15 million (3.29 ng/ml, p=0.0001) sperm, in the group with SC less than 15 million sperm and lack of IAE (3.95 ng/ml, p=0.0046), and in the group with all three pathological parameters (2.65 ng/ml, p=<0.0001). No significant difference was observed in the group with SC less than 15 million sperm and ASA positivity (11.41 ng/ml, p=0.3171). In conclusion AMH serum levels do not correlate with any of the observed parameters. AMH levels in seminal plasma positively correlate with the pathological SC and with SC pathology and IAE together.

Specific deletion of AMP-activated protein kinase (α1AMPK) in mouse Sertoli cells modifies germ cell quality.

The AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis which plays a role in fertility. Complete disruption of the AMPK catalytic subunit α1 gene (α1AMPK KO) in male mice results in a decrease in litter size which is associated with the production of altered sperm morphology and motility. Because of the importance of Sertoli cells in the formation of germ cells, we have chosen to selectively disrupt α1AMPK only in the Sertoli cells in mice (Sc-α1AMPK-KO mice). Specific deletion of the α1AMPK gene in Sertoli cells resulted in a 25% reduction in male fertility associated with abnormal spermatozoa with a thin head. No clear alterations in testis morphology or modification in the number of Sertoli cells in vivo were observed, but a dysregulation in energy metabolism in Sertoli cells occurred. We have reported an increase in lactate production, in lipid droplets, and a reduction in ATP production in Sc-α1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-α). In addition another metabolic sensor, the deacetylase SIRT1, had a reduction in expression which is correlated with a decline in deacetylase activity. Finally, expression and localization of junctions forming the blood-testis barrier between Sertoli cells themselves and with germ cells were deregulated in Sc-α1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of α1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility.

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2ß (iPLA2ß) and Group X Secreted Phospholipase A2 (sPLA2).

Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2ß and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2ß is critical for spontaneous AR, whereas both iPLA2ß and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 µm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2ß, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 µm) but not at higher P4 concentrations (~10 µm).

Carbonic anhydrases and their functional differences in human and mouse sperm physiology.

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.

Identification and characterization of a bovine sperm acrosomal matrix protein and its mechanism of interaction with acrosomal hydrolases.

Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19 kDa polypeptides. The objective of this study is to identify and characterize the 45 kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.

Lack of protein kinase C-delta (PKCδ) disrupts fertilization and embryonic development.

This study tested the function of protein kinase C delta (PKCδ) during fertilization and embryonic development using gene-knockout (Prkcd(-/-)) mice. Fertility analysis revealed that Prkcd(-/-) mating pairs produce significantly fewer pups per litter than wild-type pairs (P < 0.05), and exhibit a high incidence of embryonic loss post-implantation. Both Prkcd(-/-) male as well as Prkcd(-/-) female mice mated to Prkcd(+/+) controls also showed reduced litter sizes, with a selective loss of Prkcd-null pups. Further analysis of the females demonstrated comparable in vitro fertilization outcomes between control and Prkcd(-/-) oocytes fertilized with wild-type sperm. Pregnant Prkcd(-/-) females, however, exhibited a reduced number of total implantations, suggesting a possible disruption in early embryo quality and/or implantation. In turn, male gamete analysis revealed that Prkcd(-/-) sperm demonstrated a decreased capacity to penetrate the zona pellucida (P < 0.05), necessary for successful fertilization. Moreover, we identified phosphorylated PKCδ as a component of the sperm acrosome, indicating a potential role for this kinase in acrosome exocytosis. Therefore, loss of PKCδ disrupts key reproductive functions in both males and females that limit fertility.

Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 µm) or betulinic acid (200 µm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12-h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.