Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract.

We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.

17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, is an effective spermicide and microbicide against Neisseria gonorrhoeae.

STUDY QUESTION: Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17? SUMMARY ANSWER: At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium. WHAT IS KNOWN ALREADY: LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium; in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21-25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay. MAIN RESULTS AND THE ROLE OF CHANCE: At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment. LIMITATIONS, REASONS FOR CAUTION: For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action. WIDER IMPLICATIONS OF THE FINDINGS: Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Antimicrobial Peptide, LL-37, And Its Potential As An Anti-HIV Agent.

Human immunodeficiency virus (HIV) continues to have a profound global health impact. New infections continue at a high rate despite the development of prophylactic therapies, prompting the need for development of novel preventative approaches. Antimicrobial peptides (AMPs), such as LL-37, display broad microbicidal properties and have potential as anti-HIV agents. LL-37 has been studied for its anti-HIV activity and the limited data available suggest it can inhibit HIV infection in primary T cells as well as exert inhibitory effects on key HIV enzymes. Its immunomodulatory properties may both enhance and inhibit HIV replication. In addition, LL-37 has both 1) the ability to kill other sexually-transmitted pathogens and 2) spermicidal activity; thus, it is a good candidate for multipurpose prevention technology. Further investigation of its anti-HIV activity is warranted.

Antimicrobial peptide LL-37 and its truncated forms, GI-20 and GF-17, exert spermicidal effects and microbicidal activity against Neisseria gonorrhoeae.

STUDY QUESTION: Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37? SUMMARY ANSWER: GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37. WHAT IS KNOWN ALREADY: The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day. PARTICIPANTS/MATERIALS, SETTING, METHODS: The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method. MAIN RESULTS AND THE ROLE OF CHANCE: Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 µM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 µM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 µM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 µM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues. WIDER IMPLICATIONS OF THE FINDINGS: Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.

Clusterin in the mouse epididymis: possible roles in sperm maturation and capacitation.

Clusterin (CLU) is known as an extracellular chaperone for proteins under stress, thus preventing them from aggregation and precipitation. We showed herein that CLU, expressed by principal cells of the mouse caput epididymis, was present in high amounts in the lumen. In the cauda epididymis, CLU bound tightly to the sperm head surface and its amount on total sperm was similar to that in the bathing luminal fluid. In both immotile and motile caudal epididymal sperm, CLU was localized over the entire sperm head except at the convex ridge, although in the motile sperm population, the CLU immunofluorescence pattern was distinctively mottled with a lower intensity. However, when motile sperm became capacitated, CLU was relocalized to the head hook region, with immunofluorescence intensity being higher than that on the non-capacitated counterparts. Under a slightly acidic pH of the epididymal lumen, CLU may chaperone some luminal proteins and deliver them onto the sperm surface. Immunoprecipitation of epididymal fluid proteins indicated that CLU interacted with SED1, an important egg-binding protein present in a high amount in the epididymal lumen. In a number of non-capacitated sperm, fractions of SED1 and CLU co-localized, but after capacitation, SED1 and CLU dissociated from one another. While CLU moved to the sperm head hook, SED1 translocated to the head convex ridge, the egg-binding site. Overall, CLU localization patterns can serve as biomarkers of immotile sperm, and non-capacitated and capacitated sperm in mice. The chaperone role of CLU may also be important for sperm maturation and capacitation.

Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive.

STUDY QUESTION: Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER: Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY: LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION: Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE: Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

Rats Orally Administered with Ethyl Alcohol for a Prolonged Time Show Histopathology of the Epididymis and Seminal Vesicle Together with Changes in the Luminal Metabolite Composition.

Prolonged ethanol (EtOH) consumption is associated with male infertility, with a decreased spermatogenesis rate as one cause. The defective maturation and development of sperm during their storage in the cauda epididymis and transit in the seminal vesicle can be another cause, possibly occurring before the drastic spermatogenesis disruption. Herein, we demonstrated that the cauda epididymis and seminal vesicle of rats, orally administered with EtOH under a regimen in which spermatogenesis was still ongoing, showed histological damage, including lesions, a decreased height of the epithelial cells and increased collagen fibers in the muscle layer, which implicated fibrosis. Lipid peroxidation (shown by malondialdehyde (MDA) levels) was observed, indicating that reactive oxygen species (ROS) were produced along with acetaldehyde during EtOH metabolism by CYP2E1. MDA, acetaldehyde and other lipid peroxidation products could further damage cellular components of the cauda epididymis and seminal vesicle, and this was supported by increased apoptosis (shown by a TUNEL assay and caspase 9/caspase 3 expression) in these two tissues of EtOH-treated rats. Consequently, the functionality of the cauda epididymis and seminal vesicle in EtOH-treated rats was impaired, as demonstrated by a decreases in 1H NMR-analyzed metabolites (e.g., carnitine, fructose), which were important for sperm development, metabolism and survival in their lumen.

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein.

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca(2+) regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca(2+) oscillation without changing the basal intracellular Ca(2+) concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca(2+) concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca(2+) regulation may be a common feature among animal sperm.

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner.

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm-egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells.

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization.

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.

Accumulation of Seminolipid in Sertoli Cells Is Associated with Increased Levels of Reactive Oxygen Species and Male Subfertility: Studies in Aging Arsa Null Male Mice.

Seminolipid (also known as sulfogalactosylglycerolipid-SGG), present selectively in male germ cells, plays important roles in spermatogenesis and sperm-egg interaction. The proper degradation of SGG in apoptotic germ cells is also as important. Sertoli cells first phagocytose apoptotic germ cells, then Sertoli lysosomal arylsulfatase A (ARSA) desulfates SGG, the first step of SGG degradation. We have reported that aging male Arsa-/- mice become subfertile with SGG accumulation in Sertoli cell lysosomes, typical of a lysosomal storage disorder (LSD). Since reactive oxygen species (ROS) levels are increased in other glycolipid-accumulated LSDs, we quantified ROS in Arsa-/- Sertoli cells. Our analyses indicated increases in superoxide and H2O2 in Arsa-/- Sertoli cells with elevated apoptosis rates, relative to WT counterparts. Excess H2O2 from Arsa-/- Sertoli cells could travel into testicular germ cells (TGCs) to induce ROS production. Our results indeed indicated higher superoxide levels in Arsa-/- TGCs, compared with WT TGCs. Increased ROS levels in Arsa-/- Sertoli cells and TGCs likely caused the decrease in spermatogenesis and increased the abnormal sperm population in aging Arsa-/- mice, including the 50% decrease in sperm SGG with egg binding ability. In summary, our study indicated that increased ROS production was the mechanism through which subfertility manifested following SGG accumulation in Sertoli cells.

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt(+/⁻) mice.

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt(+/⁻) mice was quantified to be 406.06 ± 23.63 µg/g testis and 0.13 ± 0.02 µg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt⁻/⁻ males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt(+/⁻) males sire offspring. The higher than 50% expression level of SGG in Cgt(+/⁻) animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt(+/⁻) mice were sufficient for normal spermatogenesis.

Primary Sertoli Cell Cultures From Adult Mice Have Different Properties Compared With Those Derived From 20-Day-Old Animals.

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

Composition and significance of detergent resistant membranes in mouse spermatozoa.

Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction.

Interaction of arylsulfatase-A (ASA) with its natural sulfoglycolipid substrates: a computational and site-directed mutagenesis study.

Arylsulfatase A (ASA) hydrolyzes sulfate esters with a pH optimum of 5. Interactions between p-nitrocatechol sulfate (NCS, artificial substrate) and active site residues of ASA are revealed from their co-crystal structure. Since equivalent ASA interactions with its natural substrates, sulfogalactosylceramide (SGC) and sulfogalactosylglycerolipid (SGG), are not known, we computationally docked SGC/SGG to the ASA crystal structure. Our dockings suggested that Cys69 was the active site residue, and Lys302 & Lys123 as residues anchoring the sulfate group of SGC/SGG to the active site, as observed for NCS. We further confirmed these results using 2 recombinant ASA mutants: C69A and CKK (Cys69, Lys302 and Lys123-all mutated to Ala). Both ASA mutants failed to desulfate SGC/SGG, and CKK showed minimal binding to [(14)C]SGC, although C69A still had affinity for this sulfoglycolipid. However, our dockings suggested additional intermolecular hydrogen bonding and hydrophobic interactions between ASA and SGC/SGG, thus contributing to the specificity of SGC/SGG as natural substrates.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts.

Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-beta-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed.

Mammalian hyaluronidase induces ovarian granulosa cell apoptosis and is involved in follicular atresia.

During ovarian folliculogenesis, the vast majority of follicles will undergo atresia by apoptosis, allowing a few dominant follicles to mature. Mammalian hyaluronidases comprise a family of six to seven enzymes sharing the same catalytic domain responsible for hyaluronan hydrolysis. Interestingly, some of these enzymes have been shown to induce apoptosis. In the ovary, expression of three hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) has been documented. However, their precise cellular localization and role in ovarian regulation have not yet been defined. We herein investigated the possible involvement of these enzymes in ovarian atresia. First, we established a mouse model for ovarian atresia (gonadotropin withdrawal by anti-equine chorionic gonadotropin treatment) and showed that the mRNA levels of Hyal-1, Hyal-2, and Hyal-3 were significantly increased in apoptotic granulosa cells as well as in atretic follicles. Second, using ovaries of normally cycling mice, we demonstrated the correlation of Hyal-1 mRNA and protein expression with cleavage of caspase-3. In addition, we showed that expression of all three hyaluronidases induced apoptosis in transfected granulosa cells. Significantly, the induction of apoptosis by hyaluronidases was independent of catalytic activity, because enzymatically inactive Hyal-1 mutant (D157A/E159A) was as efficient as the wild-type enzyme in apoptosis induction. The activation of the extrinsic apoptotic signaling pathway was involved in this induction, because increased levels of cleaved caspase-8, caspase-3, and poly-ADP-ribose polymerase (PARP) were observed upon hyaluronidase ectopic expression. Our present findings provide a better understanding of the role of hyaluronidases in ovarian functions, showing for the first time their involvement in follicular atresia.