Impaired mitochondrial function in murine oocytes is associated with controlled ovarian hyperstimulation and in vitro maturation.

The present study was designed to determine whether controlled ovarian hyperstimulation (COH) and in vitro maturation (IVM), two common clinical procedures in human IVF treatment, have an impact on mitochondrial DNA (mtDNA) copy number and mitochondrial function in oocytes. Matured mouse oocytes recovered following COH, IVM and natural cycles (NC), which simulated those treatments in human clinic IVF treatment. The copies of mtDNA, the activity of mitochondria as determined by inner mitochondrial membrane potential and oocyte adenosine trisphosphate (ATP) content, pattern of mitochondrial distribution, reactive oxygen species (ROS) levels and the integrity of the cytoskeleton were evaluated in oocytes. Significant differences were detected between COH and NC groups in all measures, except the pattern of mitochondrial distribution and ROS levels. There were also significant differences detected between IVM and NC treatment groups in the copies of mitochondrial DNA, the level of ROS and the integrity of the cytoskeleton in oocytes. In conclusion, the results of this investigation indicate that non-physiological COH and IVM treatments inhibit mtDNA replication, alter mitochondrial function and increase the percentage of abnormal cytoskeleton and ROS production. Damage related to the mitochondria may partly explain the low efficiency of IVF and high rate of embryonic loss associated with these clinical procedures.

Multifunctional glycoprotein DEFB126--a curious story of defensin-clad spermatozoa.

During maturation, the surface of mammalian spermatozoa undergoes dramatic changes leading to the acquisition of properties vital for survival and performance in the female reproductive tract. A prominent change is the addition to the sperm surface of an atypical ß-defensin polypeptide. In primates, the ß-defensin DEFB126 becomes adsorbed to the entire sperm surface as spermatozoa move through the epididymal duct. DEFB126 has a conserved ß-defensin core and a unique long glycosylated peptide tail. The carbohydrates of this domain contribute substantially to the sperm glycocalyx. DEFB126 is critical for efficient transport of sperm in the female reproductive tract, preventing their recognition by the female immune system, and might facilitate the delivery of capacitated sperm to the site of fertilization. A newly discovered dinucleotide deletion in the human DEFB126 gene is unusually common in diverse populations and results in a null allele. Predictably, men who are homozygous for the deletion produce sperm with an altered glycocalyx and impaired function, and have reduced fertility. Insights into the biology of DEFB126 are contributing to a better understanding of reproductive fitness in humans, as well as the development of diagnostics and therapeutics for male infertility.

A common mutation in the defensin DEFB126 causes impaired sperm function and subfertility.

A glycosylated polypeptide, ß-defensin 126 (DEFB126), derived from the epididymis and adsorbed onto the sperm surface, has been implicated in immunoprotection and efficient movement of sperm in mucosal fluids of the female reproductive tract. Here, we report a sequence variant in DEFB126 that has a two-nucleotide deletion in the open reading frame, which generates an abnormal mRNA. The allele frequency of this variant sequence was high in both a European (0.47) and a Chinese (0.45) population cohort. Binding of the Agaricus bisporus lectin to the sperm surface glycocalyx was significantly lower in men with the homozygous variant (del/del) genotype than in those with either a del/wt or a wt/wt genotype, suggesting an altered sperm glycocalyx with fewer O-linked oligosaccharides in del/del men. Moreover, sperm from del/del carriers exhibited an 84% reduction in the rate of penetration of a hyaluronic acid gel, a surrogate for cervical mucus, compared to the other genotypes. This reduction in sperm performance in hyaluronic acid gels was not a result of decreased progressive motility (average curvilinear velocity) or morphological deficits. Nevertheless, DEFB126 genotype and lectin binding were correlated with sperm performance in the penetration assays. In a prospective cohort study of newly married couples who were trying to conceive by natural means, couples were less likely to become pregnant and took longer to achieve a live birth if the male partner was homozygous for the variant sequence. This common sequence variation in DEFB126, and its apparent effect of impaired reproductive function, will allow a better understanding, clinical evaluation, and possibly treatment of human infertility.

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel.

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze ("fresh") and post-thawed ("thawed") samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, "Pap"-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.

Macaque sperm coating protein DEFB126 facilitates sperm penetration of cervical mucus.

BACKGROUND: Sperm coating protein beta-defensin 126 (DEFB126) is adsorbed onto the entire surface of macaque sperm in the caudal epididymis and is retained on viable sperm collected from the cervix and the uterine lumen of mated female macaques. We investigated the role of sperm coating protein DEFB126 in cervical mucus penetration (CMP). METHODS: Cervical mucus (CM) was collected from peri-ovulatory female macaques and loaded into CMP chambers. Sperm were introduced to CMP chambers following treatment with either polyclonal antibodies raised to DEFB126 or seminal plasma proteins (SPPs), 1 mM caffeine+1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces release of DEFB126 from sperm surface), neuraminidase (NMase) or poly-L-lysine (PLP). Following removal of DEFB126 or SPPs from the sperm surface, sperm were treated with concentrated DEFB126 or concentrated SPPs prior to being introduced to CMP chambers. The numbers of sperm that penetrated and traversed CM were scored over 6 min. RESULTS: Treatment of sperm with anti-DEFB126 antibodies, 1 mM caffeine+1 mM dbcAMP, NMase, and PLP resulted in similar and significant levels of inhibition of sperm CMP, whereas addition of anti-SPPs antibodies had no effect. In experiments where DEFB126 and SPPs were removed, CMP capability of sperm was restored by addition of DEFB126 back to the sperm surface, whereas treatment of sperm with concentrated SPPs slightly inhibited sperm penetration. CONCLUSIONS: DEFB126 and its high negative charge appears to be critical for the movement of sperm through CM in the macaque, while SPPs adhered to the sperm surface offer no advantage in CMP.

Beta-defensin 126 on the surface of macaque sperm mediates attachment of sperm to oviductal epithelia.

Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.

Macaque sperm release ESP13.2 and PSP94 during capacitation: the absence of ESP13.2 is linked to sperm-zona recognition and binding.

ESP13.2 coats the entire surface of macaque sperm and remains until sperm become capacitated (Yudin et al., 2003: Biol Reprod 69: 1118-1128). Capacitation of macaque sperm is synchronized by treatment with dibutyrl cAMP (dbcAMP) and caffeine. ESP13.2 and PSP94 constituted approximately 95% of the proteins released from the sperm surface following treatment with caffeine + dbcAMP. Caffeine and dbcAMP alone induce different patterns of ESP13.2 release. As determined by ELISAs of supernatants and immuno-fluorescent labeling of sperm heads, caffeine alone and caffeine + dbcAMP induced comparable release of ESP13.2, while dbcAMP-treated sperm did not differ from controls. Sperm treated with caffeine + dbcAMP showed a reduction of ESP13.2 from the entire surface, while caffeine treatment alone induced removal of ESP13.2 from the sperm head and midpiece. As confirmed with immunofluorescence, ESP13.2 could be added back to the surfaces of sperm that had been previously exposed to caffeine. Treatment with caffeine significantly increased the number of sperm that bound tightly to the zona pellucida as compared with controls (42 +/- 9 and 13 +/- 3 sperm/zona, respectively; P < or = 0.01). This increase in binding was inhibited by "adding back" ESP13.2 to the sperm surface (12.8 +/- 3; P < or = 0.01). Alexa-conjugated anti-ESP13.2 Ig labeling of live sperm showed that only sperm lacking ESP13.2 over the head were capable of tight binding to the zona. Our results suggest that ESP13.2 masks zona pellucida ligands on the sperm surface and its release, as part of capacitation, is required for sperm-zona interaction.

Lignosulfonic acid blocks in vitro fertilization of macaque oocytes when sperm are treated either before or after capacitation.

Lignin-derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell-to-cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of ligno-sulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm-oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass "binding chambers." The number of capacitated sperm that attached to the zona over a 3-minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P <.001) or before washing (82.5%; P <.001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm-oocyte interaction, sperm-zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% +/- 17%. No LSA-treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.