Female Reproductive Fluids Research Articles

Thiol based mechanism internalises interacting partners to outer dense fibers in sperm

The sperm tail outer dense fibres (ODFs) contribute passive structural role in sperm motility. The level of disulphide cross-linking of ODFs and their structural thickness determines flagellar bending curvature and motility. During epididymal maturation, proteins are internalized to modify ODF disulphide cross-linking and enable motility. Sperm thiol status is further altered during capacitation in female tract. This suggests that components in female reproductive tract acting on thiol/disulphides could be capable of modulating the tail stiffness to facilitate modulation of the sperm tail rigidity and waveform en route to fertilization. Understanding the biochemical properties and client proteins of ODFs in reproductive tract fluids will help bridge this gap. Using recombinant ODF2 (aka Testis Specific Antigen of 70 kDa) as bait, we identified client proteins in male and female reproductive fluids. A thiol-based interaction and internalization indicates sperm can harness reproductive tract fluids for proteins that interact with ODFs and likely modulate the tail stiffness en route to fertilization.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.

Extracellular vesicles from oviductal isthmus and ampulla stimulate the induced acrosome reaction and signaling events associated with capacitation in bovine spermatozoa.

Cells can communicate with other neighboring or distant cells through the secretion of extracellular vesicles (EV), composed of a lipid bilayer and bearing surface molecules that allow them to recognize target cells. In this way, EV induce signaling via different mechanisms, modulating the physiological state of the recipient cell. EV have been identified in both male and female reproductive fluids, however, the possible role of EV isolated from female reproductive fluids has become an emerging field only recently. It is known that ejaculated mammalian spermatozoa need to undergo physiological preparation in the female reproductive tract to fertilize the egg. EV secreted by different regions of the female tract constitute signals that may have a key role in regulating sperm functions. The aims of the present study were isolating EV from different regions of the bovine oviduct and analyzing their interaction and physiological effects on spermatozoa. Here, we report the characterization of bovine oviductal fluid EV from the isthmus and ampulla region and their effect on the induced acrosome reaction and signaling events associated with sperm capacitation. EV induced an increase in sperm protein tyrosine phosphorylation, while cell survival of cryopreserved bovine spermatozoa was maintained. We also show that EV uptake regulates the sperm calcium levels by inducing an immediate increase in the intracellular calcium concentration and sperm priming, after a pre-incubation period, of the progesterone-induced intracellular calcium rise. Our data contribute to understand the role of EV in the communication between the female reproductive tract and the sperm physiology, information that may be used to improve the efficiency of reproductive assisted technologies.

Female nutritional condition affects ovarian fluid quality in guppies.

Male and female gametes are often embedded in fluids that are produced by gonads and other reproductive tissues. Female reproductive fluids, usually called ovarian fluid (OF), which often constitute a relevant volumetric component of the egg mass, are rich in ions, sugars and proteins, and are involved in several functions, from protecting gametes to facilitating fertilization, and often act as mediators of post-mating sexual selection. Despite their applied and evolutionary importance, we know virtually nothing about the costs of female reproductive fluid production. We investigated the effect of nutritional condition on OF quality by experimentally manipulating the diet of two groups of female guppies (Poecilia reticulata) which were maintained for 20 days either on a restricted diet or had ad libitum access to food. In this species, OF enhances sperm swimming longevity and velocity (a predictor of sperm competition success) and mediates post-copulatory inbreeding avoidance. We found that sperm velocity was significantly lower in the OF of diet-restricted females, indicating that OF quality is dependent on female nutritional condition. Our results demonstrate that OF represents a non-trivial component of female reproductive investment and provides a tool to investigate which OF constituents are involved in modulating OF-sperm interactions and fertilization.

Copper: a biphasic regulator of caprine sperm forward progression

Copper is essential for spermatogenesis and its presence has been demonstrated in male and female reproductive fluids in several mammalian species. However, little is known about the physiological significance of this trace element in the regulation of forward progression of mammalian sperm cells which is essential for sperm fertility potential in vivo. The purpose of this investigation was to determine the physiological role of the bivalent copper ion (Cu2+) on mammalian sperm forward motility using a chemically-defined medium and caprine cauda epididymal sperm model. Sperm forward motility was significantly enhanced by Cu2+ in a dose-dependent manner; maximal activation (approx 20%) was noted at the 5 µM level of the metal. Above 10 µM Cu2+ sperm motility decreased, showing that Cu2+ exerts a biphasic regulation on sperm motility. These findings have been confirmed using a spectrophotometric motility assay, an objective method of motility analysis. At lower concentrations (up to 5 µM), copper enhanced sperm membrane lipid peroxidation as well as the level of intra-sperm cyclic adenosine mono phosphate (c-AMP), but at a higher level it caused marked inhibition of both of the biochemical parameters. The observed correlation of Cu2+-dependent biphasic modulation of sperm membrane lipid peroxidation and intrasperm c-AMP with sperm forward motility is consistent with the view that Cu2+ regulation of sperm motility is mediated by membrane lipid peroxidation, which in turn modulates the level of intra-sperm c-AMP, a well-known activator of sperm motility.

P,p′-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations

STUDY QUESTIONIs the environmental endocrine disruptor p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters?SUMMARY ANSWERp,p′-DDE promoted Ca2+ flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization.WHAT IS KNOWN ALREADYp,p′-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known.STUDY DESIGN, SIZE, DURATIONNormozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p′-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p′-DDE incubation periods were also performed in order to monitor sperm rapid Ca2+ responses. All experiments were repeated on a minimum of five sperm samples from different individuals.PARTICIPANTS/MATERIALS, SETTING, METHODSAll healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca2+ concentration ([Ca2+]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively.MAIN RESULTS AND THE ROLE OF CHANCEp,p′-DDE rapidly increased [Ca2+]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca2+ medium demonstrated that extracellular Ca2+ influx was responsible for this Ca2+ increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p′-DDE-induced [Ca2+]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p′-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p′-DDE concentrations, suggesting that [Ca2+]i rise may cause premature acrosome reaction (P < 0.05).LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study, and caution must be taken when extrapolating the results.WIDER IMPLICATIONS OF THE FINDINGSA novel non-genomic p,p′-DDE mechanism specific to sperm is shown in this study. p,p′-DDE was able to induce [Ca2+]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors.STUDY FUNDING/COMPETING INTEREST(S)The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.

Fluid control: Orchestrating blood flow in the Durgā Pūjā

Tantric rituals are often concerned with the manipulation of life fluids. The Bengali Durgā Pūjā, an annual worship celebration for the Hindu Great Goddess, is an example of an essentially Tantric ritual with orthodox Vedic and Puranic features. Although the overt rationales for the ritual have undergone changes over the centuries, from its martial origins to socio-economic display, the liturgy offers an underlying rationale that reveals its Tantric interests. Ritual features, symbolic elements, and the states and statuses of the actors during the enactment of the Durgā Pūjā demonstrate its concern with orchestrating the flow of female reproductive fluids through a ritual manipulation of the divine feminine. This paper will articulate the most salient of those aspects. Although often overlooked, because of its non-Tantric façade, the Bengali Durga Pūjā points us to a valuable resource for the study of Hindu Śākta Tantrism.

Investigating the role of murine epididymosomes and uterosomes in GPI‐linked protein transfer to sperm using SPAM1 as a model

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation.

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.

Ionized calcium in human male and female reproductive fluids: relationships to sperm motility

The levels of ionized calcium in seminal plasma were approximately 20% of the serum levels. In contrast, cervical mucus contained a level of ionized calcium similar to both serum and follicular fluid. Titration of seminal plasma and serum with increasing concentrations of calcium chloride indicated a 10-fold higher calcium-binding capacity for seminal plasma. In a random group of men under semen investigation, concentrations of ionized calcium and citrate in semen were inversely correlated (r = 0.732; P less than 0.001), an observation which was confirmed by studies of split ejaculates. These findings supported the contention that citrate is the major regulator of the levels of ionized calcium in seminal plasma and primarily responsible for maintaining the calcium gradient between the seminal plasma and cervical mucus. No significant relationship could be demonstrated between the levels of ionized calcium in the ejaculates and any of the motility characteristics of the spermatozoa in the same sample. Furthermore, the addition of increasing quantities of calcium chloride (0.16-20.00 mM) to washed spermatozoa had no major effects on their progressive motility. These data suggest that human spermatozoa are effective in maintaining an appropriate level of internal ionized calcium, necessary for normal motility, despite fluctuations in external calcium.