Regulation of sperm storage and movement in the mammalian oviduct

The oviduct plays a vital role in ensuring successful fertilization and normal early embryonic development. The male inseminates many thousands or even millions of sperm, but this alone does not ensure that fertilization will be successful. The female tract, particularly the oviduct, provides filters that select for normal vigorously motile sperm. In conjunction with molecules in the seminal plasma and on sperm, the female tract regulates how and when sperm pass though the tract to reach the site of fertilization. Various regulatory processes control sperm passage into and through the oviduct. In some species, the uterotubal junction opens and closes to regulate when sperm may enter; furthermore, passage through the junction requires certain proteins on the sperm surface. Most of the sperm that manage to enter the oviduct soon become trapped and held in a reservoir. In marsupials and insectivores, this involves trapping sperm in mucosal crypts; while in most other mammalian species, this involves binding sperm to the oviductal epithelium. As the time of ovulation approaches, the sperm in the reservoir undergo capacitation, including motility hyperactivation. Capacitating sperm shed proteins that bind them to the mucosal epithelium, while hyperactivation assists the sperm in pulling off of the epithelium and escaping out of mucosal pockets. The process of sperm release is gradual, reducing chances of polyspermic fertilization. Released sperm may be guided towards the oocyte by secretions of the oviduct, cumulus cells, or oocyte. Hyperactivation likely assists sperm in penetrating the cumulus matrix and is absolutely required for penetrating the oocyte zona pellucida and achieving fertilization.     

Polyandry as a mediator of sexual selection before and after mating

The Darwin–Bateman paradigm recognizes competition among males for access to multiple mates as the main driver of sexual selection. Increasingly, however, females are also being found to benefit from multiple mating so that polyandry can generate competition among females for access to multiple males, and impose sexual selection on female traits that influence their mating success. Polyandry can reduce a male''s ability to monopolize females, and thus weaken male focused sexual selection. Perhaps the most important effect of polyandry on males arises because of sperm competition and cryptic female choice. Polyandry favours increased male ejaculate expenditure that can affect sexual selection on males by reducing their potential reproductive rate. Moreover, sexual selection after mating can ameliorate or exaggerate sexual selection before mating. Currently, estimates of sexual selection intensity rely heavily on measures of male mating success, but polyandry now raises serious questions over the validity of such approaches. Future work must take into account both pre- and post-copulatory episodes of selection. A change in focus from the products of sexual selection expected in males, to less obvious traits in females, such as sensory perception, is likely to reveal a greater role of sexual selection in female evolution.     

Calcitonin acts as a first messenger to regulate adenylyl cyclase/cAMP and mammalian sperm function

Calcitonin stimulates capacitation in uncapacitated mouse spermatozoa and then inhibits spontaneous acrosome loss in capacitated cells, responses similar to those elicited by fertilization promoting peptide (FPP), a peptide known to regulate the adenylyl cyclase/cAMP pathway. This study investigated the hypothesis that calcitonin also modulates this pathway. Calcitonin significantly stimulated cAMP production in uncapacitated spermatozoa and then inhibited it in capacitated cells; the magnitude of both stimulatory and inhibitory changes was similar to that obtained with FPP but the inhibitory responses to FPP preceded those of calcitonin. This possibly reflects the involvement of two different adenosine receptors in response to FPP compared with one calcitonin receptor. Calcitonin receptors were located on the acrosomal cap and the flagellum, the midpiece having a greater abundance than the principal piece. Although both calcitonin and adenosine receptors are found in the head and flagellum, there was no evidence for cross-talk between them. Chlortetracycline investigations to determine the minimum extracellular Ca(2+) requirement for responses to calcitonin revealed that calcitonin significantly stimulated capacitation in Ca(2+)-deficient medium but FPP did not. Calcitonin also significantly stimulated cAMP production under these conditions, and similarly preincubated suspensions, when diluted into +Ca(2+) medium, were significantly more fertile in vitro than untreated controls. These results indicate that calcitonin, like FPP, acts as a first messenger to regulate the production of cAMP and mammalian sperm function, but the differences in Ca(2+) requirements suggest that calcitonin and FPP may regulate different isoforms of adenylyl cyclase.     

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization

Dear Editor, Sperm cooperation has been observed in multiple species(Pizzari and Foster,2008),yet its existence and benefit for reproductive success in mammals remains underexplored.Here,combining tissue-clearing with deep three-dimen-sional (3D) imaging,we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooper-ative clusters at the utero-tubal junction (UTJ),a key physical barrier for passage into the oviduct.Reducing sperm number in male mice by unilateral vasoligation (Uni-Vas) or busulfan-treatment impairs sperm cluster formation and oviduct entry.     

Novel function of PERK as a mediator of force-induced apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.     

Regulation of nucleosome stability as a mediator of chromatin function

Interactions among regulatory proteins, enzymes and DNA are required to use the information encoded in genomes. In eukaryotes, the location and timing of interactions between these proteins and DNA is determined in large part by whether DNA at a given locus is packaged into a nucleosome. Given the central role of nucleosome formation in regulating genome function, many mechanisms have evolved to control nucleosome stability at loci across the genome. New conclusions about the role of the DNA sequence itself in the regulation of nucleosome stability have come from two new types of experiment: high-resolution mapping of in vivo nucleosome occupancy on a genomic scale; and in vivo versus in vitro comparisons of nucleosome stability on natural DNA templates. These new types of data raise intriguing questions about the evolutionary constraints on DNA sequence with regard to nucleosome formation, and at long last might enable the derivation of DNA sequence-based rules that produce reliable predictions of nucleosome behavior.     

The sperm, a neuron with a tail: 'neuronal' receptors in mammalian sperm

A number of plasma membrane receptor types originally thought to be specific to neurons have been found in other somatic cells. More surprisingly, the mammalian sperm and neuron appear to share many of these 'neuronal' receptors. The morphology, chromosome number, genomic activity, and functions of those two cell types are as unlike as any two cells in the body, but they both achieve their highly disparate goals with the aid of a number of the same receptors. Exocytosis in neurons and sperm is essential to the functions of these cells and is strongly influenced by similar receptors. 'Neuronal' receptor types in sperm may also play a role in the control of sperm motility (a function of course not shared by neurons). This review will consider the evidence for the presence of sperm plasma membrane 'neuronal' receptors and for their significance to mammalian sperm function. The persuasiveness of the evidence varies depending on the receptor being considered, but there is strong experimental support for the presence and importance of a number of 'neuronal' receptors in sperm.     

Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs

The oviducts of both oestrous and pseudopregnant rabbits can be used for the successful culture of mammalian embryos for short periods. This has alowed some selection to be made on the embryos as they are examined on at least two occasions before final transfer. Not only have pregnancy rates been normal, but in some instances they have been higher following a limited period (2–3 days) in the rabbit oviduct. It would appear that these higher pregnancy rates result from a more intensive selection of embryos at the time of transfer rather than from some substance acquired during storage in the oviduct. However, the system is not without disadvantages. There is some loss of embryos (15–30%) in the oviduct and all embryos recovered may not have developed at the normal rate.The rabbit oviduct has been used as a site of xenogenous fertilization. Initial reports indicate that success in that area is lower than when using large animals as the site of fertilization. With more widespread interest in the use of microsurgery in embryos, the rabbit oviduct has been used for the short term storage of agar cylinders and has been found to be unsuitable because of the high rate of degeneration of agar chips. However, the rabbit oviduct is still useful as an experimental tool in the manipulation of embryos from the domestic species.     

Profile of a mammalian sperm receptor

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.     

S-Nitrosohemoglobin: an allosteric mediator of NO group function in mammalian vasculature

Since the discovery of NO as the endothelium-derived relaxing factor, there has been considerable interest in how NO interacts with hemoglobin (Hb). Numerous investigations have highlighted the possibility that rather than operating as a sink to consume NO, the vasculature can operate as a delivery mechanism for NO. The principal hypothesis proposed to explain this phenomenon is that Hb can transport NO on the conserved cysteine residue beta93 and deliver that NO to the tissues in an allosterically dependent manner. This proposal has been termed the S-Nitrosohemoglobin (SNO-Hb) Hypothesis. This review addresses the experimental evidence that led to development of this hypothesis and examines much of the research that resulted from its generation. Specifically it covers the evidence concerning NO in the vasculature, the SNO-Hb Hypothesis itself, the biochemical and biophysical data relating to NO and Hb interactions, SNO-Hb in human physiology, and alternative vascular forms of NO. Finally a model of NO in the vasculature in which SNO-Hb forms the central core is proposed.     

Role of the oviduct in sperm capacitation

Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation - capacitation in particular - is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.     

Saccadic target selection as a function of time

Recent evidence indicates that stimulus-driven and goal-directed control of visual selection operate independently and in different time windows (van Zoest et al., 2004). The present study further investigates how eye movements are affected by stimulus-driven and goal-directed control. Observers were presented with search displays consisting of one target, multiple non-targets and one distractor element. The task of observers was to make a fast eye movement to a target immediately following the offset of a central fixation point, an event that either co-occurred with or soon followed the presentation of the search display. Distractor saliency and target-distractor similarity were independently manipulated. The results demonstrated that the effect of distractor saliency was transient and only present for the fastest eye movements, whereas the effect of target-distractor similarity was sustained and present in all but the fastest eye movements. The results support an independent timing account of visual selection.     

Effect of bovine oviduct epithelial cell apical plasma membranes on sperm function assessed by a novel flow cytometric approach

In the bovine, as in many mammalian species, sperm are temporarily stored in the oviduct before fertilization by binding to the oviduct epithelial cell apical plasma membranes. As the oviduct is able to maintain motility and viability of sperm and modulate capacitation, we propose that proteins present on the apical plasma membrane of oviduct epithelial cells contribute to these effects. To verify this hypothesis, the motility of frozen-thawed sperm was determined after incubation for 6 h with purified apical plasma membranes from fresh or cultured oviduct epithelial cells or from bovine mammary gland cells as a control. Analysis of intracellular calcium levels was performed by flow cytometry on sperm incubated with fresh membranes using Indo-1 to assess the membrane effect on intracellular calcium concentration. The coculture of sperm with fresh and cultured apical membranes maintained initial motility for 6 h (65% and 84%, respectively). This effect was significantly different from control sperm incubated without oviduct epithelial cell apical membranes (23%), with mammary gland cell apical membranes (23%), or with boiled epithelial cell apical membranes (21%). Apical membranes from oviduct epithelial cells diminished the percentage of sperm that reached a lethal calcium concentration over a 4-h period (18.7%) compared with the control (53.8%) and maintained lower intracellular calcium levels in viable sperm. These results show that the apical plasma membrane of bovine oviduct epithelial cells contains anchored proteinic factors that contribute to maintaining motility and viability and possibly to modulating capacitation of bovine sperm.     

DNA-fluorometry of mammalian sperm

Summary The DNA-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescence distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.