Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.

In vivo three-dimensional tracking of sperm behaviors in the mouse oviduct.

Mammalian sperm evolutionarily acquired complex mechanisms to regulate their behaviors, which are thought to be crucial in navigating through the female reproductive tract toward fertilization. However, all current knowledge of this process is largely extrapolated from in vitro and ex vivo studies, because in vivo analysis of sperm in their native fertilization environment has not been possible. Here, we report a functional optical coherence tomography approach that allows, for the first time, in vivo three-dimensional tracking of sperm behaviors in the mouse oviduct. Motile sperm are identified with their intrinsic dynamic characteristics. Sperm trajectories are reconstructed in three dimensions with a ∼5 µm spatial resolution, allowing for quantitative analysis of the sperm velocity and location relative to the oviduct. Using this method, we found different behavior patterns, including sperm collection by the oviduct epithelium, spatial dependence of sperm velocity, and sperm grouping and separation as the first in vivo evidence of sperm cooperation in the ampulla, the site of fertilization. This approach opens new avenues to study sperm-oviduct interactions in vivo toward a more complete understanding of fertility and reproductive disorders.

Active peristaltic movements and fluid production of the mouse oviduct: their roles in fluid and sperm transport and fertilization†.

To study how the oviduct behaves in relation to fluid secretion and sperm transport, ovary-oviduct-uterus complexes of the mouse were installed in a fluid-circulating chamber without disturbing the blood circulation or parasympathetic innervation. Injection of a bolus of Indian ink into the lower isthmus revealed very active adovarian peristalsis of the isthmus, which was most prominent during the periovulatory period. Oviduct fluid, secreted by the entire length of the isthmus, was rapidly transported to the ampulla and ovarian bursa before draining into the peritoneal cavity. The upper isthmus, in particular the isthmic-ampullary junction, was responsible for this adovarian fluid flow. Peristalsis of the oviduct, undisturbed flow of oviduct fluid from the isthmus to the peritoneal cavity, and the spermatozoon's own motility all contribute to efficient sperm ascent and to fertilization within the oviduct. Therefore, chemotaxis, rheotaxis, and thermotaxis of spermatozoa toward oocyte-cumulus complexes in the ampulla are all unlikely mechanisms for explaining sperm-oocyte contact and successful fertilization, given the rapid adovarian flow of oviduct fluid in this species.

The flagellar protein Enkurin is required for mouse sperm motility and for transport through the female reproductive tract.

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.

Deciphering the mechanisms involving cenexin, ninein and centriolin in sperm maturation during epididymal transit in the domestic cat.

The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin-a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%-26% and 33%-48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation.

A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila.

Innate behaviors are often executed in concert with accompanying physiological programs. How this coordination is achieved is poorly understood. Mating behavior and the transfer of sperm and seminal fluid (SSFT) provide a model for understanding how concerted behavioral and physiological programs are coordinated. Here we identify a male-specific neural pathway that coordinates the timing of SSFT with the duration of copulation behavior in Drosophila. Silencing four abdominal ganglion (AG) interneurons (INs) that contain the neuropeptide corazonin (Crz) both blocked SSFT and substantially lengthened copulation duration. Activating these Crz INs caused rapid ejaculation in isolated males, a phenotype mimicked by injection of Crz peptide. Crz promotes SSFT by activating serotonergic (5-HT) projection neurons (PNs) that innervate the accessory glands. Activation of these PNs in copulo caused premature SSFT and also shortened copulation duration. However, mating terminated normally when these PNs were silenced, indicating that SSFT is not required for appropriate copulation duration. Thus, the lengthened copulation duration phenotype caused by silencing Crz INs is independent of the block to SSFT. We conclude that four Crz INs independently control SSFT and copulation duration, thereby coupling the timing of these two processes.

Transport, Distribution and Elimination of Mammalian Sperm Following Natural Mating and Insemination.

The integrity of transport, distribution and elimination of sperm in the female genital tract plays a pivotal role for successful reproduction in mammals. At coitus, millions or billions of sperm are deposited either into the anterior vagina (human, primates), the cervix (most mammalian species) or the uterus (pig). In most species, the first anatomical barrier is the cervix, where spermatozoa with poor morphology and motility are filtered out by sticking to the cervical mucus. The second anatomical barrier is the uterotubal junction (UTJ) with its tortuous and narrow lumen. Finally, only a few thousand sperm enter the oviduct and less than 100 sperm reach the site of fertilization. As soon as the sperm enter the oviduct, they form a sperm reservoir enabling them to stay vital and maintain fertilizing capacity for 3-4 days (cow, horse) up to several months (bats). After ovulation, mammalian sperm show hyperactivation which allows them to detach from the tubal epithelium and migrate to the site of fertilization. This review will focus on recent insights of sperm transport, sperm storage and sperm-oviduct interaction in mammals which have been gained by live cell imaging in cows and mice under near in vivo conditions. Detailed knowledge of the biology of spermatozoa within the female genital tract creates the basis for new therapeutic concepts for male subfertility and infertility - an essential prerequisite to increase success rates in assisted reproduction.

New insights into epididymal function in relation to sperm maturation.

Testicular spermatozoa acquire fertility only after 1 or 2 weeks of transit through the epididymis. At the end of this several meters long epididymal tubule, the male gamete is able to move, capacitate, migrate through the female tract, bind to the egg membrane and fuse to the oocyte to result in a viable embryo. All these sperm properties are acquired after sequential modifications occurring either at the level of the spermatozoon or in the epididymal surroundings. Over the last few decades, significant increases in the understanding of the composition of the male gamete and its surroundings have resulted from the use of new techniques such as genome sequencing, proteomics combined with high-sensitivity mass spectrometry, and gene-knockout approaches. This review reports and discusses the most relevant new results obtained in different species regarding the various cellular processes occurring at the sperm level, in particular, those related to the development of motility and egg binding during epididymal transit.

Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica).

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

[Research progress in sperm mediated gene transfer technology].

With the rapid development of biotechnology, we can change the trait of organism using transgenetic technology. In recent years, there are growing interests in the establishment of sperm mediated gene transfer (SMGT) technology as an effective and convenient method to produce transgenic animals. SMGT technology is a transgenetic method, which is easy in operation and does little harm to the cell compared with the other transgenetic methods. In this review, we expound the background, development, mechanism, operation and application of SMGT.

The oocyte cumulus complex regulates mouse sperm migration in the oviduct.

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-ß ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-ß receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-ß signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.

Can the controversy about the putative role of the human female orgasm in sperm transport be settled with our current physiological knowledge of coitus?

INTRODUCTION: Spermatozoal uptake, facilitated by uterine contractions induced by oxytocin at orgasm during coitus, has been a long term concept. Studies attempting its support, however, have been poorly examined especially in the context of the changes in the female genital tract activated by sexual arousal. AIM: To examine experimental support for the concept. METHODS: Using a variety of search engines, mainly peer reviewed articles and un-reviewed books were examined relating to sperm transport and function in the human female genital tract in the absence and presence of arousal to orgasm. MAIN OUTCOME MEASURES: Identifying evidence-based data to support authority-based opinion. RESULTS: All the experimental observations of sperm or model substitute's transport have been undertaken in women who were not sexually aroused. They fail to take into account that arousal creates vaginal tenting lifting the cervico-uterine complex into the false pelvis away from the ejaculated semen. This delays sperm uptake and transport making conclusions from these observations invalid in relation to transport during coitus. Studies injecting oxytocin have not used women in their sexually aroused state and used supraphysiological doses unlikely to be comparable with coitus and orgasm. The proposal that the transport of extra sperm by oxytocin-induced uterine contractions at orgasm is needed to facilitate fertility ignores possible harm from increased sperm numbers creating polyspermy and sperm enzyme release causing ovum degeneration, leading to decreased fertility. The role of sperm motility in their uptake from the vagina into the cervix as opposed to en bloc transfer through uterine archimyometrial-mediated transport in the absence of orgasm is at present unresolvable because of conflicting studies. CONCLUSION: The bulk of the reported evidence favors the conclusion that the female orgasm, with its concomitant central release of oxytocin, has little or no effective role in the transport of spermatozoa in natural human coitus.

Sperm migration in pigs after deep intrauterine and intraperitoneal insemination.

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.

Absence of estrogen receptor alpha leads to physiological alterations in the mouse epididymis and consequent defects in sperm function.

Male mice deficient in ESR1 (ERalpha) (Esr1KO mice) are infertile, and sperm recovered from the cauda epididymis exhibit reduced motility and fail to fertilize eggs in vitro. These effects on sperm appear to result from defective epididymal function and not a direct effect on spermatogenesis, as Esr1KO germ cells transplanted into wild-type testes yield normal offspring. We hypothesized that the previously described defect in efferent duct fluid reabsorption would lead to alterations in the epididymal fluid milieu, which would negatively impact sperm function. Analysis of the epididymal fluid revealed that the Esr1KO maintains a higher luminal pH throughout the epididymis, confirming an inability of the efferent ducts and/or epididymis to properly acidify the luminal contents. Subsequent studies showed that these abnormalities were not the result of global defects in epididymal function since protein secretion by the Esr1KO epididymis appeared normal as judged by SDS-PAGE of total secreted proteins and by immunoblotting of candidate secreted proteins. To gain insight into the basis of the aberrant fluid homeostasis in the Esr1KO epididymis, the expression of several enzymes and transporters known to be involved in acid/base regulation were analyzed. The levels of SLC9A3 (NHE3) as well as carbonic anhydrase XIV and SLC4A4 (NBC1) were all reduced in the proximal portion of the Esr1KO epididymis, while other components appeared unaffected, including other ion transporters and ATP6V0A1 (V-ATPase). The altered luminal milieu of the Esr1KO epididymis was shown to lead to a corresponding increase in the intracellular pH of Esr1KO sperm, relative to sperm from control animals. Since pH and bicarbonate ions are critical regulators of sperm cAMP levels and motility, we attempted to bypass the abnormal luminal and intracellular environment by supplementing sperm with exogenous cAMP. This treatment rescued all defective motility parameters, as assayed by CASA, further showing that motility defects are not intrinsic to the sperm but, rather, result from the abnormal epididymal milieu.

Cooperation and the evolution of anisogamy.

In the lights of the concept of cooperation wholes, I discuss why the differentiation of sperm and ova can occur with a mathematical model. Most of Parker's explanations for anisogamy are not completely proper, because it is proved that sperm competition is neither sufficient nor necessary for anisogamy and cooperation to deal with fertilization risks is the real key to understand the evolution of anisogamy. According to the computer simulation results, the transport of gametes between different individuals, risks of the transport, the consequent inequality of sperm and eggs and competition among different individuals were the main causes of gamete differentiation. But these factors have different roles and effects. The transport risk is the main reason for individuals of different mating types to cooperate and differentiate into sperm and egg producers. The transported gametes have an advantage to evolve into sperm to seek for a larger gamete number over the fixed gametes, because they suffer more risks as they can encounter the same fixed gamete and less sibling competition as they can be dispersed better. Gamete competition among different individuals just causes the transported gametes to become as small as possible if they have already become smaller beyond a critical state. In the final discussion, I further put the evolution of anisogamy into a broader background of levels of selection and of the evolution of cooperation, the most important existential mode of matters that makes life as life.

Hormonal induction of spermiation, courting behavior and spawning in the southern bell frog, Litoria raniformis.

We trialled the efficacy of various exogenous hormones to induce spermiation, courtship behavior, and spawning in the "endangered" southern bell frog, Litoria raniformis. Intralymphatic administration of Lucrin(®), a synthetic nonapeptide luteinizing hormone releasing hormone (LHRH), was used successfully to induce courting behaviors and ejaculation of spermatozoa in males. Various hormones, including Lucrin(®), another synthetic LHRH analog ([des-Gly(10), D-Ala(6)]-LHRH), human chorionic gonadotropin, progesterone, and a dopamine receptor antagonist failed to promote oviposition and spawning in females. This and earlier studies indicate that in the efficacy of hormonal induction in amphibians varies between taxa, hormones, and genders. The lack of response in females may limit the use of reproduction technology in the southern bell frog and closely related species of Australian bell frogs.

Getting to and away from the egg, an interplay between several sperm transport mechanisms and a complex oviduct physiology.

In mammals, the architecture and physiology of the oviduct are very complex, and one long-lasting intriguing question is how spermatozoa are transported from the sperm reservoir in the isthmus to the oocyte surface. In recent decades, several studies have improved knowledge of the factors affecting oviduct fluid movement and sperm transport. They report sperm-guiding mechanisms that move the spermatozoa towards (rheotaxis, thermotaxis, and chemotaxis) or away from the egg surface (chemorepulsion), but only a few provide evidence of their occurrence in vivo. This gives rise to several questions: how and when do the sperm transport mechanisms operate inside such an active oviduct? why are there so many sperm guidance processes? is one dominant over the others, or do they cooperate to optimise the success of fertilisation? Assuming that sperm guidance evolved alongside oviduct physiology, in this review we propose a theoretical model that integrates oviduct complexity in space and time with the sperm-orienting mechanisms. In addition, since all of the sperm-guidance processes recruit spermatozoa in a better physiological condition than those not selected, they could potentially be incorporated into assisted reproductive technology (ART) to improve fertility treatment and/or to develop innovative contraceptive methods. All these issues are discussed in this review.

The cystic fibrosis transmembrane conductance regulator in reproductive health and disease.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel regulated by cAMP-dependent phosphorylation, which is expressed in epithelial cells of a wide variety of tissues including the reproductive tracts. Mutations in the gene encoding CFTR cause cystic fibrosis, a common genetic disease in Caucasian populations with a multitude of clinical manifestations including infertility/subfertility in both sexes. However, the physiological role of CFTR in reproduction and its involvement in the pathogenesis of reproductive diseases remain largely unknown. This review discusses the role of CFTR in regulating fluid volume and bicarbonate secretion in the reproductive tracts and their importance in various reproductive events. We also discuss the contribution of CFTR dysfunction to a number of pathological conditions. The evidence presented is consistent with an important role of CFTR in reproductive health and disease, suggesting that CFTR might be a potential target for the diagnosis and treatment of reproductive diseases including infertility.

Ciliary transport, gamete interaction, and effects of the early embryo in the oviduct: ex vivo analyses using a new digital videomicroscopic system in the cow.

Using a digital videomicroscopic analysis system in the bovine, we showed that the mechanisms of transport caused by ciliary beating are distinctly different in ampulla and isthmus of the oviduct. The average particle transport speed (PTS) in the oviduct (mean, 133 microm/sec) does not differ in the cycle (metestrus) and during pregnancy after implantation, but it is locally modulated at the site of the embryo. Using videomicroscopy, we were able to document that after entering the ampulla, the cumulus-oocyte complex (COC) is not transported by ciliary beating down the oviduct, but firmly attaches to the ampullar epithelium. This attachment is mediated by the cumulus cells. However, when a COC is degenerated, it is floating in the oviductal lumen. As soon as a vital COC is in the ampulla, the sperm bound in the sperm reservoir of the ampullar isthmic junction leave the reservoir and hurry to the oocyte. When a sperm has penetrated the zona pellucida, the COC detaches and continues its migration. Quantitative measurements showed that the early embryo is able to locally downregulate PTS during its migration down the oviduct. It locally changes the pattern of vascularization and induces the formation of secretory cells. Our studies imply that the oviductal epithelium is able to select vital oocytes. The early embryo is able to induce the formation of secretory cells, modify vascularization, and downregulate speed of transport, thus creating the prerequisite for the first embryo-maternal communication in the oviduct.

Sperm competition and maternal effects differentially influence testis and sperm size in Callosobruchus maculatus.

The evolutionary factors affecting testis size are well documented, with sperm competition being of major importance. However, the factors affecting sperm length are not well understood; there are no clear theoretical predictions and the empirical evidence is inconsistent. Recently, maternal effects have been implicated in sperm length variation, a finding that may offer insights into its evolution. We investigated potential proximate and microevolutionary factors influencing testis and sperm size in the bruchid beetle Callosobruchus maculatus using a combined approach of an artificial evolution experiment over 90 generations and an environmental effects study. We found that while polyandry seems to select for larger testes, it had no detectable effect on sperm length. Furthermore, population density, a proximate indicator of sperm competition risk, was not significantly associated with sperm length or testis size variation. However, there were strong maternal effects influencing sperm length.