Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface

In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.     

Expression and relationship of male reproductive ADAMs in mouse

A number of a disintegrin and metalloprotease (ADAM) family members are expressed in mammalian male reproductive organs such as testis and epididymis. These reproductive ADAMs are divided phylogenically into three major groups: ADAMs 1, 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the first group); ADAMs 2, 3, 5, 27, and 32 (the second group); and ADAMs 7 and 28 (the third group). Previous mouse knockout studies indicate that ADAM1, ADAM2, and ADAM3 have intricate expressional relationships, playing critical roles in fertilization. In the present study, we analyzed processing, biochemical characteristics, localization, and expressional relationship of the previously-unexplored, second-group ADAMs (ADAM5, ADAM27, and ADAM32). We found that all of the three ADAMs are made as precursors in the testis and processed during epididymal maturation, and that ADAM5 and ADAM32, but not ADAM27, are located on the sperm surface. Using sperm from Adam2(-/-) and Adam3(-/-) mice, we found that, among the three ADAMs, the level of ADAM5 is modestly and severely reduced in Adam3 and Adam2 knockout sperm, respectively. Further, we analyzed ADAM7, an epididymis-derived sperm surface ADAM from the separate phylogenetic group, in the knockout sperm. We found that the level of ADAM7 is also significantly reduced in both Adam2 and Adam3-null sperm. Taken together, our results suggest a novel expressional relationship of ADAM5 and ADAM7 with ADAM2 and ADAM3, which play critical roles in fertilization.     

Localization and expression of ADAM2 in the dromedary camel testis,epididymis and sperm during rutting season

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.     

Processing and subcellular localization of ADAM2 in the Macaca fascicularis testis and sperm

Fertilin, a heterodimeric protein complex composed of ADAM1 and ADAM2 located on the sperm surface, is involved in sperm–egg interaction. In our study, we examined the physiological processing and subcellular localization of M. fascicularis ADAM2 during spermatogenesis in the testis and epididymal tract. M. fascicularis ADAM2 was initially synthesized as a 100 kDa precursor in testicular germ cells. After passing into 50 kDa intermediate form in the epididymal tracts, the precursor form was finally processed into a 47 kDa protein in sperm. We found that M. fascicularis ADAM2 is localized on the sperm surface and contributes to the formation of a candidate fertilin complex. In particular, Far-Western blot analysis revealed that M. fascicularis ADAM2 cystein-rich domain may be related to protein–protein interaction. Therefore, the cystein-rich domain of ADAM2 could provide a mechanism to form a fertilin complex.     

Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.     

Identification of an ADAM2-ADAM3 complex on the surface of mouse testicular germ cells and cauda epididymal sperm

Male mice lacking ADAM2 (fertilin beta) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2(-/-) TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.     

Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice

Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from Clgn-/- mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn-/- and Ace-/- sperm. ADAM3 was absent from Clgn-/- sperm. In the Ace-/- mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergent-enriched phase may explain the inability of Clgn-/- and Ace-/- sperm to bind to the zona pellucida.     

Calsperin is a testis-specific chaperone required for sperm fertility

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.     

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88^Tg737Rpw mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88^−/− mice are completely sterile. They produce ∼350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.     

Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857-1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.     

Construction of a Catsper1 DNA Vaccine and Its Antifertility Effect on Male Mice

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.     

Chromosomal Assignment of Four Testis-Expressed Mouse Genes from a New Family of Transmembrane Proteins (ADAMs) Involved in Cell–Cell Adhesion and Fusion

A new gene family of multidomain membrane proteins (ADAMs) that include isintegrin nd etalloprotease domain comprises an increasing number of identified members. Two members of this family, fertilin α and fertilin β, form a heterodimeric protein that is required for sperm–egg fusion. Most recently, it has been shown that a third family member, meltrin α, is involved in myoblast fusion (Yagami-Hiromasaet al.,1995,Nature377: 652–656). Using restriction fragment length polymorphism analysis of a DNA panel from an interspecific backcross, we have determined the chromosomal locations of four mouse genes of this family that are expressed in testis: fertilin α, fertilin β, ADAM 4, and ADAM 5. These genes have been given the locus symbolsFtna(fertilin α),Ftnb(fertilin β),Adam4(ADAM 4), andAdam5(ADAM 5). They were mapped to chromosomes 5, 14, 9, and 8, respectively, revealing a dispersed localization. Human chromosome locations of these genes are predicted on the basis of the mapping results using the information provided by comparative linkage maps. Because all four of these ADAM genes are expressed in testis and fertilin α and β have been found to be important for fertilization, we compared their chromosomal locations with known mouse mutations affecting spermatogenesis and fertility.     

Male fertility in Mus musculus requires the activity of TRYX5 in sperm migration into the oviduct

Nowadays, abnormal loss of serine proteases appears very frequently in male patients with unexplained sterility. In fact, many testis-specific serine proteases, the largest family among the four protease families implicated in murine spermatogenesis, are indispensable for reproduction. In the present study, we demonstrate that the previously uncharacterized testis-specific serine protease TRYX5 (1700074P13Rik) is required for male fertility in mice. Tryx5^−/− male mice are sterile, yet they have normal spermatogenesis and normal sperm parameters. In vivo fertilization experiments showed that the fertilization rate of Tryx5^−/− sperm was almost zero. Sperm counting and analysis of paraffin sections of oviducts revealed that Tryx5^−/− sperm were unable to migrate into the oviduct, which is likely the cause of the observed infertility of the Tryx5^−/− male mice. Importantly, we also found that there was almost no mature ADAM3 present in Tryx5^−/− sperm and almost no ADAM3 precursor in Tryx5^−/− elongated spermatids of S13-16 stage, even though testes of Tryx5^−/− and wild type mice had the same amount of the total precursor ADAM3. Collectively, our results demonstrate that Tryx5 is essential for male fertility in mice and suggest that TRYX5 functions in the stability or localization of ADAM3 precursor in elongated spermatids S13-16 stage, thereby regulating the ability of sperm to migrate from the uterus into the ampulla of the oviduct, the site of fertilization.     

A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.     

Defects in secretory pathway trafficking during sperm development in Adam2 knockout mice

Adam2-null and Adam3-null male mice exhibit reduced levels of one or more ADAM proteins on mature sperm, in addition to the loss of the genetically targeted protein. ADAM protein loss was believed to occur posttranslationally, although the timing of loss and the mechanism by which the loss occurred were not explored. In this study we have found that in Adam3-null mice, fertilin beta (also known as ADAM2) is lost during the formation of testicular sperm. In Adam2-null males, most cyritestin (ADAM3) protein is also lost at this stage, but 25% of cyritestin is lost later, during sperm passage through the epididymis. Although normal levels of cyritestin are synthesized and acquire Endoglycosidase H resistance, indicating transit through the Golgi, the protein does not reach the cell surface. We also discovered that the majority of both fertilin beta and cyritestin are found in a Triton X-100 insoluble compartment on testicular sperm, when most of the cyritestin was observed on the cell surface. This insoluble compartment may represent a sorting platform, because in Adam2-knockout cells, only a small fraction of the cyritestin becomes Triton X-100 insoluble. Thus, it appears that cyritestin loss in Adam2-knockout mice may result, at least in part, from a disruption in protein trafficking.     

JAM-A is present in mammalian spermatozoa where it is essential for normal motility

Junctional adhesion molecules (JAMs) that are expressed in endothelial and epithelial cells and function in tight junction assembly, also perform important roles in testis where the closely-related JAM-A, JAM-B, and JAM-C are found. Disruption of murine Jam-B and Jam-C has varying effects on sperm development and function; however, deletion of Jam-A has not yet been studied. Here we show for the first time that in addition to expression in the Sertoli-Sertoli tight junctions in the seminiferous tubules, the ∼ 32 kDa murine JAM-A is present in elongated spermatids and in the plasma membrane of the head and flagellum of sperm. Deletion of Jam-A, using the gene trap technology, results in flagellar defects at the ultrastructural level. In Jam-A-deficient mice, which have reduced litter size, both progressive and hyperactived motility are significantly affected (P < 0.0001) before and, more severely, after capacitation. The findings show that JAM-A is involved in sperm tail formation and is essential for normal motility, which may occur via its signal transduction and protein phosphorylation properties. Detection of JAM-A in human sperm proteins indicates that its role may be conserved in sperm motility and that JAM-A may be a candidate gene for the analysis of idiopathic sperm motility defects resulting in male subfertility in the human population.     

Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm

Although fertilin is a heterodimeric complex between ADAM1 (A Disintegrin And Metalloprotease 1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface, two different ADAM1 isoforms, ADAM1a and ADAM1b, are present in the mouse testis. In this study, we have examined the localization of ADAM1a and ADAM1b in testicular germ cells and epididymal sperm. ADAM1a was restrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contained only ADAM1b on the cell surface. The precursors of ADAM1a and ADAM1b formed a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAM1b and ADAM2 was also found on the sperm surface. These data imply the potential roles of ADAM1a and ADAM1b in spermatogenesis and fertilization, respectively.     

Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa

Eight kinds of gene-disrupted mice (Clgn, Calr3, Pdilt, Tpst2, Ace, Adam1a, Adam2, and Adam3) show impaired sperm transition into the oviducts and defective sperm binding to the zona pellucida. All of these knockout strains are reported to lack or show aberrant expression of a disintegrin and metallopeptidase domain 3 (ADAM3) on the sperm membrane. We performed proteomic analyses of the proteins of these infertile spermatozoa to clarify whether the abnormal function is caused exclusively by a deficiency in ADAM3 expression. Two proteins, named PMIS1 and PMIS2, were missing in spermatozoa from Clgn-disrupted mice. To study their roles, we generated two gene-disrupted mouse lines. Pmis1-knockout mice were fertile, but Pmis2-knockout males were sterile because of a failure of sperm transport into the oviducts. Pmis2-deficient spermatozoa also failed to bind to the zona pellucida. However, they showed normal fertilizing ability when eggs surrounded with cumulus cells were used for in vitro fertilization. Further analysis revealed that these spermatozoa lacked the ADAM3 protein, but the amount of PMIS2 was also severely reduced in Adam3-deficient spermatozoa. These results suggest that PMIS2 might function both as the ultimate factor regulating sperm transport into the oviducts and in modulating sperm-zona binding.     

Glutathione S-transferase Mu 3 is associated to in vivo fertility,but not sperm quality,in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.     

Do male secondary sexual characters correlate with testis size and sperm length in the small hairy maggot blowfly?

The phenotype-linked fertility hypothesis proposes that secondary sexual characters (SSCs) advertise a male's fertility to prospective mates. However, findings from empirical studies attempting to test this hypothesis are often ambivalent or even contradictory, and few studies have simultaneously evaluated how both morphological and behavioural SSCs relate to ejaculate characteristics. Males of the small hairy maggot blowfly, Chrysomya varipes, possess conspicuous foreleg ornaments and display highly stereotyped courtship behaviour. These traits are favoured by females during pre-copulatory mate choice, but it remains unknown whether they correlate with post-copulatory traits expected to influence male fertility. The aim of this study was to investigate whether male courtship and ornamentation correlate with testis size and sperm length in C. varipes. We found that males investing more in courtship had bigger testes, and males with more extensive foreleg ornamentation released sperm with longer tails. Based on the assumption that larger testes enable males to produce more sperm, and that sperm with longer tails have greater propulsive force, our findings suggest that more vigorous and more ornamented males may be more fertile. These findings lend support to the phenotype-linked fertility hypothesis. However, a complete test of this hypothesis will require evaluating whether testis size and sperm length influence male fertilisation ability, as well as female fecundity and/or fertility.