Sperm Types Research Articles

Two pathways for spermatogenesis in Sinanodonta woodiana (Lea, 1834) (Bivalvia: Unionidae)

AbstractThe Chinese pond mussel, Sinanodonta woodiana, an invasive species indigenous to Asia, naturally colonizes new locations as a glochidium that parasitizes fish. Human activity has facilitated a continental-scale spread of S. woodiana in Europe, where it is found in artificial reservoirs and has begun to invade natural habitats. This study aimed to investigate the reproductive activity of males in a female-biased population of S. woodiana that lives in a riverine channel linked to a power plant with a cooling water system in NW Poland. Cells formed during spermatogenesis and morphological changes in reproductive follicles (acini) were analysed over a 2-year period using light microscopy. Typical and atypical spermatogenic pathways were identified. The atypical spermatozoa were released from multinucleated cysts and had significantly shorter heads than typical spermatozoa. Spermatozoa formed in the typical way occurred consistently in all months over the study period, while the atypical spermatozoa were recorded occasionally in the first year of the study and more frequently in the second study. The highest proportion of males with spent gonads was found in late summer and early autumn. Our data show that mussels produce spermatozoa year-round, but that the levels of typical and atypical spermatogenesis varied. The two pathways of spermatogenesis in S. woodiana provide an important insight into potential mechanisms underlying the evolution of doubly uniparental inheritance of mitochondrial DNA, a phenomenon observed in S. woodiana. Additionally, a detailed understanding of the reproductive cycle of this non-native species can help to understand its reproductive potential and predict the likely direction of population change in a newly colonized area. The breeding population of S. woodiana studied by us inhabits a tributary of the Odra River, one of Poland’s largest rivers, and thus poses a threat to the natural environment. Riverine habitats like our study site are local heat islands and the mussels reproducing in them over time may colonize natural waters and expand the range of their distribution.

Spermatological Morphology of Triatoma Species (Hemiptera: Reduviidae: Triatominae).

Spermatozoa are among the most diverse cell types, and their morphologies often provide data that can be used to reliably evaluate phylogenetic relationships. They can also help to clarify the nature of 'specific complexes', which are common among triatomines. In the present study, we evaluated the copulation behavior of Triatoma rubrovaria Blanchard 1843 (Hemiptera: Reduviidae: Triatominae) and the structural morphology of sperm from T. carcavalloi Jurberg Rocha & Lent, 1998, T. infestans Klug, 1834, T. pintodiasi Jurberg Cunha & Rocha, 2013, and T. rubrovaria. Copulatory behavior was described from the moment males and females genitalia joined until they separated. Insemination was confirmed by the presence of a spermatophore in the female's bursa copulatrix. To measure their sperm, males were dissected and their seminal vesicles were removed, squashed on glass slides, and then spread, fixed, and observed under a photomicroscope. The images obtained were analyzed to measure the sperm. Seminal vesicles were also prepared for transmission electron microscopy. We performed K-means clustering separately for each species to group their sperm based on morphology. The differences in spermatozoa length among species of Triatominae, sperm types, and the interaction between species and sperm type were assessed with a two-way analysis of variance (ANOVA). The copulation time in T. rubrovaria was 3 to 5 min, which was sufficiently long for spermatophore transfer. All taxa showed polymorphic (short and long) sperm, with significant differences in the lengths of sperm among taxa. Using electron microscopy, the sperm cells of the four taxa examined were found to have similar ultrastructural morphology, confirming the hypothesized synapomorphies of sperm within the suborder Heteroptera (Hemiptera).

Ecology of Italian Protura

The ecology of Protura in Italy (including Corsica) is tentatively described according to original and bibliographical data. Protura are quite common but very rarely abundant: their mean density in soil samples collected in Liguria (NW Italy) was estimated to be 372 /m² (s.d. 657 – max. 2790 /m² in a Holm oak forest). Information from the analyses of habitat and/or elevation of 3448 specimens from 269 collections and 4071 specimens from 295 collections identified to species and genus level, respectively, enabled us to outline Protura assemblages for eight different macro-habitats. The unbalanced sex ratio in favour of females observed in 12 of the dominant species of Acerentomata suggests that (based also on the sperm types known in Protura) a single male can fertilize several females through spermatophores. For one species (Proturentomon minimum) only females were collected, which may suggest the possibility of parthenogenesis in some Protura. An analysis of the phenology and population dynamics of the five dominant species, showed annual cycles with one (Acerentomon microrhinus) or more (A. gallicum, A. italicum, A. maius and Eosentomon transitorium) peaks of reproductive activity. Other ecological remarks (e.g. about the relationship between the amplitude of latitudinal/altitudinal distribution and the ecological distribution of Protura in Italy) are discussed.

Spermatogenesis of the freshwater pearl mussel Margaritifera laevis (Bivalvia: Margaritiferidae): A histological and ultrastructural study.

Spermatogenesis in the freshwater pearl mussel Margaritifera laevis was investigated using light and electron microscopy. The testes of M. laevis are composed of numerous acini. We observed type A spermatogonia, large cells of irregular shape, solely near the acinus basal lamina. Type A spermatogonia proliferate and become type B spermatogonia, which are also irregular in shape and form clusters of germ cells of the same developmental stage. The numerous clusters differ with respect to developmental stage and are arranged randomly along the acinus periphery. The central region of the acinus was observed to contain only mature spermatozoa. This germ cell arrangement contrasts that of other bivalvians and may be characteristic of Margaritiferidae and Unionidae. We noted that each germ cell cluster is entirely covered throughout spermatogenesis by Sertoli cells that are loosely bound together. This report is the first to describe the involvement of Sertoli cells in Unionoidea spermatogenesis. Mature spermatozoa of M. laevis are of the primitive sperm type, having a cylindrical head with a discoidal acrosome and a midpiece with five spherical mitochondria.

Sperm morph and remating frequency in the Indian meal moth, Plodia interpunctella.

All Lepidoptera produce two sperm types: normal, nucleated 'eupyrene' sperm and anucleate 'apyrene' sperm. One hypothesis for the evolution of apyrene sperm suggests that they act to reduce female remating rate. Apyrene sperm require less resources to produce than do eupyrene sperm, and could delay remating by females by acting as a 'cheap filler', packing the spermatheca and thereby reducing receptivity. This would reduce the risk of sperm competition, giving a potential adaptive advantage to the male producing these sperm. This leads to the prediction that the probability of a female remating should correlate with the number of stored apyrene sperm, which has previously been supported by experiments using the green-veined white butterfly, Pieris napi We repeated this experiment using the Indian meal moth, Plodia interpunctella We find that in this species, eupyrene, not apyrene sperm number is the best predictor of female remating probability, indicating that the 'cheap filler' hypothesis for the function of apyrene sperm is not well supported in Pl. interpunctella.

Ultrastructure of spermatophoral sperm in the freshwater gastropod Thiara amarula (Linnaeus, 1758) (Cerithioidea: Thiaridae): potential taxonomic features including eusperm nuclear content differentiation

The ultrastructure of spermatophoral sperm is investigated for the first time in a cerithioidean gastropod. Thiara amarula (Linnaeus, 1758), a widespread Indo-West Pacific species of the Thiaridae, produces uniaxonemal euspermatozoa and multiaxonemal paraspermatozoa. Euspermatozoa possess a laterally-flattened acrosomal vesicle (with eccentrically positioned invagination and subacrosomal material), a tubular, laterally-compressed nucleus, an elongate midpiece (with four, straight, equal-sized mitochondria each with parallel cristae), an elongate glycogen piece and an end piece. The eusperm nucleus exhibits or is associated with a number of unusual features including: (1) nuclear contents differentiated into two components (fibrous, highly electron-dense material, enclosing a pair of less-dense, finely granular tracts); (2) nuclear invagination with two elongate grooves aligned at 90 degrees to the axonemal central microtubules and parallel with the 1 and 5 axonemal doublets; (3) a periodically-banded rootiet associated with the centriolar complex near the nuclear apex; (4) an electron-dense structure attaching the nucleus to the axoneme at the nucleus-midpiece junction. Feature (1) has been reported elsewhere in Melanoides tuberculata (Thiaridae), but in no other examined gastropods (or other molluscs) and its purpose is unknown. Paraspermatozoa of T. amarula possess an elongate head region (up to 14 axonemes and large, irregular mitochondria enclosed by sheath of spherical, dense vesicles), a short 'glycogen' region and a posterior tuft of glycogen tails. The two sperm types do not form spermatozeugmata and observations suggest that neither type undergoes any noticeable structural change while inside the spermatophore. Sperm features suggest a close relationship between T. amurula and M. tuberculata and more broadly with other 'Group T cerithioidean families exhibiting four equal-sized eusperm mitochondria, including Potamididae, Modulidae, Scaliolidae, Melanopsidae, Semisulcospiridae and Pleuroceridae.

Fine structure of the ladybird spermatozoa (Insecta, Coleoptera, Coccinellidae)

The sperm structure of several ladybird species belonging to different subfamilies of Coccinellidae was studied. Three main sperm types were clearly recognized, and were characterized by differences in acrosomal length, the presence of a dense coat around the acrosome, the length of the basal body, the amount of the centriole adjunct material, and the diameter of the mitochondrial derivatives. However, the whole group shares a pattern of the posterior sperm region uncommon for insects, in which the axoneme and other flagellar components are running parallel with the nucleus. As a general conclusion, this study has revealed an inconsistency between the sperm structure and the systematics of the group, indicating that the generic concepts within the group do not reflect a natural classification, a statement also shared by molecular studies.

Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen

The experiment was conducted to study cryopreservation induced sperm cryoinjuries and the protective effect of reduced Glutathione supplementation in Murrah bull semen. A total of 20 semen ejaculates were split into two parts after initial examination and were extended in glycerolated egg yolk TRIS diluter (Control group) and glycerolated egg yolk TRIS diluter + 0.5 mM reduced Glutathione (Treatment Group). The diluted semen samples were loaded into 0.25 ml French mini straw and sealing of straws were done. Thereafter, semen straws were kept for equilibration for 4 h at 5 °C and semen was frozen using a standard cryopreservation protocol in automatic biological freezer. Post-thaw analysis was performed after 24 h of semen storage in liquid nitrogen. Fresh and post-thaw sperm assessments included sperm motility, viability (SYBR-14/PI assay), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (HOST). Cryopreservation of semen in liquid nitrogen induced a marked reduction in post-thaw sperm motility, viability, mitochondrial function and plasma membrane integrity and increased moribund type of sperm (SYBR-14/PI assay) in control group as compared to reduced glutathione treated group. There were significant (P < 0.05) cryo injuries in frozen-thawed spermatozoa following cryopreservation in buffalo bull semen. The supplementation of reduced glutathione in treatment group exhibited significantly (P < 0.05) lower cryoinjuries during cryopreservation and semen storage in liquid nitrogen. From the study it was concluded that, spermatozoa from Murrah bulls are susceptible to injuries due to cryopreservation in liquid nitrogen, but these cryo induced damage can be protected significantly (P < 0.05) by the use of reduced Glutathione.

The Inability of Human Sperm with Chromosomal Abnormalities to Penetrate the Oocyte in Assisted Reproductive Technology (ART): Risk Factors and the Role of Seminal Plasma

Abstract: The combination of ICSI and a sperm chromosome assay provides a useful opportunity to directly investigate relationships between chromosomal aberrations and head morphology, motility or in vitro aging. An increase of aneuploidy was identified in sperm with large heads. Structural chromosome aberrations were frequently found in elongated heads. These types of sperm should therefore be avoided for ICSI and can easily be identified under a microscope. In sperm with normal heads, the chromosomal aberration rate was initially low but increased gradually after removal of the seminal plasma (3.3% to 15.9%); in immotile sperm, the increase of DNA damage was more pronounced (4.5% to 48.1%). Therefore, there are motile and immotile sperm populations in human ejaculates that are potentially vulnerable to in vitro culture conditions. Since these sperm can maintain DNA integrity in the seminal plasma for at least 3 h, and even after cryopreservation, the seminal plasma appears to be an efficient suppressor o...

MPK-1/ERK regulatory network controls the number of sperm by regulating timing of sperm-oocyte switch in C. elegans germline

The precise regulation of germline sexual fate is crucial for animal fertility. In C. elegans, the production of either type of gamete, sperm or oocyte, becomes mutually exclusive beyond the larval stage. Hermaphrodites initially produce sperm and then switch to produce oocytes. This change of fate during germline development is tightly controlled by several regulators. In C. elegans hermaphrodites, FBF-1 and FBF-2 (>95% identical, members of the Pumilio RNA-binding protein family) proteins function redundantly to promote the sperm-oocyte switch. Here, we demonstrate that loss of LIP-1 (dual specificity phosphatase) in fbf-1(ok91) single mutants leads to excess sperm production due to a delayed sperm-oocyte switch. This phenotype was dramatically rescued by depletion of MPK-1 (an ERK homolog). In contrast, loss of LIP-1 in fbf-2(q738) single mutants leads to a premature sperm-oocyte switch and loss of sperm. Notably, fbf-1 fbf-2; lip-1 triple mutants produce excess sperm. These results suggest that the MPK-1/ERK regulatory network, including FBF-1, FBF-2, and LIP-1, controls the number of sperm by regulating the timing of the sperm-oocyte switch in C. elegans.

An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm.

Separation of X- and Y-chromosome bearing sperm has been practiced for selection of desired sex of offspring to increase the profit in livestock industries. At present, fluorescence-activated cell sorter is the only successful method for separation of X- and Y-chromosome bearing sperm. This technology is based on the differences in DNA content between these two types of sperm and has been commercialized for bovine sperm. However, this technology still has problems in terms of high economic cost, sperm damage, and lower pregnancy rates compared to unsorted semen. Therefore, an inexpensive, convenient, and non-invasive approach for sperm sexing would be of benefit to agricultural sector. Within this perspective, immunological sperm sexing method is one of the attractive choices to separate X- and Y-chromosome bearing sperm. This article reviews the current knowledge about immunological approaches, viz., H-Y antigen, sex-specific antigens, and differentially expressed proteins for sperm sexing. Moreover, this review also highlighted the different methods for identification of X- and Y-sperm.

Effects of pre-overwintering conditions on eupyrene and apyrene spermatogenesis after overwintering in Polygonia c-aureum (Lepidoptera: Nymphalidae)

Sperm polymorphism is widely known in invertebrates. In insects, Lepidoptera has two types of sperm: nucleated eupyrene (fertile) sperm and anucleated apyrene (unfertile) sperm. These sperm types are produced during post-embryogenesis, and eupyrene spermatogenesis precedes apyrene spermatogenesis. During overwintering, spermatogenesis stops and a portion of undifferentiated-stage spermatocytes degenerate. After overwintering, spermatogenesis restarts with unaffected spermatogonia. However, how new spermatozoa arise in the adult testes after overwintering is not known in Lepidoptera. In this study, we investigated the spermatogenesis events in the nymphalid butterfly Polygonia c-aureum after overwintering under various environmental conditions. Our results indicate that both eupyrene and apyrene spermatogenesis restart at any stopping stage and sperm of these types are regenerated in no particular order after adult insect overwintering. This suggests that the spermatogenesis occurring after overwintering proceeds without embryogenetic restrictions related to the developmental sequence.

Effects of a Protein Kinase Inhibitor on Sperm Motility in the Japanese Quail.

Sperm motility is an essential trait for successful fertilization in animals. In birds, ejaculated sperm migrate into sperm storage tubules before fertilization and are stored in a quiescent state. We previously reported that this type of sperm's flagellar quiescence was induced by lactic acid through flagellar dynein ATPase inactivation following cytoplasmic acidification (<pH 6.0). However, signal transduction in the sperm cells leading to motility inactivation is not well understood. The aim of the present study was to investigate the role of protein kinases in putative signal transduction in quail spermatozoa motility in vitro. Following incubation with bisindolylmaleimide II (BisII), a potent-competitive protein kinase C (PKC) inhibitor, sperm motility decreased in a dose related-manner. However, no such inhibitory effect was found in sperm exposed to bisindolylmaleimide V, H-89, or LY294002, a weak inhibitor of PKC, a potent inhibitor of protein kinase A (PKA) and a selective inhibitor of phosphatidylinositol 3-kinase, respectively. BisII-treated sperm exhibited no significant differences in pHi, [Ca2+]i, mitochondrial activity, intracellular cAMP or ATP concentration, as well as dynein ATPase activity, compared to the control sperm. However, when the phosphorylated substrate proteins by PKC were detected by Western blot analysis, the intensity of the band in sperm incubated in the presence of BisII decreased. Moreover, immunoreactive PKCι and µ isoforms in the sperm lysates were also detected. These results indicated that the PKC signaling pathway may be involved in sperm motility regulation, and protein phosphorylation by PKC may be required to maintain flagellar movement in the Japanese quail.

Arabidopsis male sexual lineage exhibits more robust maintenance of CG methylation than somatic tissues

Cytosine DNA methylation regulates the expression of eukaryotic genes and transposons. Methylation is copied by methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Transgenerational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of transgenerational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells-the cell types that comprise pollen-with mutations in the DRM, CMT2, and CMT3 methyltransferases. We find that DNA methylation dependency on these enzymes is similar in sperm, vegetative cells, and somatic tissues, although DRM activity extends into heterochromatin in vegetative cells, likely reflecting transcription of heterochromatic transposons in this cell type. We also show that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and similar to those of H1-depleted roots. Our results demonstrate that the mechanisms of methylation maintenance are similar between pollen and somatic cells, but the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

Clouded leopards (Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n=9) were allocated to simple washing (control; resulting in 11.7%±1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9%±0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution (HOS, 60 mOsm fructose solution) containing 5mM dithiothreitol (DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 (TX) (a detergent) was added to the HOS/DTT-treated samples. After HOS/DTT incubation, the control samples and sperm-selected samples presented 73.4%±3.1% and 73.9%±2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8%±3.8% and 26.5%±3.4%; p=.1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6%±10.4% and 21.1%±7.9%; p=.06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation.

Effect of seminal plasma and sperm of boars valued by freezability on seminal cryopreservation

The aim of this study was to determine the effect of sperm and seminal plasma (SP) on the freezability of porcine semen. Semen of eight commercial males from two farms in the central-western region of Colombia (four boars in each farm) was frozen and tested to select two males with high freezability (MHF) and two with low freezability (MLF), according to the percentage of functionally competent sperm (FCS). Immediately after the collection was completed, the SP and sperm from the males selected were separated by centrifugation to combine the two types of plasma with the two types of sperm, incubate them for three hours and then freeze them. The variables evaluated were: sperm morphology, structural and functional integrity of plasmatic membrane, progressive and total motility, DNA fragmentation, acrosome integrity, capacitated sperm and FCS. The combination of sperm and plasma of MHF recorded the highest value (P&lt;0.01) of acrosome integrity (24.3 ± 0.082 vs 6.076 ± 0.16) when compared to MLF plasma and cells. Membrane structural integrity was higher (P&lt;0.01) with MHF (53.56 ± 0.0395) than with MLF plasma (47.49 ± 0.0419). The differences in porcine semen freezability depend on interactions between seminal plasma and sperm.

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia.

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l−1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l−1) to a hypotonic solution (290 mOsm l−1), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4′-diisothiocyanatostilbene-2,2′- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.

The evolution of insect sperm − an unusual character system in a megadiverse group

Spermatozoa provide an unusual character system, with a limited number of components organized in a single cell. Similar spermatozoa occur in groups widely separated in the phylogenetic tree of Metazoa. Nevertheless, the character system contains phylogenetic information. Hexapoda have acquired spermatophores along with the switch from aquatic to terrestrial habitats, and related to this, a multitude of different sperm types. The aim of this study is a formal evaluation of the phylogenetic information content of spermatozoa. For the first time, sperm characters are coded for formal phylogenetic analyses. Different approaches are used and compared. Mainly due to a high level of homoplasy, the evaluation of sperm characters alone is insufficient for a reconstruction of the phylogeny of the group. Yet, a reliable reconstruction of the evolution of insect sperm is possible when character transformations are assessed using a phylogeny based on extensive molecular data. Important changes took place in the early evolution of Hexapoda. Sperm characters support some major clades (e.g. Hexapoda, Dicondylia, Polyneoptera, Psocodea), but important steps in the evolution are not reflected by transformations of spermatozoa, notably the rise of Pterygota or Holometabola. Important innovations are the formation of mitochondrial derivatives and the acquisition of accessory microtubules. Some features are conservative, whereas others evolved rapidly (e.g. presence or absence of the acrosome vesicle). Some groups are conservative in their sperm features (e.g. Odonata, Heteroptera), whereas the evolution of spermatozoa was distinctly accelerated in others (e.g. Ephemeroptera). The rate of evolution can change drastically in closely related groups. Profound changes in the morphologically uniform Zoraptera underline that sperm evolution can follow a pattern very different from the general somatic morphology. The mode of character reconstruction preferred here will be useful for the evaluation of specialized morphological character systems and strengthen the concept of evolutionary morphology.

Changes to sperm surface carbohydrates following exposure to seminal plasma and freezing

Epididymal, ejaculated and frozen–thawed spermatozoa are exceptionally different sperm types. When placed directly into the uterus all sperm types display high fertility. However, when forced to traverse the ovine cervix (following vaginal or cervical AI), epididymal and frozen–thawed ram spermatozoa display dramatically lower fertility than fresh ejaculated spermatozoa. We suggest that changes to the outer carbohydrate-rich glycoprotein coat of spermatozoa represent a potential reason for altered cervical transit ability, as this is the primary interface with the female environment. As such, we used lectin binding to investigate modifications of the carbohydrate portion of sperm glycoconjugates in response to seminal plasma exposure and freezing. Fresh and frozen ejaculated spermatozoa and fresh epididymal spermatozoa were subjected to a swim up to remove loosely associated glycoproteins and remnants of freezing diluent. Spermatozoa were stained with fluorescein isothiocyanate (FITC) conjugated lectins (LPA, PNA, WGA and ConA), specific for the sugars of interest (sialic acid, galactose, N-acetylglucosamine andmannose, respectively) and counter-stained with propidium iodide (PI). Median FITC fluorescence of the PI negative (live) population was compared amongst treatments using a restricted maximum likelihood approach. The results provide strong evidence that both exposure to seminal plasma and freezing are processes which alter the outermost surface of spermatozoa. Seminal plasma exposure significantly increased N-acetylglucosamine (WGA; p<0.001) on the sperm membrane. Conversely, freezing led to a significant decrease in available galactose (PNA; p<0.01) and N-acetylglucosamine (WGA; p<0.001) as well as increased availability of mannose (ConA; p<0.001), typically a carbohydrate located proximal to the membrane in glycoconjugates. The observed changes lend support to the theory that while exposure to seminal plasmamay orchestrate meaningful alteration of the sperm surface, the freezing process further modifies this important region, possibly to its detriment. This is the first evidence that industry standard freezing of ram semen leads to large and significant changes in selected sperm surface carbohydrates. Further research is required to fully elucidate the consequenceof these changesandestablishpossiblemeans to prevent or repair the damage to the glycoprotein coat of ram spermatozoa. Research support by AWI and NSWSMBA Trust.

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

AbstractLepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.