BACKGROUND:Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins.RESULTS:We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract.CONCLUSION:Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.

Le sperme est le vecteur principal de dissémination du VIH. Des travaux récents indiquent la présence de facteurs dans le liquide séminal (LS) des hommes non infectés qui modulent l'infectivité par le VIH de cellules in vitro. Nous avons préalablement montré que les principaux organes génitaux mâles qui contribuent à l'élaboration du sperme sont infectés par le VIH, ce qui modifie probablement la composition du LS des hommes VIH+. Pour preuve, des différences significatives dans les profils cytokiniques ont été mises en évidence dans le LS d'hommes VIH+ par rapport à celui d'hommes sains. Dans ce contexte, mes travaux de thèse se sont articulés autour de 3 axes : Effet du sperme d'hommes infectés par le VIH-1 sur l'infection des lymphocytes T CD4+ : nous avons comparé, pour la première fois à notre connaissance, l'effet du LS d'hommes infectés versus celui d'hommes sains sur l'infectivité par le VIH dans des modèles cellulaires. Nous avons mis en évidence un effet différentiel du LS d'hommes séropositifs par rapport au LS d'hommes séronégatifs. L'effet stimulateur observé du LS d'hommes VIH-non-infectés sur l'infection par le VIH-1 des cellules T CD4+ est significativement diminué pour les LS d'hommes VIH+. Cet effet différentiel serait au moins en partie lié à la surexpression de certaines cytokines dont RANTES et à la sous expression à la membrane du corécepteur CCR5. Effet du LS sur l'infection d'explants cervicaux et colo-rectaux : les muqueuses cervivo-vaginale et colorectale représentent des portes d'entrée majeures du virus dans l'organisme et sont donc des modèles particulièrement physiologiques. Nous avons montré que le LS induit une diminution de l'infection ex vivo des tissus exocervicaux alors qu'aucun effet n'est observé sur l'infection des tissus colorectaux. Fractionnement du liquide séminal et identification des facteurs à activité biologique sur l'infection par le VIH-1 : nous avons débuté une approche protéomique qui nous a permis de décomplexifier le LS par fractionnement grâce à des HPLC successives, de sélectionner des fractions biologiques actives et d'identifier des protéines candidates, par spectrométrie de masse, potentiellement responsables d'un effet inhibiteur sur l'infection par le VIH-1. Sur la base de la littérature et de nos résultats, il apparaît clairement que le LS est un fluide biologique complexe dont l'effet global sur l'infection résulte de la somme des activités biologiques de facteurs stimulateurs et inhibiteurs. En outre, l'effet du sperme sur l'infection est dépendant du modèle d'étude cellulaire ou tissulaire, et du statut sérologique du donneur. En effet, les différences de composition (cytokines, peptides antimicrobiens…) entre les spermes d'hommes infectés ou non aboutissent à des modifications dans la modulation de l'infection VIH-1 par le sperme via des mécanismes directs ou indirects qui ouvrent de nombreuses perspectives d'études. / Semen represents the main vector of HIV transmission. Several studies have recently shown that, in addition of being a carrier of HIV particles and infected cells, seminal fluid (SF) could modulate the efficiency of HIV infection of target cells through its intrinsic properties. These studies were all performed with SF from uninfected donors. But the composition of SF from HIV-infected men significantly differs from that of HIV-infected men, as demonstrated by studies on SF cytokines content, and microbiome. In addition, we showed that semen-producing organs are infected by HIV which most probably influences their seminal secretions. As a matter of fact, the volume of the ejaculate of HIV+ donors is generally less than that of uninfected men. In this context, my thesis was articulated around 2 axes: Effect of SF from HIV infected men on HIV-1 infection of Lymphocytes T CD4+: the effect of SF from HIV-infected men (HIV+SF) versus SF from uninfected donors (SF) on HIV infectivity of CD4+ T cells. We observed a significantly reduced infection of CD4+T cells by HIV-1 R5 in presence of HIV+SF as compared with SF, the latter displaying enhancing activity. This differential effect was not observed on the infectivity of the CD4+ cell line TZM-bl, or on PBMC infection by HIV-1 X4. We compared the composition of HIV+SF versus SF in terms of enhancing peptides, cytokines and prostaglandins, and investigated the impact of SF from both HIV-infected and uninfected men on CD4+T cell HIV receptors expression, activation and proliferation. HIV+SF vs HIV-SF was found to induce a significantly higher downmodulation of CCR5 expression shortly after exposure, which correlated with the concentrations of CCR5 ligands in semen. Effect of SF on HIV infection of cervix and colorectal tissues: the cervix and colorectal mucosa represent major front doors of the virus in the body and are thus particularly physiological models. We showed that the SF leads to a decrease of the infection ex vivo in exocervix tissues while no effect is observed on the infection of colorectal tissues. Fractionation of the seminal fluid and identification of factors with biologic activity on the HIV-1 infection : we had beginning a proteomic approach which allowed to decomplexified SF by successive HPLC, to select active biological fractions and to identify candidate proteins, by mass spectrometry, potentially responsible for an inhibitory effect on the infection by the HIV 1. Based on the literature and on our results, it clearly appears that SF is a complex biological fluid. Moreover, the effect of the semen on HIV-1 infection is dependent on the cellular or tissular model and on the HIV status of the donor. Indeed, the differences of composition (cytokines, antimicrobial peptides) between semen from infected men or SF from uninfected men lead to modifications of the modulation of HIV-1 infection by semen via direct or indirect mechanisms which which pave the way for further investigations.

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Seminal fluid proteins (SFPs) have been shown to affect the physiology,behaviour and immune responses of mated females in some species. Thisopen window for manipulation of female’s fitness allows the possibility forcomplex evolutionary dynamics between the SFPs and proteins of femalesthat would counter the effects of the former, the female reproductive proteins (FRPs). Also, the bean beetles of the Bruchinae subfamily are pests to pre-ferred species of plant hosts. The hosts have a great variety of secondary defensive metabolites between them and to detoxify those compounds, each beetle species is expected to have a well adapted arsenal of digestive proteinsfor a specific host. I carried out a comparative study with four species of bean beetles with the aim to identify patterns of selection in the proteins mentioned. Expression data for one of those species, Callosobruchus maculatus, has allowed to identify its SFPs, FRPs and digestive proteins and with orthology inference I identified their orthologues in the other three species. Then I estimated theratio of non-synonymous to synonymous substitution rates (ω) for each protein by using codeML of the PAML package and used them as a proxy for estimating selection. FRPs had about the same ω values as conserved genes found across the Arthropod phylum, while the SFPs and digestive proteins hadhigher ω values, indicating more relaxed purifying selection. I also performed tests of positive selection and have identified 92 digestive proteins, 9 FRPs and 26 SFPs as potential targets for future functional work. Finally, I examined the scenario of co-evolution between SFPs and FRPs because of direct interaction. By correlating branch-specific ω values for each possible pairs of proteins I found that SFPs are associated on average more with FRPs than with digestive or conserved genes, as expected. The same was true for the FRPs. Also I examined the possibility of factors contributing to the association such as expression levels, sex-biased expression and protein function. Using linear regression models I found that expression levels and proteinfunction do predict in some degree the ω estimates and could thus also affectthe correlations examined. High gene expression levels reduce the overall ωvalues of genes, also known as E-R anticorrelation. Sex-biased expression does not affect the overall ω values, but does affect the intensity of the E-R anticorrelation, with it being less prominent in male-biased genes and more prominent towards female-biased genes.

Sperm competition is a widely-recognized and powerful selective force. Male accessory glands are organs found across animal taxa that can influence sperm performance, and thus may be selected for in competitive contexts. In fishes, these organs are in fact rare, but display great diversity in form and function across species. Although the accessory gland is known to play a role in mate attraction, parental care, fertilization, or post-copulatory competition in a few select species, the role of this organ remains a mystery in most species. Many fishes with accessory glands also exhibit alternative reproductive tactics (ARTs), which add an extra layer of complexity to how species respond to sperm competition. Because males of different ARTs typically experience different levels of sperm competition risk, it’s possible they may differentially invest in accessory glands to overcome this competition. In this thesis, I used the plainfin midshipman fish (Porichthys notatus), a species with both ARTs and an accessory gland, to experimentally investigate the role of the accessory gland in sperm competition and uncover how this organ may differ between ARTs. Over a two-year period, I studied tactic-specific investment in the accessory gland in fish from the beaches of British Columbia. I also examined the effects of seminal fluid, produced in part by the accessory gland, on sperm performance and morphology. I found that males adopting the ‘guarder’ male tactic invested more in one region (the lobules) of this organ, while males adopting the smaller ‘sneaker’ male tactic invested more the other region of the gland (the nodes). Using data collected over five years, I found that guarder males also invested more in their whole accessory glands. Additionally, I report that sperm swam faster in the presence of seminal fluid, and seminal fluid increased sperm head size in both male tactics and increased midpiece size in guarder males. These results suggest that the plainfin midshipman accessory gland may have dual functions, one of which may be to aid sperm competitive ability through enhancements in swimming speed and potentially more successful sperm morphology. Taken together, the results of my thesis improve our knowledge of the role of non-sperm components like seminal fluid and the accessory gland in sperm competition, and demonstrate how species with ARTs can have varying physiological responses to such competition. Only a handful of studies have considered the effects of seminal fluid on sperm performance. By examining sperm competition in a more biologically relevant way and incorporating the effects of a little-studied organ that impacts sperm competition, we should be able to more generally and accurately appreciate the dynamics of post-copulatory competition and fertilization. / Thesis / Master of Science (MSc)

The aim of this diploma thesis is to compare quality of northern pike sperm collected by different methods. First method is collection of stripped sperm by abdominal massage of the belly. Second method is collection of stripped sperm with special catheter to eliminate sperm contamination by urine. The last method is collection of testicular sperm. Differently collected sperm was evaluated and compared its quality. The main observed parameters were sperm volume, spermatozoa concentration, spermatozoa motility and velocity and osmolality of seminal fluid. Sperm samples were used for eggs fertilization. In fertilized eggs, the fertility of eggs and larvae hatching rate were observed.