Effect of estrogens on boar sperm capacitation in vitro

<p>Abstract</p> <p>Background</p> <p>Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.</p> <p>Methods</p> <p>Boar sperm were capacitated in vitro in presence of estrogens. Capacitation progress in control and experimental samples was analyzed by flow cytometry with the anti-acrosin monoclonal antibody (ACR.2) at selected times of incubation. Sperm samples were analyzed at 120 min of capacitation by CTC (chlortetracycline) assay, immunocytochemistry and flow cytometry with anti-acrosin ACR.2 antibody. Furthermore, sperm samples and capacitating media were analyzed by immunocytochemistry, ELISA with the ACR.2 antibody, and the acrosin activity assay after induced acrosomal reaction (AR).</p> <p>Results</p> <p>Estrogens stimulate sperm capacitation of boar sperm collected from different individuals. The stimulatory effect depends on capacitation time and is highly influenced by differences in the response to estrogens such as E2 by individual animals. Individual estrogens have relatively same effect on capacitation progress. In the boar samples with high estrogen responsiveness, estrogens stimulate the capacitation progress in a concentration-dependent manner. Furthermore, estrogens significantly increase the number of acrosome-reacted sperm after zona pellucida- induced acrosomal reaction.</p> <p>Conclusions</p> <p>We demonstrate here the stimulatory effect of four different estrogens on boar sperm capacitation in vitro. According to our results, there is significant difference in the response to tested estrogens at different capacitation time and among individual animals. In animals with a high response to estrogens, there is a concentration-dependent stimulation of capacitation and individual estrogens have relatively the same effect. Effects of individual estrogens, differences in the response to them by individual animals, their time and concentration-dependent outcomes further contribute to our knowledge about steroidal action in sperm.</p

Absence of spermatozoal CD46 protein expression and associated rapid acrosome reaction rate in striped field mice (Apodemus agrarius)

<p>Abstract</p> <p>Background</p> <p>In rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus.</p> <p>Methods</p> <p>Reverse transcription polymerase chain reaction (RT-PCR) was used to assess whether A. agrarius transcribe testicular CD46 mRNA. RT-PCR was supplemented with 3'- and 5'-rapid amplification of cDNA ends to determine the complete nucleotide sequence of A. agrarius CD46. Fluorescence microscopy was used to assess whether CD46 protein is expressed by A. agrarius sperm. The acrosome status of A. agrarius sperm was calculated over time by immunocytochemistry using peanut agglutinin lectin.</p> <p>Results</p> <p>We demonstrate that A. agrarius mice transcribe two unique alternatively spliced testicular CD46 mRNA transcripts, both lacking exon 7, which differ from those described previously in other Apodemus species. The larger A. agrarius CD46 transcript has an insert between exons 10 and 11 which, if translated, would result in a novel cytoplasmic tail. In addition, A. agrarius CD46 transcripts have an extended AU-rich 3'-untranslated region (UTR) and a truncated 5'-UTR, resulting in failure to express spermatozoal CD46 protein. We show that A. agrarius has a significantly faster spontaneous acrosome reaction rate than A. sylvaticus and Mus.</p> <p>Conclusion</p> <p>Absence of CD46 protein expression is associated with acrosomal instability in rodents. A. agrarius mice express novel CD46 transcripts, resulting in the trade of spermatozoal CD46 protein expression for a rapid acrosome reaction rate, in common with other species of field mice. This provides a strategy to increase competitive sperm advantage for individuals, leading to faster fertilisation in this highly promiscuous genus.</p

Of Oestrogens and Sperm: A Review of the Roles of Oestrogens and Oestrogen Receptors in Male Reproduction

The crucial role that oestrogens play in male reproduction has been generally accepted; however, the exact mechanism of their action is not entirely clear and there is still much more to be clarified. The oestrogen response is mediated through oestrogen receptors, as well as classical oestrogen receptors’ variants, and their specific co-expression plays a critical role. The importance of oestrogen signalling in male fertility is indicated by the adverse effects of selected oestrogen-like compounds, and their interaction with oestrogen receptors was proven to cause pathologies. The aims of this review are to summarise the current knowledge on oestrogen signalling during spermatogenesis and sperm maturation and discuss the available information on oestrogen receptors and their splice variants. An overview is given of species-specific differences including in humans, along with a detailed summary of the methodology outcome, including all the genetically manipulated models available to date. This review provides coherent information on the recently discovered mechanisms of oestrogens’ and oestrogen receptors’ effects and action in both testicular somatic and germ cells, as well as in mature sperm, available for mammals, including humans

Sperm-Egg Fusion: A Molecular Enigma of Mammalian Reproduction

The mechanism of gamete fusion remains largely unknown on a molecular level despite its indisputable significance. Only a few of the molecules required for membrane interaction are known, among them IZUMO1, which is present on sperm, tetraspanin CD9, which is present on the egg, and the newly found oolema protein named Juno. A concept of a large multiprotein complex on both membranes forming fusion machinery has recently emerged. The Juno and IZUMO1, up to present, is the only known extracellular receptor pair in the process of fertilization, thus, facilitating the essential binding of gametes. However, neither IZUMO1 nor Juno appears to be the fusogenic protein. At the same time, the tetraspanin is expected to play a role in organizing the egg membrane order and to interact laterally with other factors. This review summarizes, to present, the known molecules involved in the process of sperm-egg fusion. The complexity and expected redundancy of the involved factors makes the process an intricate and still poorly understood mechanism, which is difficult to comprehend in its full distinction

CD9 and CD81 Interactions and Their Structural Modelling in Sperm Prior to Fertilization

Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm&ndash;egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm&ndash;egg membrane fusion

Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins &#945;6&#946;4, &#945;3&#946;1 and &#945;6&#946;1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits &#945;3, &#945;6, &#946;1 and &#946;4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the &#946;4 integrin subunit is expressed in mouse sperm and that it pairs with subunit &#945;6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of &#946;4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: &#945;6/&#946;4-inner apical acrosomal membrane and equatorial segment; &#945;3, &#945;6/&#946;1, &#946;4-plasma membrane overlaying the apical acrosome; and &#945;3/&#946;1-outer acrosomal membrane. The existence of &#945;6&#946;4, &#945;3&#946;1 and &#945;6&#946;1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of &#945;3, &#945;6, &#946;1 and &#946;4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers &#945;6&#946;4, &#945;3&#946;1 and &#945;6&#946;1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction

<i>Toxoplasma gondii</i> Decreases the Reproductive Fitness in Mice

<div><p><i>Toxoplasma gondii</i> is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for <i>Toxoplasma gondii</i>, which forms specialized vacuoles containing reproductive cysts in the formers’ tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how <i>Toxoplasma gondii</i> can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of <i>Toxoplasma gondii</i> infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in <i>Toxoplasma gondii</i> positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in <i>Toxoplasma</i> infected mice, and differences in the DNA methylation of selected genes were detected between the <i>Toxoplasma</i> positive and control group. These findings demonstrate a direct relation between <i>Toxoplasma gondii</i> infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of idiopathic infertility in humans.</p></div