Elucidating the Role of the Epididymis in Conception by Targeted Gene Inactivation

It is now well established that spermatozoa of eutherian mammals are continuously released from the seminiferous epithelium as infertile and virtually immotile gametes, that they undergo further maturation in the proximal epididymis, and that large numbers of them are subsequently stored in a quiescent state in the distal portion of the Wolffian duct until ejaculated. However, there remains a considerable lack of understanding as to the nature of sperm/epididymis relationship, nor is there any certainty about the biological significance ofthis organ. The thesis describes an attempt to address these questions in the mouse by applying the methods ofreverse genetics. . Given the potential of the CrelloxP binary transgenic technology, an attempt was made to produce a panel oftransgenic mice with regionalized Cre-recombinase expression under the transcriptional control ofthe epididymis-specific genes: RnaselO, EG627821, Crispl, Crisp4 and Dejb41. Using the knock-in approach, RnaselOCre (initial segment) and Crisp4cre (zones 2-4) mouse lines were derived. Since the initial segment is the primary site of sperm maturation, and the ~ epididymal epithelium is reliant on androgenic support, an initial segment-specific androgen receptor Ar) knock-out was generated by intercrossing the Rnase1OCre mouse with an A/oxP mouse. Consequences of this manipulation are described, as is the phenotype ofthe RnaselOCre/Cre(-/-) male. The findings challenge the reductionist model offertilization in the mouse, and the place of the epididymis in the chain of eutherian conception is discussed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Elucidating the Role of the Epididymis in Conception by Targeted Gene Inactivation

It is now well established that spermatozoa of eutherian mammals are continuously released from the seminiferous epithelium as infertile and virtually immotile gametes, that they undergo further maturation in the proximal epididymis, and that large numbers of them are subsequently stored in a quiescent state in the distal portion of the Wolffian duct until ejaculated. However, there remains a considerable lack of understanding as to the nature of sperm/epididymis relationship, nor is there any certainty about the biological significance ofthis organ. The thesis describes an attempt to address these questions in the mouse by applying the methods ofreverse genetics. . Given the potential of the CrelloxP binary transgenic technology, an attempt was made to produce a panel oftransgenic mice with regionalized Cre-recombinase expression under the transcriptional control ofthe epididymis-specific genes: RnaselO, EG627821, Crispl, Crisp4 and Dejb41. Using the knock-in approach, RnaselOCre (initial segment) and Crisp4cre (zones 2-4) mouse lines were derived. Since the initial segment is the primary site of sperm maturation, and the ~ epididymal epithelium is reliant on androgenic support, an initial segment-specific androgen receptor Ar) knock-out was generated by intercrossing the Rnase1OCre mouse with an A/oxP mouse. Consequences of this manipulation are described, as is the phenotype ofthe RnaselOCre/Cre(-/-) male. The findings challenge the reductionist model offertilization in the mouse, and the place of the epididymis in the chain of eutherian conception is discussed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa

Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species

Epididymal protein Rnase10 is required for post-testicular sperm maturation and male fertility

Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction

Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding

During epididymal maturation, sperm acquire the ability to swim progressively by interacting with proteins secreted by the epididymal epithelium. Beta-defensin proteins, expressed in the epididymis, continue to regulate sperm motility during capacitation and hyperactivation in the female reproductive tract. We characterized the mouse beta-defensin 41 (DEFB41), by generating a mouse model with iCre recombinase inserted into the first exon of the gene. The homozygous Defb41(iCre/iCre) knock-in mice lacked Defb41 expression and displayed iCre recombinase activity in the principal cells of the proximal epididymis. Heterozygous Defb41(iCre/+) mice can be used to generate epididymis specific conditional knock-out mouse models. Homozygous Defb41(iCre/iCre) sperm displayed a defect in sperm motility with the flagella primarily bending in the pro-hook conformation while capacitated wild-type sperm more often displayed the anti-hook conformation. This led to a reduced straight line motility of Defb41(iCre/liCre) sperm and weaker binding to the oocyte. Thus, DEFB41 is required for proper sperm maturation. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Dicer1 Ablation in the Mouse Epididymis Causes Dedifferentiation of the Epithelium and Imbalance in Sex Steroid Signaling

<div><h3>Background</h3><p>The postnatal development of the epididymis is a complex process that results in a highly differentiated epithelium, divided into several segments. Recent studies indicate a role for RNA interference (RNAi) in the development of the epididymis, however, the actual requirement for RNAi has remained elusive. Here, we present the first evidence of a direct need for RNAi in the differentiation of the epididymal epithelium.</p> <h3>Methodology/Principal Findings</h3><p>By utilizing the Cre-LoxP system we have generated a conditional knock-out of Dicer1 in the two most proximal segments of the mouse epididymis. Recombination of <em>Dicer1</em>, catalyzed by <em>Defb41^iCre/wt</em>, took place before puberty, starting from 12 days postpartum. Shortly thereafter, downregulation of the expression of two genes specific for the most proximal epididymis (lipocalin 8 and cystatin 8) was observed. Following this, segment development continued until week 5 at which age the epithelium started to regress back to an undifferentiated state. The dedifferentiated epithelium also showed an increase in estrogen receptor 1 expression while the expression of androgen receptor and its target genes; glutathione peroxidase 5, lipocalin 5 and cysteine-rich secretory protein 1 was downregulated, indicating imbalanced sex steroid signaling.</p> <h3>Conclusions/Significance</h3><p>At the time of the final epididymal development, Dicer1 acts as a regulator of signaling pathways essential for maintaining epithelial cell differentiation.</p> </div