Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Spermatogenesis leads to the formation of functional sperm cells. Here we have applied high-pressure freezing in combination with transmission electron microscopy (TEM) to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea, a species in which spermatogenesis had not previously been studied. We show the ultrastructure of different stages of sperm development in this species. Thorough examination of TEM data and electron tomographic reconstructions revealed interesting structural features of the nebenkern, an organelle composed of fused mitochondria that has been studied in spermatids of other insect species. We have applied serial-section electron tomography of the nebenkern to demonstrate in three dimensions (3D) that this organelle in H. membranacea is composed of two interwoven mitochondrial derivatives, and that the mitochondrial derivatives are connected by a zipper-like structure at opposing positions. Our approach will enable further ultrastructural analyses of the nebenkern in other organisms.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.

Thesis (MScMedSc (Obstetrics and Gynaecology))--University of Stellenbosch, 2005. / Chapter 1 contains a review dealing with nuclear and morphological changes during spermatogenesis and spermatozoa transport with emphasis on the maturation of spermatozoa, capacitation, acrosome reaction and the interaction with the cumulus oophorus complex (COC). The oocyte and cumulus oophorus complex is also discussed particularly on the topic of maturity (oocyte and cumulus maturity). Also presented is a review of the fluorescent binding agents, namely Fluorescein Isothiocyanate labeled with Pisum sativum (FITC-PSA), Chlorotetracycline test (CTC) and Chromomycin A3 (CMA3). Chapter II describes all the materials and methods used during this study. Routine semen analysis is described with emphasis on normal spermatozoon morphology according to strict criteria. The evaluation of capacitation and acrosome reaction (AR) using the CTC and PSA-FITC staining methods as well as the evaluation of spermatozoon nuclear chromatin packaging using the CMA3 staining method is described. Chapter III represents the results recorded in this study. Compared with those spermatozoa cultured in medium alone, spermatozoa exposed to the cumulus mass were more likely to be capacitated and acrosome reacted, with a distinct increase in chromatin packaging quality. A general discussion of the results and future applications are discussed in Chapter IV. In short An in vitro model for spermatozoa penetration through the cumulus oophorus was established. The model can be applied to investigate the effect of the cumulus oophorus on sperm functions and to assist in the selection of functional sperm for intracytoplasmic sperm injection therapy. All relevant references are presented in Chapter V .