Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm - oocyte binding sites that exhibited this actin response was reduced in pyk2(-/-) oocytes and the pyk2(-/-) oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm - oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm binding sites under conditions where sperm oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

Fertilization triggers activation of a series of pre‐programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free‐calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm–oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm–oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm–oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte. Mol. Reprod. Dev. 80: 260–272, 2013. © 2013 Wiley Periodicals, Inc.

FYNkinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated α‐tubulin (Ac‐α‐Tu) is a component of long‐lived, stable microtubules and deacetylation of α‐tubulin enhances cell motility, we hypothesized that acetylation of α‐tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac‐α‐Tu at different stages of testis development was characterized by immunohistochemistry for Ac‐α‐Tu and spermatogonia‐specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac‐α‐Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac‐α‐Tu appeared polarized toward the basement membrane. In adult pig testes, Ac‐α‐Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL−), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac‐α‐Tu. Comparison with the expression pattern of β‐tubulin and tyrosinated α‐tubulin confirmed that only Ac‐α‐Tu is specific to germ cells. The unique pattern of Ac‐α‐Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348–352, 2010. © 2009 Wiley‐Liss, Inc.

Fyn kinase is highly expressed in oocytes, with inhibitor and dominant-negative studies suggesting a role in the signal transduction events during egg activation. The purpose of the present investigation was to test the hypothesis that Fyn is required for calcium signalling, meiosis resumption and pronuclear congression using the Fyn-knockout mouse as a model. Accelerated breeding studies revealed that Fyn-null females produced smaller litter sizes at longer intervals and exhibited a rapid decline in pup production with increasing age. Fyn-null females produced a similar number of oocytes, but the frequency of immature oocytes and mature oocytes with spindle chromosome abnormalities was significantly higher than in controls. Fertilised Fyn-null oocytes frequently (24%) failed to undergo pronuclear congression and remained at the one-cell stage. Stimulation with gonadotropins increased the number of oocytes ovulated, but did not overcome the above defects. Fyn-null oocytes overexpressed Yes kinase in an apparent effort to compensate for the loss of Fyn, yet still exhibited an altered pattern of protein tyrosine phosphorylation. In summary, Fyn-null female mice exhibit reduced fertility that appears to result from actin cytoskeletal defects rather than calcium signalling. These defects cause developmental arrest during oocyte maturation and pronuclear congression.

Mouse oocytes fertilized in vivo exhibit highly localized protein tyrosine kinase (PTK) signaling events in the egg cortex which we have demonstrated by immunofluorescence with an anti-phosphotyrosine antibody. In order to detect events that occur during the early stages of sperm-egg interaction, the present study used in vitro fertilization (IVF) of zona-free eggs to enhance the synchrony of sperm-egg interaction and sperm incorporation. Oocytes were fixed at 15, 30, 45, 60, 75-90, and 120 minutes after addition of capacitated sperm and processed for confocal immunofluorescence microscopy. Tyrosine phosphorylated proteins were localized with an anti-P-Tyr antibody (4G10) and controls were incubated with 4G10 neutralized with phospho-L-tyrosine. Localized accumulation of P-Tyr-containing proteins was evident in the egg cortex underlying attached sperm within 30-45 minutes post-insemination (mpi), suggesting that sperm-egg contact or fusion triggered PTK signaling in the egg. P-Tyr was also evident in the egg cortex adjacent to maternal chromatin at anaphase and paternal chromatin after incorporation beneath the plasma membrane. Subsequently, tyrosine phosphorylation of egg cortex proteins propagated throughout the entire egg cortex by 120 mpi and persisted through meiosis completion, then declined during the pronuclear stage. Parthenogenetic activation of MII oocytes with strontium chloride triggered cortical accumulation of P-Tyr-containing proteins during late anaphase in a manner similar to that induced by fertilization, indicating that sperm were not required for the cortical signaling during meiosis resumption. Western blot analysis of eggs with 4G10 antibody detected PTK substrates of 63, 67, 82, and 120 KDa, but failed to detect any significant increases in response to fertilization or parthenogenic activation. In fact, the P-Tyr content of most bands declined during the pronuclear stage. This failure to detect biochemical confirmation of the enhanced P-Tyr signaling observed via microscopy could indicate either that fertilization-induced cortical changes involved phosphorylation of only a small fraction of the total pool of each protein, or that they resulted from concentration of P-Tyr containing proteins in regions of the egg cortex with no net increase in overall phosphorylation state. The kinase inhibitor SKI-606 blocked the fertilization-induced accumulation of P-Tyr-containing proteins observed by immunofluorescence and specifically reduced the P-Tyr content of bands at 63 and 67 KDa. This suggested that Src-family protein kinases were in part responsible for phosphorylating at least some of the egg cortex proteins during anaphase. The focal adhesion kinase (FAK) was also implicated in cortical PTK signaling since an antibody to the autophosphorylated form of FAK detected activation of this kinase in the egg cortex with a pattern very similar to that of phosphotyrosine. (poster)

Fyn kinase is highly expressed in mammalian and non-mammalian oocytes and has been implicated in the signal transduction events including calcium signaling, cell cycle events such as meiosis resumption and pronuclear congression. The results of the above experimental studies contradict other reports that the Fyn-null mice exhibit normal fertility. The purpose of the present study was to compare the fertility of B6/129S7-Fyntm1Sor/J female mice with females from the parent strain (B6/129SF2/J) (The Jackson Laboratory, Bar Harbor, ME). Western blot analysis revealed the absence of Fyn protein in Fyn-null oocytes, although these oocytes did over express Yes protein kinase, possibly in an attempt to compensate for the loss of Fyn. Fyn-null females were smaller in size than controls and produced significantly smaller litter sizes during the first five parturitions. The interval between litters was longer and the overall reproductive lifespan was shorter than controls. Superovulation of age-matched females produced oocytes that were similar in number and size to controls, but exhibited a thinner zona pellucida and a marked accumulation of dense cytoplasmic inclusions of unknown composition. Evaluation by confocal microscopy revealed that approximately 18% of the ovulated eggs recovered from the oviduct at 15hrs post hCG administration were immature (MI) which was significantly higher than controls. Fyn-null oocytes also exhibited defects in spindle organization as well as reduced ability to position the MII spindle close to the egg cortex. These spindle abnormalities correlated with defects in cortical actin cytoskeleton polarity. Fertilization in vitro with wild type sperm revealed that the Fyn-null oocytes were fertilized at a rate similar to controls, but exhibited reduced developmental potential since 73% of fertilized eggs (pronuclei) reached the 2-cell stage vs 98% from controls. Embryos that reached the 2-cell stage developed to blastocyst at a rate similar to controls. Calcium imaging studies revealed that Fyn-null oocytes fertilized with wild type sperm exhibited reduced frequency of calcium oscillations and a modified pattern of peak amplitude, although the total calcium signal produced by Fyn-null oocytes was similar to controls. The altered calcium signaling properties were correlated with a modified distribution of IP3 receptor as detected by confocal immunofluorescence. The IP3 receptor type 1 was frequently distributed in a highly irregular pattern in Fyn-null oocytes with large clumps or accumulations that were not present in control oocytes. In summary, the Fyn-null oocyte exhibits defects in meiotic maturation, meiotic spindle integrity, cortical cytoskeleton organization, and calcium signaling that may explain the reduced developmental potential. (platform)

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy‐terminal hydrolase 1 (UCH‐L1) is expressed in type A (A s , A pr , and A al ) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH‐L1 was associated with self‐renewal versus differentiation divisions of SSCs as defined by co‐localization of UCH‐L1 high and PLZF, a known determinant of undifferentiated SSCs, versus co‐localization of UCH‐L1 low/− with proteins expressed during SSC differentiation (DAZL, DDX4, c‐KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH‐L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH‐L1 and PLZF in situ in seminiferous tubules. Expression level of UCH‐L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH‐L1‐positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self‐renewal and differentiation in mammalian spermatogenesis. J. Cell. Physiol. 220: 460–468, 2009. © 2009 Wiley‐Liss, Inc.

The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally.

Rough set theory is a very useful tool for describing and modeling vagueness in ill-defined environments. Traditional rough set theory is restricted to crisp environments. However, nowadays, it has been extended to fuzzy environments, resulting in the development of the so-called fuzzy rough sets. Type-2 fuzzy sets possess many advantages over type-1 fuzzy sets, but for the general type-2 fuzzy sets, the computational complexity is severe. On the other hand, set-theoretic and arithmetic computations for the interval type-2 fuzzy sets are very simple. Motivated by the aforementioned accomplishments, in this paper, the concept of fuzzy rough sets is generalized to interval type-2 fuzzy environments. Subsequently, a method of attribute reduction within the interval type-2 fuzzy rough set framework is proposed. Lastly, the properties of the interval type-2 fuzzy rough sets are presented.

Testicular germ cell tumours (TGCTs) are prevalent cancers among young men. Currently, there is no reliable animal model for TGCTs. To establish such animal models, we have explored the possibility of intratubular testicular transplantation as means to deliver tumour cells into the seminiferous tubules of host animals. Our results demonstrated that transplanted cells could effectively populate the testis of a recipient mouse and develop into TGCTs. In addition, the donor cells could be transfected with a specific transgene before transplantation, thereby providing an approach to evaluate the specific effects of gene functions in the oncogenic processes. Hence, depending on selection of specific donor cells or mixtures of donor cells, transplantation models of TGCTs could be significant for studies on the pathogenesis, diagnosis and therapies of such a prevalent and important cancer in men.

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.

Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4-8 microM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations.

Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.

This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.

Nitrosation of dibenzylphosphoramidates 4 enables the synthesis of phosphoric acid monoesters 6 from primary n-alkylamines via 5 in good yield.