Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the β-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the α and β subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon. Mol. Reprod. Dev. 48:71–76, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Sequence analysis of cDNAs encoding precursors of starfish asterosaps

To understand how starfish sperm activating peptides (asterosaps) are synthesized in the ovary, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences. The mRNA encoding asterosaps was synthesized only in the oocytes, but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap precursors are large prepolypeptide chains with an unusual “rosary‐type” structure made of 10 successive similar stretches of 51–55 residues. Each stretch finishes with a “spacer” of 17–21 residues immediately followed by the sequence of one asterosap isoform. The N‐terminal of this precursor has 19–21 successive glutamine‐rich repeating units. Maturation of the precursor may require endopeptidases that cleave both C‐ and N‐sites of lysine‐arginine. Dev. Genet. 25:130–136, 1999. © 1999 Wiley‐Liss, Inc.     

p53 independent,region‐specific epithelial apoptosis is induced in the rat epididymis by deprivation of luminal factors

Luminal testicular factors are known to be important for the regulation of the epididymal epithelium. The present study was undertaken to test the hypothesis that complete deprivation of luminal factors by efferent duct ligation (EDL) would induce apoptosis in the epididymal epithelium, as does removal of trophic factors from other cell types. Additionally, experiments were performed to determine whether the apoptosis detected was p53 dependent or independent. Apoptosis detection was by terminal deoxynucleotidyl‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling and by DNA fragmentation studies. EDL caused loss of testicular luminal contribution in zone 1A of the rat epididymis (proximal initial segment) within 6 hr and induced epithelial apoptosis within 12 hr of the efferent duct obstruction. The wave of apoptosis in zone 1A was completed by three days after EDL and was followed by a much smaller wave in zone 1B which peaked three days after EDL. Significant apoptosis was not detected in any epididymal region distal to the initial segment for periods as long as 15 days after EDL. p53, a key apoptotic‐pathway molecule in many tissues and conditions was tested by immunohistochemical and Western blot techniques and was not upregulated in the initial segment epithelium within the time cells were undergoing apoptosis and well before the wave of apoptosis was complete. It was concluded that epithelial apoptosis in the initial segment of the rat epididymis is induced by deprivation of luminal testicular factors, is localized to the proximal and middle initial segment, and is p53 independent. Mol. Reprod. Dev. 53:188–197, 1999. © 1999 Wiley‐Liss, Inc.     

A 140‐kDa glycopeptide from the sperm ligand of the vitelline coat of the freshwater bivalve Unio elongatulus,only contains O‐linked oligosaccharide chains and mediates sperm‐egg interaction

In previous studies we found that sperm binding activity in the vitelline coat of the freshwater bivalve Unio elongatulus is located on the O‐linked oligosaccharide chains of gp273, one of the two major components of the extracellular coat, and that fucose plays a key role in this interaction. In this paper we report the partial characterization of a large glycopeptide (about 140 kDa) obtained by cyanogen bromide fragmentation of gp273, that maintains sperm binding activity. Lectin blotting revealed that the glycopeptide reacted with lectins from Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA) but not Canavalia ensiformis (ConA). No other PNA‐positive fragments could be detected in the electrophoretic pattern of fragmented gp273 but several ConA‐positive fragments of lower molecular weight were present indicating that all the O‐linked chains are clustered together in this fragment. Two‐dimensional gel electrophoresis of the fragment revealed it to be acidic in nature in contrast with the neutral character of the whole gp273 molecule. Competition binding assay showed that this fragment is a strong inhibitor of the interaction, whereas no effect was detected using the ConA‐positive peptides. This confirms that the sperm receptor activity of gp273 is related to its O‐linked chains. The immunodominance of this fragment is also discussed. Mol. Reprod. Dev. 54:203–207, 1999. © 1999 Wiley‐Liss, Inc.     

果蝠的婚配制度及繁殖策略

翼手类(Chiroptera)(俗称蝙蝠)分为小蝙蝠亚目(Microchiroptera)和大蝙蝠亚目(Megachiroptera),大蝙蝠又称果蝠或狐蝠,果蝠仅狐蝠科(Pteropodidae)1科188种。深入地了解其独特的婚配行为机制、独特的繁殖发育机制,对有效地开展果蝠的保护工作、合理地控制种群数量有积极意义。本文对果蝠的婚配制度及繁殖策略进行了阐述。     

金针菇外切-β-1,3-葡聚糖酶家族基因鉴定及在不同发育时期的差异表达

金针菇具有很高的营养与保健价值,菌柄长短决定金针菇的产量与品质,而菌柄伸长的相关作用酶及分子机理尚不清楚。前期草菇中发现外切-β-1,3-葡聚糖酶基因（exg2）可能与菌柄伸长相关,但在金针菇中尚没有exg基因的相关报道。本研究首先在金针菇全基因组中鉴定到3个外切-β-1,3-葡聚糖酶家族基因（分别命名为：Ffexg1、Ffexg2、Ffexg3）,并进行了克隆验证。进一步采用定量PCR对3个基因在金针菇不同发育时期及组织部位的差异性表达进行了分析。结果显示：Ffexg1只在菌柄中高表达,Ffexg2与Ffexg3在菌柄中表达量先上升后下降,在菌盖中呈逐渐上升趋势。3个Ffexg基因均在菌柄发生伸长的部位表达量较高,且在菇体水平放置后菌柄弯曲程度较大的部位表达量较高。结果显示金针菇exg家族3个基因存在时空差异性表达,在菌柄中伸长较快的时期及部位伴随着Ffexg基因的高表达。结合其功能预测,Ffexg家族基因可能作用于细胞壁成分β-1,3-葡聚糖链,从而在金针菇菌柄及菌盖发育中起作用。