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Identification and purification of proteins from germ cell-conditioned medium (GCCM)

Germ cells are known to regulate Sertoli cell and testicular function possibly through released factor(s) or via cell-cell contact. However, the identities of many of these putative biological factors are not known. The aim of this study is to present a strategy to identify and purify germ cell derived proteins found in germ cell-conditioned medium (GCCM) at a quantity sufficient to permit protein microsequencing. The purification scheme of a novel germ cell-derived protein from GCCM designated GC-26 is presented along with several germ cell proteins using a combination of high pressure liquid chromatography (HPLC) columns. The purity of GC-26 and other germ cell proteins were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining. The identities of GC-26, a 26-kDa polypeptide, and other proteins were determined by direct protein microsequencing. These partial NH2-terminal amino acid sequences were compared with the existing databases at Protein Identification Resource (PIR), GenBank, and BLAST. These analyses revealed that these proteins are unique. This strategy should be useful for the micropurification of proteins from other biological samples and/or fluids.link_to_subscribed_fulltex

Rat testicular N-cadherin: Its complementary deoxyribonucleic acid cloning and regulation

Using primer sets specific for mouse N-cadherin and rat testicular RNA for RT-PCR, a full-length complementary DNA (cDNA) coding for rat testicular N-cadherin was isolated. The deduced amino acid sequence of rat N-cadherin yielded a 883-amino acid polypeptide that displayed a 98.6% identity with the mouse homolog. N-Cadherin was found to be expressed by Sertoli and germ cells in the rat testis by RT-PCR. Using Sertoli-germ cell cocultures, it was found that the N-cadherin expression increased with time in culture. To assess whether this is due to a soluble factor(s) released from germ cells that affects Sertoli cell N-cadherin expression, germ cell-conditioned media (GCCM) were fractionated by preparative anion-exchange HPLC, and the resulting fractions were divided into 14 pools. Pool 4 was found to contain a factor(s) that induced a dose-dependent stimulation on Sertoli cell N- cadherin expression with a maximal stimulation at 2 μg protein/dish/4.5 x 106 Sertoli cells. At higher doses between 12 and 32 μg protein/dish, this pool relinquished its effect on Sertoli cell N-cadherin expression suggestive of a biphasic effect. This biphasic effect was confirmed using increasing doses of crude GCCM on Sertoli cell cultures. Since nonviable germ cells failed to stimulate Sertoli cell N-cadherin expression, it illustrates the observed stimulatory effect by GCCM is likely to be mediated via a soluble factor(s) releasing from viable germ cells. These results reveal the presence of a stimulatory factor(s) in GCCM that can modulate Sertoli cell N-cadherin expression in vitro. Since N-cadherin plays a crucial role in facilitating invasive capacity of metastatic tumor cells, the observation of germ cell- released factor(s) in affecting Sertoli cell N-cadherin expression may suggest its possible role in facilitating germ cell migration during spermatogenesis.published_or_final_versio

Evidence for cross-talk between sertoli and germ cells using selected cathepsins as markers

To examine whether proteases are possibly involved in cellular migration and/or spermiation when developing germ cells translocate across the seminiferous epithelium during spermatogenesis, in situ hybridization was used to localize messenger RNA (mRNA) transcripts of cathepsin L, D, and S in the epithelium at different stages of the spermatogenic cycle in the rat. Cathepsin L mRNA was found to localize almost exclusively near the basal lamina of the epithelium. At stages VI and VII of the cycle before spermiation, the signal of cathepsin L mRNA was so intense that it formed a complete dark precipitate near the basal lamina encircling the entire tubule. At stage VIII, the expression of cathepsin L was completely abolished, and no staining of cathepsin L mRNA was seen in the epithelium. The mRNA of cathepsin D and S was found near the basal lamina, a finding consistent with their localization in Sertoli cells and possibly primary spermatocytes in almost all stages, but peaked at stages VII-IX and VII-VIII of the cycle, respectively, at the time before and during spermiation. These results illustrate the possible involvement of these proteases in facilitating germ cell movement and spermiation. To examine whether germ cells express any of these cathepsin genes, spermatocytes with a purity of greater than 95% were isolated from 15-day-old rat testes by Percoll gradient centrifugation for reverse transcriptase-polymerase chain reaction. It was found that primary spermatocytes expressed multiple cathepsin genes, including cathepsin B, C, D, H, L, and S. Furthermore, the expression of cathepsin L by germ cells isolated from 15-day-old rats (largely spermatocytes and spermatogonia) can be stimulated by Sertoli cell-enriched culture medium in a dose-dependent manner, but not by germ cell-conditioned medium. These results reveal that germ cell function can be regulated by Sertoli cells. Moreover, these results suggest that germ cells may play an active role in the overall testicular protease expression. Also, we present evidence suggesting there is cross- talk between Sertoli and germ cells, since the expression of cathepsin L in each cell type is regulated by one another via either soluble factors or cell-cell contact.published_or_final_versio

Practice of breastfeeding and factors that affect breastfeeding in Hong Kong

Objectives. To describe the patterns of and factors affecting breastfeeding and to find out any significant relationship between breastfeeding and health of the child. Design. Cohort study. Setting. Postnatal ward of the Prince of Wales Hospital. Participants. A total of 243 infants born in 1998 to 2001 at the hospital. Each infant was followed up for 3 years. Home visits were carried out at 3, 15, 24, and 36 months of age by medical students from the Chinese University of Hong Kong. A questionnaire was completed at each visit. Independent sample t-tests and Pearson Chi squared tests were used. Results. Of the 243 subjects, 213 provided data on the method of infant feeding. There were 66.7% of mothers initiating breastfeeding, with a median duration of 1 month. Only 13.4% met the World Health Organization's recommendations on breastfeeding. Breastfeeding was found to have a statistically significant relationship with (i) the infant's birth order and (ii) the mother's and father's education level. During follow-up, 44.6% of the infants were hospitalised but there was no significant relationship between breastfeeding and number of hospitalisations. Conclusions. The current breastfeeding rate in Hong Kong falls below expectations when compared with other developed nations. To raise this rate, more support is needed for families with parents having a lower education level or more than two children, as they are the least likely to breastfeed. This might be achieved by encouraging antenatal class attendance, counselling of husbands, and more support for breastfeeding from doctors.link_to_subscribed_fulltex

Changes in the expression of junctional and nonjunctional complex component genes when inter-Sertoli tight junctions are formed in vitro

Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly and reassembly of intercellular junctions suggesting germ cell movement is composed of intermittent phases of junction disassembly and reassembly. A study was performed to correlate the expression of junctional-complex components (such as zonula occludens-1 [ZO-1], a tight-junction component protein) and nonjunctional complex components (such as urokinase-type plasminogen activator [uPA], a serine protease; cathepsin L, a cysteine protease; α2-macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cysteine protease inhibitor) at the time when inter-Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expression of nonjunctional component genes also correlates with the formation of inter-Sertoli tight junctions in vitro. This is part of an effort to understand the physiologic elements of germ cell movement in the epithelium. Sertoli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of apoptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 x 106 cells/cm2 for up to 7 days on bicameral culture units coated with Matrigel (Collaborative Research) and were assessed by morphologic analysis and agarose gel electrophoresis. It was noted that many of the Sertoli cells cultured at 3 x 106 cells/cm2 underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 x 106 cells/cm2 illustrating the Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO-1 increased significantly between days 2 and 3 prior to the establishment of inter-Sertoli tight junctions assessed by transepithelial resistance measurement (TER), which illustrates that ZO-1 can be used as a marker to monitor this cellular event. More interestingly, there was also a transient increase in the expression of uPA and cathepsin L between days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 x 104 cells/cm2), when a confluent monolayer of cells could not form, there were no changes in the expression of either ZO-1, uPA, or cathepsin L throughout the 7-day culture period. These results show that the establishment of specialized junctions, such as tight junctions between Sertoli cells in vitro, may require the participation of both junctional and nonjunctional complex components.link_to_subscribed_fulltex

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by sertoli and germ cells, is a potential marker for studying cell-cell interactions in the rat testis

The full-length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three ~2.9-kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gin421 and Phe422 within the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X-linked myotubular myopathy. A 22 amino acid peptide NH2-TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66-kDa protein was indeed detected in both Sertoli and germ-cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter-Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady-state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or Ionidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell-cell interactions in the testis. (C) 2000 Wiley-Liss, Inc.link_to_subscribed_fulltex