First Report of Sweet potato chlorotic stunt virus, a Component of Sweetpotato Virus Disease, in North Carolina.

Sweet potato chlorotic stunt virus (SPCSV) is the whitefly-transmitted component of the sweet potato virus disease (SPVD), a devastating disease originally described in Africa (4). Two isolates designated as G-01 and T-03 were obtained in North Carolina in July 2001 and October 2003, respectively, from plants of cv. Beauregard exhibiting symptoms typical of SPVD, including stunting, leaf narrowing and distortion, vein clearing, and chlorotic mosaic. Sap extract from symptomatic plants tested positive for SPCSV by nitrocellulose immuno-dot blot, using monoclonal antibodies specific for SPCSV obtained from the International Potato Center. Total RNA was extracted from 100 mg of symptomatic leaf tissue by using the PureLink Total RNA Purification System Kit from Invitrogen (Carlsbad, CA) with a minor modification (adding 2% PVP-40 and 1% 2-mercaptoethanol to the extraction buffer) (1). Results were confirmed by reverse transcription (RT)-PCR using primers CP1 and CP3 and HSP70-A/HSP70-B (2), corresponding to the capsid protein and 'heat shock' protein genes, respectively. HSP70 amplicons were cloned using the TOPO TA Cloning Kit (Invitrogen) and sequenced. At the nucleotide level, viral sequences from clones from both isolates were an average 99.4% similar to West Africa and 77.9% to East Africa sequences of SPCSV from Genbank (1). Although the isolates were collected from different fields, viral sequences generated from clones for T-03 and G-01 differed by only six nucleotides and were identical at the amino acid level. The neighbor-joining phylogenetic tree constructed using the HSP70 gene fragment (39 nt) delineated two major clusters with two subpopulations each: Cluster 1, "East Africa", consisted of East Africa and Peru subpopulations; Cluster 2, "West Africa", consisted of Argentina-Brazil and USA-West Africa subpopulations (1). In addition, SPCSV isolates from East Africa and West Africa clusters were sufficiently distant phylogenetically to suggest that they may correspond to two different criniviruses, with an average similarity between the populations of 78.14% and an average within the populations above 89%. Hudson's tests confirmed the presence of genetically distinct SPCSV groups with high statistical significance (1). Two groups (Peru and East Africa) were differentiated in the East Africa cluster, and three groups (Argentina-Brazil, USA, and West Africa) were differentiated in the West Africa cluster, suggesting that the USA population is not a recent introduction. Although SPCSV was previously reported in the United States, the source was a single accession of cv. White Bunch from the USDA Sweetpotato Germplasm Repository (3). Sweet potato feathery mottle virus (SPFMV) (family Potyviridae, genus Potyvirus), the other component of SPVD, was also detected in both cultivars. To our knowledge, this is the first report of SPCSV in sweetpotato fields in the United States. References: (1) J. A. Abad et al. Phytopathology (Abstr.) 96(suppl.):S1, 2006. (2) T. Alicai et al. Plant Pathol. 48:718, 1999. (3) G. Pio-Ribeiro et al. Plant Dis. 80:551, 1996. (4) G. A. Schaefer and E. R. Terry. Phytopathology 66:642, 1977.

Expression of a novel factor, short-type PB-cadherin, in Sertoli cells and spermatogenic stem cells of the neonatal rat testis.

In the rodent testis, contact-mediated interactions between gonocytes, or neonatal stem cells, and Sertoli cells are critical for development. Previously, we showed that the neural cell adhesion molecule (NCAM) serves as a Sertoli cell-gonocyte attachment factor in neonates. Its expression decreases dramatically by 1 week of age and eventually disappears in vivo, and appears to be down-regulated by thyroid hormone (tri-iodothyronine (T(3))). In this study, we used a cDNA microarray to screen for additional adhesion factors which might be important in testes of developing rats and detected expression of a novel factor, short-type PB-cadherin (STPB-C). Next, RT-PCR was used to generate cDNA for STPB-C from total RNA isolated from co-cultures, cDNA was cloned into pPCR-Script Amp SK(+) cloning vector, and plasmid DNA was isolated and sequenced to confirm the fidelity of the STPB-C cDNA portion of the plasmid. In situ hybridization analyses of testicular sections indicated that STPB-C expression in neonates is localized in the cytoplasm of many, but not all, gonocytes and in the cytoplasm of most of the surrounding Sertoli cells. Parallel hybridizations carried out on co-cultures also demonstrated a strong cytoplasmic signal in some gonocytes and in the great majority of the Sertoli cells of the underlying monolayer. With Northern analyses we found that STPB-C is expressed in vivo at high levels between days 1 and 5, with a subsequent large drop by day 10 and thereafter, suggesting that its expression may be associated with Sertoli or germ cell differentiation. Subsequent analyses of co-cultures exposed under a variety of conditions to T(3) suggest that, unlike NCAM, STPB-C is not regulated by this hormone. Next, we studied production of STPB-C protein by using an antiserum recognizing a peptide sequence unique to this factor in Western blotting and in immunolocalization. Signal was detected both intracellularly and at cell surfaces in most Sertoli cells and many gonocytes, although many of the latter cell type were also found to be negative for the protein, suggesting a potential role for STPB-C in survival and further development of some of these germ cells from which all subsequent spermatogenic cells originate.

Gonocyte-Sertoli cell interactions during development of the neonatal rodent testis.

During neonatal testicular development in the rat, events critical for subsequent germ cell development occur that set the stage for fertility later in life. Some gonocytes resume mitotic activity and/or migrate to the surrounding basal lamina, and use of a carefully defined Sertoli cell-gonocyte coculture system indicates that these crucial events occur without added factors or hormones and are hence likely to depend on interaction with adjacent Sertoli cells. Coupling of the Kit receptor protein on gonocytes to stem cell factor from Sertoli cells is vital for successful migration by gonocytes, as antagonism of the former suppresses and addition of the latter stimulates gonocyte migration. During the neonatal period, intercellular adhesion is modified in a developmental manner such that neural cell adhesion molecule (NCAM) is the main adhesive molecule expressed and functioning at birth, with a progressive decline as development proceeds. This decline in NCAM expression is supported by the addition of exogenous 3,3',5-triiodothyronine in vitro, and because this factor is recognized as supporting Sertoli cell differentiation, it seems likely that changing intercellular adhesion is a function of progressive development of Sertoli cells. Other avenues whereby maturing testicular cells influence each other doubtless exist, including secretion of growth factors and other peptides and developmentally important changes in the makeup of the extracellular matrix, which Sertoli cells and gonocytes contact. Continued investigation in these areas will be very valuable in enlarging our understanding of how neonatal testicular development provides the basis for successful spermatogenesis.

A single dose of Di-(2-ethylhexyl) phthalate in neonatal rats alters gonocytes, reduces sertoli cell proliferation, and decreases cyclin D2 expression.

In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20-500 mg/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose-response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.

Thyroid hormone down-regulates neural cell adhesion molecule expression and affects attachment of gonocytes in Sertoli cell-gonocyte cocultures.

Contact-mediated interactions between Sertoli cells and gonocytes are important for testicular development. Specifically, down-regulation of neural cell adhesion molecule (NCAM)-based intercellular adhesion during postnatal maturation is likely to be important for appropriate differentiation of testicular cells. Besides NCAM, P-cadherin is also present in neonatal testicular cords, at least in mice, and seems to disappear from the seminiferous epithelium after the first postnatal week. Another factor known to be important in regulating development of the neonatal testis is thyroid hormone (T3). T3 is involved in control of Sertoli cell proliferation and differentiation. Therefore, we examined the effect(s) of T3 on adhesive factors found within the testis using Sertoli cells and gonocytes isolated from neonates and maintained in coculture. T3 (100 nM) down-regulated NCAM expression in vitro, as assessed by Western blotting and immunofluorescent staining. This contrasted with the continued expression of NCAM in cultures without added T3 but mimicked the disappearance of NCAM from the neonatal rat testis in vivo. In addition, Western analysis confirmed that P-cadherin is highly expressed in the developing rat testes, as it is in those of mice. We found that P-cadherin is strongly expressed in gonocytes and weakly expressed in Sertoli cells. Moreover, unlike NCAM, P-cadherin expression diminishes with time in vitro in the absence of added hormones. In parallel with our observations for NCAM, expression of P-cadherin was also apparently decreased by T3 (100 nM). Subsequent quantitative analyses of cultures exposed to a range of T3 levels (0.1-100 nM) indicated that T3 causes detachment of many gonocytes in a dose- and time-dependent manner (approximately 80% detached at 100 nM). In addition, Western blotting indicated that lower concentrations of T3 down-regulate NCAM but not P-cadherin. From this we conclude that the apparent decrease in P-cadherin induced by 100 nM T3 and detected on Western blots reflects loss of gonocytes. In contrast, even low levels of T3 appear to down-regulate NCAM production before any significant detachment of gonocytes. Finally, low levels of T3 that did not affect numbers of adherent Sertoli cells nevertheless caused detachment of gonocytes. Thus, our observations identify T3 as a regulator of NCAM expression in neonatal testicular cells and as a modifier of gonocyte/Sertoli cell adhesion in vitro.

Expression of 140-kDa neural cell adhesion molecule in developing testes in vivo and in long-term Sertoli cell-gonocyte cocultures.

The basis for cell-cell adhesion in the seminiferous epithelium of the developing testis is doubtless critical in supporting events that are essential for the onset and maintenance of normal spermatogenesis. In this study, we applied immunoblotting and immunolocalization approaches for the following reasons: 1) to ask whether neural cell adhesion molecule (NCAM) underlies cell-cell interactions in vivo, as we previously showed for cells in vitro, 2) to characterize the isoform or isoforms of NCAM expressed during testicular development, and 3) to study NCAM expression in long-term Sertoli cell-gonocyte cocultures and to compare and contrast this pattern of expression with that in vivo. Our findings indicate that NCAM is found ubiquitously at cell-cell interfaces within the seminiferous cord from birth through day 10 and thereafter is restricted to interstitial cells. Moreover, only polysialic acid-negative 140-kDa NCAM is expressed in the testis or in coculture, an isoform whose properties are compatible with the concept of NCAM as both a direct modifier of cell function and an indirect influence on cell responses mediated by other external factors. In addition, we found that germ cells, potentially gonocytes or Type A spermatogonia, persist in long-term cocultures maintained for 15 days after isolation from 5-day-old rat pups and that NCAM continues to be expressed at high levels in these cultures. This observation is in marked contrast to our observation that NCAM gradually decreases and eventually disappears in vivo by postnatal day 15. Thus, our findings indicate that 140-kDa NCAM is prominent in neonatal testes but is down-regulated by as yet unidentified mechanisms thereafter. Our findings also indicate that down-regulation of NCAM fails to occur in hormone- and serum-free Sertoli cell-germ cell cocultures.

Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats.

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 microM, or to 0.5% DMSO (vehicle control), or 10 microM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 microM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 microM MEHP for 24 h of exposure, but not in cultures treated with 0.01 microM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 microM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p < 0.01), while the labeling index was unchanged by treatment with 0.01 microM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 microM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p < 0. 01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 microM DEHP. Therefore, short exposure to low levels of MEHP disrupted adhesion of gonocytes to Sertoli cells and inhibited both basal and FSH-stimulated Sertoli cell proliferation in a dose-dependent manner. The lowest effective dose of MEHP in vitro was 0.1 microM, which is about 10- to 1, 000-fold lower than the dose shown to affect Sertoli cells from prepubertal animals. Moreover, our data indicate that MEHP impairs division of neonatal Sertoli cells by acting at a post-cAMP site in the FSH-response pathway or via a mechanism independent of FSH. These data provide direct new evidence that relatively low levels of MEHP disrupt Sertoli cell-gonocyte physical interactions and suppress Sertoli cell proliferation in neonates via mechanisms specific to neonatal testis where the foundations of adult fertility are established. The results also highlight the neonatal period of testicular development as one particularly sensitive to environmental chemicals.

Use of in vitro systems to study male germ cell development in neonatal rats.

The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below.

Individual quantification of 89Sr and 90Sr in nuclear reactor effluent.

An analytical method utilizing ion chromatography, a non-radioactive strontium carrier, and liquid scintillation spectroscopy to individually quantify 89Sr and 90Sr in nuclear reactor effluent is presented. It is observed that this method is less time consuming than traditional procedures for quantifying radio-strontium, deals comprehensively with separation and subsequent isotopic quantification of strontium, and avoids difficulties reported in previous research. The equipment, solutions and operating conditions for the chromatographic separation of strontium in aqueous solution are identified, and the strontium fraction is shown to elute between 7 and 7.5 min after injection. The beta spectra of 90Sr, 89Sr and 90Y are obtained using liquid scintillation spectroscopy, and the effects of quenching are shown to be negligible. The positions of the liquid scintillation windows within the combined beta spectra facilitating isotopic analysis of 89Sr and 90Sr are identified, followed by the system of equations to quantify 89Sr and 90Sr within a sample. The performance of the method is evaluated using five solutions representing effluent containing radio-strontium at known concentrations. It is observed that when the 89Sr and 90Sr concentrations each are approximately 37 Bq mL(-1) or more, the method over-estimates the 89Sr activity by 15-20% and under-estimates the 90Sr activity by 10-30%, while yielding the total radio-strontium activity to within 1-4% of expected. The lower limit of detection of the system for either 89Sr or 90Sr is shown to be approximately 0.8 Bq mL(-1) of effluent.

Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro.

Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position crucial for their survival. These cells express the c-kit gene from the day of birth through Day 5 in vivo and develop the ability to migrate in Sertoli cell-gonocyte cocultures. In this study, we asked whether c-kit expression and synthesis of Kit protein are required for pseudopod production by gonocytes in vitro. To determine whether gonocyte migration in vitro is invariably accompanied by c-kit expression, we quantified percentages of gonocytes expressing c-kit with increasing time in vitro and correlated these data with pseudopod development by individual cells. We also determined the effect of exposure to Kit antibodies on gonocyte migration in vitro, and, conversely, asked whether addition of exogenous stem cell factor (SCF), the Kit ligand, stimulates pseudopod development. We found that 1) increasing numbers of gonocytes express c-kit with increasing time in vitro; 2) once these cells begin migrating in vitro, the appearance of a pseudopod on a gonocyte is absolutely correlated with kit expression by that cell; 3) incubating cocultures with Kit antibodies significantly reduces the number of cells with pseudopods, without any detectable decrease in numbers of gonocytes; and 4) addition of exogenous SCF to cocultures prepared on Day 5 results in a transient but significant increase in the percentage of gonocytes with pseudopods even though we found that Sertoli cells in the cultures produce endogenous SCF. Thus, our findings provide evidence to support a role for c-kit expression by neonatal gonocytes and, presumably, SCF expression by neonatal Sertoli cells in stimulating migration of these germ cells in vitro.

Hst7: a male sterility mutation perturbing sperm motility, flagellar assembly, and mitochondrial sheath differentiation.

Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.

Gonocytes in testes of neonatal rats express the c-kit gene.

Information gathered from mutant mouse models and from studies on normal puberal and adult animals points to the product of the c-kit gene, a tyrosine kinase surface receptor, and the kit-ligand (KL) as important for gametogenesis in males. In fetuses, KL serves as a survival factor for primordial germ cells, at least in vitro, and in adults activity of the c-kit gene has been indirectly related to survival and subsequent development of differentiating spermatogonia. However, because of the structural complexity of the seminiferous epithelium in adults, c-kit mRNA has not yet been definitively localized to one or more types of spermatogenic cells. In addition, no information is currently available regarding the possible involvement of the c-kit protein and its ligand in mediating germ cell development and/or Sertoli-germ cell interactions immediately after birth when events critical for later onset of spermatogenesis are ongoing. Thus, the aims of the current study were (1) to determine whether the c-kit gene is expressed in testes of neonatal and adult rats and, if so, by what specific cell types, and (2) to determine if those cells expressing the gene also produce the c-kit receptor protein. For this, we isolated total RNA from testes of pups aged days 1-5 and from adult rat testes, and probed for the presence of c-kit mRNA with Northern analysis. We identified the cells containing the c-kit message by carrying out in situ hybridization with digoxigenin-labeled probes, thus allowing the colorimetric signal to be assigned beyond doubt to individual cells in sections of testes. We also utilized Western analysis and immunolocalization to confirm the presence of the c-kit receptor protein in testes at these ages and to identify those cells types producing it. Our findings indicate that (1) neonatal gonocytes express the c-kit gene and produce the receptor protein on postnatal days 1 through 5, spanning the time when they resume dividing and migrating, and (2) spermatogonia and, to a lesser extent, spermatocytes and spermatids of adults express the gene but c-kit protein is present in detectable amounts only in spermatogonia and possibly a few early primary spermatocytes.

NCAM mediates adhesion between gonocytes and Sertoli cells in cocultures from testes of neonatal rats.

During neonatal development of the rat testis, gonocytes resume mitosis and display renewed motility to migrate toward the basal lamina, two events that occur in vitro when these cells are cocultured with Sertoli cells. However, although substantial evidence suggests that development of gonocytes depends on Sertoli cells, little is known of how these cell types interact beyond our previous observations that they communicate via gap junctions and adhere avidly to each other. In the present study, we utilized several approaches to examine the mechanism by which gonocytes adhere to Sertoli cells in vitro. First, we characterized this attachment in general by (1) determining its susceptibility to brief trypsinization in decreasing concentrations of Ca2+, (2) assessing the ability of gonocytes to adhere to Sertoli cells at reduced temperature, and (3) examining the effect of phospholipase C treatment on the number of gonocytes attached to a Sertoli cell monolayer. Because the findings suggested that a non-cadherin mechanism is involved, we used immunofluorescence to identify the presence of neural cell adhesion molecule (NCAM) at virtually all gonocyte-Sertoli cell (and Sertoli cell-Sertoli cell) boundaries and found that incubation of cocultures in the continuous presence of NCAM antibodies caused release of essentially all gonocytes (but not Sertoli cells) from the monolayer. We also found, in (3) above, that gonocyte-Sertoli cell adhesion was very susceptible to phospholipase C in cocultures isolated from newborns and maintained in vitro for 2 hours or 1 day but not in cultures maintained for 3 days. Moreover, cells isolated from pups 5 days old were as resistant to enzyme treatment at 2 hours postplating as were cultures from newborns after 3 days in vitro. Thus, the way in which gonocytes adhere to Sertoli cells appears to change during the immediate postnatal period, as reflected by the observed change in phopholipase sensitivity, perhaps indicating production of a phospholipase C-resistant NCAM isoform by several days after birth. These data constitute new information on the way in which postnatal gonocytes adhere to Sertoli cells and provide a basis for future work in our ongoing exploration of germ cell development in the neonatal rat testis.

Electret ion chamber radon monitors measure dissolved 222Rn in water.

This paper describes a simple and relatively inexpensive method of determining the concentration of dissolved 222Rn in water. The method involves a recently developed electret-passive environmental radon monitor, which uses an electret ion chamber. The procedure consists of sealing a known volume of a carefully collected water sample with one of these monitors in an exposure container and determining the average equilibrium 222Rn gas concentration in the air phase during the exposure time period. This average concentration can then be used to calculate the 222Rn concentration in the original water sample. Identical samples were analyzed both by this new method and by a standard liquid scintillation method, and the results were compared over a wide range of 222Rn concentrations. There was good agreement except that the electret ion chamber method gave results that were consistently lower by about 15%. This bias in the results was attributed to both 222Rn losses during sample handling and possibly to some errors in the assumptions made in the theoretical model. A correction factor is recommended to bring the results of this technique into agreement with the standard method. The procedures are simple and economical and can be easily employed by many primary 222Rn-measuring laboratories currently using these monitors for measuring indoor 222Rn.

Monte Carlo calculation and silicon detector measurement of the hot particle dose.

In our study, we quantitatively compared the hot particle dose calculated by our microcomputer Monte Carlo electron transport code, Eltran3, with the values calculated by the VARSKIN code. For a weightless source, the VARSKIN code overestimated the skin dose by 14% to 37% because the VARSKIN code is based on the data table for point sources in an unbounded medium. Five 60Co hot particles were measured in the current and pulse mode operations of an ion-implanted silicon detector. The measurements were converted to skin dose rates and compared by use of Eltran3 and VARSKIN calculations. Measured values of the skin dose rates for four of the five hot particles agreed well with expected values. The skin dose rate per kBq of the four hot particles ranged from 0.70 to 0.85 mGy h-1 kBq-1. One of the five hot particles showed the presence of a pure beta-emitting radionuclide, which was not detected through gamma-ray measurements.

Radioiodine speciation in the hot cell effluent gases of a radiopharmaceutical production facility.

In order to characterize the various chemical forms of airborne radioiodine species, grab-sampling measurements were conducted at the hot cell laboratory of a radiopharmaceutical production facility, using a selective-adsorbent-iodine filter system. Volatile radioiodine species were produced in the hot cell process which extracted the fission product 99Mo from the irradiated uranium target. The effluent gases were then released through the hot cell filter bank and the main filter bank. Two samplings were made, one at the inlet and one at the outlet of the hot cell filter bank. In comparison with other radioiodine isotopes detected, higher than expected concentrations of 132I were found, primarily in the form of organic iodide--an observation that could be explained by the beta decay of 132Te, the precursor of 132I, in the hot cell waste solution. The relative distribution of airborne 132I was considerably different from that of other iodine radioisotopes. An unexpected component of these distributions was radioiodine penetrating the silver zeolite filter and adsorbed on a triethylenediamine (TEDA) impregnated charcoal filter.

The development and testing of a prototype on-line radioiodine monitor for nuclear power stations.

A prototype on-line monitor has been developed which is capable of detecting radioiodine in the presence of as much as 1 X 10(6) higher concentration of noble gases. The system contains two identical radiation monitoring chambers through which the monitored air and a purging gas alternately cycle. Each chamber contains a silver zeolite filter which has a high retention of the various forms of airborne radioiodine but low retention of noble gases. During the purging cycle the radioactive noble gases are quickly purged from the filter and chamber and the lower levels of radioiodine accumulated on the filter are detected. This system has been successfully tested using short-lived radionuclides simulating vented reactor gases resulting from an abnormal condition.

Evaluation of radiation monitor effectiveness for the detection of 85Kr.

As part of the preparations for the purging of TMI Unit-2, the 85Kr sensitivity of 12 radiation detector systems or system combinations was determined. Eleven of these were evaluated using a cube-shaped polyethylene-walled room containing a volume of 5.6 m3 (200 ft3). Krypton-85 gas was added to produce a concentration of 6.7 x 10(-6) muCi/ml in the test room. It was found that none of the ion chambers and scintillation detector systems were able to detect this concentration of 85Kr. Detectors employing thin window GM pancake probes were found to be sensitive enough to monitor this gas down to the unrestrictive area maximum permissible concentration level (MPC) of 3 x 10(-5) muCi/ml, while a large window gas flow proportional counter was found to be sensitive enough to monitor down to about 09.1 MPC. At the end of this experiment, 2.3 m3 (80 ft3) of the gas in the test room was pumped into a compressed air cylinder (scuba bottle) and was used to calibrate The Pennsylvania State University (PSU) Noble Gas Monitor. The sensitivity of this system, which employs gas compression and Ge(Li) spectroscopy, was demonstrated to be between 0.1 and 0.03 times MPC, depending on the counting time employed.