Demonstration of neuropeptide Y and its precursor in plasma and follicular fluid.

The present investigation provides three lines of evidence for the presence of a pro-form of neuropeptide Y (NPY) in plasma and follicular fluid. First, by the demonstration of NPY-immunoreactive material of a size corresponding to the estimated mol wt of pro-NPY. Second, an antiserum specific for the C-terminal tyrosine amide of NPY and peptide YY does not react with this material. Third, it was possible to convert the pro-NPY extracted from plasma and follicular fluid using the protease, Endoproteinase-Lys C, to a NPY-immunoreactive form eluting slightly before NPY on a G-50 column. The size of the digested product was consistent with a cleavage of pro-NPY resulting in an immunoreactive species, NPY-Gly-Lys. Pro-NPY was also found in tissue culture media from the human neuroendocrine cell line SH-SY5Y. As in the case of plasma and follicular fluid, another NPY immunoreactive species eluted from a G-50 gel filtration column slightly before synthetic human NPY. Analysis of this material with an antibody directed against the tyrosine amide of NPY in combination with isoelectric focusing revealed that this peak consisted of at least two immunoreactive forms of NPY. In conclusion, at least three different forms of NPY immunoreactivity are likely to be present in plasma, follicular fluid, and cell tissue culture media; pro-NPY, a degradation form of pro-NPY, or a biosynthetic intermediate and NPY.

Mosquito oostatic factor: a novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut.

A peptide that inhibits egg development in mosquitoes (oostatic factor) has been purified from the ovaries of female Aedes aegypti. The factor is a decapeptide with a molecular mass of 1047.6. The primary sequence has been determined as NH2-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-COOH from mass spectra recorded on a quadrupole Fourier transform instrument. The amino acid sequence exhibits sequence correlation to mammalian, plant, and several viral proteins. Injection of synthetic analogs into mosquitoes, biting midges, flies, and fleas inhibited proteolytic enzyme biosynthesis in the midgut. Binding studies with [3H]oostatic factor indicated that the midgut epithelial cells have a factor-specific receptor.

The immunocytochemical localization of angiotensinogen in the rat ovary.

The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary.

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 1 crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31/32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.

Subunits of cyclic adenosine 3',5'-monophosphate-dependent protein kinase show differential and distinct expression patterns during germ cell differentiation: alternative polyadenylation in germ cells gives rise to unique smaller-sized mRNA species.

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases: collagenase inhibitors in human preovulatory ovaries.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.

Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary.

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.

Identification of, and development of radioimmunoassays for 17 alpha, 21-dihydroxy-4-pregnene-3,20-dione and 3 alpha,17 alpha, 21-trihydroxy-5 beta-pregnan-20-one in the ovaries of mature plaice (Pleuronectes platessa).

Ovaries from a female plaice (Pleuronectes platessa) that had been injected with human chorionic gonadotrophin were incubated in vitro with 17 alpha-hydroxy[1,2,6,7-3H]progesterone. The major steroids produced by the ovaries were tentatively identified as 17 alpha,21-dihydroxy-4-pregnene-3,20di-one (11-deoxycortisol; 17,21-P), 17 alpha,21-dihydroxy-5 beta-pregnane-3,20-dione (3 alpha, 17,21-P-5 beta). A high proportion of these steroids was found in a conjugated form (sulphates or glucuronides). Radioimmunoassays were developed for 11-deoxycortisol and for 3 alpha,17,21-P-5 beta and were applied to fractions of mature male and female plaice plasmas and plaice ovarian incubates that had been separated on thin-layer chromatography. The presence of all three steroids, in vivo and in vitro, was confirmed. Particularly high amounts of conjugated 3 alpha,17,21-P-5 beta were found in the plasma of mature females (200-400 ng ml-1). The 3 alpha,17,21-P-5 beta radioimmunoassay also identified 3 alpha,17 alpha-dihydroxy-5 beta-pregnane-20-dione in all three fluids, despite the fact that this steroid was not among the radioactive incubation products of the ovary. These findings are compared with those from another flatfish, the dab (Limanda limanda), where the major gonadal steroids have been shown to be 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and its 5 beta-pregnane (3-keto and 3 beta-hydroxyl) metabolites.

Isolation and characterization of rabbit ovarian surface epithelium, granulosa cells, and peritoneal mesothelium in primary culture.

Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplemented D-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17 beta-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3 beta hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.

Inhibitors of lipoxygenase increase the ovulation rate in the in-vitro perfused luteinizing hormone-stimulated rabbit ovary.

In order to determine whether leukotrienes, products of the lipoxygenase pathway, are involved in ovulation, pairs of rabbit ovaries were treated with the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) while being perfused in vitro. The control ovaries from each rabbit received luteinizing hormone (LH) (1.5-2.25 micrograms ml-1) while the contralateral ovaries were treated with LH + NDGA (100 microM) or LH + CA (100 microM). The numbers of ovulations from both the LH + NDGA- and LH + CA-treated ovaries were significantly higher (P less than 0.05) than from their respective LH-stimulated controls. Treatment with NDGA alone in the perfusate did not cause any ovulation, while CA alone caused one ovulation from one of six ovaries perfused. Ovarian tissue levels of prostaglandins after 7 h of perfusion with LH + NDGA or with LH alone showed that, in five of the six ovaries perfused in this group, the tissue levels of PGE2, 6-keto-PGF1 alpha and PGF2 alpha were higher in the presence of NDGA. The mean differences were significant (P less than 0.05) for prostacyclin but not significant (P greater than 0.05) for PGE2 and PGF2 alpha. Our interpretation of the findings is that, when used for blocking the lipoxygenase pathway, NDGA and CA increase the substrate availability for the cyclo-oxygenase pathway of arachidonic acid metabolism, resulting in a net increase in prostaglandins. The increased ovarian levels of prostaglandins, especially prostacyclin, may cause the observed increase in ovulation rate. Consequently, although the leukotrienes may be involved in the mechanism of ovulation in the rabbit, their effects appear to be less pronounced than those of prostaglandins.

High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody.

An antibody against basic fibroblasts growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30-33 and 46 kDa in immunoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.

Changes in rat ovarian carbonyl reductase activity and content during the estrous cycle, and localization.

Carbonyl reductase activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The enzyme activity increased after the preovulatory surge of luteinizing hormone (LH) on proestrus, and the enzyme content began to increase prior to the LH surge. Although the enzyme content reached the highest level at 2000 h and remained at a plateau for 8 h, the enzyme activity increased linearly until it reached the highest level at 0800 h on the morning of estrus. At their maximum, enzyme activity and content were approximately 1.5-fold and 2-fold greater, respectively, then basal diestrus values. The enzyme protein amounted to 1-4% of the ovarian cytosolic protein. An immunohistochemical study revealed that the enzyme was primarily localized in interstitial gland cells and theca interna cells of secondary and Graafian follicles as well as atretic follicles.

Heat protectors and heat-induced preferential redistribution of 26 and 70 kDa proteins in Chinese hamster ovary cells.

An overall increase of 40% in nuclear-associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear-associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43 degrees C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1-0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6-1.8. The normal levels of these two proteins were restored when cells were incubated at 37 degrees C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector-treated cells, suggesting that "repair" of heat-induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new protein synthesis.

Heterogeneity of rat ovarian lactogen receptor species.

Three distinct ovarian lactogen receptor species with unique and highly reproducible HPLC retention times gave corresponding peaks of binding activity and migrated as single bands of 80, 40, and 34 kDa on SDS-PAGE. Reduction of the 80 kDa protein failed to reveal any conversion to the lower molecular weight proteins by either SDS-PAGE analysis or reverse phase HPLC, suggesting that the three binding proteins are not related by disulfide bond formation. Immunological studies indicate an amino acid homology at a C terminal region among the 3 receptor forms and with the rat liver receptor. However, the 80 kDa ovarian receptor also contains a unique sequence derived from microsequencing that is not immunologically apparent in the ovarian lower molecular weight forms of the rat liver receptor. These findings substantiate the existence of at least two populations of ovarian lactogen receptors perhaps originating within the same gene, a high Mr form potentially capable of signal transduction, and truncated forms that could be involved in transport and/or clearance.

[Comparative study on human chorionic gonadotropin receptor in normal human ovary with ovarian tumors].

Human Chorionic Gonadotropin (HCG) is major physiological luteotropic factors for the human corpus luteum. The observations strongly suggest that the human ovary possesses a gonadotropin receptor in the cell membrane. We studied the HCG receptor in normal human ovary and ovarian tumors. Twenty-three human ovarian specimens and 16 ovarian tumor specimens were obtained from women patients having gynecological surgery. Ovaries were homogenized and sonicated. The homogenates were centrifuged at 2000 g for 15 min. After sucrose density gradient ultracentrifugation (78,000 g, 4 h), two fractions were collected from layer of 33% and interface between 33% and 37%. Thirty micrograms of ovarian protein, 8 ng 125I-HCG and unlabeled HCG in a final volume of 0.5 ml of 0.05 mol/L Tris buffer were incubated at 30 degrees C for 2 h. The results were shown in the table.

Detection of sex-specific proteins in chick embryo gonads and mesonephros: effects of estradiol benzoate or tamoxifen on their expression.

Gonadal and mesonephric protein patterns from 19 day old normal chick embryos were investigated by two-dimensional polyacrylamide gel electrophoresis. Under these conditions, several sex-specific polypeptides were detected. As concerns gonadal extracts, four sex-specific polypeptides, all restricted to the cytosol, were present in the testis, whereas three sex-specific polypeptides, two localized in the cytosol, the other being membrane-bound, were identified in the ovary. Among the ovary-specific polypeptides two proved to be estrogen-dependent. They appeared in the left testis of embryos after early estradiol benzoate treatment and their expression was reduced in the ovary after early exposure to the antiestrogen, tamoxifen. Mesonephros extracts of both sexes also differed in their protein composition since three additional polypeptides (one in both the cytosolic and membrane fractions, the others in the cytosol) not found in females were found to be present in males. None appeared to be affected after either estradiol or tamoxifen treatment.

Hormonal correlates of sexual behavior and ovulation in male-induced and postpartum estrus in female prairie voles.

The purpose of the present study was a description of hormonal profiles in female prairie voles (Microtus ochrogaster) in estrus that was induced by male exposure versus postpartum estrus. Hormonal profiles are reported in sexually naive females and in sexually experienced females, as a function of varying amounts of coital stimulation and as a function of time since male exposure. Ovarian estradiol levels, uterine weights and uterine protein levels increased in virgin females after exposure to a male, were highest in females that showed lordosis, declined slowly when estrous females were isolated from males and decreased sharply following mating. Ovarian progesterone levels increased more rapidly following mating in females in male-induced estrus than in females in postpartum estrus. Serum progesterone levels did not increase significantly within 24 hr following mating, but were elevated by 72 hr after mating. These findings are discussed as they relate to the hormonal control of female sexual behavior.

Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram.

Significant inter- and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5-bromodeoxyuridine (BrdUrd)-labeled cells with an anti-BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re-examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti-BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti-Brd-Urd measurements in the face of routine (and sometimes necessary) experimental variations.