Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.

Cell- and stage-specific high-level expression of TBP-related factor 2 (TRF2) during mouse spermatogenesis.

Mice lacking the TBP-related factor 2 (TRF2) gene, which is highly expressed in the testis, have a severe defect in spermiogenesis. Here we show that the expression of TRF2 is both cell type- and stage-specific. TRF2 expression was first detected in the late pachytene spermatocytes at stage VIII and increased throughout the subsequent stages. After meiotic divisions, the TRF2 expression declined continuously in round spermatids during progression from stage I to stage V. This observation is consistent with an essential regulatory role of TRF2 in male germ cell differentiation during spermatogenesis.

Spermiogenesis deficiency in mice lacking the Trf2 gene.

The discovery of TATA-binding protein-related factors (TRFs) has suggested alternative mechanisms for gene-specific transcriptional regulation and raised interest in their biological functions. In contrast to recent observations of an embryonic lethal phenotype for TRF2 inactivation in Caenorhabditis elegans and Xenopus laevis, we found that Trf2-deficient mice are viable. However, Trf2-/- mice are sterile because of a severe defect in spermiogenesis. Postmeiotic round spermatids advance at most to step 7 of differentiation but fail to progress to the elongated form, and gene-specific transcription deficiencies were identified. We speculate that mammals may have evolved more specialized TRF2 functions in the testis that involve transcriptional regulation of genes essential for spermiogenesis.

Obesity regulates bioavailable testosterone levels in women with or without polycystic ovary syndrome.

OBJECTIVE: To evaluate [1] the effects of levels of sex hormone-binding globulin (SHBG), albumin, and total testosterone on the distribution of testosterone between SHBG-bound and non-SHBG-bound fractions; [2] the independent effects of polycystic ovary syndrome (PCOS) and body mass index on serum levels of total testosterone, non-SHBG-bound testosterone, SHBG, and albumin; and [3] the usefulness of levels of total testosterone and non-SHBG-bound testosterone and of the free androgen index in the diagnosis of PCOS. DESIGN: Retrospective clinical study. SETTING: An academic research environment. PATIENT(S): Forty-three women with oligomenorrhea and PCOS. Twenty-five women with regular menstrual cycles and without hirsutism served as controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Levels of non-SHBG-bound testosterone, total testosterone, SHBG, and albumin in serum. RESULT(S): Levels of total testosterone and non-SHBG-bound testosterone, and the free androgen index were higher in patients with PCOS than in healthy controls. PCOS did not have an effect on the levels of SHBG or albumin, or on the percentage of non-SHBG-bound testosterone. Levels of SHBG and albumin were inversely related to body mass index. The percentage and concentration of non-SHBG-bound testosterone and the free androgen index were directly related to body mass index. Hirsutism did not have an effect on any outcome measure. CONCLUSION(S): The distribution of total testosterone into SHBG-bound and non-SHBG-bound fractions is associated with body mass index, not with PCOS. The high levels of non-SHBG-bound testosterone and the high free androgen index in patients with PCOS reflect mainly high levels of total testosterone. Thus, the measurement of levels of non-SHBG-bound testosterone and the calculation of the free androgen index provide no further information in the diagnosis of PCOS beyond that provided by the measurement of levels of total testosterone.

FGF-8 is expressed during specific phases of rodent oocyte and spermatogonium development.

We have studied the localization of the expression of FGF-8 mRNA in adult and developing rat and mouse gonads by in situ hybridization. The expression of FGF-8 mRNA was high in oocytes of small and large antral follicles of adult mouse ovaries. No signal was observed in fetal ovaries, or in primordial and atretic follicles of adult ovary. In mouse testis, the FGF-8 mRNA signal could be demonstrated in prespermatogonia during a short period covering the fetal days 15 to 17, but not any more on day 19 of fetal life, or in adult testis. The time course of the expression of FGF-8 mRNA in mouse testis was confirmed by RT-PCR reaction. Corresponding in situ results were obtained by studying rat tissues. The observed germ cell-specific expression of FGF-8 mRNA in maturing oocytes and fetal prespermatogonia suggests that FGF-8, which is a secretory protein, has a paracrine function during the specific phases of the maturation of the follicle and fetal seminiferous epithelium.

Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium.

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII-I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P < 0.01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I-IV were threefold higher than in stages VI-VII (P < 0.01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

Haploid gene expression: temporal onset and storage patterns of 13 novel transcripts during rat and mouse spermiogenesis.

The temporal and spatial expression of thirteen novel spermatid-specific genes corresponding to cDNA clones isolated from an adult mouse testis library was analyzed. Northern analysis of RNA from seminiferous tubules at defined stages of the rat and mouse seminiferous epithelial cycle and in situ hybridization of testis sections revealed that these mRNAs were expressed in a stage-specific manner. The expression of all mRNAs was first detected in early round spermatids, and it increased to abundance during stages VII-VIII of the epithelial cycle. Twelve out of thirteen mRNAs were found not only in round spermatids but also in transcriptionally inactive elongated spermatids, suggesting that they are stored and regulated at the translational level. The variation in the length of the poly(A) tail was detected for four of the transcripts, represented by cDNA clones MTEST70, MTEST627, MTEST641, and MTEST643 at defined stages of the cycle. Similarity in the stage-specific expression pattern displayed by this group of haploid-specific genes strongly suggests the presence of common regulatory mechanisms that act during spermiogenesis, and these genes also provide a means for further studies of these mechanisms.

Follicle-stimulating hormone regulates the expression of cyclic protein-2/cathepsin L messenger ribonucleic acid in rat Sertoli cells in a stage-specific manner.

Cyclic protein-2/cathepsin L (CP-2) is secreted by Sertoli cells in a highly stage-specific manner, maximally during stages VI-VII of the rat seminiferous epithelial cycle. We investigated FSH regulation of CP-2 mRNA expression of its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA expression and its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA levels in stages IX-I, whereas in stages II-VIII, the levels of CP-2 mRNA were reduced. A similar effect was produced by two cAMP analogs, dbcAMP (0.2 mM) and Sp cAMP (20 microM). FSH and cAMP did not affect on the levels of SGP-2 mRNA during the seminiferous epithelial cycle. The magnitude of the response was time- and dose-dependent; the maximum was obtained with 100 ng/ml of FSH. It is likely that FSH regulates Cp-2 gene transcription, since de novo RNA synthesis was required for the stimulatory FSH effect on CP-2 mRNA levels, while ongoing protein synthesis was not. In conclusion, the data suggest that FSH, via cAMP-mediated pathway, regulates CP-2/cathepsin L gene transcription in rat Sertoli cells and modulated the stage-specific expression pattern.

Expression of immediate early genes in tubular cells of rat testis.

In this study we investigated the expression of the immediate early genes (IEGs) c-fos, c-jun, and junD on mRNA and protein levels during the spermatogenic cycle of the rat using Northern blotting, in situ hybridization, and immunocytochemistry. The expression of these IEG mRNAs and proteins exhibited stage-specific variations. The results suggest that IEGs take part in transcriptional events that are involved in regulating the proliferation and differentiation of spermatogenic cells during specific stages of the cycle of the seminiferous epithelium.

Expression of the mad gene during cell differentiation in vivo and its inhibition of cell growth in vitro.

Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.

Germ cell-Sertoli cell interactions: regulation by germ cells of the stage-specific expression of CP-2/cathepsin L mRNA by Sertoli cells.

CP-2/cathepsin L mRNA is expressed primarily by rat Sertoli cells within stage VI-VIII seminiferous tubules. To test whether germ cells regulated this expression, we examined if separating Sertoli cells from specific germ cells affected expression of this transcript in Sertoli cells. First, Sertoli cells were isolated from adult (90-day-old) and immature (25-day-old) rats and levels of this transcript measured immediately or after 1, 3 and 5 days in culture. Results demonstrated that immediately upon isolation, CP-2/cathepsin L mRNA levels were significantly higher in mature cells. However, after 1 day in culture, the levels of this transcript increased in immature cells and remained high in mature cells. We therefore conclude that in vivo, a subset of germ cells inhibit the expression of CP-2/cathepsin L mRNA by immature Sertoli cells. Second, to examine the effect of specific germ cells on CP-2/cathepsin L mRNA expression, we exposed the testes of mature rats to 3 Gy of gamma-radiation and analyzed stage-specific expression of this transcript at varying times during maturation depletion and subsequent germ cell restoration. Loss of spermatogonia or spermatocytes was without effect. However, when pachytene spermatocytes through step 14 spermatids were depleted, expression at stages VI-VIII was reduced by half and expression at stages IX-I was increased 14-fold. These changes resulted in the loss of stage-specific expression of CP-2/cathepsin L mRNA by Sertoli cells. Finally, stage VI-VIII tubules, depleted primarily in step 15-19 spermatids, had levels of CP-2/cathepsin L mRNA that were 60% of control. However, stage-specific expression of this transcript was detected in these tubules. In contrast to what we noted with CP-2/cathepsin L mRNA, loss and restoration of germ cells had no effect on Sertoli cell levels of SGP-2 mRNA, indicating that testicular irradiation had no overall effect on Sertoli cell function. Taken together, these data suggest that the stage-specific expression of the CP-2/cathepsin L gene results from the sequential stimulation and inhibition of Sertoli cells by germ cells, that pachytene spermatocytes through step 14 spermatids are required for this stage-specific expression and that step 18 and 19 spermatids amplify this expression at stages VI-VIII.

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules.

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.

Expression of inhibin alpha, beta A and beta B messenger ribonucleic acids in the normal human ovary and in polycystic ovarian syndrome.

We studied the cellular distribution of inhibin alpha, beta A and beta B mRNAs in the normal human ovary and in polycystic ovarian syndrome (PCOS) by in situ hybridization. Our results show that human granulosa cells express inhibin alpha, beta A and beta B subunit mRNAs, and theca cells express inhibin alpha and beta A subunit mRNAs. The co-localization of alpha and beta A mRNAs in theca cells supports the hypothesis that inhibin also has an autocrine function in these cells. We did not detect any inhibin subunit mRNA in the granulosa cells of atretic follicles, while theca cells also expressed alpha subunit mRNA in those follicles. The present findings suggest that the expression of inhibin subunits is regulated differently in human follicular granulosa and theca cells. It has been speculated that inhibin may be involved in the development of PCOS. Our results show that the cellular localization of inhibin subunit mRNAs is not disturbed in PCOS ovaries.

Localization of urokinase- and tissue-type plasminogen activator mRNAs in rat testes.

The expressions of urokinase (uPA) and tissue-type plasminogen activators (tPA) in different stages of the rat seminiferous epithelial cycle were analyzed by in situ and Northern hybridizations combined with zymographic analysis. Irradiated rat testes were used to assess the cell localization. Both of the plasminogen activators were expressed in a strictly stage specific manner. Maximal expression of uPA mRNA was seen in Sertoli cells during stages VII-VIII of the cycle. The same expression in the basal compartment of the tubules was detected at 7 days post-irradiation (p-i), during a selective reduction of spermatogonia and preleptotene spermatocytes. Levels of tPA mRNA started to accumulate in Sertoli cells at stage VIII and were high during stages IX-XII and detectable during stages XIII-XIV. At 26 days p-i, reduction of pachytene spermatocytes, which are shown to be immunoreactive for tPA, did not have an effect on tPA mRNA expression. Catalytic activities of uPA and tPA changed concomitantly to their RNA levels in different stages of the cycle. However, at 7 days p-i, uPA activity was decreased at stages VII-VIII of the cycle suggesting that germ cell Sertoli cell interaction is important for uPA activity.

Follicle-stimulating hormone regulation of inhibin alpha-subunit mRNA in staged rat seminiferous tubules.

Steady-state levels of inhibin-alpha and -beta B mRNAs are higher in stages II-VI of the seminiferous epithelial cycle than in stages VII-VIII. We investigated follicle-stimulating hormone (FSH) regulation of inhibin alpha-subunit mRNA in stages II-VI and VII-VIII to study whether the stage specificity is due to differential hormonal regulation by FSH. Follicle-stimulating hormone caused a significant increase of inhibin-alpha mRNA levels in both stages during a 20-h incubation. The mechanism of the FSH effect was studied further in stages VII-VIII. Maximal stimulation of the inhibin-alpha mRNA level was achieved with 100 micrograms/l FSH, dibutyryl-3',5'-cyclic adenosine monophosphate (db-cAMP, 0.2 mmol/l) and Sp-adenosine-3',5'-monophosphothionate (Sp-cAMPS, 10 mumol/l) (a cAMP agonist). The presence of Rp-cAMPS (200 mumol/l) (a cAMP antagonist) abolished the stimulation, Rp-cAMPS alone had no effect. Inhibin-beta B mRNA levels in stages VII-VIII were not affected by FSH, db-cAMP, Sp-cAMPS or Rp-cAMPS. Phorbol 12-myristate 13-acetate (100 nmol/l) had no effect on inhibin-alpha or -beta B mRNA levels. Actinomycin D abolished the stimulatory effect of FSH on inhibin-alpha mRNA expression. In conclusion, FSH stimulated inhibin-alpha mRNA expression similarly both in stages II-VI and VII-VIII of the seminiferous epithelial cycle and the stimulation in stages VII-VIII was cAMP-mediated.

Interleukin-1 bioactivity and DNA synthesis in X-irradiated rat testes.

Stage-specific DNA synthesis and interleukin-1 (IL-1) bioactivity were measured in the rat testis at 2, 6, 12 and 25 days after local irradiation with 3 Gy to investigate whether there was any correlation between germ cell DNA synthesis during repopulation and IL-1-like bioactivity in the seminiferous epithelium. DNA synthesis by intermediate and type-B spermatogonia was reduced significantly at 2, 6 and 12 days after irradiation in seminiferous tubules at stages II-III and IV-V. IL-1 bioactivity was increased significantly at 6 and 25 days after irradiation at stages II-VI during repopulation of spermatogonia. At 2, 6 and 25 days after irradiation at stages VIIa-c a significant increase in IL-1 bioactivity was observed that correlated with repair synthesis of DNA. Increased IL-1 bioactivity was also observed at stages IX-XII at 6 days post-irradiation. These observations support the concept that IL-1 is a spermatogonial growth factor which might also stimulate repair synthesis of DNA.

Expression of inhibin beta A and beta B, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium.

The expression of inhibin beta A and beta B subunits, follistatin, and activin-A receptor messenger RNA (mRNAs) in different stages of rat seminiferous epithelial cycle was analyzed by in situ hybridization in order to understand their role in the regulation of spermatogenesis. Inhibin beta A mRNA was expressed in Sertoli cells in a highly stage-specific manner. The mRNA levels started to accumulate in Sertoli cells at stage VIII of the cycle and were highly expressed during stages IX-XI. Follistatin mRNA expression was identical to that of inhibin beta A, while inhibin beta B mRNA was maximally expressed in Sertoli cells at stages XIII-III. Low expression was found in stages VII-VIII. Activin-A receptor mRNA was localized mainly in spermatogenic cells. Maximal expression was seen in late primary spermatocytes at stages XIII-XIV and in early round spermatids at stages I-IV. A low even expression by Sertoli cells was also seen. Inhibin beta A and follistatin mRNAs were coexpressed in stage IX-XI Sertoli cells, suggesting close interplay between these molecules. The pattern of inhibin beta B mRNA expression was similar to that of inhibin alpha-mRNA. Localization of activin-A receptor mRNA in spermatogenic cells suggests that activin may influence meiotic divisions and early spermiogenesis.

FSH receptor mRNA is expressed stage-dependently during rat spermatogenesis.

In situ hybridization was performed on testicular tissue from adult male Sprague-Dawley rats using cRNA antisense and sense probe of the monkey FSH receptor (FSHR) cDNA to determine the cellular site of synthesis, and possible stage-dependent expression of FSHR mRNA during the cycle of the seminiferous epithelium. Using antisense probe specific binding was first detected in Sertoli cells just prior to sperm release at stage VIII. The strongest specific hybridization signal was found during stages IX and X followed by a decrease of signal intensity in stages XI-XII. No specific binding was found in stages XIII-VII. The sequence of events with the maximum expression of FSHR mRNA in Sertoli cells in stages IX and X, before FSH-binding and FSH-stimulated cAMP production reach maximum values, coincides with a new wave of spermatogenesis and indicates an effect of FSH and spermatogenic cells on the regulation of FSHR mRNA expression.

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium.

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.