Single-cell ATAC and RNA sequencing reveal pre-existing and persistent cells associated with prostate cancer relapse.

Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.

Postconditioning and remote postconditioning of ischemic rat cardiac grafts.

BACKGROUND: Global warm ischemia of transplanted cardiac grafts is associated with apoptosis and subsequent tissue necrosis. It is suggested that postconditioning (PostC) may ameliorate the early outcome of cardiac grafts after ischemia-reperfusion. We investigated whether PostC and remote postconditioning (RPostC) have a histopathologic impact after transplantation of warm ischemic rat cardiac grafts. MATERIALS AND METHODS: Thirteen rats were randomized to undergo either PostC or RPostC after heterotopic transplantation of ischemic cardiac grafts. Six rats without intervention besides transplantation served as controls (control). The recipient rats were sacrificed 24 h after transplantation to evaluate histopathology and apoptotic index. RESULTS: No significant differences were observed between the study groups in terms of graft heat shock protein 70 and oxygen radical absorbing capacity, an indicator of antioxidant capacity. While apoptosis and PCARB, a marker of protein oxidation and oxidative stress, decreased after RPostC (p < 0.05), contraction band necrosis was less prominent in both PostC and RPostC. CONCLUSION: Both PostC and RPostC have a histopathologic impact after 24 h of reperfusion of warm ischemic rat cardiac grafts.

Altered levels of Smad2 and Smad4 are associated with human prostate carcinogenesis.

Alterations have been demonstrated in ligand and cognate receptor system of the transforming growth factor beta (TGF-beta) pathway in prostate cancer (PC). Still, little is known about changes in the activity of the intracellular Smad cascade of TGF-beta signaling during prostate carcinogenesis. We used immunohistochemistry to analyze phosphorylated Smad2 (p-Smad2), nuclear Smad4 and inhibitory-Smad7 in epithelial cells of normal, hyperplastic and malignant prostate. Specimens comprised 49 tissue cores of PC, 10 benign prostate hypertrophies and three normal prostates. Nuclear p-Smad2 (P<0.001) and nuclear Smad4 (P=0.023) were significantly decreased in PC with remarkable variations in cytoplasmic Smad7 levels. Substantial decreases in p-Smad2 and Smad4 levels were found in specimens with primary Gleason grades 3 and 4, whereas in grade 5, levels were markedly higher. Our results provide the first evidence for changes and reversible attenuation in the Smad system of the TGF-beta pathway during prostate carcinogenesis.

Calcidiol and prostate cancer.

Epidemiological studies suggest that serum calcidiol (25(OH)-Vitamin D3) seems to be associated with several cancers including prostate cancer. We have made several experimental studies in order to clarify the mechanism(s) involved in the association. Calcidiol has been regarded as an inactive prohormone for calcitriol, which possesses the highest biological activity of the Vitamin D metabolites, when it is evaluated on the basis of bioactivity/nmol. However, we found recently that at the physiological concentration calcidiol (100-200 nM) is an active hormone, whereas calcitriol (1alpha,25(OH)2-Vitamin D3) (100 pM) is inactive in human primary prostate stromal cells. Calcidiol is able to inhibit cell growth and to induce or inhibit several genes including 1alpha-hydroxylase and 24-hydroxylase genes. This suggests that calcidiol might be an independent endocrine system involved in the control of cell differentiation and proliferation, whereas calcitriol might be mainly involved in the regulation of calcium and phosphorous balance. Several mechanisms may mediate the action of Vitamin D in the prostate. This is a review of some recent studies on the role of (1) Vitamin D metabolism, (2) growth factors and (3) fatty acid metabolism.

Distribution of progesterone receptor in chicken: novel target organs for progesterone and estrogen action.

Expression of progesterone receptor (PR) in various organs of sexually immature chickens and after estrogen treatment was studied by immunohistochemical and Western blotting analyses. Constitutive PR expression was observed in the mesothelium and stroma of the esophagus, proventriculus, liver, spleen, pancreas, heart and lung. In the urogenital tract, PR was expressed in the mesothelial and stromal cells and smooth muscle of blood vessels. Estrogen treatment induced PR expression in the stroma and smooth muscle of the gall bladder and in the epithelium and stroma of the trachea. In the ovary of immature chickens PR was localized in the epithelium, stroma and smooth muscle and was induced in the granulosal cells by estrogen. In most tissues there was more PR-B than PR-A expression and this PR-B dominance remained after estrogen treatment. These results suggest that progesterone and estrogen may have physiological effects on many organs outside the genital tract not previously known as steroid-target tissues.

Progesterone receptor in chicken bursa of Fabricius and thymus: evidence for expression in B-lymphocytes.

In the present work constitutive progesterone receptor (PR) expression in the chicken bursa of Fabricius was detected in the stromal, smooth muscle and follicular medullary cells and smooth muscle cells of blood vessels. PR expression was increased during sexual maturation and after estrogen treatment. Bursal medullary PR-positive cells were further characterized to be B-lymphocytes by flow cytometric analysis. In addition, estrogen induced expression of PR in the bursal FAE-cells (follicle-associated epithelial cells). In the thymus PR was expressed constitutively in the connective tissue cells of the capsule and interfollicular septa, in a few medullary cells and in vascular smooth muscle. The PR-positive medullary cells consisted of epithelial cells, large polygonal cells resembling macrophages and plasma cells. T-lymphocytes were PR-negative. Estrogen up-regulated PR expression in the thymus. Immunoblotting studies revealed that both isoforms of PR, i.e. PR-A and PR-B, were expressed in the bursa of Fabricius and thymus with PR-B dominance. These results suggest that the chicken primary lymphoid organs bursa and thymus are under regulation of estrogen and progesterone. Expression of PR in B-lymphocytes, macrophages and plasma cells in the chicken is documented for the first time and suggests evidence for direct action of progesterone on immune responses.

Heat shock protein 90 and the nuclear transport of progesterone receptor.

Steroid receptors exist as large oligomeric complexes in hypotonic cell extracts. In the present work, we studied the nuclear transport of the 2 major components of the oligomeric complex, the receptor itself and the heat shock protein 90 (Hsp90), by using different in vitro transport systems: digitonin permeabilized cells and purified nuclei. We demonstrate that the stabilized oligomeric complex of progesterone receptor (PR) cannot be transported into the nucleus and that unliganded PR salt dissociated from Hsp90 is transported into the nucleus. When nonstabilized PR oligomer was introduced into the nuclear transport system, the complex dissociated and the PR but not the Hsp90 was transported into the nucleus. If PR exists as an oligomeric form after synthesis, as suggested by the experiments with reticulocyte lysate, the present results suggest that the complex is short-lived and is dissociated before or during nuclear transport. Thus, the role of Hsp90 in PR action is likely to reside in the Hsp90-assisted chaperoning process of PR preceding nuclear transport of the receptor.

Progesterone receptor and hsp90 are not complexed in intact nuclei.

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.

The role of Vitamin D3 metabolism in prostate cancer.

Vitamin D deficiency increases risk of prostate cancer. According to our recent results, the key Vitamin D hormone involved in the regulation of cell proliferation in prostate is 25(OH) Vitamin D3. It is mainly acting directly through the Vitamin D receptor (VDR), but partially also through its 1alpha-hydroxylation in the prostate. A deficiency of 25(OH) Vitamin D is common especially during the winter season in the Northern and Southern latitudes due to an insufficient sun exposure, but Vitamin D deficient diet may partially contribute to it. A lack of Vitamin D action may also be due to an altered metabolism or Vitamin D resistance. Vitamin D resistance might be brought up by several mechanisms: Firstly, an increased 24-hydroxylation may increase the inactivation of hormonal Vitamin D metabolites resulting in a Vitamin D resistance. This is obvious in the cancers in which an oncogenic amplification of 24-hydroxykase gene takes place, although an amplification of this gene in prostate cancer has not yet been described. During the aging, the activity of 24-hydroxylase increases, whereas 1alpha-hydroxylation decreases. Furthermore, it is possible that a high serum concentration of 25(OH)D3 could induce 24-hydroxylase expression in prostate. Secondly, Vitamin D receptor gene polymorphism or defects may result in a partial or complete Vitamin D resistance. Thirdly, an overexpression or hyperphosphorylation of retinoblastoma protein may result in an inefficient mitotic control by Vitamin D. Fourthly, endogenous steroids (reviewed by [D.M. Peehl, D. Feldman, Interaction of nuclear receptor ligands with the Vitamin D signaling pathway in prostate cancer, J. Steroid Biochem. Mol. Biol. (2004)]) and phytoestrogens may modulate the expression of Vitamin D metabolizing enzymes. In summary, the local metabolism of hormonal Vitamin D seems to play an important role in the development and progression of prostate cancer.

Localization of 1,25-dihydroxyvitamin D3 receptor (VDR) expression in human prostate.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been found to have a variety of physiological functions, including effects on growth and differentiation in normal and malignant cells. The antiproliferative effects of 1,25(OH)2D3 are reported to be mediated through the genomic signaling pathway by binding to a specific high affinity receptor protein, the 1,25-dihydroxyvitamin D3 receptor (VDR). VDR has been localized in a variety of tissues, but little is known about VDR distribution in human prostate. In this study, we raised an antibody against a synthetic peptide corresponding to amino acids 10-24 of human vitamin D receptor. The sequence selected for immunization is identical in human, rat and mouse VDR. Based on this antibody, we developed an immunohistochemical method suitable for studying VDR expression in paraffin-embedded tissue. The immunohistochemical staining was verified using classical target organs for vitamin D (kidney, intestine, skin). With this method, we studied VDR localization on paraffin-embedded human prostatic tissue obtained from 8 patients undergoing radical prostatectomy for urinary bladder cancer and demonstrate VDR expression in the secretory epithelial and few stromal cells of human prostate. The nuclear staining in the secretory epithelial cells was concentrated near the nuclear membrane and in discrete foci in the nucleoplasm. This suggests that effects of 1,25-dihydroxyvitamin D3 are mediated through VDR in these cells. Moreover our result indicates that there are strong variations in VDR expression between prostatic samples.

Hormone-dependent changes in A and B forms of progesterone receptor.

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied. These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.

Vitamin D and prostate cancer.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

Evidence for enhanced ubiquitin-mediated proteolysis of the chicken progesterone receptor by progesterone.

Genomic actions of progesterone are mediated via A and B isoforms of the progesterone receptor (PR). One major factor controlling PR level is progesterone causing negative autoregulation (down-regulation) of the receptor protein. In this work we studied the mechanism whereby progesterone exerts its effects on PR level in the chicken oviduct. We found that progesterone does not markedly regulate PR mRNA expression. Furthermore, we demonstrate here for the first time that PR is a target for ubiquitylation and that the proportion of ubiquitylated PR is increased by progesterone treatment. Our data suggest that ligand-induced down-regulation of PR involves enhanced degradation of receptor protein by ubiquitin-proteasome system in vivo.

Mechanisms of action of sex steroid hormones: basic concepts and clinical correlations.

The review deals with the clinically important aspects of the basic mechanisms of sex steroid hormones. Steroids can act through two basic mechanisms: genomic and non-genomic. The classical genomic action is mediated by specific intracellular receptors, whereas the primary target for the non-genomic one is the cell membrane. Many clinical symptoms seem to be mediated through the non-genomic route. Furthermore, membrane effects of steroid and other factors can interfere with the intranuclear receptor system inducing or repressing steroid-and receptor-specific genomic effects. These signalling pathways may lead to unexpected hormonal or anti-hormonal effects in patients treated with certain drugs. Steroid receptors (SRs) are members of a large family of nuclear transcription factors that regulate gene expression by binding to their cognate steroid ligands, to the specific enhancer sequences of DNA (steroid response elements) and to the basic transcription machinery. SRs are phosphoproteins, which are further phosphorylated after ligand binding. The role of phosphorylation in receptor transaction is complex and may not be uniform to all SRs. However, phosphorylation/dephosphorylation is believed to be a key event regulating the transcriptional activity of steroid receptors. SR activities can be affected by the amount of SR in the cell nuclei, which is modified by the rate of transcription and translation of the SR gene as well as by proteolysis of the SR protein. There is an auto- and heteroregulation of receptor levels. Some of the SRs appear to bind specific protease inhibitors and exhibit protease activity. The physiological significance of this weak proteolytic activity is not clear. Some SRs are expressed as two or more isoforms, which may have different effects on transcription. Receptor isoforms are different translation or transcription products of a single gene. Isoform A of the progesterone receptor is a truncated form of PR isoform B originating from the same gene, but it is able to suppress not only the gene enhancing activity of PR-B but also that of other steroid receptors. From the clinical point of view, it is important to note that the final hormonal effect in a target tissue is dependent on the cross talk between different nuclear steroid receptors and on expression of receptor isoforms.

Progesterone receptor does not form oligomeric (8S), non-DNA-binding complex in intact cell nuclei.

We raised a polyclonal antibody, alpha D, against a synthetic peptide (amino acids 522-535) of chicken progesterone receptor (PR). The sequence is located between the DNA-binding domain and the hormone-binding domain in the region within the sequences required for stability of the oligomeric form of PR. In the immunoblot, alpha D reacted with both A and B forms of PR. In the sucrose gradient and dot-blot the antibody did not recognize the so-called 8S form of PR, which is an oligomeric complex of PR and other proteins. When the oligomeric complex was dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 4S form. To study whether a similar complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, freeze-drying-vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear staining was detected independent of the fixation technique. When receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (8S). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an increased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These results suggest that the oligomeric complex (8S), present in tissue extracts, does not exist in intact cell nuclei. They also call into question the proposed role of hsp90 in regulating progesterone receptor function.

Expression of the chicken progesterone receptor forms A and B is differentially regulated by estrogen in vivo.

The chicken progesterone receptor (cPR), like its human counterpart (hPR), exists as two isoforms, PR-A and PR-B, displaying different biological activities depending upon cellular and promoter contexts. Here we show that the ratio of PR isoforms observed in the immature chicken oviduct is changed during estrogen-induced differentiation from PR-B dominancy to that of PR-A. This is the first report describing that the expression ratio of PR isoforms is altered by upregulation of PR-A by estrogen action in vivo. This result provides a plausible explanation to the differences in oviduct's response to progesterone depending on hormonal and developmental status of the animal.

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.

Immunochemical and in situ hybridization analyses of retinoic acid receptor alpha, beta, and gamma in murine Harderian and submandibular glands.

Retinoic acid (RA), through its cognate receptors (retinoic acid receptors, RARs), plays an important role in the ontogenesis and maintenance of the normal function of murine Harderian and submandibular glands. In the present study, autoradiography was used to study RA binding to these glands. Both glands showed high radioactive labelling after [14C]-RA administration in normal and partially vitamin A-deficient (VAD) mice. The peak uptake was at 6 h after [14C]-RA administration in normal mice and at 0.5 h in VAD mice. At 24 h, RA binding remained high in normal mice, while it decreased significantly in VAD mice. In western blots with an antibody recognizing all forms of RARs, a band of molecular weight 51 kDa was seen in homogenates of both glands. Immunohistochemically, RAR staining was found in the nuclei of the glandular cells. The Harderian gland exhibited more intense staining than the submandibular gland. In the latter, the most intense staining was seen in the acinar cells, followed by the intercalated duct cells. The granular convoluted tubule showed weak immunostaining and the striated duct was negative. In the Harderian gland, RAR immunostaining was observed in both type I and II cells, but only part of them stained with RAR antibody. The expression of RAR alpha, beta, and gamma transcripts was studied by in situ hybridization using specific oligonucleotide probes. The cell-specific expression of RAR alpha mRNA in the submandibular gland corresponded to the RAR proteins detected by immunohistochemistry, while the RAR beta transcript was mainly seen in the striated duct. The transcripts of RAR alpha and beta were evenly distributed in type I and II glandular cells of the Harderian gland. RAR gamma labelling was below detectable levels in both glands. This result suggests that RA and RARs regulate the functions of Harderian and submandibular glands in a cell-specific manner.

Androgen receptor in rat Harderian and submandibular glands.

Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.

Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells.

Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis.