Activin B and Activin C Have Opposing Effects on Prostate Cancer Progression and Cell Growth.

Current prognostic and diagnostic tests for prostate cancer are not able to accurately distinguish between aggressive and latent cancer. Members of the transforming growth factor-ß (TGFB) family are known to be important in regulating prostate cell growth and some have been shown to be dysregulated in prostate cancer. Therefore, the aims of this study were to examine expression of TGFB family members in primary prostate tumour tissue and the phenotypic effect of activins on prostate cell growth. Tissue cores of prostate adenocarcinoma and normal prostate were immuno-stained and protein expression was compared between samples with different Gleason grades. The effect of exogenous treatment with, or overexpression of, activins on prostate cell line growth and migration was examined. Activin B expression was increased in cores containing higher Gleason patterns and overexpression of activin B inhibited growth of PNT1A cells but increased growth and migration of the metastatic PC3 cells compared to empty vector controls. In contrast, activin C expression decreased in higher Gleason grades and overexpression increased growth of PNT1A cells and decreased growth of PC3 cells. In conclusion, increased activin B and decreased activin C expression is associated with increasing prostate tumor grade and therefore have potential as prognostic markers of aggressive prostate cancer.

Role of activin C in normal ovaries and granulosa cell tumours of mice and humans.

Activins and inhibins play important roles in the development, growth and function of the ovary. Mice lacking inhibin develop granulosa cell tumours in their ovaries that secrete activin A, and these tumours are modulated by increased activin C expression. The aim of the present study was to identify where activin C is expressed in mouse and human ovaries and whether overexpression of activin C modulates normal follicular development in mice. Immunohistochemical staining for the activin ßC subunit was performed on sections from mouse and human ovaries and human adult granulosa cell tumours. Stereology techniques were used to quantify oocyte and follicular diameters, and the percentage of different follicular types in ovaries from wild-type mice and those underexpressing inhibin α and/or overexpressing activin C. Staining for activin ßC was observed in the oocytes, granulosa cells, thecal cells and surface epithelium of mouse and human ovaries, and in the granulosa-like cells of adult granulosa cell tumours. Overexpression of activin C in mice did not alter follicular development compared with wild-type mice, but it did modulate the development of abnormal early stage follicles in inhibin α-null mice. These results provide further evidence of a role for activin C in the ovary.

Regional localization of activin-ßA, activin-ßC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

Activins and inhibins, members of the TGF-ß superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-ßA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-ß superfamily, the activin-ßC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (ßC-ßC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.

Over-Expression of Activin-ßC Is Associated with Murine and Human Prostate Disease.

Activins are members of the TGF-ß superfamily and have been linked to prostate cancer. There are four mammalian activin subunits (ßA, ßB, ßC, and ßE) that dimerize to form functional proteins. The role of activin-A (ßA-ßA) has been relatively well characterized and has been shown to generally inhibit growth in the prostate. In contrast, little is known about the biological function of the ßC and ßE subunits. Previous work indicated activin-C (ßC-ßC) to be an antagonist of activin-A. This is important because resistance to activin-A growth inhibition occurs during prostate cancer progression. This paradox is not currently well understood. Hence, we hypothesize that local expression of the activin-ßC subunit antagonizes activin-A-dependent growth inhibition and represents a key factor contributing to acquired insensitivity to activin-A observed in prostate cancer progression. To test our hypothesis, we characterized the ventral prostate lobes of 9-month-old transgenic mice over-expressing activin-ßC and examined the expression of activin-ßA, activin-ßC, and the activin intracellular signaling factor, Smad-2, in human prostate diseases. Prostate epithelial cell hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN) lesions, alterations in cell proliferation, and reduced Smad-2 nuclear localization were evident in mice over-expressing activin-ßC. Increased activin-ßA and -ßC subunit immunoreactive scores and decreased Smad-2 nuclear localization were also evident in human prostate cancer. This study suggests that over-expression of activin-ßC is associated with murine and human prostate pathologies. We conclude that the activin-ßC subunit may have therapeutic and/or diagnostic implications in human prostate disease.

Activin A regulates microRNAs and gene expression in LNCaP cells.

BACKGROUND: Prostate cancer (PCa) is an increasing health issue worldwide. For patients with advanced castration-resistant PCa (CRPC) treatment options are limited and overall survival is relatively short. Paired with this, non-invasive diagnostic options are yet to be established. Activins are members of the TGF-ß superfamily and have been linked to prostate physiology. For instance, activin A is an inhibitor of growth in the prostate. A novel class of non-coding RNA, microRNAs (miRNAs) have been intrinsically linked to a range of cellular processes and carcinogenesis. No studies have investigated the impact of activin A on miRNA expression in PCa cell lines. Hence, the objective of this study was to determine the effect of activin A on miRNA expression and downstream target genes in PCa. METHODS: Activin-sensitive (LNCaP) and insensitive (PC3) prostate cells were treated with 50 ng/ml of activin A for 72 hr. To examine miRNA expression following treatment, SYBR RT-qPCR miRNA arrays were used in conjunction with TaqMan RT-qPCR. MiRPath-TarBase analysis was conducted using the miRNAs that were significantly altered following activin A treatment of LNCaP cells to highlight enriched target genes within biological pathways. Highlighted target genes were assessed using pathway-focused TGF-ß and cell cycle SYBR RT-qPCR arrays. RESULTS: Activin A treatment altered nine miRNAs in LNCaP cells: miR-222-3p, miR-15b-5p, miR-93-5p, miR-18a-5p, and let-7i-5p were significantly decreased, while miR-30a/30d-5p, let-7c, and miR-196b-5p were significantly increased versus media control. In PC3 cells five miRNAs were altered: miR-130a-3p, miR-7-5p, and miR-140-3p were significantly decreased while miR-191-5p and miR-26a-5p were significantly increased versus media control. MiRPath-TarBase analysis highlighted that the miRNAs significantly altered in LNCaP cells targeted genes contained in activin A-related KEGG pathways. Furthermore, when LNCaP cells were treated with activin A the expression of the targeted genes was the inverse of the expression of activin A-mediated miRNAs. CONCLUSIONS: This study demonstrated the ability of activin A to modulate miRNA expression in PCa cell lines and suggests a correlative relationship between miRNA expression and downstream target genes in LNCaP cells. This study provides impetus for further studies into activin A and miRNAs in PCa. Prostate 76:951-963, 2016. © 2016 Wiley Periodicals, Inc.

Uric acid: a modulator of prostate cells and activin sensitivity.

Elevated serum uric acid (SUA) or urate is associated with inflammation and gout. Recent evidence has linked urate to cancers, but little is known about urate effects in prostate cancer. Activins are inflammatory cytokines and negative growth regulators in the prostate. A hallmark of prostate cancer progression is activin insensitivity; however, mechanisms underlying this are unclear. We propose that elevated SUA is associated with prostate cancer counteracting the growth inhibitory effects of activins. The expression of activins A and B, urate transporter GLUT9 and tissue urate levels were examined in human prostate disease. Intracellular and secreted urate and GLUT9 expression were assessed in human prostate cancer cell lines. Furthermore, the effects of urate and probenecid, a known urate transport inhibitor, were determined in combination with activin A. Activin A expression was increased in low-grade prostate cancer, whereas activin B expression was reduced in high-grade prostate cancer. Intracellular urate levels decreased in all prostate pathologies, while GLUT9 expression decreased in benign prostatic hyperplasia, prostatitis and high-grade prostate cancer. Activin responsive LNCaP cells had higher intracellular and lower secreted urate levels than activin-insensitive PC3 cells. GLUT9 expression in prostate cancer cells was progressively lower than in prostate epithelial cells. Elevated extracellular urate was growth promoting in vitro, which was abolished by the gout medication probenecid, and it antagonized the growth inhibitory effects of activins. This study shows for the first time that a change in plasma or intracellular urate levels, possibly involving GLUT9 and a urate efflux transporter, has an impact on prostate cancer cell growth, and that lowering SUA levels in prostate cancer is likely to be therapeutically beneficial.

Activin-ßC modulates cachexia by repressing the ubiquitin-proteasome and autophagic degradation pathways.

BACKGROUND: Cancer-associated cachexia and muscle wasting are considered key determinants of cancer-related death and reduction in the quality of life of cancer patients. A crucial link has been established between activin signaling and skeletal muscle atrophy-hypertrophy. We previously showed that activin-ßC, a novel activin-A antagonist, is a tumor modulator that abolishes the cancer-associated cachexia in a mouse genetic model of gonadal tumorigenesis, in which the normal balance of inhibin/activin signalling is disrupted by a targeted mutation in the Inha gene (inhibin α-KO mouse). This study aimed to identify the molecular mechanism by which activin-ßC increases survival and abolishes cancer-associated cachexia in α-KO mice. We hypothesized that overexpression of activin-ßC modulates the cachexia phenotype by antagonizing the activin signaling pathway and repressing muscle wasting via the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways. METHODS: Male and female ActC++, α-KO, and α-KO/ActC++ mice and WT littermate controls were studied. Western blot analysis for the specific E3 ubiquitin ligases, atrogin-1 and MuRF1, markers of the autophagic-lysosomal pathway, Beclin-1, p62, and LC3A/B, effectors Smad-2, Smad-3 and myostatin was performed in the gastrocnemius of age-matched mice. Histopathology of the gastrocnemius and survival analysis were also conducted in animals from the same breeding cohort. Serum levels of activin-A, inflammatory cytokines, hormonal profile, and bone density were also assessed. RESULTS: Increased levels of atrogin-1, MuRF-1, Beclin-1, p62, LC3A/B-I, Smad-2 and serum levels of activin-A were noted in the α-KO mice. These mice developed gonadal cancers followed by severe weight loss, and reduced survival. Overexpression of activin- ßC antagonized the activin signaling cascade, attenuating the ubiquitin-proteasome and the autophagic-lysosomal degradation pathways, and reduced serum levels of activin-A. α-KO/ActC++ mice displayed a less aggressive cachectic phenotype, reduced tumor weight, and prolonged survival. CONCLUSION: Our findings show for the first time a specific effect of activin-ßC on muscle wasting and transcription factors involved in muscle protein degradation. The study indicates that activin-ßC may be a novel therapy to abrogate cancer-associated weight loss and prolong survival.

Activins and activin antagonists in the human ovary and ovarian cancer.

Activins are members of the transforming growth factor ß superfamily that play an important role in controlling cell proliferation and differentiation in many organs including the ovary. It is essential that activin signalling be tightly regulated as imbalances can lead to uncontrolled cell proliferation and cancer. This review describes the expression and function of the activins and their known antagonists in both normal and cancerous human ovaries.

Re-evaluating the role of activin-ßC in cancer biology.

Transforming growth factor-ß (TGF-ß) superfamily signaling pathway and its ligands are essential regulators of cellular processes such as proliferation, differentiation, migration, and survival. Alteration of this pathway results in uncontrolled proliferation and cancer progression. This review focuses on a specific member of the TGF-ß superfamily: activin-ßC. After its initial discovery, activin-ßC has been considered non-biologically relevant. Therefore, for years several experimental designs have ignored the potential contribution of this molecule to the final biological outcome. Here we focus on recent advances in the activin field, with a particular emphasis on activin-ßC, its antagonistic mechanism, and the physiological relevance of activin-ßC actions in reproductive and cancer biology. Covering a novel and previously unexplored function of activin-ßC on cancer associated weight loss and muscle metabolism, this review suggests an imminent need to re-evaluate the function of activin-ßC in biological systems and advances the understanding of how activin-ßC antagonizes the activin signaling pathway. Thus, challenging activin biologists to consider the impact of activin-ßC when interpreting their work.

Activin-ßC modulates gonadal, but not adrenal tumorigenesis in the inhibin deficient mice.

Activins and inhibins are involved in the regulation of several biological processes, including reproduction, development and fertility. Deregulation of the inhibin/activin signaling pathway has been implicated in the progression of reproductive and adrenal cancers. Deletion of the inhibin α-subunit results in up-regulation of the circulating levels of activins and this leads to the development of sex-cord stromal tumors followed by a cancer associated-cachexia in mice. When gonadectomy is performed, development of adrenocortical carcinomas is observed. We previously showed that overexpression of activin-ßC modulates the development of sex-cord stromal tumors and reduces cancer-cachexia in the inhibin-deficient mice by antagonizing the activin signaling pathway. The adrenal cortex and gonads share in common a large subset of genes, consistent with their common embryonic lineage. Additionally, it has been shown that adrenocortical carcinomas adopt an altered cellular identity resembling the ovary. Therefore, a study to assess the impact of overexpression of activin-ßC on the onset of adrenocortical carcinoma in gonadectomized inhibin-deficient mice was warranted. Within the current study we evaluated markers of apoptosis, proliferation, tumor burden, survival analysis and serum levels of activin-A in gonadectomized mice versus sham operated controls. Results showed that overexpression of activin-ßC modulated the development of reproductive tumors but had no effect on adrenal tumorigenesis. Our data reinforces the importance of activin-ßC in reproductive biology and suggest that activin-ßC is a tumor modulator with gonadal specificity.

The inhibin/activin signalling pathway in human gonadal and adrenal cancers.

The biological function of the inhibin-α subunit (INHA) in gonadal tumorigenesis is different in humans compared with mouse. The INHA subunit is up-regulated in most human ovarian and testicular cancers but knock-out studies in mice showed the INHA subunit is a tumour suppressor with gonadal and adrenal specificity. The INHA subunit is a component of the inhibin/activin signalling pathway, which includes activin receptors ActRIIA/IIB and intracellular Smads-2/3. To resolve the incongruity in function in humans versus mouse, we re-evaluated the inhibin/activin pathway in human gonadal and adrenal cancers using contemporary protein and mRNA expression data for multiple pathway components rather than INHA alone. We used an INHA antibody raised against the N-terminal domain to compare immunoreactivity with the more commonly used antibody raised against the C-terminal domain. This study also described, for the first time, a comprehensive protein expression profile of activin-ßC in reproductive and adrenal cancers, and its effect on a human granulosa cell line, providing evidence for a role in ovarian, testis and adrenal tumour biology. Our data show reduced INHA expression at both protein and mRNA levels, and increased activin signalling in human testicular, ovarian and malignant versus benign forms of adrenal cancer. We also found that activin-C acts as an activin-A antagonist by binding to activin receptor subunits IIA and IIB and modulating the canonical Smad pathway. In conclusion, analysis of the inhibin/activin signalling pathway helps to explain discrepancies arising from studies of only one hormone or subunit and suggests that altered expression of the inhibin and activin subunits is associated with reproductive and adrenal cancer biology.

microRNA and non-canonical TGF-ß signalling: implications for prostate cancer therapy.

The incidence of prostate cancer is increasing worldwide and marks a significant health issue. Paired with this, current therapeutic options for advanced prostate cancer, notably androgen deprivation therapy (ADT), fail to provide a consistent level of efficacy throughout the treatment period, highlighting the need for new robust therapies. Growth factors, such as Transforming Growth Factor-beta (TGF-ß), possess the ability to impede cancer development in the early stages, via alterations in either apoptosis, cell proliferation, or the promotion of cellular senescence. However, later in the pathogenesis, advanced prostate cancer cells become insensitive to the previously beneficial effects of TGF-ß. The molecular mechanisms behind this acquired insensitivity are not well understood. Thus, the aim of this review is to examine the effects of a class of small non-coding RNA, microRNA (miRNA), on TGF-ß signalling. The impact of miRNA on the canonical TGF-ß Smad signalling pathway has been well investigated, hence, in this review, we will examine whether miRNA targeting members of non-canonical TGF-ß signalling members, such as, Erk, RhoA, PI3K/Akt and JNK/p38 could provide alternate therapeutic options for advanced prostate cancer.

The therapeutic potential of blocking the activin signalling pathway.

Members of the transforming growth factor ß (TGF-ß) family regulate fundamental physiological process, such as cell growth, differentiation and apoptosis. As a result, defects in this pathway have been linked to uncontrolled proliferation and cancer progression. Here we explore the signal transduction mechanism of TGF-ß focusing on therapeutic intervention in human diseases. Like TGF-ß, another member of the TGF-ß superfamily, activin has been proven to play an important role in maintenance of tissue homeostasis and dysregulation leads to disease. Several studies showed elevated levels of activin are responsible for the development of gonadal tumours and a cachexia-like weight loss syndrome. Discussing the recent advances in approaches developed to antagonise the activin pathway and the encouraging results obtained in animal models, this review presents a therapeutic rationale for targeting the activin pathway in conditions such as cachexia, neuromuscular and/or musculoskeletal disorders.

Activin-ß(c) reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin-deficient mice.

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α-subunit and by an activin-binding protein, follistatin. The activin-ß(C) subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin-ß(C) forms heterodimers with activin-ß(A) and activin-C antagonizes activin-A in vitro. Thus, it is proposed that overexpression, rather than loss of activin-ß(C) , regulates activin-A bioactivity. In order to prove biological efficacy, inhibin α-subunit knockout mice (α-KO) were crossed with mice overexpressing activin-ß(C) (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin-A, and cancer-associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α-KO/ActC++ mice, where excessive activin-A is the underlying cause. Accordingly, a reduction in activin-A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α-KO/ActC++ mice. Overexpression of activin-ß(C) antagonized the activin signalling cascade; thus, the tumourigenic effects of activin-A were abrogated. This study provides proof of the biological relevance of activin-ß(C) . Being a regulator of activin-A, it is able to abolish cachexia and modulate reproductive tumour development in α-KO mice.

Insensitivity to the growth inhibitory effects of activin A: an acquired capability in prostate cancer progression.

Prostate cancer (PCa), the most common non-skin cancer in men, is a worldwide health concern. Treatment options for aggressive PCa are limited to androgen deprivation therapies (ADT), which are ineffective, with robust diagnostic options also being limited. The prostate specific antigen (PSA) test, for instance, is subject to high levels of false positive results and cannot distinguish between cancer confined to the prostate and aggressive metastatic cancer. As such, additional therapeutic and diagnostic options are urgently required. In recent years, a clear association between activins and prostate cancer has become evident. Activins are members of the TGF-ß superfamily and are responsible for a plethora of physiological processes, including cell proliferation, apoptosis, immune surveillance, embryonic development, and follicle stimulating hormone (FSH) regulation. Activin A normally inhibits cancer development and progression, however, cancer cell growth in high-grade PCa is not inhibited by this protein. The mechanism for this apparent acquired capability to resist activin A-mediated growth inhibition is currently not well understood. Thus, the aim of this review is to analyse the role of activin A in PCa progression and to present mechanisms by which transformed cells may escape its effects. The overarching hypothesis is that insensitivity to the growth inhibitory effects of activin A is an acquired capability in PCa progression. Therefore, local and genetic elements that may be responsible for this change in cellular sensitivity to activin A during cancer progression will be highlighted with a view to identifying potential diagnostic or therapeutic targets.

Activins and activin antagonists in the prostate and prostate cancer.

Activins are members of the TGF-ß super-family. There are 4 mammalian activin subunits (ß(A), ß(B), ß(C) and ß(E)) that combine to form functional proteins. The role of activin A (ß(A)ß(A)) is well characterized and known to be a potent growth and differentiation factor. Two of the activin subunits (ß(C) and ß(E)) were discovered more recently and little is known about their biological functions. In this review the evidence that activin-ß(C) is a significant regulator of activin A bioactivity is presented and discussed. It is concluded that activin-ß(C), like other antagonists of activin A, is an important growth regulator in prostate health and disease.

Vinclozolin exposure in utero induces postpubertal prostatitis and reduces sperm production via a reversible hormone-regulated mechanism.

Vinclozolin is an endocrine-disrupting chemical (EDC) that binds with high affinity to the androgen receptor (AR) and blocks the action of gonadal hormones on male reproductive organs. An alternative mechanism of action of Vinclozolin involves transgenerational effects on the male reproductive tract. We previously reported in utero Vinclozolin exposure-induced prostatitis (prostate inflammation) in postpubertal rats concurrent with down-regulation of AR and increased nuclear factor-kappaB activation. We postulated the male reproductive abnormalities induced by in utero Vinclozolin exposure could be reversed by testosterone supplementation, in contrast to the permanent modifications involving DNA methyltransferases (Dnmts) described by others. To test this hypothesis, we administered high-dose testosterone at puberty to Vinclozolin-treated rats and determined the effect on anogenital distance (AGD); testicular germ cell apoptosis, concentration of elongated spermatids, and the onset of prostatitis. Concurrently we examined Dnmt1, -3A, -3B, and -3L mRNA expression. Consistent with previous reports, in utero exposure to Vinclozolin significantly reduced AGD, increased testicular germ cell apoptosis 3-fold, reduced elongated spermatid number by 40%, and induced postpubertal prostatitis in 100% of exposed males. Administration of high-dose testosterone (25 mg/kg) at puberty normalized AGD, reduced germ cell apoptosis, and restored elongated spermatid number. Testosterone restored AR and nuclear factor-kappaB expression in the prostate and abolished Vinclozolin-induced prostatitis. Altered Dnmt expression was evident with in utero Vinclozolin exposure and was not normalized after testosterone treatment. These data demonstrate in utero Vinclozolin-induced male reproductive tract abnormalities are AR mediated and reversible and involve a mechanism independent of Dnmt expression.

Activin C antagonizes activin A in vitro and overexpression leads to pathologies in vivo.

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-beta(C) subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-beta(C). Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LbetaT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-beta promoter. Transgenic mice that overexpress activin-betaC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-beta(C) antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-betaC immunoreactivity. This study provides evidence that activin-beta(C) is an antagonist of activin A and supplies an impetus to examine its role in development and disease.

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice.

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.

Rat activin-betaE mRNA expression during development and in acute and chronic liver injury.

Activin-betaE mRNA expression was investigated in male and female rats using gel-based and quantitative RT-PCR, in fetal and post-natal liver during development and in a variety of tissues from 200 gm adult animals. Activin-betaE expression was also assessed in rat liver after partial hepatectomy, and after repeated toxic insult. Male Sprague Dawley rats were subjected to partial hepatectomy or sham operations. Samples were collected from the caudate liver lobe during regeneration, from 12 to 240 hr after surgery. Three groups of 5 male rats were treated with CCl(4) for 0 (control), 5 or 10 weeks, to induce liver fibrosis and cirrhosis. Activin-betaE mRNA was predominantly expressed in liver, with much lower amounts of mRNA observed in pituitary, adrenal gland and spleen, in both males and females. Low activin-betaE expression was observed in liver at fetal day 16, with higher levels seen between post-natal days 3 and 35 and a further increase noted by day 47, in both males and females. Liver activin-betaE mRNA concentrations did not change from control values 12-72 hr after PHx, but significantly increased over six fold, 168 hr post-hepatectomy, when liver mass was restored. Activin-betaE mRNA was up-regulated after 5 weeks of CCl(4) treatment, but not after 10 weeks. The changes in activin-betaE mRNA concentrations after liver insult did not always parallel those reported for the activin-betaC subunit, suggesting activin-betaE may have an independent role in liver under certain conditions.