Testicular Germ Cell Proliferation Research Articles

MOTILIPERM Ameliorates Immobilization Stress-Induced Testicular Dysfunction via Inhibition of Oxidative Stress and Modulation of the Nrf2/HO-1 Pathway in SD Rats.

It is well established that physiological stress has an adverse effect on the male reproductive system. Experimental studies have demonstrated the promising effects of MOTILIPERM in male infertility. MOTILIPERM extract is composed of three crude medicinal herbs: Morinda officinalis How (Rubiaceae) roots, Allium cepa L. (Liliaceae) outer scales, and Cuscuta chinensis Lamark (convolvulaceae) seeds. The present study aimed to investigate the possible mechanisms responsible for the effects of MOTILIPERM on testicular dysfunction induced by immobilization stress. Fifty male Sprague Dawley rats were divided into five groups (10 rats each): a normal control group (CTR), a control group administered MOTILIPERM 200 mg/kg (M 200), an immobilization-induced stress control group (S), an immobilization-induced stress group administered MOTILIPERM 100 mg/kg (S + M 100), and MOTILIPERM 200 mg/kg (S + M 200). Stressed rats (n = 30) were subjected to stress by immobilization for 6 h by placing them in a Perspex restraint cage, while controls (n = 20) were maintained without disturbance. Rats were administrated 100 or 200 mg/kg MOTILIPERM once daily for 30 days 1 h prior to immobilization. At the end of the treatment period, we measured body and reproductive organ weight; sperm parameters; histopathological damage; reproductive hormone levels; steroidogenic acute regulatory protein (StAR); biomarkers of oxidative stress; and apoptosis markers. MOTILIPERM treatment improved testicular dysfunction by up-regulating (p < 0.05) sperm count, sperm motility, serum testosterone level, StAR protein level, Johnsen score, and spermatogenic cell density in stressed rats. MOTILIPERM decreased oxidative stress by increasing (p < 0.05) testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione peroxidase-4 (GPx 4), catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) levels and decreasing (p < 0.05) malondialdehyde (MDA) and reactive oxygen species/reactive nitrogen species (ROS/RNS) levels. Furthermore, MOTILIPERM down-regulated (p < 0.05) cleaved caspase 3 and BCL2 associated X protein (Bax) levels; increased pro caspase-3 and B-cell lymphoma 2 (Bcl-2) levels; and upregulated testicular germ cell proliferation in stressed rats. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels also significantly (p < 0.05) decreased after pretreatment with MOTILIPERM in stressed rats. Collectively, our results suggest that, in immobilization-mediated stress-induced testicular dysfunction, MOTILIPERM sustains normal spermatogenesis via antioxidant and anti-apoptotic activities by activating the NRF/HO-1 signaling pathway.

Insulin-like growth factors 1 and 2 are associated with testicular germ cell proliferation and apoptosis during fish reproduction.

To support sperm production, fish testes undergo intense tissue remodelling, with endocrine, paracrine and autocrine signals regulating gonad physiology. The aim of this study was to investigate the testicular expression of insulin-like growth factor (Igf) 1 and Igf2 during spermatogenesis, and their relationship with cell proliferation and apoptosis throughout the reproductive cycle. The study was performed in male Hypostomus garmani, a catfish living in headwater rivers of the São Francisco River basin, Brazil. Spermatogenesis was analysed using histology, morphometry, immunohistochemistry and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) analysis at different maturity stages. The results showed the proliferation of spermatogonia throughout the reproductive cycle, with a higher rate during the ripe stage. Germ and Sertoli cells expressed Igf1 at all stages of testicular maturity, Igf2 was predominant at the ripe stage and both Igf1 and Igf2 occurred at the spent stage. Caspase-3 and TUNEL analysis revealed a higher rate of apoptosis at the spent stage associated with reduced expression of Igf1 and Igf2. Sertoli cell proliferation was associated with spermatogonia and spermatocyte cysts at different stages of the reproductive cycle. Together, the data support a proliferative role for Igf1 and Igf2 in regulating testicular apoptosis in H. garmani, with cyclical variation in their expression during gonad maturation.

Malaysian Bee Bread Attenuates Apoptosis and Improves Cell Proliferation in Testis of High-Fat Diet-Induced Obese Rats

Introduction: Obesity has been reported to impair male reproductive function and testicular apoptosis while bee bread has been traditionally consumed to enhance male fertility.Objective: The objective of this study was to determine the effects of Malaysian bee bread on apoptosis and proliferation of testicular germ cells in high-fat diet-induced obese rats.Methods: Twenty-four adult male Sprague Dawley rats weighing between 250-300 g were randomised into four groups (n=6/group), namely normal control (NC), high-fat diet (HFD), HFD plus bee bread (HFD+B) and HFD plus an anti-obesity drug orlistat (HFD+O) groups. Bee bread (0.5g/kg/day) and orlistat (10 mg/kg/day) were suspended in distilled water and given by oral gavage for 12 weeks. Markers for apoptosis and proliferation of testicular germ cells were assessed.Results: mRNA transcript levels of caspase-3, caspase-8, caspase-9, p53 and Bax/Bcl2 ratio in the testis of HFD group were significantly increased while immunohistochemical staining of cleaved caspase-3 increased and proliferating cell nuclear antigen (PCNA) immunoexpress ions decreased relative to NC group. Treatment with bee bread significantly decreased the apoptotic markers, significantly decreased mRNA transcript levels of anti-apoptotic markers (Bcl2) and increased PCNA immunoexpression in HFD+B, relative to NC group and HFD+O.Conclusion: Bee bread improved proliferation of testicular germ cells by attenuating apoptosis in high- fat diet-induced obese male rats.International Journal of Human and Health Sciences Supplementary Issue: 2019 Page: 44

Oxidative Stress, NF-κB-Mediated Inflammation and Apoptosis in the Testes of Streptozotocin-Induced Diabetic Rats: Combined Protective Effects of Malaysian Propolis and Metformin.

Oxidative stress, inflammation and apoptosis are major complications that trigger organ failure in diabetes mellitus (DM), and are proven to adversely affect the male reproductive system. Clinical and experimental studies have demonstrated the promising protective effects of propolis in DM and its associated systemic effects. Herein, we investigated the effect of Malaysian propolis (MP) on testicular oxidative stress, inflammation and apoptosis in diabetic rats. Further, the possibility of a complementary effect of MP with the anti-hyperglycaemic agent, metformin (Met), was studied with the idea of recommending its use in the event that Met alone is unable to contain the negative effects of DM on the male reproductive system in mind. Male Sprague-Dawley rats were either gavaged distilled water (normoglycaemic control and diabetic control groups), MP (diabetic rats on MP), Met (diabetic rats on Met) or MP+Met (diabetic rats on MP+Met), for 4 weeks. MP decreased oxidative stress by up-regulating (p < 0.05) testicular mRNA levels of nuclear factor erythroid 2-related factor 2, superoxide dismutase, catalase and glutathione peroxidase; increasing (p < 0.05) the activities of antioxidant enzymes; and decreasing (p < 0.05) lipid peroxidation in the testes and epididymis of diabetic rats. Further, MP down-regulated (p < 0.05) testicular mRNA and protein levels of pro-inflammatory mediators (nuclear factor kappa B, inducible nitric oxide synthase, tumour necrosis factor-α and interleukin (IL)-1β), decreased (p < 0.05) the nitric oxide level, and increased (p < 0.05) IL-10 mRNA and protein levels. MP also down-regulated (p < 0.05) Bax/Bcl-2, p53, casapase-8, caspase-9 and caspase-3 genes, and increased (p < 0.05) testicular germ cell proliferation. MP’s effects were comparable to Met. However, the best results were achieved following co-administration of MP and Met. Therefore, we concluded that administration of the MP+Met combination better attenuates testicular oxidative stress, inflammation and apoptosis in DM, relative to MP or Met monotherapy, and may improve the fertility of males with DM.

Comparison of PLZF Gene Expression between Pluripotent Stem Cells and Testicular Germ Cells.

Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05). ConclusionThese results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is down- regulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.

The Effect of Diazinon on Cell Proliferation and Apoptosis in Testicular Tissue of Rats and The Protective Effect of Vitamin E.

BackgroundDiazinon (DZN) is an organophosphate pesticide, and nowadays this pesticide is mostly used in agri- culture. In this study, we analyzed the effects of DZN and vitamin E (Vit E) on apoptosis and the proliferation of germ cells in rat testis.Materials and MethodsIn this experimental study, 30 male Wistar rats were divided into five groups (n=6 per group) consisting of control, sham (received olive oil), experimental group i (60 mg/kg DZN), experimental group ii (60 mg/kg DZN and 200 mg/kg Vit E), and experimental group iii (200 mg/kg Vit E). After six weeks, left testis of rats was removed for the detection of proliferative cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase end-labeling (TUNEL).ResultsCompared with the control group, DZN in the experimental group i decreased the number of PCNA-positive cells and increased the number of TUNEL-positive cells (P<0.001). Vit E improved detrimental changes by the de- crease in the rate of apoptosis and the increase in the proliferation of testicular germ cells (P<0.001).ConclusionVit E can decrease the number of TUNEL-positive cells and increase the number of PCNA-positive cells by the neutralization of the toxicity caused by DZN in the testicular tissue.

Vitamin D deficiency impairs testicular development and spermatogenesis in mice

Vitamin D deficiency is prevalent especially in men. Nevertheless, whether vitamin D deficiency impairs male reproduction remains under debate. The aim of this study is to investigate whether vitamin D deficiency has an impact on testicular development and spermatogenesis in mice. In the control group and vitamin D deficient (VDD) diet group, dams and their pups were fed with standard-chow diet and VDD diet, respectively. Interestingly, testicular weight and sperm quality are reduced, testicular germ cell proliferation is suppressed, and the percentage of mature seminiferous tubules is decreased in VDD diet-fed mice. Moreover, testicular testosterone (T) synthesis enzymes are down-regulated in VDD diet-fed mice. Correspondingly, serum and testicular T levels are reduced in VDD diet-fed mice. Importantly, fertility index is reduced and live fetuses are decreased when both males and females are fed with VDD diet. These results provide evidence that vitamin D deficiency impairs testicular development and spermatogenesis.

Proliferation and apoptosis processes in the seasonal testicular development of the wild Daurian ground squirrel (Citellus dauricus Brandt, 1844).

The aim of the present study was to elucidate the regulatory role of cell proliferation and apoptosis in testicular development of wild Daurian ground squirrels during the breeding season (April), the non-breeding season (June) and before hibernation (September). Gross mass and hormonal analysis showed that the testis:body mass ratio and plasma testosterone concentration fluctuated seasonally, with a peak in April and lowest values in June. Similarly, spermatogenesis was fully developed in April but suppressed in June and September. Testicular decellularisation and vacuolisation was seen during the transition from the breeding to the non-breeding season. Furthermore, testicular levels of proliferating cell nuclear antigen, cyclin D2 and caspase-3 protein were significantly increased in June and September. Intriguingly, positive terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling staining and nuclear translocation of caspase-3 in testicular germ cells appeared only during the prehibernation period, whereas accumulation of cyclin D2 in spermatocyte nuclei occurred in September. These findings demonstrate, for the first time, that both cell proliferation and apoptosis are stimulated during the prehibernation period, indicating that a hormonal-regulated balance of testicular germ cell proliferation and apoptosis may play a pivotal role in preparing for testicular recrudescence of wild Daurian ground squirrels.

Heat stress decreases testicular germ cell proliferation and increases apoptosis in short term: an immunohistochemical and ultrastructural study

Scrotal hyperthermia has been known as a cause of male infertility but the exact mechanism leading to impaired spermatogenesis is unknown. This work was aimed to investigate the role of scrotal hyperthermia on cell proliferation and apoptosis in testes. The rats were randomly allotted into one of the four experimental groups: A (control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), and D (35 days after scrotal hyperthermia); each group comprised 7 animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia-exposed rats were killed under 50 mg/kg ketamine anaesthesia and tissue samples were obtained for biochemical and histopathological investigations. Hyperthermia treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione level, superoxide dismutase, and glutathione peroxidase activities. Moreover, exposure to hyperthermia resulted in lipid peroxidation increase in testes. Our data indicate a significant reduction in the expression of proliferating cell nuclear antigen and an enhancement in the activity of terminal deoxynucleotidyl transferase dUTP nick end labelling after scrotal hyperthermia. In scrotal hyperthermia, the mitochondrial degeneration, dilatation of smooth endoplasmic reticulum, and enlarged intercellular spaces were observed in both Sertoli and spermatid cells. Scrotal hyperthermia is one of the major factors that impair spermatogenesis in testis. This heat stress is shown to be closely associated with oxidative stress, followed by apoptosis of germ cells.

TSPY and Male Fertility

Spermatogenesis requires the concerted action of thousands of genes, all contributing to its efficiency to a different extent. The Y chromosome contains several testis-specific genes and among them the AZF region genes on the Yq and the TSPY1 array on the Yp are the most relevant candidates for spermatogenic function. TSPY1 was originally described as the putative gene for the gonadoblastoma locus on the Y (GBY) chromosome. Besides its oncogenic properties, expression analyses in the testis and in vitro and in vivo studies all converge on a physiological involvement of the TSPY1 protein in spermatogenesis as a pro-proliferative factor. The majority of TSPY1 copies are arranged in 20.4 kb of tandemly repeated units, with different copy numbers among individuals. Our recent study addressing the role of TSPY1 copy number variation in spermatogenesis reported that TSPY1 copy number influences spermatogenic efficiency and is positively correlated with sperm count. This finding provides further evidence for a role of TSPY1 in testicular germ cell proliferation and stimulates future research aimed at evaluating the relationship between the copy number and the protein expression level of the TSPY1 gene.

Toxic and hormonal effects of polychlorinated biphenyls on cultured testicular germ cells of embryonic chickens

The toxic and hormonal effects of polychlorinated biphenyls (PCBs) on testicular germ cell development were revealed with a germ-Sertoli cell co-culture model from embryonic chickens. Testicular cells were dispersed from 18-day-old embryo and exposed to Aroclor 1254 (A1254) alone and combined with α-tocopherol, flutamide and tamoxifen for discretion of the toxic and hormonal actions of A1254. Cell damage was evaluated by determinations of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage. Results showed that 10 μg/ml A1254 induced condensed nuclear and vacuolated cytoplasm; cell exfoliated and broke into pieces as a sign of cell degeneration after treatment for 6 h. The morphological cytotoxicity was confirmed by MTT reduction and LDH leakage assays. α-Tocopherol attenuated the toxic effect of A1254. After culture for 48 h, A1254 (0.1–1 μg/ml) manifested obvious hormonal effect to induce germ cell proliferation, while 10 μg/ml A1254 displayed obvious toxic and hormonal effects on germ and somatic cells. Blocking of either androgen receptor by flutamide or estrogen receptor by tamoxifen inhibited hormonal effect of A1254 on germ cell proliferation and increased the cytotoxicity. The above results indicated that A1254 exposure imposed both toxic and hormonal effects on embryonic testicular germ cell proliferation, which may cause reproductive disorder and even infertility at adulthood.

Effects of follicle-stimulating hormone and androgen on proliferation of cultured testicular germ cells of embryonic chickens

A germ–Sertoli cell coculture model was established to study effects of follicle-stimulating hormone (FSH) and testosterone (T) on testicular germ cell proliferation of the embryonic chickens. Germ and somatic cells were dispersed from 18-day-old embryonic testes and cultured in 96-well plates. Germ cells were characterized by expression of stem cell factor receptor c-kit. Germ cell proliferation was assessed by an increase in cell number and expression of proliferating cell nuclear antigen (PCNA). Results showed that the germ and Sertoli cells kept alive in serum-free McCoy’s 5A medium supplemented with insulin, transferrin, and selenite (ITS medium). Germ cells adhered to the free surface of Sertoli cells that spread the filopodia and formed a monolayer in ITS medium. In the serum-containing medium, Sertoli cells displayed an increment with a flat squamous form and only a few very large germ cell masses were found in the free surface of Sertoli cells. Many germ cells showed apoptosis in the McCoy’s 5A medium without ITS or serum. Only germ cells showed positive staining for c-kit in the coculture. Ovine FSH (0.25–1.0 IU/ml) significantly increased the number of germ cells, and PCNA-labeling index ( P < 0.05). FSH also induced stronger c-kit expression compared with the control. In the FSH-treated groups, germ cells were manifested distinct knob-like form. Similar stimulating effect was found in the germ cell number by T treatments (10 −7–10 −6 M). Furthermore, FSH (0.5 IU/ml) combined with T significantly promoted higher testicular germ cell proliferation ( P < 0.05) compared with either FSH or T alone, which indicated that interaction of FSH and T might be additive. The above results showed that the serum-free germ–Sertoli cell coculture model allowed evaluating hormonal regulation of testicular germ cell proliferation. FSH and T promoted testicular germ cell proliferation probably through indirect effects on Sertoli cells.

Dynamics of testicular germ cell proliferation in normal mice and transgenic mice overexpressing rat androgen-binding protein: a flow cytometric evaluation.

Transgenic mice carrying rat androgen-binding protein (ABP) genomic DNA express high amounts of testicular ABP and develop a progressive impairment of spermatogenesis. To understand the mechanism of these changes, we have studied the pattern of testicular germ cell proliferation from 7 to 360 days of age in wild-type (WT) control and transgenic homozygous (ABP-TG) mice by flow cytometry after labeling DNA in isolated germ cells with propidium iodide. At all ages studied, the body weight of the ABP-TG mice was lower than that of age-matched WT controls. Significantly reduced testicular weight and total germ cell number in the ABP-TG mice were evident from Day 30 and Day 60, respectively. Flow cytometric analysis of isolated germ cells revealed that the number of germ cells undergoing proliferation (S-phase cells) was identical in WT control and ABP-TG mice up to Day 14. Subsequently, the number of germ cells in S-phase was consistently higher in ABP-TG than in WT mice. The number of primary spermatocytes was significantly increased starting from Day 60, and the numbers of round and elongated spermatids were significantly reduced in the ABP-TG animals from Day 21 and Day 60 onwards, respectively. Immunocytometry for intracellular ABP at 90 days of age revealed that the percentage of ABP-containing germ cells was greater in ABP-TG than in WT mice. The continuous presence of ABP in mouse seminiferous tubules at greater than physiological concentrations facilitates the formation of primary spermatocytes but impairs subsequent transformation to round and elongated spermatids. Based on our observations and the analysis of the available literature, the most likely mechanism for production of these effects is sustained reduction in the bioavailability of androgens.

Spermatogenesis by Sisyphus: proliferating stem germ cells fail to repopulate the testis after 'irreversible' injury.

2,5-Hexanedione is the toxic metabolite resulting from oxidation of the commonly used solvents n-hexane and methyl n-butyl ketone. Exposure to 2,5-hexanedione or its precursors results in a slowly progressive peripheral polyneuropathy and testicular injury. The chemical basis of the injury involves reaction of 2,5-hexanedione with protein amines, such as the epsilon-amine of lysine, to form pyrroles which further react to form protein-protein crosslinks. The target cell of injury in the testis is the supportive cell in the seminiferous epithelium, the Sertoli cell. A major function of the Sertoli cell is to nurture the dependent germ cell population by secreting seminiferous tubule fluid. 2,5-Hexanedione-induced crosslinking of the microtubule subunit protein, tubulin, leads to altered Sertoli cell microtubule-dependent transport and deficient formation of seminiferous tubule fluid, compromising germ cell viability. In an established model of testicular injury, rats are exposed to 1% 2,5-hexanedione in the drinking water for a period of 3-5 weeks. Three weeks after initiating exposure, decreased seminiferous tubule fluid secretion initiates a wave of germ apoptosis which peaks during the 5th week. The germ cell content of the injured testis continues to decline after cessation of the exposure, reaching a nadir during the 12th week. From this time onward, the testis is severely atrophic with less than 1% of seminiferous tubules in a testicular cross section containing germ cells more advanced than spermatogonia. Interestingly, this persistent state of post-injury 'irreversible' atrophy in the rat is characterized by the presence of a proliferating stem germ cell population which produces differentiating spermatogonia which then die by apoptosis. Serial cross sections of bromodeoxyuridine-labeled testis were analyzed to determine the kinetics of stem germ cell proliferation. Approximately 40% of stem cells (identified as single cells in the seminiferous epithelium) were actively proliferating with a cell cycle time of 8-14 days. Analysis of the total germ cell population present and modeling using the known cell cycle times of differentiating spermatogonia indicated a block in differentiation at the level of type A3/A4 spermatogonia. Quantitation of the frequency of apoptosis indicated that all of the germ cells died prematurely by this mechanism. Leuprolide is a gonadotropin-releasing hormone agonist which produces a profound suppression of testosterone levels with chronic administration. When delivered as a series of 3 depot injections 24 days apart, leuprolide resulted in a partial reversal of the 2,5-hexanedione-induced persistent atrophy. The reinitiation of spermatogenesis follows a lowering of the intratesticular testosterone concentration, indicating that intratesticular testosterone is at least partially responsible for the persistent atrophy. The efficacy of leuprolide-induced reversal of the persistent atrophy decreases with time after injury, suggesting that atrophic seminiferous tubules are initially capable of recovery and then enter a state of irreversible injury. Injection of ethane dimethane sulfonate at the beginning of leuprolide treatment eliminated Leydig cells during therapy and ablated the recovery of spermatogenesis, indicating that a Leydig cell-associated paracrine factor is required to restart spermatogenesis. The rat, therefore, has multiple states of testicular germ cell proliferation: normal spermatogenesis and at least two forms of persistent atrophy (leuprolide reversible and leuprolide non-reversible). Partial reversal of the persistent atrophy can be achieved by lowering intratesticular testosterone. Ongoing experiments are designed to address the role of the Leydig cell in post-injury recovery, and to further characterize the molecular events contributing to the different states of persistent atrophy.

Follistatin modulates activin activity in a cell- and tissue-specific manner.

The high affinity activin-binding protein, follistatin, has recently been shown to block activin-stimulated activities in several in vitro systems. In the present study we sought to extend these observations and investigate the effects of follistatin on the activity of activin in stimulating the re-aggregation of Sertoli cell monolayers and proliferation of testicular germ cells, as measured by incorporation of [3H]-thymidine in vitro. Germ-Sertoli cell cocultures prepared from 21 day old rats were treated with media alone or media containing recombinant human (rh) activin A or rh activin B with or without follistatin, the low affinity activin-binding protein, alpha 2 macroglobulin, or a monoclonal antibody (mAB) known to block activin B activity. Follistatin blocked the ability of activin A to stimulate reaggregation of Sertoli cell monolayers when present at a 2-fold ratio (wt/wt) to activin. However, in these same cultures, follistatin had no effect on the ability of activin A to stimulate [3H]-thymidine incorporation. In activin B-treated cultures, both responses could be blocked by the addition of a neutralizing mAB directed against activin B. These results suggest that follistatin can modulate activin action in a cell-type specific fashion, and that this protein may play an important role in regulating the bioavailability of activin.