Expression of the GM-CSF receptor in ovine spermatozoa: GM-CSF effect on sperm viability and motility of sperm subpopulations after the freezing-thawing process

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of haematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed in myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other non-haematopoietic cells. We have previously demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and vitamin-C uptake and enhance several parameters of sperm motility in the presence of glucose or fructose substrates. In this study, we have analyzed the expression of GM-CSF receptors in ovine spermatozoa and studied the effect of GM-CSF on sperm viability and motility after the freezing-thawing process. Immunolocalization and immunoblotting analyses demonstrated that ovine spermatozoa (Xisqueta race) expressed GM-CSF receptors. In addition, GM-CSF partially counteracted the impairing action of freezing/thawing on the percentage of total motility, as well as on the specific motility patterns of each of the separate, motile sperm subpopulations of ram ejaculates subjected to this protocol. These results suggest that GM-CSF can play a role in the resistance of ram spermatozoa to environmental thermal stress.     

Expression of GM-CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport

We studied the expression and function of the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in male germ cells. RT-PCR showed expression of mRNAs encoding the alpha- and beta-subunits of the GM-CSF receptor in human testis, and the presence of the alpha- and beta-proteins was confirmed by immunoblotting with anti-alpha and anti-beta-antibodies. Immunolocalization studies showed the level of expression of GM-CSF alpha- and beta-subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM-CSF revealed that bull spermatozoa have about 105 high-affinity sites with a K(d) of 222 pM and approximately 1100 low-affinity sites with a K(d) of 10 nM. GM-CSF signaled, in a time- and dose-dependent manner, for an increased uptake of glucose and vitamin C.     

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression,Functionality and Involvement in Motility Regulation

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.     

Granulocyte macrophage colony stimulating factor (GM-CSF) biological actions on human dermal fibroblasts.

Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.     

Reg I protein in healthy and seminoma human testis

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation.     

Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.     

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.     

TWEAK/Fn14 interaction stimulates human bronchial epithelial cells to produce IL-8 and GM-CSF

TNF-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family, is a multifunctional cytokine that regulates cellular proliferation, angiogenesis, inflammation, and apoptosis. In this study, we investigated the effect of TWEAK on human bronchial epithelial cells. A human bronchial epithelial cell line, BEAS2B, expressed a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), and produced IL-8 and GM-CSF upon TWEAK stimulation in a dose-dependent manner, which was abrogated by anti-Fn14 blocking antibody. TWEAK induced phosphorylation of IkappaBalpha and BAY11-7082, a selective inhibitor of IkappaBalpha phosphorylation, inhibited the TWEAK-induced IL-8 and GM-CSF production by BEAS2B cells. Moreover, primary cultured human bronchial epithelial cells also expressed Fn14 and produced IL-8 and GM-CSF upon TWEAK stimulation. Collectively, TWEAK stimulated human bronchial epithelial cells to produce IL-8 and GM-CSF through Fn14. Because IL-8 and GM-CSF are associated with inflammatory conditions, these results suggest that TWEAK/Fn14 interaction may play some roles in airway inflammatory responses.     

The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

Endothelium-derived GM-CSF influences expression of oncostatin M

During and after transendothelial migration, neutrophils undergo a number of phenotypic changes resulting from encounters with endothelium-derived factors. This report uses an in vitro model with human umbilical vein endothelial cells and isolated human neutrophils to examine the effects of two locally derived cytokines, granulocyte (G)-macrophage (M) colony-stimulating factor (GM-CSF) and G-CSF, on oncostatin M (OSM) expression. Neutrophils contacting activated HUVEC expressed and released increased amounts of oncostatin M (OSM), a proinflammatory cytokine known to induce polymorphonuclear neutrophil adhesion and chemotaxis. Neutrophil transendothelial migration resulted in threefold higher OSM expression and protein levels compared with nontransmigrated cells. Addition of anti-GM-CSF neutralizing antibody reduced OSM expression level but anti-G-CSF was without effect. GM-CSF but not G-CSF protein addition to cultures of isolated neutrophils resulted in a significant increase in OSM protein secretion. However, inhibition of β(2) integrins by neutralizing antibody significantly reduced GM-CSF-induced OSM production indicating this phenomenon is adhesion dependent. Thus cytokine-stimulated endothelial cells can produce sufficient quantities of GM-CSF to influence in an adhesion-dependent manner, the phenotypic characteristics of neutrophils resulting in the latter's transmigration. Both transmigration and adhesion phenomenon lead to increased production of OSM by neutrophils that then play a major role in inflammatory response.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Molecular modeling of the GM-CSF and IL-3 receptor complexes.

A model for the structure of the cytokine interleukin-3 (IL-3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin-5 and granulocyte macrophage colony stimulating factor (GM-CSF). In addition, models of the receptor complexes of GM-CSF and IL-3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM-CSF and IL-3 to their receptors.     

The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.     

Casein α s1 is expressed by human monocytes and upregulates the production of GM-CSF via p38 MAPK

Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4(+) and CD8(+) T cells, but not in CD19(+) B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14(+) monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF.     

A quantitative focus assay for titration of retroviruses that encode human granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a haematopoietic growth factor that regulates proliferation, differentiation, and effector functions of monocyte-macrophages and granulocytic cells. Because of the ability of this cytokine to enhance immune functions of antigen-presenting cells, retroviruses encoding GM-CSF have been used to transduce GM-CSF into murine and human tumour cells as part of autologous tumour vaccine strategies. We have previously shown that NIH 3T3 cells engineered to express functional human GM-CSF receptors (hGMR-NIH 3T3), become fully transformed when these cells are incubated in the presence but not in the absence of human GM-CSF. In this study we have used these hGM-CSF conditional transformants to devise a sensitive focus assay to titrate retroviruses encoding hGM-CSF, using MFG-hGM-CSF/Psi-CRIP as our model virus. This helper-free amphotropic retrovirus, which has been frequently used to transduce hGM-CSF into tumour cells, was quite transforming in our indicator cell line, exhibiting virus titres well above 10(5)FFU/ml. The transformation-based assay described here allows rapid determination of the titre of hGM-CSF-viruses, and may serve as a model for development of quantitative assays for other cytokine-encoding viruses of clinical importance.     

GM-CSF triggers a rapid,glucose dependent extracellular acidification by TF-1 cells: Evidence for sodium/proton antiporter and PKC mediated activation of acid production

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic “cell capsule” that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionmycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1–2h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA. © 1993 Wiley-Liss, Inc.     

Localization of gamma-butyrobetaine hydroxylase in the rat testis

The activity of gamma-butyrobetaine hydroxylase in testes is comparable on a protein basis to the amount of activity found in liver and accounts for a significant amount of the total activity found in the rat. The enzyme is localized to the seminiferous tubules of the testes. In attempting to further localize this enzyme, activity remained the same when reported on a protein basis in cryptorchid testes as compared to controls, and activity was found in bovine sperm cells obtained from the caput epididymis. These results are consistent with the localization of this enzyme in both the Sertoli cells and the germ cells.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.