Renewal of spermatogonia in man

The spermatogonia of normal adult human testis were investigated in view of clarifying their mode of proliferation and renewal. Three main types of spermatogonia were identified: the dark type A spermatogonia (Ad) tentatively considered as the stem cells, the pale type A spermatogonia (Ap) and the type B spermatogonia (B), these being the more and more differentiated elements giving rise to preleptotene spermatocytes. The dark and pale type A spermatogonia were present in all stages of the cycle of the seminiferous epithelium, the type B spermatogonia were found in stages VI, I and II of the cycle and the preleptotene spermatocytes in stages III and IV of the cycle. The type A spermatogonia divided preferentially in stage V of the cycle and the type B spermatogonia in stage II of the cycle. Quantitative data on spermatogonia and preleptotene spermatocytes revealed that the cell ratio Ad: Ap: B: Pl was equal to 1:1:2:4. This indicated that the spermatogonial stem cells divided to produce equal numbers of new stem cells (Ad) and of the more differentiated pale type A spermatogonia (Ap). Each one of the latter gave rise to two type B spermatogonia which in turn produced four spermatocytes. The arrangement in pairs of the dark and pale type A spermatogonia throughout the duration of the cycle indicated that the mitoses of spermatogonial stem cells are “equivalent” in nature; therefore, the possibility of having “differential” mitoses to explain the renewal of spermatogonial stem cells should be abandoned. Lastly, the frequent arrangement of the two classes of type A spermatogonia in homogeneous clusters indicated that the impetus which facilitates the differentiation of stem cells into the more differentiated elements (Ap) may affect homogeneous and compact groups of stem cells.     

Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides

Monkeys were sacrificed at three hours and at 12 days-3 hours after an intraperitoneal injection with ³H-thymidine. Radioautographs were prepared of periodic acid-Schiff-hematoxylin-stained sections of the testes. At three hours, the most evolved labeled germ cells were leptotene spermatocytes in stage VIII of the cycle; at 12 days-three hours the most evolved labeled cells were pachytene spermatocytes in stage IX of the succeeding cycle. Thus, over a 12-day interval, the most evolved labeled spermatocytes had advanced through slightly more than one complete cycle. A quantitative analysis of tubular cross sections containing labeled spermatocytes permitted the calculation of the duration of one cycle of the seminiferous epithelium which turned out to be 11.6 days. The whole process of spermatogenesis which begins with the first spermatogonial mitoses taking place in stage VIII and terminates with the release of spermatozoa taking place in stage VI of the cycle, extends over the duration of 3.8 consecutive cycles and therefore requires approximately 44 days. On the same histological material counts of resting and dividing spermatogonia were performed at various stages of the cycle to determine their mode of proliferation and renewal. Six mitotic peaks were disclosed and were located in stages VIII, X, XII, II, IV and VI of the cycle. Cell counts and labeling indices indicated that the pale type A spermatogonia were the cells dividing during the first two of these six peaks of mitoses while the type B spermatogonia divided during the remaining four peaks of mitoses. The dark type A spermatogonia were not seen to divide during the cycle and may be considered as “reserve stem cells.” Cell counts and cell ratios indicated further that the pale type A spermatogonia divided in stage VIII of the cycle (and some in stage IX) to yield about twice their number of pale type A spermatogonia: of several possible schemes the authors believe that half of these entered a long interphase and became stem cells for the spermatogonia to be formed during the next cycle; these may be referred to as “renewing stem cells”; we consider that the other half of the pale type A spermatogonia arising from stage VIII peak of mitoses divided in stage X to produce type B spermatogonia. The latter cells then could enter the series of four consecutive mitoses during which they doubled their number each time to yield a generation of primary spermatocytes.     

Stem cells in the testis

The origin and development of the spermatogenic cell lineage is reviewed, as well as spermatogonial kinetics in adult nonprimate mammals in relation to the cycle of the seminiferous epithelium, the emphasis being on spermatogonial stem cells. A hypothesis is presented for the transition from foetal germ cells, gonocytes, to adult type spermatogonia at the start of spermatogenesis. An overview is given of the present knowledge on the proliferation and differentiation of undifferentiated spermatogonia (spermatogonial stem cells and their direct descendants) and the regulation of these processes. It is concluded that the differentiation of the undifferentiated into differentiating type spermatogonia is a rather vulnerable moment during spermatogenesis and the models for studying this are described. Research into the molecular basis of the regulation of spermatogonial proliferation, differentiation and apoptosis is at its infancy and the first results are reviewed. An exciting new research tool is the spermatogonial stem cell transplantation technique which is described. Finally, reviewing the nature of human germ cell tumours it is concluded that at present there are no animal or in vitro models to study these tumours experimentally.     

Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.     

Spermatogonial stem cells in the albino rat

The existence of two classes of spermatogonial stem cells in the rat testis, i.e., reserve type A₀ spermatogonia and renewing, types A₁-A₄ spermatogonia, postulated by Clermont and Bustos-Obregon (′68), was reexamined in a quantitative analysis of type A spermatogonia in both whole mounts of tubules and in radioautographed sections of testes from animals killed at various times, up to 26 days, after one or multiple injections of ³H-thymidine. The cell counts obtained from whole mounts of tubules revealed that the number of isolated type A₀ cells per unit area of limiting membrane remained constant throughout the cycle of the seminiferous epithelium. Paired type A₀ spermatogonia also remained unchanged in number per unit area of basement membrane from stage I to stage VIII of the cycle. The low mitotic index of type A₀ spermatogonia (0.1%) indicated that these cells were not actively involved in the production of spermatogonia or spermatocytes during each cycle of the seminiferous epithelium and thus were considered as reserve stem cells. The type A₁ spermatogonia, which are formed during stage I of the cycle, remained resting until stage IX, when they undertook a series of four successive divisions resulting in the production of new type A₁ and Intermediate-type spermatogonia. An analysis of the labeling indices of type A spermatogonia obtained from cell counts in radioautographed testicular sections after a single or multiple ³H-thymidine injections indicated that the percentages of labeled type A cells corresponded to the percentages of type A₁-A₄ at each stage, whereas the percentages of unlabeled type A cells corresponded to the percentages of type A₀ spermatogonia obtained from counts of cells in whole mounts. This confirmed that type A₀ cells were generally non-proliferative throughout the cycle of the seminiferous epithelium while the type A₁-A₄ spermatogonia underwent complete renewal during each cycle. The present results thus support the concept of the existence of two classes of spermatogonial stem cells in rats.     

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones. Testis tubule diameter is directly correlated with testicular weight during the annual cycle. Seminiferous tubule stages found during testicular activity start with sperm release and round spermatids in the Golgi stage (I). Then they progress through the cap and acrosome stages (stages II to VI) until elongate spermatids are formed. During these stages preleptotene, leptotene and zygotene cells develop into pachytene cells which mature with the long spermatids (stage VII). Two distinct tubule associations (stages VIII, IX) follow during which the first and second meiotic metaphases occur. These stages are correlated with the middle and late phases of residual lobe retraction and condensation. The last stage (X) has final sperm development and is present with round spermatids that have no Golgi development. During regression changes are initially associated with the seminiferous tubule stages of active testes and end with relocation of Sertoli cell nuclei to a position above the basal layer of spermatogonia. Out of season testes are characterized by few spermatogonial mitoses and absence of viable spermatocytes. In recrudescent testes, Sertoli cell nuclei again become basal, spermatogonia resume mitoses and spermatocytes and spermatids progressively develop. After each cycle of proliferation of germ cells there is sloughing of the most differentiated spermatocytes and spermatids until the final tubule associations of the active testis are present. Anat. Rec. 247:355–367, 1997. © 1997 Wiley-Liss, Inc.     

Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.     

Relative volume of sertoli cells in monkey seminiferous epithelium: A stereological analysis

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.     

Limited survival of adult human testicular tissue as ectopic xenograft

BACKGROUND: Grafting of testicular tissue into immunodeficient mice has become an interesting and promising scientific tool for the generation of gametes and the study of testicular function. This technique might potentially be used to generate sperm from patients whose testes need to be removed or are destroyed due to therapeutic intervention or as a consequence of disease. Here we explore whether adult human testicular tissue from patients with different testicular pathologies survives as xenograft. METHODS AND RESULTS: Testis tissue from adult patients with varying degrees of spermatogenesis was grafted into two strains of immunodeficient mice (severe combined immunodeficiency, Nu/Nu). Tissue with active spermatogenesis prior to grafting largely regressed. However, testicular tissue survival was better in cases where spermatogenesis was suppressed prior to grafting and occasionally spermatogonial stem cells survived. Cases with spermatogenic disruption were not corrected by the xenografting. CONCLUSION: Superior survival of the germinal epithelium and spermatogonia when spermatogenesis was suppressed prior to grafting could provide a novel strategy for germline preservation in pre-pubertal cancer patients. This approach could also be valuable to study the early stages of human spermatogenesis.     

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

BACKGROUND: Successful cryopreservation of gonadal tissue is an important factor in guaranteeing the fertility preservation via germ cell or testicular tissue transplantation. The aim of this study was to evaluate the effects of cooling and cryopreservation on spermatogonial stem cell survival and function of immature non-human primate testicular tissue xenografted to nude mice. METHODS: Group 1 (control group) received subcutaneous grafts of fresh immature rhesus monkey testes. The treatment groups received grafts after 24 h cooling in ice-cold medium (Group 2), after 24 h of cryopreservation without cryoprotectant (Group 3), with ethylene glycol (Group 4: 1.4 M) or with dimethylsulphoxide (DMSO) (group 5: 1.4 M; group 6: 0.7 M), using cooling rates of 0.5 degrees C/min. The graft number, weight and histology were examined 3-5 months later. RESULTS: After xenografting, grafts from fresh and cooled tissue showed good survival and spermatogenic induction to spermatocytes. Cryopreservation in 1.4 M DMSO also allowed grafts to initiate spermatogenesis. In contrast, 0.7 M DMSO and ethylene glycol showed inferior protection. CONCLUSIONS: Our observations suggest that cryopreservation of immature primate testis is a feasible approach to maintain spermatogonial stem cells and may serve as a promising tool for fertility preservation of prepubertal boys. The possibility to delay the transplantation of cooled samples suggests an option for clinical centralization of testicular tissue cryopreservation.     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative to Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy.     

Transcription and imprinting dynamics in developing postnatal male germline stem cells

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1⁺; high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1⁺; moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT⁺). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor–ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles.     

Spermatogonial stem cells: questions, models and perspectives

This review looks into the phylogeny of spermatogonial stem cells and describes their basic biological features. We are focusing on species-specific differences of spermatogonial stem cell physiology. We propose revised models for the clonal expansion of spermatogonia and for the potential existence of true stem cells and progenitors in primates but not in rodents. We create a new model for the species-specific arrangements of spermatogenic stages which may depend on the variable clonal expansion patterns. We also provide a brief overview of germ cell transplantation as a powerful tool for basic research and its potential use in a clinical setting.     

Use of confocal microscopy for the study of spermatogenesis

Spermatogenesis consists of spermatogonial proliferation, meiosis and spermatid differentiation. Laser scanning confocal microscopy (LSCM) may be used as an advanced analytical tool to follow spermatogenesis inside the seminiferous tubules without performing histological sections. For this purpose, separated seminiferous tubules are fixed in 0.5% paraformaldehyde, stained for DNA with propidium iodide and analyzed by LSCM. By producing longitudinal optical sections in the layer of spermatogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followed within the tubules by LSCM. Longitudinal z-sections may be obtained to produce three-dimensional images of the seminiferous tubules. In addition, different proteins may be followed during spermatogenesis in a stage specific manner within the tubule by incubation of the fixed seminiferous tubules with appropriate antibodies.As an example of the spermatogenesis studies using described LSCM techniques, detailed examination of spermatogonia, spermatocytes and spermatids during golden hamster spermatogenesis is presented. LSCM analysis of c-kit and SC3 protein expression at different stages of hamster spermatogenesis is demonstrated.     

Spermatogonial transplantation: the principle and possible applications

Spermatogenesis is the process of male germ cell proliferation and differentiation that begins at puberty and lasts throughout life. Spermatogonia, especially stem spermatogonia, are the cells essential for the continued maintenance of spermatogenesis. Although many studies of spermatogonia have been performed with morphological methods, the very nature of spermatogonia still remains unknown. The technique of spermatogonial transplantation, developed in 1994, made it possible to study functional aspect of spermatogonial stem cells. Many new developments, such as cryopreservation, xenotransplantation, purification, and culturing of spermatogonial stem cells, have been achieved and are still under investigation. The techniques could be used not only for basic research but also for medicine and other disciplines.     

A study of germ cell morphology and duration of spermatogenic cycle in the baboon,Papio anubis

Biopsy and orchiectomy specimens were collected from two adult baboons (Papio anubis) at different intervals after intratesticular injection of H³-thymidine. Zenker-formol or Bouin's fixed materials were stained with PASWeigert-Hematoxylin and radioautographed using the H.S.R. (Harleco Synthetic Resin) coating technique. Morphological features of most germ cells appeared similar to those of other monkeys, except that the spermatids in steps 9 to 11 showed a spike-like projection of the acrosome. Also, the type A spermatogonia showed some resemblance to the human type A spermatogonia. The cell associations consisted of 12 stages and a large number of tubular cross sections showed the presence of two or more stages. In Papio anubis, the zygotene spermatocytes are formed in stage VIII, and spermatozoa are released during stages V and VI. H³-thymidine labeling of germ cells indicated that one cycle of spermatogenesis in this species takes approximately 11 days, and complete spermatogenesis occupies 3.8 cycles, or approximately 42 days. The presence of labeled B type spermatogonia 35 days after H³-thymidine injection indicated the existence of some stem cells. The presence of some labeled Ad spermatogonia in these specimens could not be explained. The data indicated that spermatogenesis in this monkey is somewhat different from that in other monkey species described.     

Spermatogonial stem cells: characteristics and experimental possibilities

The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFRalpha-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers.     

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.     

Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes

The expression and localization of mRNA's for tissue plasminogen activator (tPA), urokinase PA (uPA), uPA receptor (uPAR) and inhibin subunits, alpha, beta A and beta B in monkey testes was investigated. Using in-situ hybridization with digoxigenin-labelled cRNA probes (dig- cRNA), we demonstrated that tPA and plasminogen activator inhibitor type 1 (PAI-1) were expressed in testes of both immature and mature rhesus monkeys. tPA mRNA was localized predominantly in Sertoli cells. Expression level was low in immature testis, increased dramatically in the adult and varied with seminiferous cycle. PAI-1 mRNA was localized mainly in germ cells except late spermatids. uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis. uPA receptor mRNA was localized in germ cells of mature testis but not in spermatogonia or late spermatids. Assayed by fibrin overlay technique, PA activity in conditioned media of purified Sertoli cells (Sc) was negligible, PA activity in media obtained from co-cultured Sertoli and Leydig cells (LS), however, was significantly increased, although Leydig cells alone were not capable of producing any PA activity. Addition of follicle stimulating hormone (FSH) to the incubation medium remarkably increased PA secretion in both Sc and LS cultures. Human chronic gonadotrophin (HCG) had no significant effect on PA activity in the Sc culture but dramatically stimulated PA activity in the co-culture system. Dihydrotestosterone (DHT) did not mimic the effect of HCG. PAI-1 activity was secreted mainly by germ cells and did not differ between the two culture systems. FSH and forskolin inhibited PAI-1 secretion. Inhibin alpha, beta A and beta B subunit mRNAs were localized in Sertoli cells of adult monkey testes, with no obvious difference in the expression levels. These data suggest that PA/PAI-1 and other related factors are expressed in rhesus monkey testis under the control of various hormones, seminiferous cycle and cell-cell interactions through paracrine or autocrine regulation. Locally generated fibrinolysis may play an important role in the process of spermatogenesis.