Rat Seminiferous Epithelium Research Articles

Stage-Specific Expression and Cellular Localization of the Heat Shock Factor 2 Isoforms in the Rat Seminiferous Epithelium

Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, we examined HSF2 expression at specific stages of the rat seminiferous epithelial cycle. We found that expression of the alternatively spliced HSF2-α and HSF2-β isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrated that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I–IV and in the nuclei of round spermatids at stages V–VIIab. In contrast a strong HSF2 immunoreactivity was detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis revealed that these structures are mainly cytoplasmic bridges between germ cells. Our results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells.

Sertoli cell nuclear pore number changes in some stages of the spermatogenic cycle of the rat seminiferous epithelium

In an earlier study we described changes in the number and distribution of nuclear pores during maturation of germ cells at given stages of the spermatogenic cycle; these changes were related to the activity of nucleus-cytoplasm transport. Similarly, the present work was performed by combining freeze-fracture techniques with Sertoli nuclei identification criteria, using fragments of tubules isolated by transillumination under stereo-microscopy. We studied the density of nuclear pores in freeze-fracture replicas of the Sertoli nuclear envelope at stages XIII-XIV-I compared with stages IX-XII. Pore counts were carried out on photographs of the platinum replicas using a digitalized morphometric board. The results were statistically analyzed using Student's t test. The difference in density (pore number/μm2 ± SEM) was significant between stages IX-XII (8.25 ± 0.63) and XIII-XIV-I (10.80 ± 0.60). We postulate that this density appears to be increased at the time of increased metabolic requirements of the Sertoli cell.

Junctional contacts between Sertoli cells in normal and aspermatogenic rat seminiferous epithelium contain alpha6beta1 integrins, and their formation is controlled by follicle-stimulating hormone.

The distribution of alpha6beta1 integrins at the level of cell-to-cell contacts within the rat seminiferous epithelium was investigated. Double fluorescence experiments using phalloidin staining of actin filaments and anti-integrin subunit antibodies showed that the receptor belongs to the Sertoli cell lateral domains engaged in the characteristic junctional structures known as ectoplasmic specializations (ES), at the level both of inter-Sertoli junctions and of the contacts between Sertoli cells and elongating spermatids. In the seminiferous epithelium of aspermatogenic testes, obtained through X-irradiation in utero (Sertoli-cell-only testes), at the level of inter-Sertoli junctions both ES and alpha6beta1 integrins are present. In order to study the dependence of alpha6beta1 receptors and ES formation upon FSH stimulation during development, 9-day-old testes were grown in organ culture in basal as well as FSH-supplemented conditions. FSH stimulation, which is necessary for the progression of spermatogenesis to early meiotic stages, appears to be required for the development of inter-Sertoli junctional structures containing ES and alpha6beta1 integrins. These observations indicate that the receptor belongs to the inter-Sertoli junctional machinery and that its expression at that level is not dependent on active spermatogenesis but requires FSH stimulation.

Stage-specific expression and phosphorylation of retinoblastoma protein (pRb) in the rat seminiferous epithelium

To assess the potential role of retinoblastoma protein (pRb) in the regulation of cell cycle during spermatogenesis, the expression of retinoblastoma (Rb) mRNA and protein, as well as the phosphorylation states of pRb, in the rat seminiferous epithelial cycle, were studied. Two transcripts, 5.4 kb and 3.4 kb long, were detected in total RNA from the adult rat testis and only the 5.4 kb transcript was detected in poly (A) +-RNA from 8, 14 and 23-day old rat testes by Northern hybridization. Polysome analysis revealed that only a small portion of both Rb transcripts could be efficiently translated. By in situ hybridization, Rb mRNA was localized to germ cells from stage V pachytene spermatocytes to step 13 spermatids along the epithelial cycle. pRb immunoreactivity was detected in Sertoli cells and spermatogonia at all stages, as well as in the elongated steps 14–19 spermatids by immunohistochemistry. The amount of pRb and the phosphorylation status varied in a stage-specific manner in Western blots. These results show that pRb is expressed in the rat seminiferous epithelium in a cyclic fashion and suggest that it is involved in the regulation of proliferation of spermatogonia and maintenance of the differentiation status of Sertoli cells and spermatids.

Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats.

We have identified multiple fucosyltransferases (FTs) (alpha[1-2]-, alpha[1-3]-, alpha[1-4]-FTs) on cells of the rat seminiferous epithelium as demonstrated by fucose incorporation into phenyl-beta-D-galactoside (Ph-beta-D-Gal), 2'-fucosyllactose (2'-FL), and lacto-N-fucopentaose-l (LNF I), respectively. Now, using fluorescence laser scanning cytometry, we report that multiple FTs are implicated in germ cell-Sertoli cell adhesion in vitro. Sertoli cells were isolated from 19- to 21-day-old CD rats and cultured for 6-10 days. Mixed germ cells were obtained by enzymatic dispersion of adult rat testis and cultured overnight before labeling with 10 microM acetoxymethyl ester derivative of the fluorescent indicator, calcein. The adherent cell analysis and sorting 570 interactive laser cytometer was used to determine the number of labeled adherent germ cells on Sertoli cell monolayers in the presence or absence of a variety of low molecular weight acceptors for fucose. Coincubation of labeled germ cells with Sertoli cell monolayers in the presence of GDP-fucose, UDP-galactose, Ph-beta-D-Gal, 2'-FL, LNF I, and Lewis-X and 3'-sialyl-Lewis-X oligosaccharides resulted in significant reduction of germ cell binding when compared to that of the untreated controls or of control samples incubated with cellobiose, melibiose, and alpha-D-mannopyranose, which do not serve as fucose acceptors. Our results suggest that multiple FTs and their lectin/selectin ligands are involved in mediating germ cell-Sertoli cell adhesion to form a cohesive epithelium and thus aid germ cell adluminal translocation within the seminiferous epithelium.

Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium.

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII-I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P < 0.01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I-IV were threefold higher than in stages VI-VII (P < 0.01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion

In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion.

The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage-specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation.

Cell kinetics of the rat seminiferous epithelium following lead acetate treatment

Cell kinetics of the rat seminiferous epithelium following lead acetate treatment

In‐situ quantification of stage‐specific apoptosis in the rat seminiferous epithelium: effects of short‐term experimental cryptorchidism

Two techniques have been combined for quantification of apoptotic germ cells in defined stages of the cycle of the seminiferous epithelium: the improved transillumination method and nonradioactive in-situ end-labelling of DNA (ISEL). Segments of rat seminiferous tubules were squashed between a microscope slide and coverslip, and the stage identified under a phase-contrast microscope. After fixation, apoptotic cells were detected by ISEL and scored per 1 mm tubule. In the normal testis apoptotic cells were found in all stages, the highest frequency occurring in stages XII-XIV (19 cells/mm). In short-term (24 and 48 h) experimentally cryptorchid testes, a significant increase in number of apoptotic germ cells was evident in all stages, except for VI and VIII. Apoptosis of germ cells was confirmed by electrophoresis of radioactively labelled DNA from stages VII-VIII and XIII-I. It is proposed that apoptosis is a means of eliminating the most sensitive germ cells after short-term experimental cryptorchidism.

Changes in Protein Prenylation and Prenyltransferase Activity in the Rat Seminiferous Epithelium during Early Stages of Spermatogenesis1

Changes in protein prenyltransferase activity, levels of prenylated protein, and the type of isoprenoid modification was described in cells of rat seminifereous epithelium and correlated with differentiative events of spermatogenesis. The activity of protein farnesyltransferase (PFT) was at least 10-fold higher than that for protein geranylgeranyltransferase-I (PGGT-I) in seminiferous epithelium and spermatogenic cells of prepubertal rats of different ages. Both activities increased during the meiotic stages of differentiation and peaked at 23 days of age. The activity of farnesyltransferase in seminiferous epithelium was the same as that in mixed spermatogenic cell populations from animals aged 9 and 23 days, indicating that the activity of this enzyme in somatic cells and germ cells was similar at these ages. Farnesyltransferase activities were similar and low in both pachytene spermatocytes and round spermatids from adult rats; however, the activity in pachytene spermatocytes from 23-day old animals was 2-fold higher than in adults. The highest activity was associated with intermediate-sized spermatocytes appearing late during meiosis. PGGT-I activity was at least 10-fold lower than farnesyltransferase activity and was not significantly different among all cell populations. Differentiation-dependent in vivo protein prenylation was demonstrated by labeling of seminiferous epithelium with [3H]mevalonic acid at different prepubertal ages. Total protein prenylation and the ratio of geranylgeranylated to farnesylated protein, in contrast to prenyltransferase activity, decreased with increasing age. Although 20-30-kDa proteins were the most highly labeled at all ages, [3H]-proteins from different-aged prepubertal rats showed age-dependent changes in the level of prenylation of at least 14 proteins as determined by two-dimensional (2D) electrophoresis. Prenylated proteins of round spermatids were distinguished from those of the spermatocytes by the lack of many 20-30-kDa proteins and by low geranylgeranyl/farnesyl (GG/F) ratios. These results show that independent changes in prenyltransferase activity and protein prenylation accompany the differentiation events during the premeiotic and meiotic stages of spermatogenesis. This suggests that prenylation in the seminiferous epithelium may be more dependent on available protein substrate than on protein prenyltransferase activity.

Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate.

The quantitative effects of ethane dimethane sulfonate (EDS), a Leydig cell toxin, on apoptosis in adult rat seminiferous epithelium were studied by the improved transillumination method. Nonradioactive in situ end labeling of fragmented DNA in squash preparations revealed significant increases in apoptotic cells in stages II-XI, whereas controls showed 0.5-2.3 apoptotic cells/mm tubule. Seven days post-EDS treatment, the highest numbers of apoptotic cells were scored in stages VIIab and VIIcd (74.7 +/- 23.8 and 61.3 +/- 16.0 cells/mm, respectively). The effects were suppressed by testosterone (T) supplementation, except in stages II-III and VIIcd. An opposite effect was found in stage XII, where the number of apoptotic cells decreased 1, 3, and 7 days after EDS treatment and returned to control levels in T-supplemented rats. Electrophoretic analysis of internucleosomal DNA fragmentation revealed a biphasic apoptotic process after 1 and 5-7 days due to Leydig and germ cell apoptosis, respectively. The specific germ cell apoptosis was also confirmed by electron microscopic analysis. The results suggest that T withdrawal induces apoptotic cell death in most stages of the cycle and that the effects are largely preventable. In stage XII, however, T seems to promote apoptosis in premeiotic cells.

Expression of Mitochondrial Heat Shock Protein 60 in Distinct Cell Types and Defined Stages of Rat Seminiferous Epithelium1

Changes in the level of the gene transcript of heat shock protein (hsp)60, a mitochondrial chaperonin, during the cycle of rat seminiferous epithelium and its cellular localization were studied. The seminiferous epithelium showed a cell type-specific expression of hsp60. Immunostaining of adult rat testis revealed localization in Sertoli and Leydig cells. In germ cells, mitochondria of spermatogonia and early primary spermatocytes were immunoreactive for hsp60. Mitochondria of all other germ cell types were completely negative for hsp60. Stage-specific expression of hsp60 was determined from pooled segments of stage-specific microdissected tubules by a combination of Western blotting and polymerase chain reaction (PCR). High concentrations of hsp60 were found in stages I-V and IX-XIV, and low levels were detected in the other stages, i.e., VI-VIII. In stages with high hsp60 expression, spermatogonia divide mitotically, whereas in stages lacking mitosis, the hsp60 level was much weaker. In seminiferous epithelium, two different types of mitochondria are present. Therefore, immunoelectron microscopy was used to differentiate these two morphologically distinct types of mitochondria. The crista type of mitochondria (e.g., in Sertoli cells and spermatogonia) reacted with the antibody against hsp60, whereas hsp60 was negative in so-called "condensed"-type mitochondria found in midpachytene spermatocytes and more advanced germ cells. It could be shown for the first time that expression of the hsp60 gene is regulated during the cycle of the seminiferous epithelium. The results indicate that the gene product is primarily needed during the initial steps of spermatogenesis in which most of the cell divisions occur, while its expression during the differentiation of spermatids and sperm is obviously not necessary. The presence of hsp60 in stages with mitotic activity suggests a very active mitochondrial protein import and protein assembly machinery that generates further mitochondria for the dividing cells.

Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium.

Four genes (ratPDE1/IVc, ratPDE2/IVa, ratPDE3/IVd, and ratPDE4/IVb) encoding different isoforms of phosphodiesterase that specifically hydrolyze the second messenger cAMP (cAMP-PDEs) are present in the rat. Previous data from our laboratory indicated that these genes are differentially expressed in the somatic and germ cells of the seminiferous epithelium of the testis. To further characterize their spatial and temporal expression in the seminiferous tubules, in situ hybridization was used to monitor the expression of the four cAMP-PDE messenger RNAs (mRNAs). The signals corresponding to ratPDE1/IVc and ratPDE2IVa mRNAs were localized in two restricted layers of the seminiferous epithelium. The ratPDE1/IVc mRNA was present in a region of the epithelium corresponding to the location of middle-late pachytene spermatocytes. Conversely, the ratPDE2/IVa signal was confined to a more adluminal area corresponding to the location of maturing round spermatids. The ratPDE3/IVd and ratPDE4/IVb mRNAs were distributed throughout the span of the seminiferous epithelium, indicating a localization in the Sertoli cell cytoplasm. Although the intensity of the signal corresponding to ratPDE4/IVb was similar in all seminiferous tubule stages, the ratPDE3/IVd signal varied in intensity in tubules at different stages of the seminiferous cycle. Maximal expression was present in tubules at stages I-V and XI-XIII of the cycle and minimal at stages VIII-IX of the cycle. The expression of the ratPDE3/IVd mRNA positively correlated with the ability of specific inhibitors of the cAMP-PDEs to potentiate the FSH-dependent cAMP accumulation in tubules at different stages of the seminiferous cycle, with maximal potentiation observed at stages II-VI of the cycle. These data demonstrate that different cAMP-PDE genes are active in different cells of the seminiferous tubules and that the ratPDE3/IVd gene is expressed in the Sertoli cell in a cyclical fashion during the seminiferous cycle.

Stage and cell-specific expression of the adenosine 3',5' monophosphate- phosphodiesterase genes in the rat seminiferous epithelium

Stage and cell-specific expression of the adenosine 3',5' monophosphate- phosphodiesterase genes in the rat seminiferous epithelium

Function of interleukin-6 as an inhibitor of meiotic DNA synthesis in the rat seminiferous epithelium

Interleukin-6 bioactivity (IL-6) has been shown to be present in Sertoli cells. To further characterize the IL-6 in the seminiferous epithelium, the IL-6 like-antigen was detected, stage-specific basal distribution of IL-6-like bioactivity and its regulation by FSH, cAMP and TPA was characterized in isolated, rat seminiferous tubule segments. In addition, the effects of human recombinant IL-6 on stage-specific DNA synthesis was investigated. Both monoclonal and polyclonal antibodies recognized M r 22 and 23 kDa of IL-6 like immunoreactivity in the seminiferous epithelium. The basal IL-6 production showed high levels during stages XIII-XIV-I-V, low during VII and VIII. FSH stimulated IL-6 production at nearly all stages and most significantly at stage VII of the cycle. Human recombinant IL-6 dose-dependently inhibited the onset of meiotic DNA synthesis of preleptotene spermatocytes, and a minor inhibition was found on advanced (A 3-type B) spermatogonia. These results support the hypothesis that IL-6 is a stage-specific paracrine regulator of the seminiferous epithelium exerting a specific inhibitory action on meiotic DNA synthesis.

Integrin Receptor α6β1 is Localized at Specific Sites of Cell-to-Cell Contact in Rat Seminiferous Epithelium1

With the aim of investigating the presence and role of integrin receptors in cell-to-cell interactions during spermatogenesis, we have immunolocalized alpha 6A integrin chain in the rat seminiferous epithelium. In both prepubertal and adult seminiferous epithelium, the antigen was found to be restricted to definite sites of intercellular contact, following a stage-specific distribution almost invariably identical to that known for beta 1 chain. In the adult seminiferous epithelium, positivity for both antigens was found exclusively around the profiles of elongating and maturing spermatids and, in most but not all stages, at a characteristic suprabasal position. In the prepubertal rat, the antigen is localized along a very regular suprabasal line of intercellular contacts. In immunoprecipitation experiments on both seminiferous epithelium explants and Sertoli cell cultures from 3-wk-old rats, anti-alpha 6A antibody coprecipitates a polypeptide of 118 kDa, presumably corresponding to beta 1 chain. These data strongly suggest that the integrin heterodimer alpha 6A beta 1 is expressed at sites of intercellular contact in the rat seminiferous epithelium. The stage-specific and restricted pattern observed by immunofluorescence suggests that this integrin is involved in regulatory interactions between Sertoli cells and germ cells during spermatogenesis.

Hormonal regulation of plasminogen activator in rat and mouse seminiferous epithelium.

To elucidate the possible role of plasminogen activator (PA) in spermatogenesis and spermiation in mammals, we studied the hormonal regulation of PA secretion in cultured rat and mouse seminiferous tubules during defined stages of spermatogenesis. Results indicated that: (1) under basal conditions, segments of rat seminiferous tubules released primarily urokinase-type PA (uPA) at all stages of the cycle. The highest level of PA secretion occurred at stages VIIab, VIIcd and VIII. FSH, 8-bromo cyclic AMP and forskolin (FK) stimulated PA secretion, predominantly tissue-type PA (tPA). (2) In contrast, mouse seminiferous tubules secreted only tPA under basal conditions. In the presence of 50 microM MIX, seminiferous tubules at stages VII and VIII secreted higher levels of both types of PA than at the other stages. Both tPA and uPA secretion was enhanced by addition of FSH and FK to the organ culture media. (3) Segments of both rat and mouse seminiferous tubules at stages IX-XII in which the sperm residual bodies are absorbed into the Sertoli cells were also very sensitive to the addition of FSH to the organ culture. These results suggest that tPA in rat and mouse testes may play an essential role in the process of spermatogenesis and spermiation as well as in sperm residual body absorption.

Localization of mRNAs by in-situ hybridization to the residual body at stages IX-X of the cycle of the rat seminiferous epithelium: fact or artefact?

Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in-situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB). In the present study testicular tissue, perfusion-fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII-X were clearly distinguishable. RNA content of the RBs was demonstrated at stages VIII-X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using 35S-labelled and digoxigenin-labelled riboprobes (activin receptor-II, alpha-inhibin, transferrin, androgen-binding protein (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and 2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), and digoxigenin-labelled oligonucleotide probes (transition protein-1 (TP-1), TP-2 and protamine-1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2, cystatin C and alpha-inhibin) showed hybridization to basally located residual bodies in tubules at stages IX-X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies. A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK- vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC-rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB. It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC-rich fragments of RNA.

MIP-1α is a regulator of mitotic and meiotic DNA synthesis during spermatogenesis

To find out whether macrophage inflammatory protein-1α (MIP-1α) has a role in the regulation of germ cell development, we studied its effects on spermatogenic stage-specific DNA synthesis in vitro. MIP-1α increased the DNA synthesis of primitive type A 2–4 spermatogonia and of premeiotic cells, whereas the DNA synthesis of more differentiated intermediate and type B spermatogonia was inhibited when cultured in the presence of MIP-1α. An antibody against MIP-1α cross-reacted with a protein of 15 kDa from every spermatogenic stage of rat seminiferous epithelium. Immunohistochemical studies with the same antibody revealed a complex pattern of MIP-1α localization both in primitive and advanced spermatogenic cells. These observations suggest that MIP-1α is a local regulator of mitotic and meiotic DNA synthesis.