Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.     

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.     

Seasonal changes in the expression of PACAP,VPAC1, VPAC2, PAC1 and testicular activity in the testis of the muskrat (Ondatra zibethicus)

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the steroidogenesis and spermatogenesis in the testis through its receptors PAC1, VPAC1 and VPAC2. In this study, we investigated the seasonal expressions of PACAP, PAC1, VPAC1, VPAC2, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP17A1 in the testis of male muskrat during the breeding season and non-breeding season, respectively. Histologically, we observed the presence of Leydig cells, Sertoli cells and various types of germ cells in the testis during the breeding season, yet only Leydig cells, Sertoli cells, spermatogonia and primary spermatocyte during the non-breeding season. In addition, the immunohistochemical localizations of PACAP and VPAC1 were identified in the Leydig cells, spermatogonia and spermatozoa during the breeding season, while only in the Leydig cells and spermatogonia during the non-breeding season, and PAC1 and VPAC2 were localized in the Leydig cells in both seasons, among which LHR, StAR, 3β-HSD and CYP17A1 were also expressed. Meanwhile, the protein and mRNA expression levels of PACAP, PAC1, VPAC1, VPAC2, LHR, FSHR, StAR, 3β-HSD and CYP17A1 in the testis during the breeding season were significantly higher than those during the non-breeding season. These results suggested that PACAP is involved in the regulation of steroidogenesis and spermatogenesis via an endocrine, autocrine or paracrine manner in the testis of muskrat.Key words: Pituitary adenylate cyclase-activating peptide (PACAP), PACAP receptors, steroidogenesis, testis, Ondatra zibethicus.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Dual neofunctionalization of a rapidly evolving aquaporin-1 paralog resulted in constrained and relaxed traits controlling channel function during meiosis resumption in teleosts

The preovulatory hydration of teleost oocytes is a unique process among vertebrates. The hydration mechanism is most pronounced in marine acanthomorph teleosts that spawn pelagic (floating) eggs; however, the molecular pathway for water influx remains poorly understood. Recently, we revealed that whole-genome duplication (WGD) resulted in teleosts harboring the largest repertoire of molecular water channels in the vertebrate lineage and that a duplicated aquaporin-1 paralog is implicated in the oocyte hydration process. However, the origin and function of the aquaporin-1 paralogs remain equivocal. By integrating the molecular phylogeny with synteny and structural analyses, we show here that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication rather than WGD and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut, a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. We demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. We further show that the rapid structural divergence of the C-terminal regulatory domain causes ex vivo loss of function of halibut Aqp1ab when expressed in amphibian oocytes but not in zebrafish or native oocytes. However, by using chimeric constructs of halibut Aqp1aa and -1ab and antisera specifically raised against the C-terminus of Aqp1ab, we found that this cytoplasmic domain regulates in vivo trafficking to the microvillar portion of the oocyte plasma membrane when intraoocytic osmotic pressure is at a maximum. Interestingly, by coinjecting polyA(+) mRNA from postvitellogenic halibut follicles, ex vivo intracellular trafficking of Aqp1ab is rescued in amphibian oocytes. These data reveal that the physiological role of Aqp1ab during meiosis resumption is conserved in teleosts, but the remarkable degeneracy of the cytoplasmic domain has resulted in alternative regulation of the trafficking mechanism.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.     

Ultrastructural changes in the seminiferous epithelium of two seasonally reproducing bats (Mammalia: Chiroptera)

The Sertoli cells of the Cape horseshoe bat (Rhinolophus capensis) and Schreiber's long-fingered bat (Miniopterus schreibersii) undergo marked changes in ultrastructure related to stages in the spermatogenic cycle. The amount of lipid stored in the Sertoli cells varies annually and is at a maximum from just after spermiation to early in the following spermatogenic cycle. During spermatogenesis, the diameter of the lipid droplets decreases, reaching a minimum prior to spermiation. Sertoli cells exhibit a marked apicobasal differentiation, particularly in the vicinity of developing late spermatids, where the cytoplasm of the Sertoli cell is packed with smooth endoplasmic reticulum. The possible roles of lipid droplets and smooth endoplasmic reticulum. The possible roles of lipid droplets and smooth endoplasmic reticulum in steroidogenesis by Sertoli cells are discussed. Junctional complexes occur between Sertoli cells and spermatogonia, are apparently absent from between Sertoli cells and spermatocytes, and are restricted to the region of the developing acrosome in the spermatids. Annulate lamellae, which occur commonly in the developing germinal cells and less frequently in the Sertoli cells, may be associated with the production of microtubules, which are present in both spermatids and Sertoli cells.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

A testis-specific serine protease,Prss41/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis

The function of protease during male meiosis has not been well studied. We previously cloned and characterized four testis-specific serine proteases in the mouse testis. One of the proteases, Prss41/Tessp-1, was expressed in the germ and Sertoli cell. This time, to examine the involvement of Prss41/Tessp-1 in spermatogenesis, we conducted the organ culture of testis fragments in the presence of the anti-Prss41/Tessp-1 antibody. Because in the Sertoli cell, the Prss41/Tessp-1 protein was mostly associated with the membrane of intracellular organelles by glycosylphosphatidylinositol, the antibody was expected to affect Prss41/Tessp-1 at the plasma membrane of spermatogonia. By adding the antibody, the number of germ cells was decreased in some seminiferous tubules. The marker genes expression strongly suggested that meiosis was arrested at spermatogonia, and the number of apoptotic germ cells increased by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. These data indicated that Prss41/Tessp-1 was necessary for the progression of meiosis at the stage of spermatogonia during in vitro spermatogenesis. Together with our previous study, the current results suggest that the Prss/Tessp proteases are important for the progression of meiosis at each stage.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.     

Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post-natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/W ^v mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild-type mice with an anti-c- kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage- and developmental-specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Ameboid cells in spermatogenic cysts of caecilian testis

Sertoli cells constitute a permanent feature of the testis lobules in caecilians irrespective of the functional state of the testis. The developing germ cells are intimately associated with the Sertoli cells, which are adherent to the basal lamina, until spermiation. There are irregularly shaped cells in the cores of the testis lobules that interact with germ cells at the face opposite to their attachment with Sertoli cells. These irregularly shaped (ameboid) cells first appear in the lumen of the cysts containing primary spermatocytes and are continually present until spermiation. We did not observe any cytoplasmic continuity between a Sertoli cell and an ameboid cell. Both light microscopic and TEM observations reveal a phagocytic role for the ameboid cells: they scavenge the residual bodies shed by spermatozoa. Organization of the ameboid cells is grossly different from that of the spermatogenic and Sertoli cells. They appear to develop from the epithelium at the juncture of the collecting ductule with the testis lobule.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.