Unscheduled DNA synthesis assay in mammalian spermatogenic cells: an update

The unscheduled DNA synthesis (UDS) assay measures DNA repair in response to DNA damage. To date, 59 chemicals plus UV and X rays have been tested for UDS in spermatogenic cells of humans, rabbits, rats, and mice. In vivo, in vitro, and combined in vivo/in vitro procedures have been used. UDS has been shown to occur in spermatogonia, meiotic spermatocytes, and early spermatid stages. Fifty-nine percent of the agents tested gave a positive UDS response in one or more germ-cell stages. Results show 95% concordance (positive or negative) between different mammalian species. Some well-known genotoxic chemicals, for example, aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), did not induce significant levels of UDS. Possible explanations are discussed. Results from the UDS assay were compared with those from the mouse specific-locus mutation (SLM) test to determine correlations between the two assays. Only two chemicals, ethyl- and methyl-nitrosourea (ENU and MNU), have been tested for UDS and SLM induction in spermatogonial stages. Results show full concordance between the two assays. In postspermatogonial stages, 25 chemicals and X rays have been tested for UDS and SLM induction. Seventy-seven percent of these agents showed similar results (positive or negative) in these germ-cell stages. Although the UDS assay cannot replace the SLM test, the strong correlations between the two assays suggest the usefulness of the UDS assay as a predictor of germ-cell mutations in mammalian systems.     

In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n‐ethyl‐n‐nitrosourea (ENU), 6‐mercaptopurine (6‐MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration‐dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration‐related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6‐MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real‐time PCR results showed that the mRNA level expression of p53 increased and bcl‐2 decreased significantly with the increasing ENU, 6‐MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6‐MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6‐MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99–107, 2017. © 2017 Wiley Periodicals, Inc.     

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.     

Early G1 in the male rat meiotic cell cycle is hypersensitive to N-methyl-N-nitrosourea-induced micronucleus formation

The effects of the known carcinogenic and teratogenic agentN-methyl-N-nitrosourea (MNU) were studied on male rat meiosis.To examine possible cell-cycle delay, an immunohistochemicaltechnique based on 5-bromo-2'-deoxyuridine (BrdU) labellingof S-phase cells was developed. BrdU tablets were implantedsubcutaneously in adult male rats. A single i.p. injection of10 mg/kg of MNU was given simultaneously. After 16–22days, preparations of stage 1 of the seminiferous epitheliumwere made and stained immunohistochemically using anti-BrdUantibodies. MNU did not cause any significant meiotic delay,but did cause a slight non-significant reduction of the percentagesof BrdU-labelled step 1 spermatids at 18 days (80%) comparedto controls (95%). In addition, the induction of meiotic micronucleiwas studied after short (1–3 days: late meiotic stages)and long (16–22 days: early spermatocytes and B spermatogonia)exposure times. The peak induction occurs between 21 and 20days, indicating that the M–G₁ transition or the verybeginning of G₁ of the cell cycle of primary spermatocytes arethe most sensitive stages to the action of MNU. The number ofstep 1 spermatids decreased dramatically in animals treatedfor 22 days, denoting a highly toxic effect on type-B spermatogonia.No unscheduled DNA synthesis was detected in any meiotic stageof spermatogenesis by using this BrdU labelling method. Theresults indicate that the spermatid micronucleus test basedon microdissection of seminiferous tubules can accurately pointout the most sensitive stage for chemically induced clastogenesis.More-over, the BrdU-immunohistochemical application enablesthe simultaneous study of cell cycle kinetics. The probablereasons for hypersensitivity of spermatocytes to MNU in thebeginning of G₁ are discussed. ¹To whom correspondence should be addressed     

Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA

A study of meiotic and postmeiotic germ-cell-stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid-spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg. By using 14C-labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.     

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.     

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.     

Validation studies with the micronucleus test for early spermatids of rats. A tool for detecting clastogenicity of chemicals in differentiating spermatogonia and spermatocytes.

Male Wistar rats were given a single i.p. injection with different doses of ethylnitrosourea, mitomycin C, methyl methanesulphonate, cyclophosphamide or vincristine sulphate. Clastogenic damage induced in differentiating spermatogonia and spermatocytes was measured by counting micronuclei in derived early spermatids. At dose levels not resulting in cell death of resting spermatocytes, all chemicals--with the exception of vincristine--induced most of the damage in G1- and S-phase of primary spermatocytes (also called resting, pre-leptotene or pre-meiotic spermatocytes). However, at doses causing death of G1- and S-phase spermatocytes, high frequencies of micronuclei may be observed in early spermatids derived from spermatocytes treated in diplotene, diakinesis and MI and II. This is exemplified by our results with ethylnitrosourea. In our experience, the most sensitive stage of primary spermatocyte development (i.e. G1- and S-phase cells) can best be sampled 20 days after treatment. This is the optimal time interval for demonstrating the clastogenic potential of low or moderate doses of a test chemical in meiotic male germ cells of rats. The optimal sampling time for the detection of typical spindle poisons remains to be established. In general, at low or moderate dose levels, smaller or negligible amounts of chromosomal damage were induced in differentiating spermatogonia, in spermatocytes in meiotic prophase and in dividing primary or secondary spermatocytes. For obvious reasons, the micronucleus test for early spermatids cannot be used to detect clastogens which act exclusively on postmeiotic male germ cells.     

Degeneration of germ cells in normal,hypophysectomized and hormone treated hypophysectomized rats

In normal adult rats some germ cells degenerate at several vulnerable steps of spermatogenesis. These are the type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which degenerate during their respective maturation divisions and step 7 and 19 spermatids. In the present study, these degenerating cells were examined under the electron microscope, and their frequency was determined in toluidine blue stained semithin sections of testes from normal, hypophysectomized (at 5.5 days after operation) and hypophysectomized rats injected with FSH and LH separately or in combination. With the exception of the step 19 spermatids, the degenerating germ cells underwent necrosis in vacuolated spaces delimited by Sertoli cells. In the case of the affected step 19 spermatids, an apical cytoplasmic process of the Sertoli cell initially ensheathed a long segment of their flagellum, and then each degenerating cell was drawn deep in the seminiferous epithelium where it was phagocytozed by the Sertoli cell. Soon after hypophysectomy the incidence of degenerating mid-pachytene spermatocytes, step 7 and 19 spermatids which are present in stages VII or VIII of the cycle of the seminiferous epithelium, increased significantly. In contrast the number of degenerating primary or secondary spermatocytes during the meiotic divisions seen in stage XIV of the cycle or of any other germinal cell was not significantly modified. While the injection of FSH alone had no influence on the number of degenerating cells in hypophysectomized rats, injections of LH at the two doses administered (0.7 μg or 20 μg) reduced significantly the number of degenerating cells seen in stages VII-VIII of the cycle; combined injections of FSH and LH (20 μg) reduced the number of these degenerating cells to the normal low values. Thus it appeared that the mid-pachytene spermatocytes and the step 7 and 19 spermatids, all present in the adluminal compartment of the seminiferous epithelium in stages VII or VIII of the cycle, were more sensitive to the presence of absence of gonadotropic hormones than the other germ cells present in the seminiferous epithelium.     

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Protein expression and cell organelle behavior in spermatogenic cells

Spermatogenic cells stage-specifically produce a wide variety of proteins during spermatogenesis, wherein protein expression is coordinated with cell organelle behavior. It has been shown that the Golgi apparatus and the endoplasmic reticulum (ER) are uniquely coordinated with the expression of an immunoglobulin super-family protein, flagellar plasma membrane MC31 (MC31/CE9), and a molecular chaperone, calmegin, respectively. When the Golgi apparatus begins to generate sperm components in the primary spermatocytes, it actively engages in producing proteins for the acrosome in round spermatids and for the flagellum in elongating spermatids. Structurally, the Golgi apparatus is reduced in size during meiotic division, moves from the apical to the basal region (cytoplasmic lobe) when spermatids differentiate from round to elongating phase, and then collapses in the late maturation phase. The ER is distributed uniformly over the entire cytoplasm of spermatocytes and round spermatids, and then moves distally toward the cytoplasmic lobe along the bundles of microtubule, called the manchette, in elongating spermatids. The ER is resorbed into the radial body in late maturation spermatids. MC31/CE9 expresses strong immunostaining twice on the Golgi apparatus during spermatogenesis, first in early pachytene spermatocytes and then in early elongating spermatids. Calmegin expression exactly parallels ER behavior. This mini-review focuses on the unique relationships in spermatogenic cells, particularly those between protein expression and cell organelle behavior.     

一种利用吖啶橙染色快速鉴定重力密度梯度沉降法中生精细胞类型的方法

目的建立一种重力梯度沉降法分离粗线期精母细胞,圆形及长形精子细胞的快速精确的镜检方法。方法利用重力密度梯度沉降法分离,将收集到的每管细胞分别放入96孔板中,每孔加入一定浓度的吖啶橙染液,于荧光显微镜下观察。结果通过分析对应荧光通道下3种生精细胞的细胞核/细胞质染色结果,可快速精确的分辨出粗线期精母细胞,圆形及长形精子细胞。结论建立了一种在重力密度梯度沉降法分离生精细胞过程中快速镜检检测粗线期精母细胞,圆形及长形精子细胞的方法。     

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.     

CD147 is required for matrix metalloproteinases-2 production and germ cell migration during spermatogenesis

Spermatogenesis is a highly programmed process that requires the degradation of the extracellular matrix and the remodeling of tight junctions (TJ) to facilitate differentiating germ cell migration. Matrix metalloproteinases (MMPs) are essential in regulating Sertoli cell TJ in the testis. CD147 is known to stimulate the production of MMPs in tumor metastasis and its knockout mice are infertile. However, the functional relationship between CD147 and MMPs in spermatogenesis has not been investigated. In the present study, we examined the expression profile of CD147 and MMPs during mouse testicular development by RT-PCR, western blot and immunofluorescence staining. We also examined CD147 involvement in the production of MMP-2 and the migration of germ cells (GC-1 and GC-2 cells) using CD147 antibody or synthetic microRNA mimics-mediated knockdown. The results showed that CD147 was present at all stages of testicular development from 7 to 56 days post-partum (dpp). CD147 expression was found to increase after 21 days from moderate levels in 7 and 14 days. Of the eight MMPs studied, MMP-2, MMP-7, MMP-9 and MMP-23 were detected to have changes in expression during testicular development, with MMP-2 showing the largest change. CD147 and MMP-2 were co-localized in spermatogonia, spermatocytes and round spermatids in mouse testis, while in human testis, they were co-localized in spermatocytes and round spermatids. MMP-2 expression and migration of GC-1 and GC-2 cells were reduced by interfering with CD147 expression and function in vitro. These data suggest that CD147 regulates migration of spermatogonia and spermatocytes via induction of MMP-2 production during spermatogenesis.     

The ultrastructure of the pre-meiotic and meiotic stages of spermatogenesis in Plagioscion squamosissimus (Teleostei,Perciformes, Sciaenidae)

Spermatogenesis of 'corvina' P. squamosissimus starts from a stem cell that gives rise to germ cells. These cells are enveloped by Sertoli cells, forming cysts. The germ cells in the cysts are all at the same stage of development and are interconnected by cytoplasmic bridges. Spermatogonia are the largest germ cells. In the cysts, these cells differentiate into primary spermatogonia and secondary spermatogonia. The primary spermatogonia are isolated in the cyst and give rise to the secondary spermatogonia. After several mitotic divisions, they produce spermatocytes I, which can be identified by synaptonemal complexes in the nucleus. The spermatocytes I enter the first phase of meiosis to produce the spermatocytes II. These are not very frequently seen because they rapidly undergo a second phase of meiosis to produce spermatids.     

DNA-damaging potential of diethylstilbestrol evaluated in the germ cell unscheduled dna synthesis assay

Despite the fact that the nonsteroidal estrogen diethylstilbestrol (DES) exerts its toxic effects primarily on the reproductive system, little is known about the possible interference of this compound with germ cell DNA. The measurement of unscheduled DNA synthesis (UDS) in spermatocytes and early spermatids of mice germ-cells is a valid indicator for the DNA-damaging potential of a compound. UDS occurrence was thus determined after IP administration of 10, 30, 60 or 180 mg/kg DES to male mice. Tritiated thymidine ([³H]dThd) was then injected into the testes, the spermatozoa were serially collected, the sperm heads isolated, and UDS determined by the amount of [³H]dThd incorporation. [³H]dThd measurements in germ cells of mice which were treated with 10 mg/kg DES were comparable to those of the controls. Higher incorporation of [³H]dThd, indicating UDS, was measured in sperm cells which had been spermatocytes at the time of treatment with 30 and 60 mg/kg DES; this increase was statistically significant at 60 mg/kg. Administration of 180 mg/kg DES caused [³H]dThd incorporation which was comparable to that of the controls, suggesting that DES interfered with repair mechanisms or delayed spermatogenic cycles at high dose levels. General toxicity was manifested in a dose-dependent decrease of the sperm cell numbers in the spermatogenic stages investigated. This study provides evidence that DES, or its metabolite(s), reached the germ cells of adult mice in sufficient amounts to produce DNA damage. The levels of radioactivity measured were comparable to those measured after cyclophosphamide treatment, but [³H]dThd incorporation was about 10 times less than in methylmethane sulfonate-treated animals.     

Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.     

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4±3.6 µm (February) and 216.8±9.2 µm (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm² of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.