Rat spermatogenesis in vitro traced by quantitative flow cytometry

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Immunohistochemical localization of urokinase-type plasminogen activator in Sertoli cells and tissue-type plasminogen activator in spermatogenic cells in the rat seminiferous epithelium

The occurrence of plasminogen activators of the urokinase-type (u-PA) and tissue-type (t-PA) at various stages of the epithelial cycle was studied immunohistochemically in rat seminiferous tubule segments. u-PA immunoreactivity was detected exclusively at stages VII and VIII in Sertoli cells, displaying a distinct granular cytoplasmic staining. t-PA immunoreactivity was found during mid- and late pachytene and diakinesis (stages VII-XIII) in spermatogenic cells, displaying a granular cytoplasmic staining with maximal intensity in stages IX-XIII. The specificity of the stainings was supported by staining controls, including absorption of the antibodies with purified preparations of the activators. It was also supported by zymographic studies of the occurrence of u-PA and t-PA in extracts of tubular segments at different stages of the cycle, isolated by transillumination-assisted microdissection. The possible functions of the two types of plasminogen activators in the seminiferous epithelium are discussed.     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

Seasonal changes in spermatogenic cell degeneration in the seminiferous epithelium of adult Japanese macaques (Macaca fuscata fuscata)

The degenerating pattern of spermatogenic cells in the seminiferous tubule of Japanese macaques was studied to clarify a relationship between seasonal changes of reproductive performances and cytological findings in the Japanese macaque. For light microscopy, testis samples were obtained from five adult animals by biopsy in April (nonmating season) and October (mating season). For electron microscopy, specimens from four additional macaques were used. Degenerating cells were found in all steps of spermatogenesis. In stages I to V of the cycle of the seminiferous epithelium, morphologically atypical pachytene spermatocytes were observed in 14.7 and 10.0% of the cells in the nonmating and mating seasons, respectively, although the difference in percentage was not significant. Mature spermatids with atypical features in those stages occupied 59.6% of the cells in the nonmating season, which significantly decreased to 34.1% in the mating season. These results imply that the seasonal change of sperm production is related, at least in part, to the process of degeneration of the spermatogenic cells in this species.     

DNA flow-cytometric analysis of testicular germ cell populations of the bonnet monkey (Macaca radiata) as a function of sexual maturity

Testicular germ cell populations of biopsies from 32 male bonnet monkeys in 5 different age groups were quantitated in a flow cytometer after labelling of germ cell DNA with the specific fluorochrome, 4,6-diamidino phenyl indole. The 5 quantifiable populations were spermatogonia (2C), preleptotene spermatocytes (S phase), primary spermatocytes (4C), round spermatids (1C) and elongate spermatids (HC). The seminiferous tubules of immature 3-4-year-old monkey had only Sertoli cells and spermatogonia (2C). At 5-6 years, germ cells in S-phase (9.5%), 4C (11.1%), 1C (41.8%) and HC (17.1%) stages of maturation appeared for the first time but at 7-8 years of age and beyond all cell types except HC decreased while 1C remained relatively constant. Histometric analysis correlated well with the flow-cytometric data. The decrease in cells of 2C, S-phase and 4C stages was associated with an increase in mitotic index, signifying acceleration in the kinetics of germ cell transformation into subsequent cell types. The total turnover in cell transformation (1C:2C) was significantly (P less than 0.01) increased at and beyond 7-8 years. Maximum transition from 2C to 4C occurred at 5-6 years (4C:2C ratio 0.8 at 5-6 years and 0.6 at 7-8 years). The ratio HC:1C (kinetics of cell transformation during spermiogenesis) attained near total efficiency only by 10 years of age (1.08 at 10-14 years; 0.9 at 18-20 years). Also, the cell associations within the seminiferous tubules of monkeys greater than or equal to 10 years of age were better defined than those of younger animals. The changes in germ cell ratios correlated well with alterations in testicular volume, sperm numbers in the ejaculate and surges of testosterone and increments in FSH in the serum, characteristic of development of sexual maturity. It is apparent from this study that DNA flow cytometry of testicular germ cell populations reveals subtle changes in spermatogenic status of bonnet monkeys with a high degree of sensitivity.     

Factors affecting spermatogenesis in the stallion

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells) and germ cells (spermatogonia, spermatocytes, and spermatids). Activities of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number to haploid and yield four spermatids. Spermiogenesis is the differentiation without division of spherical spermatids into mature spermatids which are released from the luminal free surface as spermatozoa. The spermatogenic cycle (12.2 days in the horse) is superimposed on the three major divisions of spermatogenesis which takes 57 days. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest seasonal impact on horses occurs during spermatocytogenesis. Daily spermatozoan production is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells and Leydig cell number are related to spermatozoan production. Seminiferous epithelium is sensitive to elevated temperature, dietary deficiencies, androgenic drugs (anabolic steroids), metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases. However, these different factors may elicit the same temporary or permanent response in that degenerating germ cells become more common, multinucleate giant germ cells form by coalescence of spermatocytes or spermatids, the ratio of germ cells to Sertoli cells is reduced, and spermatozoan production is adversely affected. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon. Several extrinsic factors can influence spermatogenesis to cause a similar degenerative response of the seminiferous epithelium and reduce fertility of stallions.     

Seasonal changes in the spermatogenic epithelium of adult Japanese macaques (Macaca fuscata fuscata)

A histological study was undertaken to clarify seasonal changes in the spermatogenic epithelium of Japanese macaques. Testicular tissue samples were excised by biopsies from five adult laboratory-maintained males in mating and non-mating seasons. The samples were fixed with Bouin's solution, embedded in paraffin, and stained with PAS and hematoxylin. Microscopic observations on cross-sections of seminiferous tubules revealed that the seminiferous epithelium in the mating season was thicker than in the non-mating season. PAS-stained granules were found in some of the dark A-type spermatogonia, which significantly increased in the non-mating season. Spermatids of the steps preceding the appearance of the acrosomic cap in stages I to III were observed significantly more often than those in the step coinciding with the formation of the acrosomic cap in stage IV. In stage I, the ratio of mature spermatids or spermatozoa to immature spermatids in the mating season was higher than that in the non-mating season. These findings suggest that spermiogenesis, as well as spermatocytogenesis, is inhibited in the non-mating season.     

Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca)

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by “A” spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.     

The cycle of the seminiferous epithelium in Landrace boars

The present study describes the morphological features of the eight stages of the seminiferous epithelium in Landrace boars according to the tubular morphology method, as well as their relative frequency, length, and duration. In Landrace boars the pre-meiotic stages occupied the 31.9 +/- 19.9% of the spermatogenic cycle and had a total length of 1788.8 +/- 1153.0 microm and a duration of 2.78 days; they were mainly characterised by the presence of leptotene and pachytene spermatocytes and round spermatids. Meiotic stages, with a relative frequency of 16.4 +/- 6.8%, a length of 787.1 +/- 603.1 microm and a duration of 1.41 days, contained spermatocytes in advanced meiosis I and/or in meiosis II and elongating spermatids grouped in bundles. Post-meiotic stages occupied the 50.6 +/- 20.4% of the spermatogenic cycle and had a length of 2096.8 +/- 1175.0 microm and a duration of 4.37 days; the most important event of these stages was the spermiation, which included the complete remodelling of sperm head and tail and the releasing of spermatozoa into the lumen, as well as the formation of residual bodies. From data obtained we concluded that both germ cell associations of the stages maintain constant among Landrace boars, and that the relative frequency, length and duration of the stages were directly dependent of the cytological transformations on the seminiferous tubules.     

Characterization of the testicular cell types present in the rat by in vivo 31P magnetic resonance spectroscopy.

Testes of vitamin A-deficient Wistar rats before and after vitamin A replacement, of rats irradiated in utero, and of control rats were investigated by in vivo 31P magnetic resonance (MR) spectroscopy. The testicular phosphomonoester/ATP (PM/ATP) ratio ranged from 0.79 +/- 0.05 for testes that contained only interstitial tissue and Sertoli cells to 1.64 +/- 0.04 for testes in which spermatocytes were the most advanced cell types present. When new generations of spermatids entered the seminiferous epithelium, this ratio decreased. The testicular phosphodiester/ATP (PD/ATP) ratio amounted to 0.16 +/- 0.06 for testes in which Sertoli cells, spermatogonia, or spermatocytes were the most advanced cell type present. When new generations of spermatids entered the seminiferous epithelium, the PD/ATP ratio rapidly increased and finally reached a value of 0.71 +/- 0.06 for fully developed testes. Taken together, specific patterns of the PM/ATP ratio, the PD/ATP ratio, and pH were obtained that were correlated to the presence of spermatogonia, spermatocytes, round spermatids, and elongated spermatids or to the absence of spermatogenic cells. Hence, a good impression of the status of the seminiferous epithelium in the rat can be obtained by in vivo 31P MR spectroscopy.     

Regulated expression and ultrastructural localization of galectin-1, a proapoptotic beta-galactoside-binding lectin,during spermatogenesis in rat testis

Galectin-1, a highly conserved beta-galactoside-binding protein, induces apoptosis of activated T cells and suppresses the development of autoimmunity and chronic inflammation. To gain insight regarding the potential role of galectin-1 as a novel mechanism of immune privilege, we investigated expression and ultrastructural localization of galectin-1 in rat testis. Galectin-1 expression was assessed by Western blot analysis and immunocytochemical localization in testes obtained from rats aged from 9 to 60 days. Expression of this carbohydrate-binding protein was developmentally regulated, and its immunolabeling exhibited a stage-specific pattern throughout the spermatogenic process. Immunogold staining using the anti-galectin-1 antibody revealed the typical Sertoli cell profile in the seminiferous epithelium, mainly at stages X-II. During spermiation (stages VI-VIII), a strong labeling was observed at the luminal pole of seminiferous epithelium, localized on apical stalks of Sertoli cells, on heads of mature spermatids, and on bodies of residual cytoplasm. Moreover, spermatozoa released into the lumen showed a strong immunostaining. Following spermiation (stage VIII), galectin-1 expression was restored at the basal portion of Sertoli cells and progressively spread out through the whole cells as differentiation of germinal cells proceeded. Immunoelectron microscopy confirmed distribution of galectin-1 in nuclei and cytoplasmic projections of Sertoli cells and on heads and tails of late spermatids and residual bodies. Surface localization of galectin-1 was evidenced in spermatozoa from caput epididymis. Thus, the regulated expression of galectin-1 during the spermatogenic cycle suggests a novel role for this immunosuppressive lectin in reproductive biology.     

Quantity rather than quality in teratospermic males: a histomorphometric and flow cytometric evaluation of spermatogenesis in the domestic cat (Felis catus)

Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs within the Felidae, including certain domestic cats, but the cellular and molecular mechanisms that give rise to this phenomenon remain unknown. This study quantified spermatogenesis to identify differential dysfunctions in teratospermic versus normospermic (>60% normal sperm/ejaculate) domestic cats. Sperm used were from electroejaculates and cauda epididymides. Testes from 10 normo- and 10 teratospermic males were obtained by castration and then evaluated by histomorphometry, flow cytometry, and testicular testosterone enzyme immunoassay. Some morphometric traits (tubular diameter, epithelium height, interstitial area, number of Leydig cells, and blood vessels per cross-section) as well as testicular testosterone concentrations were similar between groups, but testicular volume was greater in teratospermic males. Stage frequencies differed also between both cat populations, suggesting possible dysfunctions in spermiation. Quantification of cell populations in most frequent stages revealed more spermatogenic cells and fewer Sertoli cells per tubule cross-section as well as per tissue unit in teratospermic donors. Hence, the ratio of spermatogenic cells per Sertoli cell was elevated in the teratospermic cat. DNA flow cytometry confirmed higher total spermatogenic and meiotic transformations in teratospermic males. In summary, compared with normospermic counterparts, teratospermic cats have a higher sperm output achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during spermatogenesis. Gains in sperm quantity are produced at the expense of sperm quality.     

Single administration of di(n-butyl) phthalate delays spermatogenesis in prepubertal rats

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3 h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2-4, while those of the control were steps 12-13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8-9, while those of the control were steps 15-19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.     

兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

Cytology of the human seminiferous epithelium

The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.     

Gonadotropin receptor regulation in hypophysectomized rat Leydig cells.

The number of gonadotropin receptors decrease in the Leydig cells following hypophysectomy (hypox). The receptor number is reduced to 52, 48, 11, 10 and 12 % of the control 8 days following hypophysectomy in 40, 50, 60, 70 and 80 days old rats respectively. hCG injection (0.6 or 30 μg) produces a decrease in the receptor number in 58 days old hypox rat. Receptors remain almost undetectable between 24 to 72 hours following hCG injection. Desensitization to hCG is observed between 12 and 48 hours and full responsiveness to hCG is obtained at 60 hours following hCG injection (0.6 μg).The results demonstrate that LH is not a necessary condition for the presence of gonadotropin receptors in the Leydig cells and that hCG induces the “down regulation” of the receptors and the desensitized state as well in the hypox as in the intact animal. They also indicate that a variation in the number of gonadotropin receptors is probably not the major biochemical alteration esponsible for steroidogenic refractoriness in Leydig cells.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.