Compensatory upregulation of myelin protein zero-like 2 expression in spermatogenic cells in cell adhesion molecule-1-deficient mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.     

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.     

Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis

Spermatogenesis is a complex process of terminal differentiation by which mature sperms are generated,and it can be divided into three phases:mitosis,meiosis and spermiogenesis.In a previous study,we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility.Here,we further investigated the localization and the role of flotillin-2 in spermiogenesis.Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting,immunohistochemistry and immunofluorescence.Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA(siRNA),and sperm abnormalities were assessed three weeks later.Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis.Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome.Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids,indicating that flotillin-2 was localized to the Golgi apparatus.Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis,compared with control siRNA-injected testes.This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis.     

Successive increases in human cyclin A1 promoter activity during spermatogenesis in transgenic mice

The human cyclin A1 gene is highly expressed in pachytene spermatocytes and is essential for spermatogenesis. To analyze mechanisms of cyclin A1 gene expression in vivo, we cloned a 1.3 kb fragment of the promoter upstream of the cDNA of enhanced green fluorescent protein (EGFP). Four lines of transgenic mice were generated that carried the transgene. Cyclin A1 promoter activity in the organs of the transgenic mice was analyzed using fluorescence microscopy and flow cytometry. Expression of EGFP was seen in male germ cells of all four murine lines. Spermatogonia at the basal membrane expressed low levels of EGFP, but bright green fluorescence was present in spermatocytes entering meiosis. Interestingly, a further sharp increase in EGFP expression was found in spermatocytes approximately at the stage of the first meiotic division. EGFP levels stayed high thereafter and EGFP was present in mature spermatozoa. A portion of c-kit expressing cells in the testis also expressed EGFP indicating cyclin A1 promoter activity in a subpopulation of spermatogonia. These data suggest that cyclin A1 is active not only in pachytene spermatocytes but also in earlier phases of spermatogenesis.     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

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

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.     

Importin alpha mRNAs have distinct expression profiles during spermatogenesis.

Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin alpha family genes. We first clarified the identity of the five known mouse importin alpha genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin alpha mRNAs were detected in spermatogonia. In the adult mouse testis, importins alpha1 and alpha3 were detected in spermatogonia and early pachytene spermatocytes. Importin alpha4 mRNA was identified in pachytene spermatocytes, alpha6 mRNA in round spermatids, and alpha2 mRNA in both of these. The distinct importin alpha expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis.     

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.     

Decreased expression of cold-inducible RNA-binding protein (CIRP) in male germ cells at elevated temperature.

Physiological scrotal hypothermia is necessary for normal spermatogenesis and fertility in mammals. Cirp is a recently identified cold-inducible RNA-binding protein that is inducible at 32 degrees C in mouse somatic cells in vitro. Cirp is constitutively expressed in the testis of mouse and structurally highly similar to RBM1, a candidate for the human azoospermia factor. To elucidate the role played by Cirp in spermatogenesis, we investigated its expression levels during spermatogenesis and after heat stress. In the mouse testis, cirp mRNA was detected in the germ cells, and the level varied depending on the stage of differentiation. Also, a high level of Cirp protein was detected immunohistochemically in the nucleus of primary spermatocytes. Expression of Cirp was decreased in the GC-2spd(ts) mouse germ cell line when culture temperature was raised from 32 degrees C to 37 degrees C. When mouse testis was exposed to heat stress by experimental cryptorchidism or immersion of the lower abdomen in warm (42 degrees C) water, the expression of Cirp was decreased in the testis within 6 hours after either treatment. In human testis with varicocele analyzed immunohistochemically, germ cells expressed less Cirp protein than those in the testis without varicocele. These results demonstrated that CIRP expression is down-regulated at elevated temperature in male germ cells of mice and humans. Analysis of Cirp expression in the testes will help elucidate the molecular mechanisms leading to male infertility.     

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.     

Expression of growth differentiation factor 9 (GDF9) and its receptor in adult cat testis

Oocyte-secreted growth differentiation factor (GDF) 9 plays an essential role during follicle maturation through actions on granulosa cells. Despite its critical role in female reproduction, GDF9 expression, signalling and function are less well characterized during spermatogenesis. The purpose of this study was to investigate temporal and spatial expression and potential cellular targets of GDF9 in the adult cat testis. Our result confirmed that GDF9 is stage-specifically localized in the cytoplasm of round spermatids and pachytene spermatocytes of the cat seminiferous epithelium. In particular, activin receptor-like kinase (ALK) 5, the type I receptor of GDF9, is principally localized in the cytoplasm of round spermatids. Smad2/3, signal transducers for GDF9 signalling pathway, is mainly immunolocalized in the cytoplasm of germ cells, Sertoli cells and Leydig cells, but the expression in germ cells are weaker than in Sertoli cells. The expression pattern of ALK5 and Smad2/3 show that GDF9-ALK5-Smad2/3 may not be the only signalling pathway for testicular cell to respond to GDF9. Overall, our results demonstrate that GDF9 is a germ cell-specific factor in the adult cat testis, and that GDF9 regulates the tight junctions of Sertoli cells by paracrine secretion, and regulates the germ cells by autocrine secretion.     

中心体蛋白centrin在大鼠精子发生过程中的表达

目的：研究中心体蛋白centrin在大鼠生精细胞中的表达情况,以深入了解centrin在精子发生过程中的作用。方法：通过重力沉降法分离大鼠不同发育阶段的生精细胞,用免疫荧光和蛋白印迹实验检测各级生精细胞中centrin蛋白的表达,用定量RT－PCR检测centrin同源基因centrin1和centrin2mRNA的表达水平。结果：间接免疫荧光和蛋白印迹显示精母细胞、圆形、长形精子细胞均有centrin蛋白存在,位于中心粒上,而在附睾的成熟精子中centrin则消失。RT－PCR研究发现,centrin在睾丸组织中特异性表达,centrin2在多种组织中均有表达。在睾丸中,centrin1仅在生精细胞进入减数分裂后转录,其mRNA水平在圆形精子细胞中最高,而centrin2在精原细胞中即有表达,减数分裂后其mRNA难以检测到。结论centrin蛋白在大鼠雄性配子的发育过程中最终丢失;该基因家族中同源基因centrin1和centrin2表达呈现组织特异性和发育阶段特性,在精子发生过程中发挥不同功能,centrin1蛋白可能与减数分裂及鞭毛生成相关,centrin2则参与细胞有丝分裂过程。     

Localization of the germ cell-specific protein,hnRNP G-T,in testicular biopsies of azoospermic men

The increasing interest in the application of in vitro fertilization techniques in human reproduction has led to a wide use of testicular biopsies to identify the presence of spermatogenic foci in testes of azoospermic men. Histopathologic evaluation of these testicular biopsies is required to determine the spermatogenic state with respect to fertility potential and to rule out preinvasive testicular lesions. Heterogeneous nuclear ribonucleoprotein G-T (hnRNP G-T) is a germ cell-specific protein expressed most prominently during meiosis. We studied the usefulness of hnRNP G-T antibody in the evaluation of these biopsies and reasoned that its germ cell-restricted expression pattern might provide a marker to improve accuracy of diagnosis. Testicular biopsies with various spermatogenic impairments were evaluated immunohistochemically for hnRNP G-T expression. In biopsies exhibiting normal spermatogenesis (obstructive azoospermia), hnRNP G-T was localized in meiotic pachytene spermatocytes and round spermatids. Immunostaining was barely detected when maturation was arrested at the spermatocyte level and not at all in cases of Sertoli cell-only syndrome. Biopsies with a mixed histologic phenotype and minute concentrations of spermatogenesis demonstrated strong immunostaining only in tubules with full spermatogenesis. This distribution pattern of hnRNP G-T enabled instant identification of spermatogenic foci. Thus, exploitation of the hnRNP G-T marker, which is expressed preferentially as meiosis proceeds, enhances sensitivity and accuracy of diagnosis in the histologic evaluation of testicular biopsies.     

Testis structure in the sys (symplastic spermatids) mouse

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.     

Localisation cellulaire de la sécrétion de l’inhibine B testiculaire

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an α and βB subunit. In adult testis, the cellular site of production of these subunits is still controversial: Leydig cells, Sertoli cells and/or germ cells. The immunohistological localization (monoclonal antibodies anti α and anti βB) of both sub-units and the expression patterns of their mRNA (in situ hybridization with RNA probes) were examined in adult testicular biopsies with normal spermatogenesis or spermatogenetic arrest. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin α subunit and expressed inhibin α subunit mRNA. Conversely, germ cells expressed the βB peptide (located from pachytene spermatocytes to round spermatids) and the βB subunit mRNA (located from spermatogonia to round spermatids). These results agree with the recent opinion that inhibin B is possibly a joint product of Sertoli cells and germ cells in adult men and it may be used as a serum marker of spermatogenesis.     

Retinoic acid receptor alpha is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.     

Stage-specific expression of mouse germ cell-less-1 (mGCL-1), and multiple deformations during mgcl-1 deficient spermatogenesis leading to reduced fertility

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1), is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-1 in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum et al., 2003), postacrosomal sheath, and posterior ring apparently formed. In addition, MN7 and MN13, essential substances for fertilization that are located in sperm heads, were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.     

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.