Change in expression of a 90-kDa glycoprotein GP90-MC301 during prenatal and postnatal testicular development: a differentiation marker for rat germ cells

GP90-MC301, a 90-kDa glycoprotein recognized by the monoclonal antibody MC301, is a reliable stage-specific marker for preleptotene to pachytene spermatocytes in adult rat testes. In this study we confirmed that the glycoprotein is also useful as a marker for germ cells in prenatal and postnatal testes. Immunohistochemical analysis showed a dramatic change in GP90-MC301 expression in germ cells during testis development. Strong expression was detected in primordial germ cells at embryonic day (E) 13 and in gonocytes at E16, and the expression was then markedly reduced at around the time (E18) gonocytes undergo G1/G0 arrest, and was not restored in gonocytes or spermatogonia afterward. Thereafter, it reappeared in primary spermatocytes in the prepubertal period. Testicular somatic cells such as Sertoli cells, Leydig cells, and peritubular myoid cells expressed GP90-MC301 during specific periods which were largely correlated with periods of active proliferation of these testicular somatic cells. Western blotting showed that GP90-MC301 was expressed during testis development without a change in its molecular size. Thus, GP90-MC301 is potentially useful for the analysis of not only spermatogenesis but also early testis development.     

Efficiency of the process of meiosis in scrotal testes of healthy boars and unilateral abdominal cryptorchid boars.

Unilateral abdominal cryptorchidism has usually been correlated with abnormalities in the spermatogenic activity of the scrotal testis. The present study describes the effects of unilateral abdominal cryptorchidism on the meiotic process in scrotal testes from postpubertal boars. The percentage of primary spermatocytes, secondary spermatocytes, and round spermatids was evaluated in testicular smears from scrotal testes of healthy boars and of right-sided unilateral abdominal cryptorchid boars. As compared to the scrotal testes of healthy boars, the scrotal testes of unilateral abdominal cryptorchid boars showed low transformation from primary to secondary spermatocytes (meiosis I), but normal transformation from secondary spermatocytes to round spermatids (meiosis II). The data obtained indicate that spontaneous unilateral abdominal cryptorchidism on the right side induced partial arrest of spermatogenesis at the primary spermatocyte stage that was attributed to anomalies in Sertoli-cell activity. Abnormal paracrine signals from altered Sertoli cells could have resulted in either disturbed mitosis, which led to the formation of spermatocytes with an abnormal DNA content, or abnormalities in the metabolic activity and the organization of the cytoskeleton of primary spermatocytes.     

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.     

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

Seasonal patterns of ornithine decarboxylase activity and levels of polyamines in relation to the cytology of germinal cells during spermatogenesis in the sea star, Asterias vulgaris

The activity of ornithine decarboxylase (ODC) and levels of polyamines were measured in the testes of Asterias vulgaris collected throughout an annual spermatogenic cycle. Samples of the testes were prepared for light and electron microscopy to observe the associated changes in the cytology of germinal cells. The specific activity of ODC increased at the onset of testicular growth, decreased during the coldest period of the winter when testicular growth was minimal, and increased again early in the spring when testes grew maximally. Increased activity of ODC resulted in increased levels of polyamines and was correlated with either mitotic or meiotic germinal cell divisions, or both. Spermine levels were always greater than putrescine, followed by spermidine. Highest levels of polyamine synthesis coincided with the onset of spermatogonial proliferation during the fall and with the period of meiotic differentiation and spermiogenesis in the spring. Mid-summer (July) testes were small (0.3-0.5 gonad index (GI)) and contained amitotic spermatogonia arrested in G(1) of the cell cycle. Mitotic and pre-meiotic testes (October/November) increased slightly in size (0.3-1.4 GI) and contained actively dividing spermatogonia, most of which differentiated into primary spermatocytes. Testes from February and March were large (1-6.75 GI), but the proliferative status of their spermatogonia and primary spermatocytes varied. Spermatogonia and primary spermatocytes from early February testes were not dividing. In testes obtained in March, both spermatogonial mitosis and meiosis of spermatocytes resumed, coincident with increased field water temperatures.     

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     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.     

LDH-C：睾丸重要的特异表达基因

乳酸脱氢酶C(LDHC)是目前已知的最早在男性生精细胞中发现的睾丸特异同功酶。LDHC最早通过凝胶电泳技术在人精子及睾丸生精细胞中被发现。免疫组化结果显示LDHC最早出现在早期的粗线期初级精母细胞中,其数量随着减数分裂逐渐增加。成熟精子中LDHC主要定位在精子尾部的主段区域。研究显示,乳酸脱氢酶家族的同功酶在哺乳动物细胞中无所不在,他们受到发育的调控,具有组织细胞的特异性,且功能多样。本文就LDHC的发展史及他们在帮助精子完成受精过程中的作用作一综述。     

Meiosis of Primary Spermatocytes and Early Spermiogenesis in the Resultant Spermatids in Newt, Cynops pyrrhogaster in vitro

Dissociated spermatogenic cells were cultivated within the collagen matrix at low cell density. The largest cell type in the culture was identified as the primary spermatocytes by their size and the morphological characteristics revealed by ultra-thin sections. Chromosome analysis showed that about 90% of the cells examined were either in first or second meiosis. Within the collagen matrix, the fates of 282 single primary spermatocytes at meiotic stage in diakinesis or metaphase were followed. In a few days, most of them gave rise to four spermatids, passing through first and second meiotic divisions. About 80% of the spermatids formed motile flagella. They grew about 20–60 μm a day. The final state of the differentiation attained in our culture conditions was the spermatids with localized spherical nuclei and motile flagella, about 500 μm in length after 1-month's culture. Ultra-thin sections of the spermatids show that the rings, neck-pieces, and acrosomes developed in the cells.     

In situ localization of male germ cell-associated kinase (mak) mRNA in adult mouse testis: specific expression in germ cells at stages around meiotic cell division.

Biochemical analysis of the male germ cell-associated kinase (mak) gene, which was isolated recently by using weak cross-hybridization with the v-ros tyrosine kinase gene, revealed that the gene was highly expressed in mammalian testicular germ cells, but not in ovarian cells. In order to identify the cells which express the mak gene in more detail, we localized mak mRNA in frozen sections of mouse testis by non-radioactive in situ hybridization. In this study, we utilized thymine-thymine (T-T) dimerized mak cDNA as a haptenic, non-radioactive probe, and the signal was detected enzyme-immunohistochemically by using an anti-T-T antibody. As a result, mak mRNA was localized intensely in late pachytene (stage X) and diplotene (stage XI) spermatocytes, and faintly in dividing spermatocytes (stage XII) and early round spermatids (stage I-II), suggesting that the gene may play an important role in the phase around meiotic cell division, but not throughout the entire meiosis.     

Induction of meiosis in fetal mouse testis in vitro

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a "meiosis-preventing substance" (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a "meiosis-inducing substance" (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.     

Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage

To begin to examine the function of the A-type cyclins during meiosis in the male, we have examined the developmental and cellular distribution of the cyclin A1 and cyclin A2 proteins, as well as their candidate cyclin-dependent kinase partners, Cdk1 and Cdk2, in the spermatogenic lineage. Immunohistochemical localization revealed that cyclin A1 is present only in male germ cells just prior to or during the first, but not the second, meiotic division. By contrast, cyclin A2 was expressed in spermatogonia and was most abundant in preleptotene spermatocytes, cells which will enter the meiotic pathway. Immunohistochemical detection of Cdk1 was most apparent in early pachytene spermatocytes, while staining intensity diminished in diplotene and meiotically dividing spermatocytes, the cells in which cyclin A1 expression was strongest. Cdk2 was highly expressed in all spermatocytes. Notably, in cells undergoing the meiotic reduction divisions, Cdk2 appeared to localize specifically to the chromatin. This was not the case for spermatogonia undergoing mitotic divisions. In the testis, cyclin A1 has been shown to bind both Cdk1 and Cdk2 but we show here that cyclin A2 binds only Cdk2. These results indicate that the A-type cyclins and their associated kinases have different functions in the initiation and passage of male germ cells through meiosis.     

Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: Evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro

Summary Dispersed testicular cells prepared from 14-d-old rats were cultured on type 1 collagen gels using a medium composed of a 1∶1 mixture of Ham’s F12 medium and Leibovitz’s L15 medium (F12-L15 medium) containing 10% (vol/vol) fetal bovine serum. The viability of the spermatogenic cells was facilitated by supplementing a rat adrenal extract into the medium. The effective substance(s) (the survival factor) was purified from acid extracts of adrenals by molecular sieve high performance liquid chromatography and identified as epinephrine and norepinephrine. Both epinephrine and norepinephrine promoted the survival of the spermatogenic cells with a half saturating dose of 10 ng/ml. The spermatogenic cells, which could be cultured for 2 wk on a collagen gel by supplementing with the survival factor (epinephrine or norepinephrine), were subjected to Giemsa staining and to DNA flow cytometry. The following results were obtained: a) The spermatogenic cells from 14-d-old rats did not contain spermiogenic cells (lc-cells). b) During a culture period of 2 to 7 d the ratio of meiotic prophase spermatocytes (4c-cells) to premeiotic cells (2c-cells) increased. On Day 7, more than 90% of the surviving cells were meiotic prophase spermatocytes. c) On Day 10, spermatids (lc-cells) appeared for the first time. The time of the first appearance of spermatids in the culture was consistent with that in vivo. These results suggest that both epinephrine and norepinephrine facilitated the viability of meiotic prophase spermatocytes and that a part of the meiotic prophase spermatocytes completed the meiotic divisions in the testicular cell culture.     

Acid glycohydrolases in rat spermatocytes, spermatids and spermatozoa: enzyme activities, biosynthesis and immunolocalization

Mammalian sperm acrosomes contain several glycohydrolases that are thought to aid in the dispersion and digestion of vestments surrounding the egg. In this study, we have used multiple approaches to examine the origin of acrosome-associated glycohdyrdolases. Mixed spermatogenic cells, prepared from rat testis, were separated by unit gravity sedimentation. The purified germ cells (spermatocytes [SC], round spermatids [RS], and elongated/condensed spermatids [E/CS]) contained several glycohydrolase activities. Metabolic labeling in the cell culture, immunoprecipitation, and autoradiographic approaches revealed that β-D-galactosidase was synthesized in SC and RS in 88/90 kDa forms which undergo processing in a cell-specific manner. Immunohistochemical approaches demonstrated that the enzyme was localized in Golgi membranes/vesicles, and lysosome-like structures in SC and RS, and forming/formed acrosome of E/CS. Published December 3, 2001