层黏连蛋白受体在小鼠睾丸和附睾中的表达

目的 探讨层黏连蛋白受体(LAMR1)在小鼠睾丸和附睾中的表达.方法收集3只正常成年昆明小鼠睾丸和附睾.采用原位杂交和免疫组织化学方法,检测LAMR1 mRNA及蛋白在成年小鼠睾丸和附睾中的分布.结果 LAMR1 mRNA在附睾头和附睾尾表达最强;在睾丸生精细胞中也有表达.免疫组织化学结果显示,LAMR1蛋白从附睾头到...     

Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies

The incidence of Y/autosome translocations is low. Whereas involvement of non-acrocentric chromosomes often leads to infertility, cases related with acrocentric chromosomes are usually familial with no or minimal effect on fertility. A de novo (Yp/13p) translocation was found in a 32-year-old male referred for severe oligozoospermia. Conventional cytogenetic procedures (GTG, CBG and NOR banding) and molecular cytogenetic techniques (Fluorescence In Situ Hybridization, FISH) were performed on high-resolution chromosomes obtained after peripheral blood lymphocyte culture as also on interphase nuclei of spermatogenic cells from semen samples. Screening of AZF microdeletions in the Yq11.2 region known to be involved with spermatogenesis defects was also performed. GTG banding showed a (Yp/13p) translocation in all scored metaphases. CBG and NOR staining of the derivative chromosome revealed the maintenance of Yq heterochromatin and of the 13p NOR region. FISH with centromeric Y and 13/21 probes, SRY specific probe and X/Y (p and q arms) sub-telomeric probes gave the expected number/location of fluorescent signals. Hybridisation with a pan-telomeric repeat (TTAGGG) probe showed an absence of the telomeric sequences at the fusion point of the rearranged chromosome. FISH analysis with probes to chromosomes X, Y, 13 and 18 showed an abnormal segregation of the translocated chromosome during meiosis I, which explains that only 13.6% of the secondary spermatocytes were normal. Most of these became arrested, as after meiosis II the large majority of the round spermatids were normal (70%), as were in consequence most of the sperm (85.1%). Multiplex-PCR confirmed the intactness of the SRY region and showed absence of AZF microdeletions. We report a novel de novo (Yp;13p) translocation characterised by loss of the 13p and Yp telomeres. Meiotic studies using FISH demonstrated meiosis I chromosome unpairing and mal segregation that justifies the severe oligozoospermia. Although most sperm have a normal chromosomal constitution, preimplantation genetic diagnosis should be considered an option for this patient.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Expression of sperm antigens during spermatogenesis and maturation detected with monoclonal antibodies

The expression of hamster sperm antigens was investigated during spermatogenesis and sperm maturation with the use of monoclonal antibodies generated in culture from mice immunized with hamster cauda epididymal spermatozoa or sperm heads. Antigens were localized by immunofluorescence and immunoperoxidase techniques which were first visualized on isolated spermatids over the developing acrosome. In one case, antibody inhibited fertilization in vitro although localization on testicular or epididymal spermatozoa was minimal compared with the early spermatid. Antibodies also recognized surface antigens first expressed in the epididymis whose localization on the spermatozoon altered during epididymal transit or incubation in capacitating medium. The results were discussed in relation to the expression and function of surface determinants on the haploid germ cell.     

Ultrastructural localization of a nuclear autoantigenic sperm protein in spermatogenic cells and spermatozoa

This study demonstrates the ultrastructural localization of rabbit nuclear autoantigenic sperm protein (NASP) in spermatogenic cells and spermatozoa. NASP is present in rabbits, rats, mice, and human testes and spermatozoa. It has recently been sequenced in rabbits and humans and characterized as an acidic, histone binding protein. Currently it has been proposed that NASP may play a role in regulating early events of spermatogenesis through its ability to bind and translocate testicular histone variants to nucleosomes. The ultrastructural localization of NASP confirms that it is initially present in primary spermatocytes in their Golgi regions and nucleus. In round spermatids it is present in the nucleus as well as in the acrosome and subacrosomal space. In later spermatids, testicular spermatozoa, and ejaculated spermatozoa, NASP is concentrated over the nucleus, although some is still present in the acrosome. It is likely that NASP would be carried into the ovum with the sperm nucleus at fertilization. © 1993 Wiley-Liss, Inc.     

三例体细胞染色体异常男性的单倍染色体分析

为了解体细胞异常男性的精子染色体变化情况，对３例有反复流产史的体细胞染色体异常男性单倍染色体进行了分析。其中１例４６，ＸＹ，ｔ（１３；１６），＋１６ｐ病例的精子染色体异常率为７６．３６％；１例４５，ＸＹ，Ｒｏｂ（２２；２２）病例仅见两种异常核型，即２３，Ｘ（Ｙ），－２２，＋Ｒｏｂ（２２；２２）为５８．８２％，２２，Ｘ（Ｙ），－２２为４１．１８％；１例４６，ＸＹ，ＹＰ＋病例发现３类Ｙ染色体核型，即２３，ＹＰ＋、２３，Ｙｐ－、２３，Ｙ；Ｘ染色体无变化。上述结果表明相互平衡易泣染色体携带者的精子染色体变化类型较复杂；罗伯逊易位携带者的精子染色体变化较简单；而Ｙ染色体短臂增加者的精子染色体变化有３类，并未见有关报道，是否造成有害的遗传效应尚有待进一步结合体细胞、生殖细胞与子代细胞的染色体进行深入研究。     

FISH analysis of chromosome X,Y and 18 abnormalities in testicular sperm from azoospermic patients

BACKGROUND: Sperm extracted from testicular biopsies of azoospermic men can successfully be used for ICSI. The concern exists that testicular sperm from azoospermic men suffering from severe testicular failure may have a higher frequency of aneuploidy, which may lead to an increased risk for chromosomally abnormal offspring. METHODS: Testicular sperm from patients showing spermatogenic failure (n = 17) and from patients with normal spermatogenesis (n = 26) were analysed by fluorescence in-situ hybridization (FISH). Numerical chromosomal abnormalities for chromosomes X, Y and 18 were evaluated by FISH in a total of 1697 testicular sperm derived from 43 azoospermic patients. RESULTS: No difference was observed between the frequency of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis (5.6%) and from patients with spermatogenic failure (8.2%). However, the frequency of aneuploidy for chromosome 18 was higher in the group of azoospermic patients with spermatogenic failure than in the group with normal spermatogenesis (3.2 versus 1.3%). Within the obstructive group, sex chromosome aneuploidy (4.5%) occurred more frequently than chromosome 18 aneuploidy (1.3%; P < 0.001). Among testicular sperm derived from patients with spermatogenic failure, sex chromosomal aneuploidy (5.8%) was similar to that for chromosome 18 (3.2%). CONCLUSIONS: So far, no difference in the total frequency of chromosomal abnormalities has been observed between patients with normal spermatogenesis and patients with severe testicular failure. However, aneuploidy for chromosome 18 was higher in the group with spermatogenic failure.     

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.     

SA-30抗原在小鼠睾丸、附睾、精子及早期胚的定位

目的 检测ＳＡ－３０（ｓｐｅｒｍ ａｎｔｉｇｅｎ－３０）原在小鼠精子发生、成熟、获能及受精后的分布变化。方法 免疫组织化学ＬＳＡＢ法。结果 ＳＡ－３０在小鼠睾丸内的各级生精上皮均有分布,尤其是造近管腔的精子细胞染色最深;附睾各段的上皮细胞也有ＳＡ－３０存在,而且主要集中在核上区;在附睾头和附睾尾的精子,ＳＡ－３０主要分布在顶体区,两者我明显区别,获能后的精子顶体区有阳性染色外,尾部和顶体后区也出现     

Fertilization with human testicular spermatids: four successful pregnancies

Between July 1995 and May 1996, 36 patients with non-obstructive azoospermia of secretory origin underwent intracytoplasmic injection of spermatids. A previous histological biopsy was performed on all patients: 15 had spermatogenic arrest, a further 13 had Sertoli cell- only syndrome, and the remaining eight had post-cryptorchidism tubal atrophy. The ejaculate was duly examined and a complete absence of spermatozoa and spermatids was confirmed, with only bacteria and debris being found. Testicular sperm extraction (TESE) was then performed. In 19 out of 36 cases round spermatids only were found, while elongated spermatids were found in the remaining 17. Both round and elongated spermatids were isolated and used for injection. A total of 135 oocytes at metaphase II were recovered from 19 partners and injected with round spermatids, while 123 mature oocytes from 17 partners were injected with elongated spermatids. The number of oocytes fertilized, as judged by the presence of two pronuclei, was 75 (55.5%) and 71 (57.7%) respectively. By 34 h after injection, the number of embryos which had cleaved to the 2-cell stage was 56 (74.6%) with round spermatids and 55 (77.4%) with elongated spermatids. All cleaved embryos were transferred into the uterus of the partners. Clinical pregnancies were established in two cases of round spermatid cycles (10.5%) (both are still ongoing), and three cases of elongated spermatid cycles (17.6%) (two are still ongoing; one was lost after 8 weeks of gestation). Chromosomal analysis showed that all fetuses had a normal karyotype (three male and one female) with no chromosomal abnormalities.      

Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation

Spermatid microinjection into oocytes has proven to be a successful assisted reproduction procedure in the animal model and in the human species, since in the latter a few full-term pregnancies were actually obtained. Patients entering our spermatid injection study included those with a total absence of spermatozoa in the testicular tissue notwithstanding previous positive biopsies (n = 29): an obstructive problem (n = 3), secretory azoospermia (n = 26), and those with total arrest at the spermatogenesis level in previous explorative biopsies (n = 15). In the latter group, absence of spermatids was recorded in four cases. Mature, elongated, elongating and round spermatids (ROS) were injected in respectively 3, 2, 3, and 32 attempts. A total of 260 metaphase II oocytes were injected with ROS, 36 oocytes with spermatids at other stages of maturity. The rates of oocytes showing two pronuclei (2PN) and two polar bodies reached 22% and 64% respectively after injection of round or elongated-mature spermatids. The fertilization rate after ROS injection was influenced by the percentage of spermatozoa observed in a previous biopsy. Patients with a positive preliminary biopsy had significantly more 2PN (33%) when compared to those with a severe spermatogenic dysfunction and in whom no spermatozoa were found (only 11%) (P < 0.05). Incubation of oocytes in calcium ionophore after ROS injection had a positive effect on the rate of 2PN formation (36 versus 16%). Ninety per cent of all the normally fertilized oocytes cleaved. The percentage of grade A and B embryos depended on the type of injected cells: 12% after ROS and 30% with the other types of haploid cells. A total of 39 transfers resulted in five pregnancies: three full term with healthy babies delivered (one after ROS injection, and two after injection of an elongating and a mature spermatid), one 4 months ongoing (after elongating spermatid injection) and one miscarriage at 4 weeks (after elongated cell injection). Compared to our conventional intracytoplasmic sperm injection- testicular sperm extraction (ICSI-TESE) programme, the implantation rate after ROS injection was very low (5.5 versus 10.5%).      

Histological changes of acrylamide-induced testicular lesions in mice

The effects of a single oral dose of 150 or 100 mg/kg acrylamide on the testis were studied histopathologically in prepubertal and adult mice over a period of 10 days following treatment. In the prepubertal mice, severe testicular damage such as vacuolation and swelling of the round spermatids, necrosis of the late elongated spermatids in stages I to VIII, abnormal meiosis in stage XIX, and a marked cellular exfoliation into the lumen were detected 1 day after administration of the 150 mg/kg dose. On Day 2, the damage was more pronounced. However, the testicular damage was repaired 7 to 10 days after treatment. In the adults, many tubules from stages I to VIII were affected, showing nuclear vacuolation in most of the round spermatids 1 day after administration of the 150 mg/kg dose. Meiosis in stage XIV was normal. In the prepubertal mice, nuclei of the round spermatids in stage I-III, especially step 1 spermatids had degenerated 1 day after administration of the 100 mg/kg dose. In the adults, the round spermatids had degenerated in stages I-II and IV-VIII 1 day after administration of the 100 mg/kg dose. These results suggested that the most vulnerable cell type among the spermatogenic cells was the round spermatid, especially that in the Golgi phase (stage I-III). Other stages and spermatogenic cells were relatively resistant to acrylamide testicular toxicity.     

Molecular chaperone calmegin localization to the endoplasmic reticulum of meiotic and post-meiotic germ cells in the mouse testis.

Calmegin is a testis-specific Ca(2+)-binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultrastructural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.     

Chromosomal segregation in spermatozoa of 14 Robertsonian translocation carriers

Male carriers of Robertsonian (Rob) translocations can havefertility problems associated with low sperm counts and abnormalsperm morphology. In this study, spermatozoa from 14 Rob translocationcarriers, seven der(13;14), two der(13;15), two der(14;15),two der(14;21) and one der(21;22), were tested by fluorescencein-situ hybridization (FISH) for the chromosomes involved, tostudy meiotic segregation behaviour. It was shown that in eachtype of Rob translocation, meiotic segregation behaviour issimilar, comparable and occurs non-randomly. Most of the spermatozoaresults from alternate segregation (range: 76–89.47%).There is, however, still much unbalanced spermatozoa resultingfrom adjacent segregation mode (range: 10.24–23.41%).These data provide useful information for genetic counsellingpurposes. Moreover, aneuploidy for chromosomes 13,18, 21, Xand Y was studied in five patients and suggested an inter-chromosomaleffect.     

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids—but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.     

The rate of aneuploidy is altered in spermatids from infertile mice

BACKGROUND: It is now possible for infertile males to father their own genetic children through the technique of ICSI. This prospect has consequently prompted several investigations into the quality of sperm being retrieved from infertile males. One potential risk is the use of aneuploid sperm or spermatids, which might then be transferred to the fertilized oocyte. METHODS: In this investigation, aneuploidy of spermatids was assessed through immunocytochemistry using antibodies directed against chromosome centromeric regions and complexes. Three different types of infertile male mice with phenotypes closely resembling those described in human non-obstructive azoospermia [PP1cgamma-deficient mice, CREM-deficient mice and C57BL/6J.MAC-17(0--23) mice] were examined for chromosome numbers by counting the number of kinetochores in round spermatids using a CREST antiserum. RESULTS: PP1cgamma(-/-) and CREM(-/-) spermatids from infertile mice showed highly significant elevated levels in the rate of aneuploidy compared with wild-type animals (P < 0.0001). Thus infertile males with independent genetic mutations resulting in different histopathologies showed a high risk in the level of aneuploidy in their spermatids. CONCLUSIONS: These results suggest that impaired spermatogenesis may lead to production of aneuploid gametes. Analysis of aneuploidy in gametes from infertile men, coupled with appropriate genetic counselling, is recommended prior to ICSI.     

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.