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The fine structural distribution of histones H2B and H3, andprotamines were localized by means of specific antibodies andultrastructural immunocytochemistry in nuclei of human spermatidsand spermatozoa. The antibodies were used to detect the nuclearbasic proteins on sections of testis and ejaculated spermatozoaby immunoelectron microscopy. A quantitative analysis of labellingdensity was performed on micrographs using an interactive imageanalysis system. The labelling density of somatic-type histonesH2B and H3 and of their testis-specific variants was constantin the nuclei of young spermatids with round nuclei (stages12), and then increased in intermediate spermatids (stages34). Histone H3 labelling decreased at the end of theelongation phase (stage 5) while histone H2B labelling decreasedin mature spermatids (stage 6) only. Spermatozoa were foundto be weakly labelled by the anti-histone antibodies. The firstsigns of labelling of protamines and basic intermediate proteinsappeared in spermatid nuclei at stage 4, increased further instage 6 spermatids and persisted in all sperm nuclei. The presentwork shows that histone-to-protamine replacement occurs at thebeginning of the spermatid maturation phase in human. However,histones are partially retained in mature spermatids and spermnuclei. histones/immunocytochemistry/nuclear proteins/protamines/spermatozoa 相似文献
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Schlicker M.; Schnulle V.; Schneppel L.; Vorob'ev V.I.; Engel W. 《Human reproduction (Oxford, England)》1994,9(12):2313-2317
During spermiogenesis, the successive replacement of the somatichistones by basic proteins, the transition proteins and protamines,allows normal sperm nuclear condensation. It was suggested thatdisturbances in nuclear condensation may result in male infertility.Here we report the first molecular analysis of the structureof three genes which code for germ cell-specific nuclear proteins,namely protamine 1 (PRM1), protamine 2 (PRM2) and transitionprotein 1 (TNP1) in infertile men with disturbed sperm chromatincondensation. In 36 infertile men whose spermatozoa showed apositive reaction with aniline blue, which is an indicationfor the presence of histones in the nuclei, the complete nucleotidesequences of the coding regions and 5 and 3 untranslatedregions of the three genes were evaluated. In addition, 10 infertilepatients with oligoasthenoteratozoospermia were studied in thesame way, as well as nine infertile patients whose spermatozoashowed a reduction of the protamine 2 content. We did not detectany mutation in the three genes in any of the patients. We assumethat the disturbances in the sperm chromatin condensation ofour patients, and those described in the literature, are notprimarily due to mutations in the genes for PRM1, PRM2 and TNP1. 相似文献
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Steger K; Klonisch T; Gavenis K; Drabent B; Doenecke D; Bergmann M 《Molecular human reproduction》1998,4(10):939-945
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Steger K Failing K Klonisch T Behre HM Manning M Weidner W Hertle L Bergmann M Kliesch S 《Human reproduction (Oxford, England)》2001,16(4):709-716
During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI. 相似文献
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The present study aims to evaluate the injection of testicular round spermatids from patients with complete failure of spermiogenesis compared with that of mature epididymal and testicular spermatozoa. Over a period of 8 months, 188 azoospermic patients were evaluated with a view to their inclusion in our intracytoplasmic sperm injection (ICSI) programme. All patients had had a previous testicular biopsy; 38 had pure obstructive azoospermia, while 150 had non-obstructive azoospermia. Mature spermatozoa were found in 93 patients, whereas spermatozoa were entirely absent, with a predominance of round spermatids in 87. In eight patients, spermatids could not be found and therefore their cycles were cancelled. There was an early appearance of the two pronuclei stage in the round spermatid group compared with the mature spermatozoa group of patients (10.2 and 16 h respectively). The fertilization rate was also significantly lower (P = 0.00001) in the round spermatid group. The numbers of embryos developed and of embryo transfers in the round spermatid injection group were significantly lower compared with the mature spermatozoa injection group (P = 0.05 and 0.0001 respectively). No pregnancies resulted from round spermatid injection, while 18 pregnancies were achieved from the injection of mature spermatozoa. In conclusion, injection of round spermatids from patients with complete failure of spermiogenesis resulted in a significantly lower fertilization rate and a higher developmental arrest compared with injection of mature spermatozoa. With no pregnancies achieved, one may question the unusual variability of reported success rates and stress the need for further research in order to improve the outcome of this novel technique. 相似文献
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Immunoexpression of testis-specific histone 2B in human spermatozoa and testis tissue 总被引:2,自引:1,他引:2
van Roijen HJ; Ooms MP; Spaargaren MC; Baarends WM; Weber RF; Grootegoed JA; Vreeburg JT 《Human reproduction (Oxford, England)》1998,13(6):1559-1566
During mammalian spermatogenesis, the chromatin of the spermatogenic cells
is profoundly reorganized. Somatic histones are partly replaced by
testis-specific histones. These histones are then replaced by transition
proteins and finally by protamines. This series of nucleoprotein
rearrangements results in a highly condensed sperm cell nucleus. In
contrast to spermatozoa from other species, human spermatozoa still contain
a significant amount of histones, including testis-specific histone 2B
(TH2B). In the present study it is shown that an antibody targeting
tyrosine hydroxylase, which has been found previously to cross-react with
rat TH2B, also specifically immunoreacts with human TH2B on Western blots,
in immunohistochemistry of human testis tissue, and in immunocytochemistry
of decondensed human spermatozoa. In human testis tissue, TH2B
immunostaining first apparent in spermatogonia, shows marked variation,
especially at the pachytene spermatocyte stage, and then reaches an intense
signal in round spermatids. Shortly before spermatid elongation, a portion
of the spermatid nucleus, corresponding to the acrosomal region, loses its
immunoreactivity. During condensation of the spermatid nucleus, the
immunodetectability of TH2B disappears gradually, from the anterior region
of the nucleus onwards. At the final stages of spermiogenesis, the
immunostaining is completely absent. Immunocytochemical staining of
spermatozoa revealed no TH2B immunosignal, but immunostaining was observed
when spermatozoa obtained from semen were decondensed to make nuclear
proteins accessible to the antibody. There was, however, a striking
intercellular variability in the intensity of staining of spermatozoa
within an ejaculate. In a population of 35 men attending our Andrology
Clinic, we observed interindividual differences in total sperm TH2B
content, which showed a significant, although not very pronounced, negative
correlation with normal morphology (P = 0.05).
相似文献
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Lassalle B Ziyyat A Testart J Finaz C Lefèvre A 《Human reproduction (Oxford, England)》1999,14(2):388-394
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Human fertilization with round and elongated spermatids 总被引:2,自引:15,他引:2
Fishel S; Green S; Hunter A; Lisi F; Rinaldi L; Lisi R; McDermott H 《Human reproduction (Oxford, England)》1997,12(2):336-340
Human spermatids from ejaculate and testicular tissue have been utilized
for evaluating human fertilization by intracytoplasmic sperm injection
(ICSI) and, where possible, compared with spermatozoa utilizing sibling
oocytes. Round and elongated spermatids obtained from ejaculates were
either prepared through Percoll gradients or isolated and washed
individually using subzonal insemination needles (SUZI; 10- 14 microm
internal diameter). Seminiferous tubules obtained after biopsy were placed
into HEPES-buffered Earle's medium and dissected using 21-gauge needles.
Spermatogenic cells and spermatozoa were isolated and washed individually
using SUZI needles. Spermatozoa were subsequently injected into the ooplasm
using 5 microm (internal diameter) ICSI needles, whereas 8-9 microm
(internal diameter) needles were used for spermatid injection. Only
metaphase II oocytes (n = 207) were injected: 64 with round spermatids, 92
with elongated spermatids and 51 with spermatozoa; the fertilization rate
was 30, 24 and 67% respectively. There was a significant (P < 0.001)
increase in the fertilization rate using spermatozoa compared with
spermatids. The fertilization rate was not different between round and
elongated spermatids, although the fertilization rates for round and
elongated spermatids in the ejaculate were 33 and 18% respectively,
compared with 22 and 38% respectively when testicular spermatids were
utilized. In three patients sibling oocytes were used to compare round and
elongated spermatids found in the ejaculate with spermatozoa extracted from
seminiferous tubules. The fertilization rate was 24% for spermatids and 79%
for testicular spermatozoa. This result suggests that, should only
spermatids be available in the ejaculate, a testicular biopsy in the hope
of obtaining testicular spermatozoa would be worth while.
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Successful pregnancy after spermatid injection 总被引:9,自引:7,他引:2
Bernabeu R; Cremades N; Takahashi K; Sousa M 《Human reproduction (Oxford, England)》1998,13(7):1898-1900
We present nine cases of spermatid intracytoplasmic injection for the
treatment of non-obstructive azoospermia. In eight cases, no elongated
spermatids or spermatozoa were found in previous spermiograms or testicular
biopsies. In these patients, treatment was performed using ejaculated (n =
6) and testicular (n = 2) retrieved round spermatids (Sa type). In cases
where ejaculated round spermatids were used, they were isolated on the day
before oocyte retrieval and left in culture for 24 h before
intracytoplasmic sperm injection (ICSI). No pregnancy was obtained in
either group, although culturing seemed to increase the fertilization rate.
In one other case, elongated spermatids were observed in the previous
spermiogram and thus a normal ICSI procedure was scheduled. However, on the
day of oocyte retrieval, no spermatids could be recovered from fresh
sequential ejaculates, and a testicular open biopsy was then performed.
Both round and elongated spermatids were found in the testicular tissue,
but only the more mature germinal cells (Sb2) were injected. From this
case, a normal pregnancy was obtained which resulted in the birth by
Caesarean section at 37 weeks of gestation of a normal healthy baby girl,
weighing 2700 g.
相似文献
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Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection 总被引:18,自引:16,他引:2
Kahraman S; Polat G; Samli M; Sozen E; Ozgun OD; Dirican K; Ozbicer T 《Human reproduction (Oxford, England)》1998,13(1):104-110
Recent studies have shown that the injection of spermatid cells into the
human oocyte can result in normal fertilization, embryo development and
even delivery of live, healthy offspring. In our study, 23 azoospermic
cases with severe spermatogenetic defects in their testicular biopsy are
presented. The serum follicle stimulating hormone (FSH) concentrations and
histopathological results of these males have been documented and compared
in terms of fertilization and embryo development. The mean FSH value of the
azoospermic males was 15.8 +/- 2.3 mIU/l, ranging from 1.6 to 39 mIU/l.
Elongated spermatids were used in three cases only, as these more mature
forms were mostly present in the testicular sample. In the remaining 20
cases, only round spermatids were found for use in intracytoplasmic sperm
injection (ICSI). The fertilization rate with two pronuclei was 31.3%. The
fertilization rate was found to be as high as 71% in three patients in the
elongating and elongated spermatids group and as low as 25.6% in the round
spermatid group. A few immature, non-motile spermatozoa were seen in only
two cases from the elongated spermatid group. However, in the remaining
cases, no spermatozoa were observed. The number of pronuclear (PN) arrest
was quite high when only round spermatids were used (36.1%). Total
fertilization failure was observed in two cases from the round spermatid
group with Sertoli cell only and germ cell aplasia. A total of three
pregnancies was achieved in 23 cases (13.0%), two from the elongated
spermatid group and one from the round spermatid group. One biochemical
pregnancy with a round spermatid resulted in an early spontaneous abortion
and surprisingly, the remaining pregnancies were achieved with elongated
spermatids resulting in multiple pregnancies. One twin and one triplet
pregnancy were established following four embryo transfers in each patient.
The twin pregnancy resulted in a live birth with two healthy babies;
unfortunately, the triplet pregnancy ended in an abortion at 11 weeks. The
use of testicular spermatids in the treatment of non-obstructive
azoospermia may give hope by offering a novel treatment model. In cases
with very severe spermatogenetic defect, even multiple pregnancies can be
achieved with elongated spermatid cells by yielding a high implantation
rate. However, the efficiency of round spermatids in achieving
fertilization and pregnancy was disappointing.
相似文献