Subhuman primates as models for the development of male contraceptives.
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Acrosome Reaction↗
Biomedical subjects
G van der Horst
Publications and source records attributed to G van der Horst.
The male vervet monkey: sperm characteristics and use in reproductive research.
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Animals↗
Short-term effects of N'N-bis(dichloroacetyl)-1,8-octamethylenediamine (WIN 18446) on the testes, selected sperm parameters and fertility of male CBA mice.
N'N-bis(dichloroacetyl)-1,8-octamethylenediamine (WIN 18446), the most potent of the diamines and one of the least amoebicidal agents, was shown to exert a specific effect on the testes of CBA mice, while the Leydig cells were unaffected. Spermatogenesis was severely affected after a 42-day treatment period with 125 mg WIN 18446/kg body weight. Large multinucleated cells, vacuolization and the absence of sperm within the testes were evident in most seminiferous tubules. After 15 days of withdrawal of WIN 18446, there was a slight recovery of spermatogenesis and after withdrawal of 42 days a marked recovery of spermatogenesis. The normality or abnormality of this spermatogenic cycle could be evaluated using the semi-quantitative Stages program. There was a significant decrease in the diameters of seminiferous tubules of WIN 18446 treated mice, however an almost complete recovery was evident after 42 days of withdrawal of WIN 18446. A significant decrease in sperm concentration and sperm morphology was observed for the WIN 18446 treated mice. Various sperm motion parameters were assessed for the different treatment groups and compared to the control group. The female and male fertility indices were assessed and compared for the different treatment groups. Complete recovery of the above-mentioned parameters was evident after 42 days of withdrawal from WIN 18446, and this confirms its potential as a possible contraceptive for animal populations.
Animals↗
Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation.
In this study, we examine the role of bone morphogenetic protein (BMP) signaling during differentiation of the murine preosteoblastic KS483 cell line, which formed alkaline phosphatase (ALP)-positive and mineralized nodules during a 3 week culture period. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of various BMPs (BMP-2, -3, -4, -6, -7, and -8A and -8B), BMP type I and II receptors (ALK2, ALK3, ALK4, BMPR-II, and ActR-IIA and -IIB), BMP antagonists (DAN, gremlin, chordin, cerberus, noggin, and tsg), and Smads 1-8. mRNA expression of these genes did not change during differentiation, except for BMP-3, BMP-8a, and noggin. BMP-3 increased gradually, particularly in the matrix formation phase; BMP-8a was induced from the onset of matrix maturation and mineralization, in parallel to the expression of osteocalcin; and noggin tended to decline during the mineralization phase. Treatment of KS483 cells with the BMP antagonists noggin or soluble truncated BMPR-IA, either continuously or during distinct periods of osteoblast differentiation; that is, matrix formation or matrix maturation and mineralization phase, decreased ALP-positive and mineralized nodule area independent of the phase of osteoblast differentiation. Notably, the antagonists inhibited mineralization of already existing nodules. Similarly, BMP-4 stimulated differentiation not only at the beginning of the culture period, but also at late stages of differentiation. These data indicate that autocrine BMP signaling is involved in KS483 osteoblastic differentiation not only during the early phase of differentiation, but also during matrix maturation and mineralization. The different expression patterns of components of BMP signaling in the KS483 cells suggest distinct functions of individual BMPs during osteoblast differentiation. In summary, our data suggest that BMP activity is required not only for initiation of osteoblast differentiation and further development of early osteoblasts, but is also involved in late-stage osteoblast differentiation and matrix mineralization.
Activin Receptors, Type I↗
Endometrial function in vervet monkeys (Cercopithecus aethiops): morphology, beta3 integrin and insulin-like growth factor binding protein-1 expression during the menstrual cycle and pregnancy in the normal and disrupted endometrium.
The expression of endometrial beta3 integrin and insulin-like growth factor binding protein-1 (IGFBP-1) was studied in cycling and pregnant vervet monkeys. There were clear changes of beta3 integrin expression during the menstrual cycle, with the strongest immunostaining observed on day 26. Moderate to strong expression was observed during pregnancy. The expression of IGFBP-1 during the menstrual cycle was weak but upregulated during pregnancy with moderate to strong staining. The administration of a single dose of onapristone at 10 mg/kg on days 17, 21 and 22 of the menstrual cycle, followed by a biopsy on days 22, 22 and 26, respectively, and during pregnancy (34-44 days menstrual age) 24 h before the biopsy, disrupted and desynchronized the endometrium. However, no effect on beta3 integrin expression could be observed and staining reflected the untreated patterns. The same applied to IGFBP-1 except that during pregnancy the expression of this protein was reduced or abolished. The results suggest that beta3 integrin is associated with endometrial receptivity in vervet monkeys and that IGFBP-1 plays an important role during pregnancy in this species. The administration of onapristone appeared to only influence IGFBP-1 expression. To our knowledge, this is the first time that these endometrial proteins have been investigated in vervet monkeys. This study should therefore contribute to improving our understanding of the reproductive function of this species.
Animals↗
The effect of oleanolic acid on sperm motion characteristics and fertility of male Wistar rats.
The study was designed to examine the effect of oleanolic acid on cauda epididymal sperm motion using a computer-aided sperm analysis system and to elucidate the relationship between sperm motion and fertility, as a tool for contraceptive studies. Oleanolic acid-polyvinylpyrrollidone suspension was orally administered to adult male Wistar rats for 30 days, followed by a 14-day drug withdrawal from half of the rats in the group. Control rats received only polyvinylpyrrollidone. All males were mated with untreated females. Treated males failed to impregnate females, whereas control and oleanolic acid withdrawn males achieved 100% pregnancies. Sperm motion analysed on the Sperm Motility Quantifier (SMQ) showed significant differences in linearity (P < 0.001) and wobble (P < 0.01) between control and treated groups. However, the curvilinear velocities were not significantly different (P > 0.05) among all the groups. Sperm motility patterns verified differences among kinematic parameters.
Administration, Oral↗
c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route.
Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.
Animals↗
The detection and monitoring of early pregnancy in the vervet monkey (Cercopithecus aethiops) with the use of ultrasound and correlation with reproductive steroid hormones.
Twenty early pregnancies were diagnosed and monitored in vervet monkeys by ultrasonography. Non-gravid uteri became increasingly echogenic from cycle days 7 to 26. The first definite sign of pregnancy was a gestational cavity of 2 mm (+/- 0.80) at 33.0 (+/- 1.48) days menstrual age, which was also used to date all subsequent features. Earlier signs, such as an endometrial line swelling or endometrial 'pregnancy' ring, as reported for other non-human primate species, could not be reliably and consistently used to diagnose pregnancy in vervet monkeys. A rapid increase of the gestational cavity size from days 37 to 49 corresponded closely to a rapid increase in plasma progesterone concentration from day 39 to 49. The first yolk sac was recognizable at 38.0 days (+/- 3.10) and measured 3.3 mm (+/- 0.40) in diameter. A heart beat could be detected at 45.5 (+/- 1.73) days and the size of the first measurable embryo at 35 days was 2 mm. The dating of most features was within the range reported for other non-human primate species.
Animals↗
Sperm viability and morphology of two genetically diverse Merino lines.
Microscopically evaluated sperm parameters, as well as computer-aided sperm motility analysis (CASMA), were used to assess sperm quality and the effect of cryopreservation on ram semen obtained from two genetically diverse Merino lines. These lines were divergently selected on maternal ranking values for multiple rearing ability from the same base population since 1986. Replacements in the high (+) line were preferentially the progeny of ewes rearing >1 lamb per joining. Progeny of ewes rearing <1 lamb per joining was preferred as replacements in the low (-) line. Sperm quality, as assessed by percentages of live, abnormal and acrosome-intact spermatozoa as well as by motility, was independent (P < or = 0.20) of line, time of sampling and their interaction in ejaculated samples obtained from the eight rams used as sires in 1995. Sperm quality of frozen-thawed samples was adversely affected (P < or = 0.01) by cryopreservation and thawing at 35 degrees C for 30 s relative to fresh ejaculated samples. No consistent differences between lines were found in epididymal sperm samples obtained from 12 slaughtered rams (6 from each line). The adverse effect (P < or = 0.05) of cryopreservation and thawing at 35 degrees C for 30 s on sperm viability and motility was also demonstrated for these samples.
Animals↗
The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa.
The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation.
Acrosome↗
The effect of p-nonylphenol, an environmental toxicant with oestrogenic properties, on fertility potential in adult male rats.
Infertility is a sad reality and it is now evident that several aspects of male reproductive health have changed for the worse over the past 30-50 years. para-nonylphenol (p-NP), an environmental toxicant with oestrogenic properties, was tested for its effect on male fertility potential. When adult male rats were exposed to 100 mg kg-1 p-NP the histological parameters of the seminiferous tubules were adversely affected. Although spermatogenesis was already established in these males at the time exposure commenced, p-NP still had an effect on the histology of the seminiferous tubules. Increasing the level to 250 mg kg-1 additionally resulted in a smaller weight gain and signs of epididymal toxicity, while 400 mg kg-1 also impaired testicular mass and sperm count. In the last two groups spermatogenesis was also affected in some animals. Because p-NP had an effect on established spermatogenesis in the rat, one could speculate that the same effects might also occur in humans. It would appear that p-NP had toxic effects on both the testis and epididymis and both structures might be important in impairing male fertility. Bio-accumulation may enhance the negative effects at even lower p-NP concentrations over longer exposure periods than reported here.
Animals↗
Formation of Shc/Grb2- and Crk adaptor complexes containing tyrosine phosphorylated Cbl upon stimulation of the B-cell antigen receptor.
B-cell antigen receptor (BCR) stimulation induces tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2. The Shc/Grb2 complex may be involved in Ras activation, since Grb2 interacts with the guanine nucleotide exchange factor Sos. We reveal here an additional complexity of the BCR-induced Shc/Grb2 complex: it contains tyrosine phosphorylated proteins of 130, 110 and 75 kDa. The 130 kDa molecule inducibly associates with Shc, while the 75 kDa protein interacts with the carboxy-terminal SH3 domain of Grb2. The 110 kDa molecule is defined as Cbl, the product of the c-cbl oncogene, which is strongly phosphorylated on tyrosine upon BCR stimulation. Cbl constitutively interacts with the SH3 domains of Grb2, with a preference for the amino-terminal domain, and is in this way recruited to Shc upon BCR stimulation. Immunodepletion studies showed that Grb2-associated Cbl can be phosphorylated by BCR-induced tyrosine kinases independent of a Shc/Grb2 interaction. This indicates that the BCR can also couple to a Grb2 complex without the involvement of Shc. Cbl not only interacts with Grb2, but also with the adaptor protein Crk. In contrast to its constitutive interaction with Grb2, tyrosine-phosphorylated Cbl only associates with Crk after BCR stimulation. In summary, we observe that the BCR activates Shc/Grb2-, Grb2- and Crk adaptor complexes of distinct composition, which may allow selective coupling to different signal transduction cascades. Cbl participates in all three adaptor complexes and is tyrosine phosphorylated upon BCR stimulation, pointing to a central role for this molecule in the regulation of antigen receptor-induced B cell responses.
Adaptor Proteins, Signal Transducing↗
Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors.
B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.
Adaptor Proteins, Signal Transducing↗
The effect of pentoxifylline on sperm movement characteristics and zona pellucida binding potential of teratozoospermic men.
This study investigates the effect of pentoxifylline on sperm movement and zona pellucida binding. Spermatozoa from nine teratozoospermic patients (10.2 +/- 6% normal forms) were included in the study. Test samples were diluted with a 4 nM solution of pentoxifylline, and control samples with Ham's F-10 culture medium only. Zona pellucida binding potential was measured under hemizona assay conditions (HZA). Sperm motility was evaluated at the start (0 h post-swim-up) and end (4 h) of the HZA, under test-tube conditions and under HZA conditions (50 microliters droplet under oil). Motility parameters tested included the curvilinear velocity (VCL), straight line velocity (VSL) and linearity (LIN). Compared to the controls, pentoxifylline-treated samples showed an immediate stimulation of sperm motility, under test-tube conditions, with a significant elevation of VCL at 0 h incubation (102.77 +/- 14.4 versus control value 84.60 +/- 10.6 microns/s; P = 0.005), which however was reversed after 4 h incubation (73.16 +/- 4.6 versus control value 85.47 +/- 12.8 microns/s; P < 0.005). A decline in sperm motility from 0 to 4 h incubation was noted for all the pentoxifylline-treated samples, under both test tube conditions (VCL: 102.77 +/- 14.4 versus 73.16 +/- 4.6 microns/s, P < 0.005; VSL: 27.2 +/- 10 versus 10.66 +/- 2.2 microns/s, P < 0.005; LIN: 23.65 +/- 7.1 versus 11.86 +/- 1.8%, P < 0.005) and HZA conditions (VCL: 100.04 +/- 13.1 versus 76.00 +/- 7 microns/s, P < 0.005; VSL: 26.40 +/- 8.7 versus 9.14 +/- 4.5 microns/s, P < 0.005; LIN: 26.2 +/- 12 versus 11.05 +/- 4.3%, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)
Female↗
Interlocked circle formation by group I introns: structural requirements and mechanism.
Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.
Base Sequence↗
Requirements of a group I intron for reactions at the 3' splice site.
Structural elements of the Tetrahymena group I intron have been implicated in directing the exon-ligation transesterification reaction (the second step of self-splicing) to the correct 3' splice site. The 3' splice site also serves as the reaction site in a distinct transesterification reaction known as G-exchange and in a specific hydrolysis reaction. The dependence of the reactions on the internal guide sequence (IGS), an exon-binding element, was examined. Hydrolysis did not require the IGS, while G-exchange did, suggesting that one of the guanosine-binding sites employed during G-exchange requires the IGS. For the exon ligation reaction, the specification of the 3' splice site is thought to be influenced by the 3' exon-IGS pairing P10 and three structural elements in the intron: the universally conserved guanosine residue preceding the 3' splice site, the long-range pairing P9.0, and the triple stem-loop region composed of P9, P9.1 and P9.2. A systematic mutational study of the three structural elements was performed in order to clarify the mechanism of the specification of the 3' splice site for the reactions. For both transesterification reactions, the elements are important, with some superfluity in function. The specific hydrolysis reaction, however, shows even more dependence on the individual elements, especially the conserved guanosine. The results are discussed in terms of models for the mechanism of self-splicing.
Animals↗
Cloning and expression of murine CD27: comparison with 4-1BB, another lymphocyte-specific member of the nerve growth factor receptor family.
CD27 is a member of the nerve growth factor receptor family, that includes two types of tumor necrosis factor receptor, CD40 and Fas/Apo-1. Human CD27 has been found only on lymphocytes. In T cells, its expression strongly increases in a transient fashion upon antigenic stimulation, suggesting that CD27 plays a role during T cell activation. To analyze the function of CD27, we have identified the murine CD27 at the cDNA and protein level. Murine CD27 shows an identity of 65% compared with human CD27. The amino-terminal cysteine-rich region, i.e. the putative ligand-binding domain, and the carboxy-terminal part of the cytoplasmic domain are approximately 80% identical in man and mouse. Murine CD27 has 29% identity to 4-1BB, another lymphocyte-specific member of the receptor family defined only at the cDNA level. Murine CD27 and 4-1BB have 39% homology in the cysteine-rich domain and share a conserved region in the cytoplasmic tail. Expression studies identified murine CD27 mRNA in thymus and spleen, but not in non-lymphoid tissues, while 4-1BB mRNA was not detected in any tissue tested. In resting T cells, only murine CD27 mRNA was found, while in activated T cells murine CD27 as well as 4-1BB were present at high levels. Murine CD27 and 4-1BB mRNA are expressed with different kinetics during T cell activation, suggesting that these molecules play different roles in this process. Peptide antisera identified murine CD27 as a 45-kDa protein on thymocytes and activated T cells, while 4-1BB was precipitated as a 35-40-kDa protein from activated T cells.
Amino Acid Sequence↗
T cell receptor-alpha beta lacking the beta-chain V domain can be expressed at the cell surface but prohibits T cell maturation.
A TCR-beta gene lacking V domain sequences (delta V-TCR-beta) was inserted into the germline of mice. Expression of the transgene inhibited endogenous TCR-beta, but not TCR-alpha gene rearrangement and expression. The mutated TCR-beta gene affected alpha beta T cell development: the common thymocyte pool was normal in cell number, with cells expressing CD4 and CD8, but the mature, "CD3bright" population expressing either CD4 or CD8 molecules was reduced by 90%. To help understand these effects on TCR-beta gene rearrangement and T cell development, biosynthesis of the delta V-TCR-beta protein was analyzed in a tumor cell line derived from a transgenic mouse. Despite absence of the V domain, the delta V-TCR-beta chain paired with endogenous TCR-alpha chains and assembled with CD3 gamma, -delta, -epsilon, and -zeta components in the endoplasmatic reticulum, followed by transport through the Golgi complex to the plasma membrane. Therefore, assembly of the complex, and even cell surface expression, may be relevant for allelic exclusion of the TCR-beta gene. In the common thymocyte population, the CD3 components, endogenous TCR-alpha, and the delta V-TCR-beta gene product were expressed at the RNA level, but endogenous TCR-beta was not. The TCR-alpha delta beta/CD3 complex was present at the cell surface at low levels and was functional in terms of anti-CD3-induced Ca2+ mobilization. The observed arrest of alpha beta T cell development at the CD4+8+ thymocyte stage indicates that ligand recognition by the TCR, with contribution of the beta-chain V domain, is not required for transition of CD4-8- thymocytes to the CD4+8+ phenotype, but necessary for entry into the "single positive," CD3bright differentiation stage.
Animals↗