Sperm chromatin integrity of bucks transgenic for
the WAP bGH gene
Piotr Gogol
∗, Michał Bochenek, Zdzisław Smor¸ag
Department of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice/Kraków, Poland
Received 8 February 2000; received in revised form 20 June 2000; accepted 18 July 2000
Abstract
The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13–18 months old. Semen was collected at 1-week intervals and 3–7 ejaculates from each rabbit were examined in total.
Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chro-matin Structure Assay method: after chroChro-matin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes.
The mean value of COMPαtwas 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant
difference, t-test). The mean values of S.D.αtfor the TG and NTG rabbits were 10.94 and 10.40 (no
significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (−0.68,P <0.05) was found between S.D.αtof NTG males and percentages of pregnant does.
The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with dam-aged chromatin structure. This suggests that the mere presence of the introduced gene construct does
∗Corresponding author. Tel.:+48-12-285-67-11; fax:+48-12-285-67-33. E-mail address: [email protected] (P. Gogol).
not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.
The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Sperm chromatin; Transgenesis; Rabbit; Fertility
1. Introduction
Reproductive disorders have been observed in transgenic mice, pigs and sheep (Rexroad et al., 1989; Pursel et al., 1990; Bartke et al., 1992; Meliska and Bartke, 1997; Maleszewski et al., 1998). Decreased fertility or infertility of transgenic males was related to altered copulatory behaviour (Meliska and Bartke, 1997) or to structural defects and defective sper-matozoa function (Maleszewski et al., 1998). One abnormality found in the spersper-matozoa of transgenic mice was that chromatin was non-homogeneous and unstable (Maleszewski et al., 1998). Condensation of nuclear heterochromatin is one of the most important changes during spermatogenesis. This process involves the exchange of chromatin’s histon proteins into protamins, and then the oxidation of the sulhydril groups (–SH) of these protamins into the form of disulfide bridges (–S–S). This process is accompanied by a strong condensation of chromatin which protects genetic material from mechanical and chemical damage. Stud-ies on spermatozoa of mice (Evenson et al., 1985), bulls (Evenson et al., 1980; Ballachey et al., 1986, 1987), boars (Evenson et al., 1994) and humans (Evenson et al., 1980; Tejada et al., 1984; Potts et al., 1999) showed that increased damage of chromatin structure may be related to spermatogenesis disorders, increased percentage of morphologically abnormal spermatozoa, and decreased fertility. It is supposed that sperm chromatin structure abnor-malities after some kinds of mutation are related to susceptibility of DNA to denaturation and decreased male fertility (Potts et al., 1999). It cannot be ruled out that genome change obtained by the introduction of a gene construct, as is the case with transgenic animals, leads to changes in chromatin properties. One method for an accurate analysis of chromatin struc-ture is the cytometric method developed by Evenson (1990) (Sperm Chromatin Strucstruc-ture Assay). The method is based on the assumption that structurally abnormal sperm chromatin is more susceptible to acid or heat denaturation. The SCSA method takes advantage of the metachromatic properties of acridine orange. This dye fluoresces in the green band when combined with the intact double DNA helix, and in the red band when combined with RNA and denatured DNA. Following mild denaturation of chromatin through decreased pH, the fluorescence of spermatozoa with structurally nabnormal chromatin is strengthened in the red band. This makes it possible to determine an increase in denatured DNA since almost no RNA is found in spermatozoa. This method also makes it possible to investigate chromatin changes during spermatogenesis.
Table 1
Composition of A and B solutions used in two-stage SCSA protocol
Solution A Solution B
Triton (X-100) 0.1 ml Citric-phosphate buffera 100 ml
HCl (1.0N) 8 ml
NaCl 0.877 g NaCl 0.877 g
H2O (2×distilled) Up to 100 ml EDTA-Na2 34 mg
Acridine orange (1mg/ml) 0.6 ml
aMixture of 37 ml of 0.1 M citric acid solution+3 ml of 0.2 M Na
2HPO4solution.
2. Material and methods
2.1. Semen
Twenty males transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13–18 months old. Semen was collected at 1-week intervals and 3–7 ejaculates from each rabbit were examined in total.
After determining sperm concentration using the Coulter counter, the samples were di-luted in PBS to obtain 1×106ml−1spermatozoa.
For chromatin denaturation and staining, we followed the two-stage SCSA protocol (Evenson, 1990): to 0.2 ml of diluted semen, we added 0.4 ml of solution A, and after 30 s, 1.2 ml of solution B (Table 1). Cytometric analysis was made following a 3 min incubation at 4◦C.
2.2. Flow cytometer
Coulter Epics Elite (Coulter, Miami, USA) flow cytometer was used. Acridine orange was excited at 488 nm and 35 mW laser beam. Fluorescence was read out through 550DL and 525BP filters in the green band and through 675BP filter in the red band. The parameters measured were forward scatter, side scatter and fluorescence in the green and red bands (linear scales).
Semen sample analyses were computer recorded in separate files as measurements of the fluorescence of 5000 spermatozoa.
2.3. Data analysis
Data files were analysed using WinList32 software (Verity SH, Topsham, USA). The number of spermatozoa with structurally abnormal chromatin was analysed on the basis of histograms that showed red-band fluorescence, and also on the basis of an artificial parameter
αtcalculated for each spermatozoon, whereαt =red/(green+red)fluorescence. For each
sample, percentage of spermatozoa outside the main populationαt(COMPαt) and standard
2.4. Fertility test for transgenic males
Eight ejaculates from six transgenic males were used in the experiment. Females were inseminated with semen in which, after dilution with Galap extender (IMV, France), the per-centage of progressive-motile spermatozoa was at least 60%. The number of spermatozoa was 15×106per insemination dose. Five or six New Zealand White does were inseminated at 5–7 months old and 3–4 kg b.w. in eight groups. Superovulation was induced by intra-muscular injection of 100 IU PMSG (Serogonadotropin, Biowet, Poland). After 72 h, the does were inseminated and intravenously injected with 100 IU HCG (Biogonadyl, Biomed, Poland). About 24 h, after insemination, eggs were flushed from oviducts isolated after slaughter. Fertilised eggs were cultured in vitro in B2-Inra medium (Laboratoire C.C.D., France) in a CO2incubator to the blastocyst stage. Fertility was assessed from the
percent-age of fertilised eggs and percentpercent-age of embryos which developed to the blastocyst stpercent-age in in vitro culture.
2.5. Fertility test for non-transgenic males
Nine ejaculates from 7 non-transgenic males were used in the experiment. Semen was
diluted with Galap extender to make the insemination dose of 0.4 ml contain 5×106
progressive-motile spermatozoa. Primiparous New Zealand does 4.5–5 months old and at 2.9–3.5 kg b.w. were chosen for the insemination. Prior to insemination, hormonally non-stimulated females were intramuscularly injected with 0.3 ml of the chemical analogue of GnRH Receptal (Hoechst, Germany) to induce ovulation. A total of 212 females (from 17 to 28 per group) were inseminated in nine groups. Fertility was evaluated from the percentage of fertilised females and from mean litter size.
2.6. Statistical analysis
The differences between groups and correlations between fertility and COMPαt and
S.D.αtparameters were calculated using the Statistica package (StatSoft, Tulsa, USA).
3. Results
The results of cytometric analysis of chromatin structure for transgenic and non-transgenic males are presented in Tables 2 and 3. The mean value of COMPαtwas 3.71 for TG rabbits
and 2.89 for NTG rabbits. The mean values of S.D.αt for the TG and NTG rabbits were
10.94 and 10.40, respectively. No statistically significant differences were found between
average values of COMPαt and S.D.αtparameters calculated for TG and NTG males.
Table 2
Results of chromatin structure analysis of transgenic rabbits
Rabbit No. of ejaculates analysed COMPαt(mean±S.D.) S.D.αt(mean±S.D.)
Results of chromatin structure analysis of non-transgenic rabbits
Rabbit No. of ejaculates analysed COMPαt(mean±S.D.) S.D.αt(mean±S.D.)
Correlation of SCSA variables (COMPαtand S.D.αt) and fertility of transgenic rabbits
Variable Percentage of fertilised oocytes Percentage of embryos developed to the blastocyst stage
COMPαt −0.42 −0.33
Table 5
Correlation of SCSA variables (COMPαtand S.D.αt) and fertility of non-transgenic rabbits
Variable Percentage of fertilised does Mean litter size
COMPαt −0.62 0.26
S.D.αt −0.68∗ 0.45
∗P <0.05.
After insemination of non-stimulated females with the semen of non-transgenic males, pregnancy rates ranged from 71.4 to 90.9 % and litter sizes from 4.9 to 7.8. A statistically significant correlation (−0.68,P < 0.05) was found between S.D.αt of NTG males and
pregnancy in the inseminated does. No correlation was found between COMPαtparameter
and percentage of fertilised does and between S.D.αt and mean litter size (Table 5).
4. Discussion
The present studies of sperm chromatin in transgenic and non-transgenic rabbits showed both groups of males to have a relatively low (3%) rate of spermatozoa with an abnormal chromatin structure. A higher rate of spermatozoa with damaged chromatin structure was found in the semen of non-transgenic rabbits by Courtens et al. (1994). Using the electron microscopy method without any experimental treatments or procedures, he demonstrated spontaneous decondensation of the chromatin in about 6% of spermatozoa in rabbit ejac-ulates. The presence of chromatin decondensation in such a large number of spermatozoa was not confirmed in the present study, despite the fact that the spermatozoa were exposed to decreased pH level prior to cytometric analysis of chromatin. It is supposed that the results obtained by Courtens et al. (1994) could have been affected by the breed of rabbits and/or by the process of sample fixing prior to microscopic analysis.
The stability of sperm chromatin in transgenic males was only slightly lower compared to non-transgenic males. In mice, incompletely condensed chromatin was found in 80% sper-matozoa in the semen (Rhim et al., 1995). These studies involved animals in which gene expression resulted in protamine characteristic of mouse chromatin being partly replaced with bird protamine (gallin) which is unable to form disulfide bridges. The above observa-tions and the lack of significant differences in sperm chromatin stability between transgenic and non-transgenic rabbits found in the present study suggest that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.
A significant relationship between the parameters of sperm chromatin stability and fertil-ity was shown by many other authors. An especially high correlation was observed between S.D.αtparameter and fertility (Evenson et al., 1994; Bochenek et al., 2000). This observa-tion was confirmed by the results of fertility testing of NTG males semen. The fact that the correlation was significant for S.D.αtbut statistically non-significant for COMPαtsuggests
In a review paper, Ward and Coffey (1991) suggest that chromatin structure is an important factor affecting fertility and viability of embryos. In the case of fertility, testing involving TG males semen, such a relationship was not found. This could result from a relatively high stability of sperm chromatin in the ejaculates used for the insemination of superovulated females. In an extreme case, the percentage of sperm with damaged chromatin structure was 7.8. However, the results obtained may indicate that sperm chromatin stability is not the es-sential condition of normal fertilisation and embryo development. Rhim et al. (1995) stated that despite chromatin condensation abnormalities detected using the microscopic method and acridin orange staining, transgenic mice were of normal fertility. He suggested that pre-cise packaging of sperm chromatin is not necessary for the normal process of unpackaging in the pronucleus of the fertilised oocyte and for normal embryonic development. Therefore, chromatin condensation abnormalities in the sperm of males with abnormal fertility are perhaps not the primary cause of their decreased fertility. Normal embryonic development obtained after the introduction of spermatids into oocytes (Yanagimachi, 1994) is evidence that the conversion of a histone-complexed DNA to a protamine-complexed spermatozoal DNA is not a prerequisite. In our studies the relationships between chromatin structure and fertility are not clear (significant for NTG rabbits and non-significant for TG rabbits) and require further investigation.
Our work have shown that the presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. The fertility of transgenic rabbits under study was normal.
Acknowledgements
This work was supported by the State Committee for Scientific Research, Grant no. 5 PO6D 035 14.
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