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Gamete Biology |
Institute of Human Genetics3
Department of Biochemistry,4 University of Göttingen, 37073 Göttingen, Germany
Department of Anatomy and Cell Biology,5 University of Giessen, 35378 Giessen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gametogenesis, sperm, sperm motility and transport, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various modifications of proteins associated with the DNA occur during spermatid differentiation, and the result is the progressive condensation of the chromatin [10, 11]. This process includes the gradual displacement of testis-specific and remaining somatic histones by transition protein 1 and 2 (Tnp-1 and Tnp-2), which are thought to participate in the initial condensation for the spermatid nucleus [11–13]. Shortly thereafter, the transition proteins are replaced by the protamines 1 and 2, which are characteristic for the mature sperm nucleus [11]. For elucidation of Tnp-2 function in spermiogenesis, knockout mice were generated and analyzed [3]. Breeding of Tnp-2-/- males revealed normal fertility on the mixed background C57BL/6J x 129/Sv but infertility on the inbred 129/Sv background. On this background, electron microscopy showed that the spermatogenic cells were undergoing chromatin condensation, although many spermatozoa exhibited head abnormalities with the acrosome not attached to the nuclear envelope [3].
Proacrosin is located in the sperm acrosome and has long been believed to be responsible for proteolysis of the zona pellucida of the oocyte, thus enabling the sperm to penetrate this extracellular matrix and to gain access to the oocyte plasma membrane [13–15]. However, it was shown that sperm from male mice homozygous for a targeted mutation in the Acr gene can penetrate the zona pellucida and the mice are fertile [4, 5].
Histones represent the major protein component of eukaryotic chromatin and play an important role in the organization of chromatin structure [10, 16]. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle [17]. Mice lacking the histone H1.1 gene show normal testicular morphology and spermatogenesis and are fertile [6]. The histone H1t gene is expressed exclusively in spermatocytes [16, 18]. The protein represents the major part of the total H1 histone complement in pachytene spermatocytes and is the early linker histone in round and elongating spermatids [19]. Mice homozygous for the mutated H1t gene locus develop normally, and testicular morphology and spermatogenesis remain unaffected in the absence of H1t [7, 8].
In the present study, two triple knockout mouse lines, Tnp-2-/-/Acr-/-/H1.1-/- and Tnp-2-/-/Acr-/-/H1t-/-, were generated on the mixed background CD-1 x C57Bl/6J x 129/Sv, and different fertility parameters were studied. It turned out that male mice of both lines did not differ from controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments with animals were performed at the Animal Facility of Institute of Human Genetics, Goettingen, Germany. The animals were housed under a 12L:12D cycle with free access to standard mouse chow and tap water. All of the experimental procedures complied with national regulations for the care and use of laboratory animals (similar to the U.S. National Research Council guidelines).
Mouse Crosses
In the past, we generated knockout mice for Tnp-2 (3), Acr (5), H1.1 (6), and H1t (7) genes. The genetic background of Acr-/- mice is CD-1 x 129/Sv and that for Tnp-2-/-, H1.1-/-, and H1t-/- is C57BL/6J x 129/Sv. To obtain double heterozygous animals for each combination, except H1.1 and H1t, which are closely linked on chromosome 13 [9], five breeding pairs (one male with two females) were used. Double heterozygous males and females (10 breeding pairs for each combination) were bred to obtain double homozygous animals. Further breeding was performed with 20 males and females: (Tnp-2-/-/Acr-/-/H1t+/+) x (Tnp-2+/+/Acr-/-/H1t-/-) and (Tnp-2-/-/Acr-/-/H1.1+/+) x (Tnp-2+/+/Acr-/-/H1.1-/-). The resulting males and females (20 animals each), Tnp-2+/-/Acr-/-/H1t+/- and Tnp-2+/-/Acr-/-/H1.1+/-, respectively, were bred to obtain homozygous triple knockout mice. Both triple knockout mouse lines on mixed genetic background CD-1 x C57BL/6J x 129/Sv are now bred in our laboratory for more than 2 years. For genotyping of the mutant mice, primers and previously established polymerase chain reaction (PCR) conditions were used [3, 5, 6, 7].
Northern Blot Analysis
Total RNA was extracted from mouse testes using the RNA Now kit (ITC Biotechnologies, Heidelberg, Germany) according to the manufacturer's recommendation. Twenty micrograms of RNA was size fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and transferred to nylon membranes. The membranes were hybridized with 32P-labeled probes for Tnp-2, Acr, H1.1, and H1t, and reprobed with elongation factor 2 cDNA [20] to ensure RNA integrity and equal loading.
Fertility Test
To study the fertility of the triple knockout animals, 10 males and 10 females of each line were bred with wild-type mice. Furthermore, 15 triple knockout males and females of each line were crossed together. The time between breeding and birth of offspring and the litter sizes were monitored. The respective data were also evaluated from breeding of wild-type animals.
Seven to eight-week-old CD-1 female mice were superovulated by intraperitoneal injections of 5 IU of eCG (Intergonan, 5 IU; Intervet, Tönisvorst, Germany) followed by 5 IU of hCG (Predalon, 5 IU; Organon, Oberschleissheim, Germany) 46–48 h later and mated with wild-type, Tnp-2-/-/Acr-/-/H1t-/-, or Tnp-2-/-/Acr-/-/H1.1-/- male mice. For each experiment, 10 sexually mature male mice were used. Oocytes from females with a copulatory plug were isolated. The oviducts were dissected out and flushed with M2 medium (Sigma, St. Louis, MO). The oocytes were treated with M2 medium containing hyaluronidase (300 µg/ml) to remove the cumulus cells, washed in M2, further maintained in M16 (Sigma-Aldrich Chemie, Deisenhofen, Germany), and subsequently examined for the presence of male and female pronuclei. Furthermore, the eggs were cultured in M16 covered with mineral oil and checked for embryonic development.
In Vitro Competition Assay
Sexually mature Tnp-2-/-/Acr-/-/H1t-/-, Tnp-2-/-/Acr-/-/H1.1-/- and Acr-deficient male mice (Tnp-2+/+/Acr-/-/H1t+/+/H1.1+/+) were used for these experiments. Female CD-1 wild-type mice were superovulated as described before and oocytes were collected 10–12 h after hCG administration. The cumulus cells were removed by hyaluronidase treatment, and the oocytes were washed and subsequently cultured in fertilization medium (MediCult, Jyllinge, Denmark). Spermatozoa were isolated from the cauda epididymis and capacitated in in vitro fertilization (IVF) medium at 37°C for 1.5 h. Three Acr-deficient male mice and three males from each triple knockout mouse line were used. Equal amounts of sperm (5 x 105 each) from each triple knockout mouse line and from Acr-deficient mice, respectively, were mixed and then added to the oocytes in 100-µl drops of fertilization medium and incubated for 6 h at 37°C in 5% CO2 covered with mineral oil. Using a large-bore micropipette, eggs were washed and further cultured in M16 to blastocyst stage. Single blastocysts were frozen in 10 µl of water, then boiled for 5 min and subjected to the PCR analysis using Tnp-2 primers [3].
Sperm Analysis
From individual male mice of both triple knockout lines and from wild-type male mice, epididymides were collected and dissected in IVF medium. Sperm number in corpus and cauda epididymis was determined using the Neubauer cell chamber. To investigate the acrosome reaction, spermatozoa were capacitated for 1.5 h in Tyrode medium and then incubated for 5 min at 37°C in 5% CO2 with Tyrode medium plus 20 µM calcium ionophore A23187 (Sigma-Aldrich Chemie). To determine the percentage of acrosome reacted spermatozoa, sperm were fixed and stained with Coomassie brilliant blue R250 as previously described [21]. At least 200 spermatozoa from three to five males were examined for the presence of the characteristic dark blue acrosomal crescent. To investigate the sperm migration in the female reproductive tract, five mutant males from each line and wild type were mated with two mature, CD-1 females each. Uteri and oviducts from females with vaginal plug were flushed with M2 medium, and the sperm number was determined.
Sperm Motility Analysis
Epididymides of wild-type and mutant homozygous mice were dissected in IVF medium (Medi-Cult). Spermatozoa were allowed to swim out of the epididymides and incubated for 1.5, 3.5, or 5.5 h at 37°C. A drop of the sperm suspension was transferred to the incubation chamber, which was preset to 37°C. Sperm movement was quantified using the CEROS computer-assisted semen analysis system (version 10, Hamilton Thorne Research, Beverly, MA). Then, 6000–10<008>000 spermatozoa from three mice of each mutant line and wild type were analyzed using the following parameters: negative phase-contrast optics; recording, 60 frames per second; minimum contrast, 60; minimum cell size, 6 pixels; straightness (STR) threshold, 
50%; average path velocity (VAP) and straight line velocity (VSL), 25 µm/sec and 30 µm/sec, respectively: minimum progressive VAP, 75 µm/sec; and minimum static contrast, 15 pixels. Slow cells motile was not used as this limit avoids counting sperm moved by other sperm, Brownian motion, and low velocity nonprogressive cells.
For statistical analysis, frequencies of the six sperm motility parameters (VAP, VSL, curvilinear velocity [VCL], amplitude of the lateral head displacement [ALH], beat cross frequency [BCF], and STR) were examined by probability plots categorized by mouse type (wild type/mutant) and time of observation (1.5, 3.5, and 5.5 h after preparation). VAP, VSL, VCL, and BCF were log-normally distributed, but ALH and STR were not. For statistical calculation, sperm motility measurements of each parameter were pooled for each individual mouse type and for each time point. The Student t-tests for independent observations were applied to define differences in VAP, VSL, SCL, and BCF means (normalized by natural logarithms) comparing wild-type and mutant mice. For the same purpose, the nonparametric ALH and STR distributions were tested by Friedman analysis of variance. Statistical analyses were performed using the Statistica software package (StatSoft, Inc., Tulsa, OK).
Electron Microscopy
Testes and epididymides were fixed with 5% glutaraldehyde in 0.2 M phosphate buffer, postfixed with 2% osmium tetroxide, and embedded in epoxy (Epon) resin. Selected areas were sectioned and examined by electron microscopy.
Statistical Analysis
Statistical analysis was performed using a computer software program (Statistica software program, StatSoft Inc., Tulsa, OK). The data of fertility and sperm analysis were compared using the Mann-Whitney U-test and Student t-test. The data of competition assay were compared using the chi-square test. The P values are given in the Results section or in the legends to the figures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The triple mutant mice Tnp-2-/-/Acr-/-H1t-/- and Tnp-2-/-/Acr-/-/H1.1-/- were genotyped by PCR, using primers for wild-type and mutant alleles for all four loci (Fig. 1A). Male and female mice of both triple knockout lines were viable and developed normally.
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Northern blot analyses with testicular total RNA were performed to confirm the inactivation of the genes in both triple knockout lines. These analyses revealed that Tnp-2, Acr, H1t, and H1.1 transcripts are absent in Tnp-2-/-/Acr-/-/H1t-/- and Tnp-2-/-/Acr-/-/H1.1-/- mice, respectively.
Fertility of Triple Mutant Mice
To investigate the consequences of combined disruption of Tnp-2, Acr, H1t, and H1.1 genes for fertility, mating tests were performed as described in Materials and Methods. All matings between triple mutant males with wild-type females were productive, and the average litter size was not significantly different (P > 0.05) from that of wild-type breeding (Table 1). Similar results were obtained from breeding of male and female triple knockout mice (P > 0.05) (Table 1). To further evaluate the fertility of Tnp-2-/-/Acr-/-/H1t-/- and Tnp-2-/-/Acr-/-/H1.1-/- male mice, we performed in vivo fertilization assays. Wild-type females were mated with wild-type or mutant males, and oocytes were collected 16 h later and scored for the presence of male and female pronuclei. No significant difference (P > 0.05) was found between fertilization rates of wild-type and mutant mice (Table 2). To study embryonic development, oocytes were cultured to blastocyst stage. Our findings indicate that the simultaneous lack of Tnp-2, Acr, histone H1t, or histone H1.1 does not affect the ability of sperm to penetrate the zona pellucida of the oocytes and to fertilize the eggs and has no influence on early embryonic development (Table 2).
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Sperm Analysis of the Mutant Males
Sperm numbers were determined in the corpus and cauda epididymis of wild-type and mutant males and in uteri and oviducts of wild-type females inseminated by Tnp-2-/-/Acr-/-/H1t-/-, Tnp-2-/-/Acr-/-/H1.1-/-, and wild-type males, respectively. No significant differences (P > 0.05) in sperm numbers were found (Table 3). Furthermore, wild-type and mutant mice did not differ in the rates of in vitro acrosome reacted sperm (P > 0.05) (Table 3). Sperm motility was measured after 1.5, 3.5, and 5.5 h of incubation in vitro. No significant differences (P > 0.05) between the motility of spermatozoa of wild-type and mutant males were observed. The following parameters were evaluated in more detail: VCL, VAP, VSL, BCF, STR, and ALH. For all parameters, no significant alterations were found (P > 0.05) (Fig. 2).
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To determine whether the spermatozoa from Tnp-2-/-/Acr-/-/H1t-/- and Tnp-2-/-/Acr-/-/H1.1-/- mice have structural abnormalities, light and electron microscopical analyses were performed. Structural defects were observed neither during spermatogenesis nor in single spermatozoa (data not shown).
In Vitro Competition Assay
Sperm from Acr-deficient mice (Tnp-2+/+/Acr-/-/H1t+/+/H1.1+/+) were mixed with equal amounts of sperm of the triple knockout mice Tnp-2-/-/Acr-/-/H1t-/- and Tnp-2-/-/Acr-/-/H1.1-/-, respectively. Sperm mixture was incubated with oocytes from wild-type mice (Tnp-2+/+/Acr+/+/H1t+/+/H1.1+/+) in an in vitro competition assay. After culture of the fertilized oocytes to blastocyst stage, genomic DNA was extracted and subjected to PCR with Tnp-2 primers [3]. Blastocysts (53.6% of 97) derived from oocytes fertilized with sperm only deficient for Acr and blastocysts (46.4% of 97) derived from oocytes fertilized with sperm lacking Tnp-2, Acr, and Hlt (Fig. 3). This difference is not significant (P > 0.05). In contrast, a significant competition (P < 0.05) between Acr-deficient sperm and those lacking Tnp-2, Acr, and H1.1 is evident. In in vitro competition assays using a mixture of equal amounts of Acr-deficient sperm and sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1.1-/-, 70.7% of 58 blastocysts were genotyped as Tnp-2+/+/Acr+/-/H1.1+/+ and 29.3% of 58 blastocysts were genotyped as Tnp-2+/-/Acr+/-/H1.1+/- (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several explanations for functional redundancy of these proteins are possible. Genes that belong to the same family of parallel pathways could be functionally redundant. Mice deficient for H1.1 and H1t are fertile, which suggests that both histones are dispensable for normal germ cell development [6–8]. Other H1 subtypes might have overlapping or redundant functions and compensate for the loss of these histone proteins. In H1t-deficient mice, it was demonstrated that H1.1, H1.2, and H1.4 histone gene expression in testis is enhanced [7]. In H1.1-deficient mice compared with wild-type litter mates, expression of the H1.2 through H1.4 genes is increased [6]. The similarity of the phenotypes of spermatozoa from Tnp-1- and Tnp-2-deficient mice and the elevated level of Tnp-1 and Tnp-2 in testis of Tnp-2- and Tnp-1-deficient mice, respectively, raise the possibility that a deficiency created through the disruption of the Tnp-2 can be compensated by recruitment of Tnp-1 and vice versa [3, 29, 30]. Therefore, it can be concluded that normal phenotype and function of sperm in our triple knockout mice result from overexpression of genes belonging to the respective families.
Another possible explanation is that some sperm proteins are involved in fine tuning of certain fertilization processes or in overcoming specific fertilization barriers rather than in the control of major developmental and/or physiological events during sperm differentiation and fertilization [2, 21]. Therefore, fertility disturbance could only become apparent in triple knockout mice exposed to a less protected environment than under standard laboratory conditions. We showed previously that Acr-deficient mice are fertile and show no impairment of fertilization. However, in a mouse model system, in which the lack of Acr in the male and modifications of the zona pellucida in the female were combined, a significant reduction of the fertilization rate in vitro was observed [21].
An additional possibility is that some of the sperm proteins perform functions that confer selective advantages through sperm competition during evolution [31, 32]. Comparing gene sequences within and between closely related species has shown that the genes encoding sperm proteins are more divergent than the genes that are expressed in nonreproductive tissues and show a rapid evolution [33, 34]. Although nonreproductive proteins exhibit a divergence of less than 10%, a divergence of more than 30% was calculated for Tnp-2, Acr, and histones H1t and H1.1 [34]. This widespread phenomenon might have important consequences, such as the establishment of fertilization barriers between species or in overcoming specific fertilization barriers within species that might lead to speciation during evolution. It was proposed that the selective forces of sperm competition, sexual selection, and sexual conflict could individually or in combination provide the selective force that drives the rapid evolution of reproductive proteins. Sperm competition occurs because each sperm competes with all the other sperm to be the first to fuse with the egg. We demonstrated previously that Acr-deficient sperm have a selective disadvantage in the presence of wild-type sperm in in vitro competition assays [5]. In the present study, it was shown that sperm from Tnp-2-/-/Acr-/-/H1.1-/- exhibit selective disadvantages in the presence of Acr-deficient sperm. It can be concluded from these results that some sperm proteins are essential for competition. Therefore, it is possible that these proteins have a role in a selection process and give the sperm the ability for competition during evolution. Functional analysis, proteomics, and sequence analysis of sperm proteins will help to clarify the function of reproductive proteins during evolution.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wolfgang Engel, Institute of Human Genetics, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany. FAX: 49 551 399303; wengel{at}gwdg.de
Received: 24 April 2003.
First decision: 12 May 2003.
Accepted: 6 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), Tnp-2-/-/Acr-/-/H1t-/- (
), and Tnp-2-/-/Acr-/-/H1.1-/- (
) spermatozoa are shown. VAP, VSL, and VCL (micrometers per second), STR (percent), ALH (micrometers), and BCF (Hertz) were measured after 1.5, 3.5, and 5.5 h. For VAP, VSL, VCL, and BCF, the means and appropriate SDs are illustrated. For ALH and STR, the medians and quartiles are shown. Spermatozoa from both triple knockout mice show no significant differences in VAP, VSL, VCL, BCF, ALH, and STR compared with wild-type sperm (P > 0.05)