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Gamete Biology |
Genome Information Research Center,3
Faculty of Pharmaceutical Sciences,4 Osaka University, Osaka 565-0871, Japan
Institute of Applied Biochemistry,5 University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan
Department of Biomedical Sciences,6 Cornell University, Ithaca, New York 14853
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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XY chimeras were produced by aggregating wild-type embryos with embryos of transgenic mice lacking functional calmegin genes and expressing enhanced green fluorescent protein (EGFP) in their acrosomes. Chimeric ejaculates contained wild-type, nonfluorescent sperm as well as sperm with EGFP-tagged acrosomes and the defective calmegin gene. Transgenic, wild-type, and chimeric males were mated to wild-type females; however, only wild-type sperm were ever found within the oviducts. Calmegin-knockout sperm, even when they were combined in chimeric ejaculates with wild-type sperm, remained outside of the uterotubal junction. These findings indicate that the presence of wild-type sperm cannot compensate for the inability of calmegin-knockout sperm to enter the oviduct and that successful ascent into the oviduct depends on the capabilities of individual sperm.
fallopian tubes, oviduct, sperm, sperm motility and transport, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies indicate that sperm from mice with disrupted calmegin genes (Clgn–/–) are impaired in their ability to ascend into the oviduct [3]. After mutant males are mated to wild-type females, very few sperm can be found in or above the uterotubal junction (UTJ). This defect in phenotype is not due directly to calmegin, which disappears from spermatogenic cells late in spermatogenesis, but may be attributed to the loss of its protein chaperone function; that is, to ensure proper folding of nascent proteins in the endoplasmic reticulum. Without the aid of chaperones, nascent proteins may form aggregates and fail to assume their proper position and function in the cell. For example, mice lacking functional calmegin produce sperm that lack ADAM2, also known as fertilin ß, on their plasma membranes [3], and ADAM2–/– sperm also fail to ascend into the oviduct [4]. Mature sperm of both Clgn–/– and ADAM2–/– males also lack additional plasma membrane proteins [5], one or more of which could be involved in enabling sperm to enter the oviduct.
In wild-type mice, sperm ascent into the oviduct is rapid. Substantial numbers of sperm are found in histologic sections of the oviductal isthmus within 30 min of mating. The ostium of the colliculus tubarius, the uterine portal to the UTJ, closes within an hour of coitus in early estrus in the mouse [6]. Transillumination of freshly dissected oviducts 1–2 h after coitus in early estrus revealed that the initial intramural region of the UTJ had constricted, while the extramural UTJ, closer to the ovary, was patent and contained many motile sperm [7]. Most of those sperm were stuck by their heads to the mucosal epithelium, a phenomenon believed to be responsible for forming a sperm reservoir prior to ovulation, but which might also enable sperm to gain a foothold in the UTJ. Little is known about the regulation of the patency of the UTJ, nor about the means by which sperm enter the oviduct.
Our study was undertaken to determine why sperm from calmegin-knockout mice cannot enter the oviduct and thus to learn more about how sperm normally enter the oviduct. We tested whether the presence of wild-type sperm in the ejaculate could compensate for the defect in the knockout sperm, enabling them to enter the UTJ. This could happen if sperm from the knockouts lack a protein that would stimulate the opening of the UTJ. On the other hand, the presence of knockout sperm could prevent all sperm from entering the oviduct. This could happen, for example, if sperm from the knockouts are recognized as "foreign" by some mechanism that triggers closing of the UTJ. In order to test these hypotheses, chimeric males were constructed that ejaculated a mixture of wild-type and calmegin-knockout sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The handling and surgical manipulation of all experimental animals were carried out according to the guidelines of the Committee on the Use of Live Animals in Teaching and Research of Osaka University. A transgenic mouse line, C57BL/6 Tg [Acro/Act-EGFP] C3-N01-FJ132 Osb that expresses enhanced green fluorescent protein (EGFP) ubiquitously as well as within the acrosomes of sperm was used. The line had been created by coinsertion of the transgene pCAGGS-EGFP (CX-EGFP) with a transgene containing the mouse proacrosin promotor and the code for the first 24 amino acids of the N terminus of prepro-acrosin signal peptide region fused to the 5' end of the EGFP gene with 6 linker peptides in between (Acr-EGFP) [8, 9]. The ubiquitously expressed EGFP was used to facilitate the identification of transgenic males carrying Acr-EGFP in their sperm by examining pups with a hand-held UV light [10]. The EGFP transgenic mice were bred with B6C3F1 mice (Japan SLC, Inc., Shizuoka, Japan) and the resulting male offspring were mated with female Clgn–/– mice [11] to generate "double" transgenic mice that lack a functional calmegin gene and produce sperm with Acr-EGFP. The F1 offspring were crossed, and nonfluorescent F2 offspring were culled. The Acr-EGFP-tagged F2 offspring that were heterozygous for calmegin (Clgn+/–) were distinguished from Acr-EGFP-tagged Clgn–/– offspring using polymerase chain reaction, as described previously [3]. Briefly, two primers were used to identify the disrupted calmegin gene (5'-TCTTACCACA AAgCACCTCC-3' and 5'-gCACAACAgg ATggATgATT-3'), and another two were used to identify the functioning calmegin gene (5'-CCTTCCTgCg gCTTgTTCTC T-3' and 5'-TATCATCCTT CTTTgCTTTT g-3').
Another transgenic mouse line that has CX-EGFP on its X chromosome, B6C3F1 Tg [Act-EGFP] CX-139-FM139Osb, was bred with B6C3F1 to facilitate selection of male embryos, as described below. This line had been identified by using fluorescence in situ hybridization to locate the insertion site of a transgene containing the coding sequence for EGFP and the CAG promoter (ß-actin promoter and hCMV enhancer) [10]. One of the six lines carrying the transgene on the X chromosome [12] was used in this experiment.
Computer-Assisted Sperm Analysis of Motility of Sperm from Transgenic Mice
Sperm were obtained from the epididymides of Clgn–/– and Clgn+/– mice by puncturing the epididymal tubules and allowing sperm to disperse into TYH medium [13] to make approximately 3 x 107 sperm/ ml. After 15, 60, and 120 min of incubation, 10 µl of sperm suspension were transferred to a Makler counting chamber (Sefi Medical Instrument, Haifa, Israel) for analysis of sperm movement using an HTM-IVOS Ver.10 (Hamilton Thorne Research, Beverly, MA). At least 200 sperm were analyzed per sample to evaluate the percentage of motile sperm, straight line velocity, curvilinear velocity, amplitude of lateral head displacement, and linearity. The following settings were used: temperature, 37°C; 10x negative phase-contrast optics; frames acquired, 30; frame rate, 60 Hz; minimum contrast, 60; minimum cell size, 3 pixels; minimum static contrast, 30; low average path velocity cutoff, 5.0 µm/sec; static head size, 0.32 to 2.99; static head intensity, 0.42 to 1.60; and magnification, 1.95. During analysis, the playback feature was used to delete sperm mistracked due to collision. Tracks of less than 16 points were eliminated. Values obtained for sperm from three Clgn+/– and three Clgn–/– males were compared using the Student t-test.
Selection of Male Wild-type Embryos
B6C3F1 females were superovulated by i.p. injection of 5 IU of eCG (Teikoku Zoki, Co. Ltd., Tokyo, Japan), followed 48 h later by 5 IU of hCG (Teikoku Zoki Co. Ltd). Oocytes were recovered from the oviducts 12 h after hCG, placed in TYH medium, and inseminated with sperm (1– 2 x 105 sperm/ml) from B6C3F1 Tg [Act-EGFP] CX-139-FM139Osb males, which carry EGFP on the X chromosome (X*). Fertilized eggs were incubated in KSOM medium until the 8-cell-morula stage, and were evaluated for X-chromosome-associated EGFP fluorescence, using a fluorescein epifluorescence filter set (U-MNIBA: BP470-490 BA515-550 DM505; Olympus, Tokyo, Japan) in an inverted microscope. Embryos with no fluorescence were considered to be wild-type males and were used to produce aggregation chimeras.
Partial Zona Dissection and In Vitro Fertilization
To assist the sperm from Clgn–/– mice to fertilize eggs, a partial zona dissection was performed. Clgn–/– and B6C3F1 oocytes were collected from oviducts of superovulated females 15 h after hCG injection and placed into a drop of TYH medium under mineral oil. The eggs were freed from cumulus cells by incubating with hyaluronidase (100 µg/ml, Sigma, St. Louis, MO) for 5 min, followed by washing. Partial zona dissection was performed according to the method described by Nakagata et al. [14]. Briefly, cumulus-free oocytes were transferred into 0.3 M sucrose in PBS in a tissue culture dish. This medium caused the oocytes to stick to the bottom of the dish and to shrink away from the zona pellucida, enabling a 30-gauge needle to be used to open a slit in the zona. Following the partial zona dissection, eggs were detached from the dish by introducing BSA-containing medium into the drop. The eggs were then washed with TYH medium [13] and fertilized with sperm (1–2 x 104 sperm/ml) from Clgn–/– male mice carrying Acr-EGFP. Six hours after insemination, the fertilized eggs were transferred into KSOM medium and incubated for 2 days to reach the 8-cell-morula stage.
Production of Chimeric Mice
Homozygous (Clgn–/–), heterozygous (Clgn+/–), and wild-type male embryos in the 8-cell-morula stage were incubated in acidic Tyrode solution (Sigma) for about 30 sec to remove the zona pellucida. The zona-free Clgn–/– and Clgn+/– embryos were each transferred with a zona-free wild-type male embryo into wells containing KSOM medium [15]. The embryo pairs were aggregated together to form chimeric blastocysts, which were transferred to pseudopregnant females that had been mated with vasectomized males. Chimeric mice arising from these transferred aggregated embryos are referred to as Clgn+/– wild-type and Clgn–/– wild-type.
Histological Evaluation of Chimeric Mice
Chimeric mice were killed by cervical dislocation, and the testes were fixed in 4% paraformaldehyde for 12 h, then rinsed with PBS for 1 h, dehydrated in acetone for 1 h, and embedded in glycol methacrylate (Technovit 8100; Heraeus Kulzer GmbH, Wehrheim, Germany). Serial sections of the entire testes were prepared at a thickness of 5 µm. Fluorescent photomicrographs were obtained using a fluorescein epifluorescence filter set, and then the sections were stained with hematoxylin and photographed again using brightfield optics.
Sperm Migration Analysis
Superovulated females were caged together with Clgn–/–, Clgn+/–, or chimeric males (Clgn–/– wild-type and Clgn+/– wild-type) 12 h after hCG injection, and were checked for the presence of vaginal plugs every 30 min. About 2 h after copulation, oviducts were excised together with the proximal part of the uterine horns. Some oviducts were carefully separated from the uterine horns and straightened out by cutting the mesosalpinx. They were transferred to slides as whole mounts, covered with coverslips supported by pillars of paraffin:petroleum jelly (1:10), and examined using epifluorescence microscopy with a fluorescein filter set to determine the presence of sperm containing the acrosomal EGFP marker. Other oviducts were prepared by fixation and sectioning to identify all sperm (fluorescent and nonfluorescent) within the UTJ. This was performed by fixing the coiled oviduct and proximal uterus for 6 h in 4% formaldehyde in PBS, washing in PBS, then freezing in OCT compound and sectioning. The sections were photographed using a fluorescein epifluorescence filter set, then stained with hematoxylin, and photographed again using brightfield illumination.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Utilizing Acr-EGFP-tagged sperm, the results of mating nonchimeric Clgn+/– and Clgn–/– males with wild-type females were examined to confirm that sperm from Clgn–/– males could not ascend into the oviduct. All sperm from Clgn+/– males behaved as though they had inherited the wild-type calmegin gene, because calmegin is shared through cytoplasmic bridges in spermatids [16]. Similarly, all sperm had Acr-EGFP in their acrosomes even when the mice had inherited the transgene hemizygously; thus, all sperm from heterozygous males showed identical acrosomal fluorescence as sperm from Clgn–/– males [9]. Three Clgn+/– males were each mated to three wild-type females, and the oviducts were removed from the females about 2 h after coitus. One oviduct from each female was uncoiled by cutting the mesosalpinx and examined as a whole mount. Hundreds of fluorescent Acr-EGFP-tagged sperm could be seen through the walls of each oviduct using epifluorescent transillumination. In contrast, when three Clgn–/– males with Acr-EGFP-tagged sperm were each mated to three wild-type females, no sperm were found in the oviducts (Fig. 1). The other oviduct from each female was processed for serial sectioning. All sections of the UTJ and isthmus were examined. Sperm were seen only in sections of oviducts taken from females that had been mated to Clgn+/– males. Comparison of brightfield and epifluorescent images of these oviducts revealed that nearly all sperm found within had intact acrosomes, determined by the presence of green fluorescence.
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In order to determine whether the failure of sperm from Clgn–/– males to ascend into the oviduct was due to a motility deficit, motility of epididymal sperm from Clgn–/– and Clgn+/– males was evaluated by computer-aided semen analysis. As shown in Table 1, no statistically significant difference in various characteristics of sperm movement was detected; therefore, the deficit in sperm transport was not due to abnormal sperm motility.
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Production of Calmegin-Knockout XY Wild-Type XY Chimeric Males
Clgn–/– XY wild-type XY chimeric mice were produced by aggregating embryos. In order to generate the chimeras efficiently, we utilized a transgenic mouse line whose X chromosome was tagged by ubiquitously expressed EGFP (X*) [12]. When sperm from this line (X*Y) were used to fertilize BCF1 wild-type oocytes, the resultant female embryos were green fluorescent from the X*, whereas the male embryos inheriting the Y chromosome were nonfluorescent (Fig. 2A). These wild-type male embryos were aggregated with Clgn+/– or Clgn–/– embryos of unknown sex, carrying Acr-EGFP (Fig. 2B). Even if the Acr-EGFP transgene was hemizygous in Clgn+/– or Clgn–/– embryos, all of the acrosomes of the sperm from Clgn+/– or Clgn–/– germ cells were green due to transfer of Acr-EGFP protein through cytoplasmic bridges between developing germ cells in the testis [9]. The chimeras were mated with females, and the semen recovered from the uterus was examined to determine whether it contained both Acr-EGFP-tagged sperm (indicating their origin from Clgn–/– or Clgn+/– males) and nonfluorescent (wild-type) sperm. As a result, we obtained two XY XY chimeras of wild-type and Clgn–/– mice, and three XY XY chimeras of wild-type and Clgn+/– mice.
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Transport of Sperm from Chimeric Males into the Oviduct
The three Clgn+/– wild-type and two Clgn–/– wild-type males were each mated with three to four wild-type females. Two hours after coitus, whole mounts of one oviduct from each female were made and examined as described above. Hundreds of fluorescent sperm were found within the lumens of oviducts from females mated with Clgn+/– wild-type males, whereas no fluorescent sperm were seen within the oviducts from females mated with Clgn–/– wild-type males (Fig. 3). Serial sections were made from the remaining oviducts and examined using brightfield and epifluorescence microscopy. Close examination of sections of the UTJ revealed that sperm derived from wild-type spermatogonia had migrated through the ostium of the colliculus tubarius, and were found within the UTJ. In contrast, no fluorescent sperm derived from Clgn–/– spermatogonia were found within the UTJ beyond the ostium at 2 h postcoitus, despite the presence of numerous fluorescent sperm at the colliculus tubarius, and only a few sperm within the epithelial folds surrounding the ostium of the UTJ in the uterus (Fig. 4).
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After completion of the sperm migration analysis, the XY XY chimeric males were killed for evaluation of the testes. The testes of the chimeric mice were similar in size and gross morphology to those of nonchimeric mice. All five of the XY XY chimeras exhibited normal testicular histology. Fluorescence microscopy revealed that some segments of the testes contained green fluorescent germ cells, derived from Clgn–/– or Clgn+/– embryonic cells, while others contained nonfluorescent germ cells, derived from wild-type embryonic cells (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For this study, XY XY chimeras were required for production of mixed sperm populations. XY XX chimeras usually develop as phenotypic males, but their postnatal germ line cells are formed exclusively from the XY cells [22]; therefore, we were unable to use the sperm from XY XX chimeras. If chimeras are generated from embryos of unknown sex, only about one in four would be XY XY. To increase the chance of obtaining XY XY chimeras, we used a mouse line in which the X chromosome had been tagged with EGFP, so that we could eliminate female embryos when choosing embryos for aggregation. This shortcut could be used for the wild-type embryos but not for the double transgenic line. Therefore, double-transgenic embryos of unknown sex were aggregated with the male wild-type embryos and XY XY males among the resultant chimeras were identified by mating them and examining the semen recovered from the uteri of the females. Acr-EGFP, Clgn–/– (or Clgn+/–) XY wild-type XY males would produce a mixture of fluorescent and nonfluorescent sperm. In addition, because Clgn–/– males are sterile [11], if we had tried to generate Clgn–/– mice by mating, we would have been required to use Clgn+/– males with Clgn–/– females, which reduces the incidence of Clgn–/– embryos by 50%. Therefore, we used partial zona dissection to assist sperm from Clgn–/– males to fertilize eggs in vitro. These two shortcuts enabled us to generate chimeras with the maximum efficiency possible using currently available tools.
It is difficult to identify all of the sperm within the oviduct. Most are held in a sperm reservoir in the extramural UTJ and adjacent oviductal isthmus. Although some sperm may be detected by looking through the semitransparent wall of the oviduct, not all can be seen. Furthermore, a visible marker is needed to distinguish the wild-type from the Clgn–/– derived sperm. We placed our marker, Acr-EGFP, in the sperm from Clgn–/– males, because these sperm were less likely to get into the oviduct and we wanted to detect any that did. As shown in Figure 2, we could easily observe Acr-EGFP-tagged sperm through oviductal wall using fluorescence microscopy.
At least three independent gene knockout mouse lines are known to produce sperm that are unable to ascend into the isthmus of the oviduct. These are mice with disrupted genes for calmegin [3], ADAM2 [4], or angiotensin-converting enzyme [23]. Sperm from these mouse lines display normal motility and can fuse with oocytes [24]; however, they cannot ascend into the oviduct in vivo, nor can they bind to the zona pellucida in vitro. The similarity of phenotypes may indicate that there is something in common missing from these sperm. It is interesting that cyritestin-disrupted sperm show impaired zona binding but are reported to reach the oviductal isthmus [5, 25]. This indicates that at least one of the molecules responsible for enabling sperm to enter the oviduct is different from those required for zona binding.
In designing these experiments, we asked whether the presence of wild-type germ cells in the testis could rescue the calmegin-disrupted germ cells from infertility or, conversely, whether the calmegin-disrupted germ cells would make the wild-type germ cells infertile. We also asked whether the calmegin-disrupted sperm could be rescued in the female by concomitantly ejaculated wild-type sperm, enabling them to pass through the UTJ.
Zamboni [6] reported that, in the mouse, the UTJ is open for only a short while after mating; however, he did not investigate whether the patency of the UTJ is controlled by signals from the female or from the sperm themselves. If UTJ patency is controlled by stimuli from sperm, the concomitantly ejaculated wild-type sperm could stimulate the UTJ to open, enabling the sperm from Clgn–/– spermatogonia to enter as well. Alternatively, the dilution of wild-type sperm by sperm from Clgn–/– spermatogonia could reduce the signal below the threshold required for opening the UTJ. Our results demonstrate that neither of these scenarios is the case.
Our results imply that individual sperm are responsible for getting themselves into the oviduct. It had been established that dead sperm rarely pass through the UTJ [26, 27]. Live sperm of c-ros knockout mice also fail to pass through the UTJ, apparently because the tails of these sperm are kinked and motility is abnormal [28]. However, our findings demonstrate that normal motility is not sufficient to enable sperm to pass through the UTJ, because the motility of calmegin-knockout sperm is normal (Table 1).
The additional required factor may be the ability of sperm to attach to oviductal epithelium. Many mouse sperm within the UTJ have been observed attaching by their heads to the epithelium [7]. There is evidence that the attachment creates a reservoir of sperm, preserving their viability until ovulation and guarding against polyspermy by allowing only a few sperm to ascend in the periovulatory period [1]. However, in addition, attachment might enable sperm to gain a foothold at the entrance to the UTJ. Within the oviduct, movement of mouse sperm toward the oocyte has been observed to consist of a series of detachments and reattachments [29]. In other mammalian species, attachment of sperm to oviductal epithelium in the reservoir is mediated by specific carbohydrate moieties [30–34]. Considering these observations, it could be the case that the attachment of sperm to the epithelium enables them to gain entry into the oviduct. If lack of calmegin results in failure of sperm to acquire certain cell surface proteins during spermatogenesis, a deficiency in cell surface proteins involved in attaching sperm to epithelium lining the UTJ of the oviduct could account for failure of sperm to gain entry.
In summary, sperm from Clgn–/– mice are unable to pass through the UTJ, even in the presence of wild-type sperm from the same ejaculate. Thus, sperm must be individually responsible for their passage into the oviduct. However, the mechanism of the selection is yet to be determined.
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FOOTNOTES |
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2 Correspondence. FAX: 81 6 6879 8376; okabe{at}gen-info.osaka-u.ac.jp
Received: 19 February 2004.
First decision: 12 March 2004.
Accepted: 13 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß heterodimerization and sperm fertility. Dev Biol 2001 240:254-261[CrossRef][Medline]XY chimaeric mouse testes: Sertoli cells are predominantly, but not exclusively, XY. Development 1991 112:265-268[Abstract]This article has been cited by other articles:
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  M. Inagaki, K. Irie, H. Ishizaki, M. Tanaka-Okamoto, J. Miyoshi, and Y. Takai Role of cell adhesion molecule nectin-3 in spermatid development. Genes Cells, September 1, 2006; 11(9): 1125 - 1132. [Abstract] [Full Text] [PDF]
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 N. Satake, R. M. A. Elliott, P. F. Watson, and W. V. Holt Sperm selection and competition in pigs may be mediated by the differential motility activation and suppression of sperm subpopulations within the oviduct J. Exp. Biol., April 15, 2006; 209(8): 1560 - 1572. [Abstract] [Full Text] [PDF]
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 R. Stepanyan, K. Day, J. Urban, D. L. Hardin, R. S. Shetty, C. D. Derby, B. W. Ache, and T. S. McClintock Gene expression and specificity in the mature zone of the lobster olfactory organ. Physiol Genomics, April 13, 2006; 25(2): 224 - 233. [Abstract] [Full Text] [PDF]
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 E. Kim, M. Yamashita, T. Nakanishi, K.-E. Park, M. Kimura, S.-i. Kashiwabara, and T. Baba Mouse Sperm Lacking ADAM1b/ADAM2 Fertilin Can Fuse with the Egg Plasma Membrane and Effect Fertilization J. Biol. Chem., March 3, 2006; 281(9): 5634 - 5639. [Abstract] [Full Text] [PDF]
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 S.S. Suarez and A. A. Pacey Sperm transport in the female reproductive tract Hum. Reprod. Update, January 1, 2006; 12(1): 23 - 37. [Abstract] [Full Text] [PDF]
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M. Pieren, C. Galli, A. Denzel, and M. Molinari The Use of Calnexin and Calreticulin by Cellular and Viral Glycoproteins J. Biol. Chem., August 5, 2005; 280(31): 28265 - 28271. [Abstract] [Full Text] [PDF]
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