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a Department of Urology, Tokyo Medical and Dental University School of Medicine, Bunkyo-ku, Tokyo 113–8519, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gonads were obtained from Japanese white rabbits from 23 to 51 days postcoitum (dpc). Gonadal thin sections were stained with hematoxylin and eosin. Germ cell alkaline phosphatase and apoptosis were detected with histochemical and immunohistochemical methods, respectively.
In the ovary, meiotic germ cells were initially recognized at 29 dpc and arrested after enclosure within follicles. Similarly, meiotic germ cells were recognized outside seminiferous tubules at 29 dpc, but no meiotic figures were identified in intratubular spaces. Apoptotic germ cells were not recognized in the intratubular spaces before 35 dpc, and no apoptotic figures were recognized in the ovary during the period studied.
In conclusion, the initiation of meiosis in testicular interstitial tissue at the time comparable to that in the ovary indicates that germ cells of both sexes have the ability to enter meiosis during the same stage of fetal development; and it appears most likely that delayed initiation of meiosis in the intratubular space is attributable to meiosis-inhibiting substance(s) present in seminiferous tubules.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore mechanisms of sexual dimorphism in initiation of germ cell meiosis, the fetal and postnatal ovary and testis of Japanese white rabbits were studied morphologically to characterize and compare the onset of meiosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Japanese white rabbits at 15–20 wk old were used. Coitus with one male at approximate 20 wk old was visually confirmed. Gonads of both sexes from fetal and postnatal rabbits were obtained at 12-h intervals from Day 23 to 34 and on Days 35, 37, 39, 41, 44, and 51 postcoitum (dpc) as indicated in Figure 1. Four to seven gonads of each sex were examined at each dpc. Since ovulation occurs about 10 h after coitus in rabbits [16, 17], dpc was used as the age of postnatal as well as fetal rabbits. Gonads were obtained from fetuses removed from the uteri of pregnant female rabbits killed by i.v. administration of pentobarbital sodium. Postnatal gonads were obtained from newborn rabbits killed by i.p. administration of pentobarbital sodium. Immediately after removal, gonads were weighed and placed on a back-lit glass parallel to their long axis and gonadal vessels. Then the shape of each was traced, and the area calculated using image analyzing software (NIH Image 1.58, National Institutes of Health, Bethesda, MD) was defined as the maximum cut surface areas. Rabbits were handled in the laboratory according to institutional guidelines for the care of laboratory animals as well as the Guide for Care and Use of Laboratory Animals of the National Research Council, and with the approval of the Institutional Animal Care and Use Committee.
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Tissue Preparation and Histological Examination
For histological observation, 4–7 gonads at each dpc were cut in half longitudinally. One half of each gonad was fixed in Bouin's fluid for 24–36 h, embedded in paraffin, serially sectioned at 6 µm, and stained with hematoxylin and eosin for histological observation of germ cell differentiation. The remaining half was fixed with ice-cold 10% formaldehyde calcium fixative (10% formaldehyde in distilled water with a saturated amount of CaCl2, pH 7.1–7.2) for 24 h, stored in ice-cold 0.88 M gum sucrose solution (10 g of Arabic gum and 300 g of sucrose in 1000 ml of distilled water) overnight, embedded in Tissue-Tek embedding medium (Sakura Finetechnical Co., Ltd., Tokyo, Japan), and frozen in liquid nitrogen. Then the gonadal tissues were serially sectioned at 8 µm and histochemically stained with alkaline phosphatase for identification of germ cells [18]. Cytoplasm of cells that stained positively with alkaline phosphatase was dark blue under the light microscope.
For in situ detection of apoptotic cells with DNA strand breaks, contralateral gonads were cut in half longitudinally. One half was fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), embedded in paraffin, and sectioned at 6 µm for the immunohistochemical staining of apoptotic cells. For transmission electron microscopic examination to confirm apoptosis, the remaining half, or gonads obtained at the same dpc, were cut into small pieces and also fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The tissues were postfixed with 1% phosphate-buffered osmic acid with 0.1 M sucrose, dehydrated in ethanol, and embedded in epoxy resin. Sections 70–90 nm thick were placed on 150-mesh copper grids and stained with uranyl acetate followed by lead citrate.
Identification of Apoptotic Cells
Apoptotic cells were stained using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) method developed by Gavirieli et al. [19], according to directions provided in the ApopTag-peroxidase kit (Oncor, Gaithersburg, MD). Sections were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted with Permount (Fisher Scientific, Fairlawn, NJ). Nuclei of TUNEL-positive cells were brown under the light microscope.
As negative controls, tissue sections were processed together in a manner identical to that described above, except that distilled water was substituted for terminal deoxynucleotidyl transferase.
Morphological signs of apoptosis in the tissue were also confirmed under transmission electron microscopy [20–22].
Germ Cell Identification
Presence of germ cells was confirmed by histochemical staining for alkaline phosphatase. Gonocytes, primary spermatocytes, and oocytes were clearly identified by hematoxylin and eosin staining from their size, huge nuclei, and chromatin appearance. Gonocytes, spermatogonia, and oogonia are large pale cells with interphase nuclei. Germ cells in each meiotic stage were characterized as follows. Preleptotene cells were characterized by a spherical nucleus with disperse chromatin, with few chromatin condensations attached to nuclear membrane. Leptotene cells had spherical nuclei with a few chromatin condensations on the nuclear membrane, which were connected by filamentous structures. These attachments form the bouquet arrangement of chromosome structure in leptotene cells. Zygotene cells were characterized by thickening of the chromosomal elements in the nucleus, while further thickening and shortening of chromosomes characterized pachytene cells. Diplotene cells were characterized by fine chromosomal elements without attachment to the nuclear membrane because of the desynapsis of the paired chromosomes.
Enumeration of Germ Cells
Sections were examined with x40 objective and x10 eyepiece. A square grid fitted within one eyepiece provided a reference area of 250 µm x 250 µm. Germ cells in mitosis, meiosis, and apoptosis within the frame of the grid were counted. For each gonad, at least 100 separate areas were examined, and the percentage ratio of germ cells in each condition per total germ cells was calculated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fetal testis was clearly distinguished from the ovary by its striped appearance due to the developing testicular cords. The fetal testis was also larger, and its blood supply was greater than that of the fetal ovary. The testis had higher weight and wider maximum cut surface area than the ovary. Both gonads developed gradually, and the development of the testis accelerated after 44 dpc (Fig. 1).
Separation of the mesonephric connection to the testis and ovary had already started at 23 dpc. At that time the gonadal and mesonephric tissues were being separated by connective tissue (Fig. 2, A and C). The testis and ovary then separated from the mesonephric tissue by 24 dpc and 24.5 dpc, respectively (Fig. 2, B and D).
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Location of Germ Cells in the Ovary and Testis
In the ovary, germ cells with positive alkaline phosphatase staining were scattered throughout (Fig. 3A1). On the other hand, the majority of male germ cells were located within the testicular cord (Fig. 3B1), while a few were observed outside the seminiferous tubule (Fig. 3C1).
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Evaluation of Mitotic and Meiotic Germ Cells
The preleptotene-like figure of the nucleus is difficult to distinguish from the second gap phase in the mitotic interphase, which is followed by the mitotic prophase. Therefore, germ cells with preleptotene-like figures were counted as cells under meiosis when germ cells at leptotene, pachytene, zygotene, or diplotene stages were present together or subsequently, and as cells under mitosis when meiotic cells beyond the preleptotene stage were not present. On the basis of this criterion, germ cells with preleptotene-like figures present from 29 to 34 dpc in the intratubular space were not counted as meiotic cells.
Mitosis of germ cells was observed in the ovary and intra- and intertubular spaces at 23.5 dpc (Fig. 3, A2, B2, and C2). Germ cells with preleptotene or preleptotene-like figures were recognized at 29 dpc in the ovary and testis (Fig. 3, A3, B3, and C3). In the ovary, germ cells beyond the preleptotene phase were identified at 29.5, 34, and 44 dpc (Fig. 3, A3, A4, and A5) and capsulated in follicles at 44 dpc (Fig. 3A5). In the intertubular space of the testis, germ cells beyond preleptotene stage were also recognized at 34 dpc, and germ cells with concentrated nuclei were recognized at 44 dpc (Fig. 3C5). In contrast, no germ cells beyond the preleptotene stage were observed in the intratubular space of the testis at 29.5, 34, and 44 dpc (Fig. 3, B3, B4, and B5); therefore, intratubular germ cells with preleptotene-like figures were counted as cells at the second gap phase of mitosis.
Apoptosis of Germ Cells in the Gonads
Apoptotic germ cells stained brown with the ApopTag kit; this finding was confirmed with electron microscopic observation (Fig. 4). Apoptotic figures of germ cells were recognized in both intra- and intertubular spaces of the testis (Fig. 4C) but not detected in the ovary (Fig. 4A). These apoptotic figures were confirmed by transmission electron microscopy. Cellular shrinkage, chromatin condensation, and the formation of spherical bits of nuclear membrane of germ cells were absent in the ovary (Fig. 4B) but present in the testis (Fig. 4D).
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Mitosis, Meiosis, and Apoptosis of Ovarian and Testicular Germ Cells in Relation to Development of Gonads from 23 to 51 dpc
Based on the above criteria, mitosis, meiosis, and apoptosis of germ cells in the ovary and testis were analyzed in relation to fetal and neonatal age as shown in Figure 5.
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Mitosis of germ cells had begun by 23.5 dpc in both the ovary and testis, and mitotic figures were more abundant in the ovary than in the testis. Mitotic figures of germ cells continued to exist in seminiferous tubules throughout the observation period but were not recognized in the ovary and intertubular space after 39 and 41 dpc, respectively.
In the ovary, meiotic germ cells were initially recognized at 29 dpc and had increased to 100% by 44 dpc. Meanwhile, enclosure of germ cells within follicles started around 37 dpc and was almost completed by 41 dpc. In germ cells enclosed within follicles, meiosis was arrested at the diplotene stage. Similarly, germ cells outside the seminiferous tubule or testicular cord entered meiosis at 29 dpc, but such meiotic figures disappeared by 41 dpc. In contrast, no meiotic figures were identified in the intratubular space throughout the observation period.
Apoptosis of intratubular germ cells was not recognized before 35 dpc, while germ cells with preleptotene-like figures were observed from 29 dpc. Further, apoptosis was observed in germ cells outside the tubular space only beyond the diplotene stage. In the ovary, neither apoptotic germ cells nor atrophic follicles were observed during the period studied.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In germ cell identification, germ cells in the meiotic preleptotene stage are difficult to distinguish from those at the second gap phase of mitotic interphase [25–27]. In previous studies such intratubular germ cells, with preleptotene-like figures, have been counted as cells undergoing meiosis [4, 15]. In the current study, however, no meiotic cells beyond leptotene stage were detected in the intratubular space. Furthermore, intratubular germ cells with preleptotene-like figures were observed during only a short and transitory period after 29 dpc, and no apoptotic figures were recognized until 37 dpc in the intratubular space. Therefore, in the current study, germ cells with preleptotene-like figures in the intratubular space were not counted as meiotic but as mitotic ones.
The current results indicate that meiosis of germ cells in the ovary and intertubular space of the testis starts at the same developmental stage, at 29 dpc, while germ cells inside seminiferous tubules are unable to enter meiosis at this time. It has been reported consistently that meiosis of male germ cells occurs in the adrenal [13] and intertubular spaces [4, 9, 15] of the mouse. In the present study, meiosis began approximately 5 days after complete separation of the mesonephric connection to the gonad, while meiosis has been reported to begin while the presumptive gonads remain connected to the mesonephros in the mouse [28, 29]. The current results provide further evidence that germ cells in both sexes enter meiosis after mesonephric connections to the gonads have been severed. In the ovotestis of a human infantile true hermaphrodite, germ cells in the seminiferous tubule never entered meiosis, but those in the primordial follicle were at the diplotene stage [12]. These observations suggest that the intratubular space of the testis provides a microenvironment that inhibits the meiotic potential of germ cells. Furthermore, it has been proposed that a meiosis-inhibiting substance is concentrated within the testicular cords. The enclosure of germ cells within cords in the presence of a meiosis-inhibiting substance prevents the action of meiosis-inducing substance in mice [4]. Therefore, meiosis-preventing substance(s) appear to be present in the intratubular space of the testis.
The present results show that meiosis of oocytes is arrested after oocytes have been encapsulated in the supporting cells of the primary follicle. Similar to what occurs in the testis, meiosis progresses in female germ cells while they are contact with the interstitial tissue but becomes arrested when the cells are enclosed within the follicles. In both gonads, isolation of germ cells from the interstitial tissue appears to affect the process of meiosis; the female germ cell has not proceeded beyond the diplotene stage, and the intratubular male germ cell has not entered meiosis. It has been suggested that the intimate contact between female germ cells and follicular or granulosa cells is responsible for maintaining the germ cells in the diplotene stage [30]. Furthermore, it has been presumed that immature granulosa cells of the porcine ovary produce an inhibitory factor that interferes with female germ cell maturation [8, 31]. However, it has been reported that oocytes at the diplotene stage are present in the adrenal tissue of the female mouse at 17 dpc and that all germ cells in the adrenal tissue are able to mature to the diplotene stage by birth, where they remain arrested until 3 wk after birth [13]. Further, primary oocytes mature to secondary oocytes when surrounding granulosa cells mature and form graafian follicles under hormonal stimulation. These facts suggest that oocytes are unable to complete meiosis beyond the diplotene stage without proper hormonal stimulation.
In conclusion, germ cells in the two sexes have the ability to enter meiosis during the same stage of fetal development. However, the microenvironment formed in seminiferous tubules appears to delay the initiation of meiosis of germ cells. Apoptosis occurs only in male germ cells, but its physiological role remains to be determined.
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FOOTNOTES |
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1 Correspondence: Tetsuo Hayashi, Department of Urology, Tokyo Medical and Dental University School of Medicine, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan. FAX: 81 3 5803 5295. 
Accepted: February 4, 2000.
Received: October 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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