Nucleolus and nuclear body in prophase spermatocytes of Helix Aspersa

NUCLEOLUS AND NUCLEAR BODY IN PROPHASE SPERMATOCYTES OF HELIX ASPERSA P. ESPONDA and J. C. STOCKERT* Departamento de Citologfa, Instituto de Biologfa Celular (C.S.I.C.), Madrid-6, Spain Received: 24 1" October 1970 INTRODUCTION Most ultrastructural studies of the nucleus during meiosis have been undertaken with the object of analysing the morphology and evolution of the chromosomes. Studies dealing with the development of other nuclear structures during the process of meiosis generally describe the morphological modifications in the nucleolar material and the appearance of certain bodies which are apparently specific in the first prophase of meiosis in the course of oogenesis. The existence of both numerous peripheral nucleoli (MILLER 1966) and of a DNA-body (LIMA-DE-FARIA 1969) and the appearance of the so-called «secondary nucleoli» (ALLEN and CAVE 1969) seem to be connected with the processes of vitellogenesis. But no similar morphological studies appear to have been carried out in the course of spermatogenesis. We here present a structural analysis of the nucleolus and of a characteristic nuclear body observed during the prophase of meiosis in spermatocytes of Helix aspersa. MATERIAL AND METHODS Ovotestes from adult specimens of Helix aspersa were fixed in 2% glutaraldehyde with a buffer phosphate at a pH of 7.2 and post-fixed in 2% osmium tetroxide, and then included in Epon 812. Sectioning was done with an LKB ultramicrotome, staining with uranyl acetate and lead citrate, and observation of the specimens with a Philips 300 electron microscope. The study was carried out on primary spermatocytes at various stages of meiosis after optical analysis of their chromosomal characteristics and morphology in each case, carried out on thick sections stained with 2% methylene blue. * Present address: Institute de Investigaciones sobre Reproducci6n, Facultad de Medicina, Buenos Aires, Argentina. [Caryologia, Vol. 24, n. 3, 1971 294 ESPONDA and STOCKERT RESULTS In spermatocytes, the nucleolus shows characteristic structural peculiarities throughout the prophase of meiosis, and is generally very near the nuclear membrane. Under the light microscope, it is observed to consist of two areas of generally variable size and distribution, but always clearly delimited, distinguished by the greater and lesser intensity with which they are stained (Fig. 1 a). Fig. 1. - a) Nucleolus of a spermatocyte during pachytene. Thick section stained with methylene blue. b) Electron micrograph of an early pachytene nucleolus showing segregation between the pars fibrosa (PF) and the pars granulosa (PG); the peripheral localization of the nucleolus can also be observed. ne : nuclear envelope. x 50.000. Under the electron microscope, the nucleolus shows two different areas from the early stages of prophase. They differ in texture and are usually clearly distinguished from one another although they both form part of the common nucleolar mass. One, the pars fibrosa, consists of a compact web of fibrils, while the other, the pars granulosa, is seen to consist of granules with a diameter of about 150 A, grouped together into strings measuring about 700-800 A (Fig. 1 b). In the course of pachytene the nucleolus remains in close connexion with the nuclear membrane, and the segregation of the two parts is more clearly seen. NUCLEOLUS IN SPERMATOCYTES OF HELIX ASPERSA 295 Fig. 2. - a) Nucleolus and nuclear body (arrow) from a spermatocyte during pachytene. Thick section stained with methylene blue. b) Prepachytene spermatocyte showing the nucleolus (Nu}, the nuclear body (arrow) and the chromatin mass surrounding these structures. x 31.920. 296 ESPONDA and STOCKERT In pre-pachytene prophase nuclei, we observe the appearance of a basophilic body which is normally more or less round, with a diameter of about 0.8 ~-tm (Fig. 2a) and which continues to be observable up to diplotene Under the electron microscope it is seen to be of mainly fibrillary texture and less dense than the nucleolus, and small masses of chromatin can be seen surrounding it, as with the nucleolus itself (Fig. 2 b). In the pachytene Fig. 3. -Part of the nucleus showing the nuclear body, in proximity to the chromosomes in which the remains of the synaptinemal complexes can be observed. Spermatocyte in a early diplotene stage. x 166.000. and diplotene stages, this body shows the same characteristics, as to size, density and texture, as we have just described, lying in all case!' in close proximity to the chromosomes (Fig. 3 ). Spermatocytes observed under the NUCLEOLUS IN SPERMATOCYTES OF HELIX ASPERSA 297 light and electron microscop~s ar stages subsequent to the first prophase showed no such body: hence we may regard it as peculiar to the prophase of meiosis. DISCUSSION It is known that there is a clear separation in space between the pars fibrosa and the pars granulosa of the nucleolus during the first meiotic phophase in many organisms, and this suggests a « natural nucleolar segregation» (GIMENEZ-MARTIN and STOCKERT 1970). In the case we are dealing with, the fibrillar and granular components take up separate areas suggesting the segregated arrangement found in other species (HAY 1968; MILLER and BEATTY 1969; STOCKERT et al. 1970) and the peripheral situation of the nucleolus in spermatocytes of Helix aspersa also resemble the situation described in other organisms (WETTSTEIN and SoTELO 1967; HEss and MEYER 1968; MOENS 1968; WESTERGAARD and WETTSTEIN 1970). A number of authors have observed nuclear bodies distinct from the nucleolus during the prophase of meiosis in plants and animals, and these have been called « spherical bodies » (JOHANSEN and FLINT 19 59); « circular bodies» (SARVELLA 1961 ); «nuclear bodies» (WALTERS 1965; KHIDIR 1969); «nucleolus-like bodies» (DAs and ALPERT 1966); «secondary nucleolus» (ALLEN and CAVE 1969); « lamellate bodies» (HALKKA et al. 1969 ), etc. But few of these structures have been studied under the electron microscope. In principle, there seems to be a certain resemblance (basophilia and mainly fibrous composition) between the body found in Helix aspersa and that described in insect oocytes as a « secondary nucleolus » (ALLEN and CAVE 1969), which appears, from autoradiographic evidence, to be connected with the synthesis of a soluble RNA (HALKKA and HALKKA 1968 ). In the case under study, it is not yet clear whether the body observed represents the secondary nucleolus found in oocytes or not, nor do we know what the other structures mentioned represent. Proximity to the chromosomes, as in our own case, has also been observed under the light microscope in «nuclear bodies», found in microsporocytes (SARVELLA 1961; WALTERS 1965 ). Although these nuclear structures, including that found in spermatocytes of Heli'X aspersa, seem to represent forms through which the activities proper to the prophase of meiosis are expressed, nothing can yet be said about their specific function. Acknowledgements. -This work was partially supported by a grant from the Fonda de Ayuda a Ia Investigaci6n, 1968. We wish to thank M. C. PARTEARROYO and A. PARTEARROYO for technical assistance. P. EsPONDA is a member of the Biology Department staff of the Uni· versity of Chile (Valparaiso) and J. C. SToCKERT has a fellowship from the Consejo Superior de Investigaciones Cientfficas, Spain. 298 ESPONDA and STOCKERT REFERENCES ALLEN E. R. and CAVE M. D., 1969. Cytochemical and Ultrastructural studies of Ribonucleoprotein containing structures in oocytes of Acheta domesticus. Z. Zellforsch, 101: 63-71. DAs N. and ALPERT M., 1966. -Nucleolar RNA synthesis during mitotic and meiotic prophase. Nat. Cancer lnst. Monogr., 26: 337-351. GIMENEZ-MARTIN G. and STOCKERT ]. C., 1970. -Nucleolar structure during the meiotic prophase in Allium cepa anthers. Z. Zellforsch., 107: 551-563. HALKKA L. and HALKKA 0., 1968. - RNA and protein in nucleolar structures of dragonfly oocytes. 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Electron microscope serial reconstitution of the spermatocyte I nuclei at pachytene. ]. Microscopie, 6: 557-576. SUMMARY In Helix aspersa spermatocytes, the nucleolus can be observed throughout the first meiotic prophase, with a « natural segregation » between « pars fibrosa >> and « pars granulosa >>. It also shows a close relationship with the nuclear envelope. From pre-pachytene stages to diplotene, the nuclei of the spermatocytes show a roughly spherical basophilic body, constituted by fibrillar material with an electronic density which is lower than that of the nucleolus. During pachytene and diplotene, this body is in close proximity to the chromosomes. The morphology of this structure is discussed in relation to other bodies found during meiotic prophase in different species.