Mykhailova et al. (2011) A Study of Spermatogenesis in Diploid Pelophylax esculentus

Mykhailova O. V., Kechedzhi A. E., Shabanov D. A. A Study of Spermatogenesis in Diploid Pelophylax esculentus (Amphibia, Anura) using Karyoanalysis in Squashed Preparations // Proceedings of the Ukrainian Herpetological Society. – 2011. – No. 3. – P. 120-127. UDC 597.851(576.354.4) A Study of Spermatogenesis in Diploid Pelophylax esculentus (Amphibia, Anura) using Karyoanalysis in Squashed Preparations O. V. Mykhailova, A. E. Kechedzhi, D. A. Shabanov Kharkiv V. N. Karazin National University, Freedom Square, 4, Kharkiv, 61077 Ukraine E-mail: d.a.shabanov@gmail.com A Study of Spermatogenesis in Diploid Pelophylax esculentus (Amphibia, Anura) using Karyoanalysis in Squashed Preparations. Mykhailova O. V., Kechedzhi A. E., Shabanov D. A. – The results of a study of spermatogenesis in 14 sexually mature diploid male Pelophylax esculentus from the Seversky Donets center of diversity of green frogs (Ukraine, Kharkiv region) are presented. Karyoanalysis in squashed macerated testis preparations was employed. The typical course of spermatogenesis corresponds to the stages of gametogenesis described for P. esculentus from Western Europe; a significant number of aneuploid cells are recorded in many individuals. In one diploid individual, both diploid cells — from which haploid spermatozoa are expected to arise — and tetraploid cells — which should give rise to diploid spermatozoa — were registered in the testes. In the same individual, cells whose chromosome complement considerably exceeds the tetraploid level were also detected in the testes. Keywords: meiosis, Pelophylax esculentus, spermatogenesis, hemiclonal inheritance, diploid and tetraploid chromosome set. A Study of Spermatogenesis in Diploid Pelophylax esculentus (Amphibia, Anura) using Karyoanalysis in Squashed Preparations. Mykhailova O. V., Kechedzhi A. E., Shabanov D.A. – The results for gametogenesis of 14 mature diploid Pelophylax esculentus males from Seversko-Donetskiy center of diversity of green frogs (Ukraine, Kharkiv region) are shown. For the research caryoanalysis in squashed macerative testicles was used. Typical way of spermatogenesis is similar to the same in ovogenesis, which for P. esculentus from Western Europe were shown. Many individuals have a big amount of aneuploid cells. For the one of all examined diploid males in testicles, both diploid, giving the rise to haploid sperm, and tetraploid cells were registred. Cells with number of chromosomes largely exceeding tetraploid one were shown for the same individual. Keywords: meiosis: Pelophylax esculentus, spermatogenesis, hemiclonal inheritance, diploid and tetraploid chromosome set. The first report of interspecific hybridization in green frogs was published nearly half a century ago (Berger, 1964). Throughout this time, green frogs have attracted enduring scientific interest. Only 10 years after Berger's discovery, H. Tunner demonstrated that the persistence of hybrid green frogs is maintained through hybridogenesis — the clonal inheritance of individual genomes (Tunner, 1974). Using current nomenclature (Frost et al., 2006) and terminology, the edible frog, Pelophylax esculentus (Linnaeus, 1758), is a hemiclonal hybrid of the pool frog, Pelophylax lessonae (Camerano, 1882), and the marsh frog, Pelophylax ridibundus (Pallas, 1771). Although the edible frog does not constitute a true species, it is referred to by a species-equivalent name for a number of reasons. Various members of the hybridogenetic complex of green frogs are capable of coexisting, forming systems proposed to be termed hemiclonal population systems (HPS) (Shabanov, Litvinchuk, 2010). In some HPS, polyploid hybrids occur alongside diploid hybrids (Plötner, 2005). The persistence of HPS in green frogs is maintained by the specific character of gametogenesis in hybrid individuals. In diploid hybrids, one of the parental genomes — the clonal one — passes into the gametes; the other genome is typically eliminated. Cytogenetic investigations of the mechanisms of this inheritance were initiated by H. Tunner, the discoverer of the unusual mode of reproduction in hybrid green frogs. One review publication summarizing the work of Tunner and his co-author (Tunner, Heppich-Tunner, 1991) emphasizes that the long-term independent existence of interspecific hybrids is possible only through aberrant (relative to typical sexual reproduction) reproductive mechanisms. These include parthenogenesis (development of a zygote from an egg without a spermatozoon), gynogenesis (activation of an egg by a spermatozoon without nuclear fusion), and hybridogenesis (nuclear fusion followed by elimination of one of the parental genomes). Using fluorescence microscopy and electrophoresis to study gametogenesis in hybrid frogs from Western Europe (predominantly females), Tunner and Heppich-Tunner demonstrated that the non-clonal genome is removed from germ-line cells before the onset of meiosis (Tunner, Heppich-Tunner, 1991, et al.). The remarkable finding of these Austrian researchers is that hemiclonal inheritance is maintained through two distinct anomalies of gametogenesis. Elimination of the non-clonal genome occurs during mitotic divisions of germ-line cells. After this, the clonal genome undergoes duplication, which may occur either during mitotic divisions of oogonia or already during meiosis, prior to the diplotene stage of prophase I. In 2004, HPS of green frogs were described in eastern Ukraine in which triploid frogs constitute a substantial proportion of hybrids and individual tetraploid hybrids were also recorded (Borkin et al., 2004). The region of their distribution was designated the Seversky Donets center of diversity of green frogs (Shabanov, Litvinchuk, 2010). In this center, sexually mature P. lessonae are absent, and all genomes of this species are transmitted clonally through hybrid generations. Among diploid males, individuals have been recorded that produce P. ridibundus gametes, P. lessonae gametes, and, finally, individuals that produce a mixture of P. ridibundus and P. lessonae gametes in proportions characteristic of each individual. This demonstrates that the study of male gametogenesis in hybrid frogs from the Seversky Donets center of diversity is of considerable scientific interest. The present publication reports the results of a study of gametogenesis in diploid hybrids. The method of karyoanalysis in squashed preparations according to V. V. Klymenko (Klymenko, 2001) with certain modifications was employed. Materials and Methods The testes of 14 sexually mature diploid male Pelophylax esculentus from the Seversky Donets center of diversity, collected in 2010–2011 in the vicinity of the Biological Station of Kharkiv V. N. Karazin National University (village of Haidary, Zmiyiv district, Kharkiv region), were examined. Preliminary identification of green frogs was based on a complex of external morphological characters (Shabanov et al., 2006). Final determination relied on flow DNA cytometry performed by S. N. Litvinchuk and Yu. M. Rozanov at the Institute of Cytology, Russian Academy of Sciences (St. Petersburg). For several individuals, ploidy determination was carried out on the basis of erythrocyte size measurements (Bondareva, Shabanov, 2011); in these cases the determination was cross-checked using the method described below in somatic tissue (small intestine). The method of karyoanalysis in squashed preparations according to V. V. Klymenko (Klymenko, 2001) with certain modifications was used. Testis tissue samples were placed for at least 2 days in a fixative consisting of one part by volume of glacial acetic acid and three parts of 96% ethanol, with a mordant of iron(III) acetate. When necessary, samples were stored in this fixative. A tissue fragment (approximately one quarter of a testis) was transferred to a 40% solution of haematoxylin in glacial acetic acid for staining. The sample was held in the dye for 1.5 h at 62–64 °C. The specimen frequently disintegrated during staining. After staining, an even smaller tissue sample (on the tip of a dissecting needle) was transferred to a watch glass containing a small amount of acetochloralhydrate (a 40% solution of chloral hydrate in glacial acetic acid). In this solution, excess dye is washed out and cells swell. Within 5–10 minutes of the start of washing, the tissue becomes lighter and looser, which is a necessary condition for preparing a squashed preparation. Complete dye removal to the point of chromosome decolorization takes approximately 3 h, after which chromosomes are practically invisible. Under visual control using a stereomicroscope, the tissue sample in the drop of acetochloralhydrate was cut into the smallest possible fragments (of several hundred cells) using microsurgical instruments (dissecting needles sharpened on a fine-grained whetstone). The resulting tissue fragment was placed in a drop of acetochloralhydrate on a glass slide and covered with a 1/4 or 1/16 fragment of a coverslip. The coverslip was gently pressed with the tip of a dissecting needle to squash the sample to a single-cell layer. Squashing was performed under a microscope at 160× magnification. On the prepared slide, cells containing chromosomes at various stages of mitotic or meiotic division were selected. Selected plates were examined at 160×, 640×, and 1600× magnification with oil immersion and photographed using a digital USB microscope camera. Results and Discussion Typical spermatogenesis in diploid P. esculentus. During examination of karyotypes from testes of diploid males, chromosomes were observed at various stages corresponding to the course of gametogenesis described for hybrid frogs from Western Europe. Germ cell formation begins with the mitotic division of spermatogonia, the diploid cells of the germ line (Fig. 1). Presumably, as in the frogs studied by Tunner, elimination of the non-clonal genome occurs during the numerous mitotic divisions of spermatogonia. This stage was not investigated in our material. [IMG_1] Fig. 1. Mitotic division of a diploid cell in the testis of P. esculentus. Metaphase stage showing 26 chromosomes Fig. 1. Mitotic fission of the diploid cell in a P. esculentus testicle. Metaphase, showing 26 chromosomes Spermatogonia undergo a growth phase and differentiate into primary spermatocytes. By metaphase I of meiosis, the germ cell precursors in hybrids already contain a double chromosome complement (2n = 26) carrying two copies of one parental genome. Meiotic division begins with prophase, which is subdivided into five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. Prophase I of meiosis is characterized by the synapsis of homologous chromosomes and crossover — reciprocal exchange of segments between them (Ayala, Kiger, 1987). Leptotene (the thin-thread stage) is characterized by the onset of chromosomal spiralization and condensation; chromosomes acquire a filamentous form. Zygotene marks the initiation of synapsis, beginning with individual regions of homologous chromosomes and completing along their full length by the end of the stage. The pairing of homologous chromosomes is referred to as synapsis. This stage is characterized by the appearance of the synaptonemal complex, which forms part of the bivalent — the pair of synapsed chromosomes. The pachytene stage (thick-thread stage) is characterized by a haploid number of bivalents, i.e., figures formed by synapsed chromosomes. Chromosomes thicken and shorten, and crossover occurs between them. At the diplotene stage (double-thread stage), the structure of bivalents is most clearly visible. Homologous chromosomes retain one or several contact zones called chiasmata. Chromosomes show comparatively greater spiralization than at the pachytene stage. Diakinesis is characterized by maximum chromosomal thickening and spiralization. Chiasmata migrate from centromeres towards the chromosome ends and disappear (Fig. 2); this process is termed terminalization of chiasmata (Ayala, Kiger, 1987). [IMG_2] Fig. 2. Meiosis in a P. esculentus testis. Prophase I stage: diakinesis. Thirteen ring-shaped structures are visible: homologous chromosomes connected in pairs at their terminal regions during terminalization of chiasmata Fig. 2. Meiosis in a P. esculentus testicle. Late prophase – diakinesis, showing the terminalization of 13 ring-shaped pairs of homologous chromosomes, connected with their ends In the hybrid green frogs examined, manifestations of crossover are clearly evident: chiasmata at the diplotene stage and terminalization of chiasmata during diakinesis. It is important to emphasize that since crossover occurs between copies of the same parental genome, it should not lead to genetic recombination. During prophase I, precise chromosome counting is possible only at the diakinesis stage, and very rarely at pachytene. Prophase I is followed by metaphase I of meiosis, at which homologs are maximally paired and form 13 bivalents. Subsequently at anaphase, bivalents separate and homologous chromosomes migrate to the poles. A nuclear membrane forms around each set of homologs, and the cell divides into two daughter cells. This is telophase I. Two identical haploid cells are produced — secondary spermatocytes — which, bypassing interphase, proceed directly to the second meiotic division. Prophase II of meiosis is very brief, and cells at this stage are encountered extremely rarely. At metaphase II, the cell contains a haploid complement, which in green frogs normally comprises 13 chromosomes. Typically, two such cells, formed after the reduction division, lie adjacent to one another (Fig. 3).

Fig. 1. Mitotic division of a diploid cell in the testis of P. esculentus. Metaphase stage with 26 chromosomes

Spermatogonia undergo a growth phase and transform into primary spermatocytes. By metaphase I of meiosis, the germ cell precursors of hybrids already contain a doubled set of chromosomes (2n = 26), which carries two copies of one of the parental genomes. Meiotic division begins with prophase, which is divided into 5 stages: leptotene, zygotene, pachytene, diplotene, diakinesis. Prophase I of meiosis is characterized by synapsis of homologous chromosomes and crossing over, the exchange of segments between them (Ayala, Kiger, 1987). Leptotene (thin thread stage) is characterized by the beginning of coiling and condensation of chromosomes; they become thread-like. Zygotene is marked by the synapsis of initially separate regions of homologous chromosomes, which is completed along their entire length by the end of the stage. The connection of homologous chromosomes is called synapsis. This stage is characterized by the appearance of the synaptonemal complex, which is part of the bivalent – a pair of synapsed chromosomes. The pachytene stage (thick thread stage) is characterized by a haploid number of bivalents, i.e., figures formed by synapsed chromosomes. Chromosomes thicken and shorten, and crossing over occurs between them. In the diplotene stage (double thread stage), the structure of bivalents is most clearly visible. Homologous chromosomes retain one or more contact zones, called chiasmata. A noticeable increase in chromosome coiling is observed compared to the pachytene stage. Diakinesis is characterized by maximum thickening and coiling of chromosomes. Chiasmata move from the centromere towards the chromosome ends and disappear (Fig. 2); this process is called chiasma terminalization (Ayala, Kiger, 1987).

Fig. 2. Meiosis in a P. esculentus testicle. Late prophase – diakinesis, showing the terminalization of 13 ring-shaped pairs of homologous chromosomes, connected with their ends

In the green frog hybrids we studied, crossing-over events are clearly visible: chiasmata at the diplotene stage, terminalization of chiasmata during diakinesis. It is important to emphasize that since crossing-over occurs between copies of the same parental genome, it should not lead to genetic recombination. During prophase I, an accurate chromosome count is possible only at the diakinesis stage, and very rarely at the pachytene stage. Prophase I is followed by metaphase I of meiosis, at which homologs are maximally aligned and form 13 bivalents. Then, in anaphase, bivalents separate, and homologous chromosomes move to opposite poles. A nuclear membrane forms around each set of homologs, and the cell divides into two daughter cells. This is the telophase I stage. As a result, two identical haploid cells are formed – secondary spermatocytes, which, bypassing the interphase stage, immediately enter the second meiotic division. Prophase II of meiosis is very short, and cells at this stage are encountered extremely rarely. In metaphase II, the cell contains a haploid set, which in green frogs normally consists of 13 chromosomes. Usually, two such cells, formed after the reductional division, lie next to each other (Fig. 3).

Fig. 3. Meiosis in a P. esculentus testicle. Metaphase II stage. 2 plates with 13 chromosomes each (1n) are visible

In anaphase II, the centromere of each chromosome divides, and it splits into two chromatids (daughter chromosomes), which move to opposite poles of the cell. As a result, in early telophase, "bunches" of 13 daughter chromosomes are visible. The resulting haploid cells are called spermatids. They then differentiate into spermatozoa. Thus, as a result of meiotic division of a diploid cell in males, 4 haploid gametes are formed. In the case of hemiclonal reproduction of P. esculentus, all gametes of one individual carry an identical clonal genome. Spermatogenesis anomalies. The typical course of spermatogenesis described above is subject to various deviations in some cases. A significant number of aneuploid cells, i.e., cells with a number of chromosomes not a multiple of the haploid set, are registered in the testes of hybrid green frogs. Such anomalies indicate instability in the formation of sex cells in hybrid forms of green frogs. Of particular interest is the course of meiosis in the testes of one of the P. esculentus males caught in the spring of 2011 near the village of Haidary. This individual was diagnosed as diploid based on the size of its erythrocytes. In the small intestine of this frog, we observed exclusively diploid cells. In the testes of this male, along with typical mitotic metaphases with a diploid set of chromosomes, cells were found in which the number of chromosomes exceeded the doubled set. In particular, we recorded tetraploid metaphase plates (Fig. 4).

Fig. 4. Meiosis in a P. esculentus testicle. Metaphase I, showing a tetraploid chromosome set (4n)

Tetraploid chromosome sets were also recorded at the pachytene and metaphase I stages of meiosis (Fig. 5). The appearance of tetraploid cells in the germline of diploid P. esculentus can be explained in several ways. It can be assumed that elimination did not occur and both parental genomes doubled, or that after the removal of the non-clonal genome, the doubling of the clonal genome occurred twice.

Fig. 5. Meiosis in the testes of P. esculentus. Late metaphase I. A doubled set of bivalents (4n) is visible. Homologous chromosomes are well distinguishable and separated, but still connected in pairs before separation.