FULL PAPER Anatomy

The Cycle of the Seminiferous Epithelium in the Greater Japanese Mole, Urotrichus talpoides

Takuo MIZUKAMI1)*, Sachi KUWAHARA1), Masako OHMURA1), Yasuko IINUMA1), June IZUMIKUBO1), Mio HAGIWARA1), Masamichi KUROHMARU2), Yoshihiro HAYASHI2) and Takao NISHIDA1)

1)Laboratory of Anatomy and Physiology, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252–8510 and 2)Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan

(Received 24 May 2000/Accepted 12 September 2000)

ABSTRACT. Spermatogenesis and acrosomal formation in the greater , Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed. KEY WORDS: cycle of the seminiferous epithelium, greater Japanese shrew mole, insectivore, spermatogenesis, spermiation. J. Vet. Med. Sci. 63(1): 31–35, 2001

Spermatogenesis is one of the most fascinating processes [20], rats [18], and other mammalian species. At present, it in the male reproduction. In the seminiferous epithelium, the is still obscure whether this feature is common throughout cells are organized in a series of well-defined cellular associ- the Order Insectivora, and it is therefore necessary to investi- ations, called “stages” [16, 17, 24]. The cycle of the semin- gate the seminiferous epithelium of other species belonging iferous epithelium has been defined in a number of to this order. Greater Japanese shrew moles (Urotrichus mammalian species [1–3, 5–16, 19–23, 27]. The stage is talpoides), distributed only in Japan and belonging to the very useful to compare the expression and localization of Order Insectivora, are relatively easy to obtain in the field some important genes and proteins in the seminiferous epi- and belong to a different family (Family ) from the thelium among mammalian species. While, insectivores musk and Watase’s (Family Soricidae) [4]. In the have been thought to be a primitive and do not form present study, an attempt was made to classify the cycle of a scrotum. There is no critical information why spermatoge- the seminiferous epithelium of the greater Japanese shrew nesis normaly occurs in these ascrotal . In order to mole. This research aims to establish the criteria to evaluate clarify the mechanism of spermatogenesis in ascrotal mam- the spermatogenic activity and male reproductive state of mals, it must be needed to classify the stages of the seminif- greater Japanese shrew mole, to observe its acrosomal for- erous epithelium in these mammals at the first step. In mation and to compare these features with those of the musk insectivores, however, there are only a few reports on the cel- shrew, Watase’s shrew and other mammalian species. lular association of seminiferous epithelium [1, 8, 27]. For example, in the musk shrew, the cycle of the seminiferous MATERIALS AND METHODS epithelium has been divided into 13 stages and developing spermatids have been subdivided into 13 steps [8]. In the Five adult greater Japanese shrew moles, weighing 14–20 Watase’s shrew ( watasei), the cycle of the semin- g, were collected at the university forest in Chichibu, Saitama iferous epithelium has been divided into 12 stages [1]. Addi- Prefecture. Most of greater Japanese shrew moles were cap- tionally, during acrosomal formation, the spermatid nucleus tured with a Sherman’s trap baited with flour paste or soy- remained in the middle region of the seminiferous epithelium bean. The were anesthetized with pentobarbital (5 and only the acrosome extended towards the basement mem- mg/100 g body weight, i.p). Then, the testes were surgically brane in each species. This characteristic acrosomal elonga- excised and immersed in Bouin’s fixative. The samples were tion in these two species is quite different from that in mice cut into slabs, dehydrated in a graded series of ethanol, and embedded in paraffin wax. They were sectioned at 4 µm, *PRESENT ADDRESS: MIZUKAMI, T., Department of Veterinary Anatomy, Graduate School of Agricultural and Life Sciences, stained with hematoxylin-eosin or periodic acid Schiff The University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan. (PAS)-hematoxylin [17] and observed by light microscopy. 32 T. MIZUKAMI ET AL.

Cycle of the seminiferous epithelium (Figs. 2, 3): Based on evaluations in this study, the acrosomal formation in the greater Japanese shrew mole was quite characteristic. The spermatid nucleus remained in the middle region of the sem- iniferous epithelium and only the acrosome extended towards the basement membrane, though the elongation of the acrosome was not prominent. The cycle of the seminifer- ous epithelium was divided into 12 stages, and developing spermatids could be subdivided into 15 steps (Figs. 2,3) based on acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid, and period of spermiation. The mean relative fre- quencies of the stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1, and 7.1%, respectively. The characteristics of each stage are described below. Stage I Two kinds of spermatids were present in stages I to VII. No acrosomal system was recognized at step 1 spermatids. While step 13 spermatids were located at the luminal surface of the seminiferous epithelium, pachytene spermatocytes were located near the basement membrane. They were observed in stages from I to X. Type A spermatogonia, attached to the basement membrane, were seen throughout all stages. Stage II Fig. 1. Gross anatomy of the male genital organ Step 2 spermatids are larger than step 1 spermatids. A of the greater Japanese shrew mole. 1: testis, 2: proacrosomal vesicle with some proacrosomal granules was epididymis, 3: spermatic cord, 4: penis, 5: first observed at step 2 spermatids. Pachytene spermatocytes glans penis, 6: guctus deferens, 7: seminal ves- increased in size in comparison with those in the previous icle, 8: prostate gland, 9: urinary bladder, 10: stage. kidney, 11: testicular artery. The testis is oval Stage III in shape and located within a cremasteric scro- One large and round granule was seen within the vesicle at tum. The ligament of the tail of epididymis, which is attached to the testis, connects to the step 3 spermatids. The vesicle had no contact with the cremasteric scrotum. Ductus deferens forms a nucleus. meshed plexus. Stage IV The acrosomal vesicle became flattened on the surface of Five hundred round seminiferous tubules were selected from the nucleus at step 4 spermatids. The expanding rim of the each testis. A total of 5000 round seminiferous tubules (500 vesicle circumscribed a maximum angle approaching 40°C tubules × 10 testes) were evaluated and classified into each on the nuclear profile. Pachytene spermatocytes gradually stage. increased in size. Step 13 spermatids were deeply embedded within the crypts of the Sertoli cell. The acrosome continued RESULTS to protrude forward from the nucleus. Stage V Gross anatomy of the male reproductive tract in the The acrosomic system spread over the nucleus of step 5 greater Japanese shrew mole: In the greater Japanese shrew spermatids. mole, each testis descends into the cremasteric scrotum. The expanding rim of the vesicle circumscribed an angle Three kinds of accessory glands–the seminal vesicle, pros- from 40°C to a maximum of 90°C. The acrosome and sub- tate gland, and bulbourethral glands–are clearly visible. In acrosomal space continued to protrude forward from the the musk shrew and Watase’s shrew, the testis is greenish in nucleus and embedded within the crypts of the Sertoli cell at color, whereas in the greater Japanese shrew mole, it shows step 14 spermatids. no peculiar color. The bulk of the spermatic cord is associ- Stage VI ated with the testicular artery and vein. The artery branches Spreading of the acrosomic system was still recognized at from the abdominal aorta and pursues a fairly direct course step 6 spermatids. The expanding rim of the vesicle circum- towards the testis. Although the testicular veins form a pam- scribed an angle from 90°C to a maximum of 120°C. Prelep- piniform plexus, in most mammalian species including the totene spermatocytes were clearly seen in this stage. musk shrew [3, 24, 25], the veins do not form such a pampin- Stage VII iform plexus in the greater Japanese shrew mole. Early in stage VII, step 15 spermatids were located at the CYCLE OF SEMINIFEROUS EPITHELIUM IN SHREW MOLE 33

Fig. 2. Light micrographs of 12 stages of the seminiferous epithelium in the greater Japanese shrew mole. Stage I: Pachytene spermatocytes are located near the basement membrane. They are observed in stages from I to X. Type A spermatogonia attached to the basement membrane are seen throughout all stages. Stage II: A proacrosomal vesicle with some proacroso- mal granules is first observed at step 2 spermatids. Stage III: The vesicles in the spermatid proacrosomal vesicle have no contact with the nucleus. Stage IV: The acrosomal vesicle becomes flattened on the surface of the nucleus at step 4 sperma- tids. Step 13 spermatids are deeply embedded within the crypts of the Sertoli cell (arrow). The acrosome continues to pro- trude forward from the nucleus. Stage V: The acrosomic system spreads over the nucleus of step 5 spermatids. Stage VI: Spreading of the acrosomic system is still recognized at step 6 spermatids. Stage VII: In early stage VII, step 15 spermatids are located at the luminal surface of the seminiferous epithelium (arrow). A cytoplasmic lobe of step 15 spermatids is small, but very densely stained. In late stage VII, spermiation occurs. The nucleolus and Golgi complex of pachytene spermato- cytes become more prominent (arrowhead). Stage VIII: Matured spermatids completely disappear from the seminiferous epithelium. Zygotene spermatocytes are seen in this stage. Stage IX: The ovoid nucleus of spermatids becomes flattened and more elongated than that in the previous stage. Stage X: Chromatin condensation begins at the apex of the nucleus (arrow). Stage XI: Diplotene spermatocytes differentiate from pachytene spermatocytes in this stage. Stage XII: Meiotic anaphase or telophase of meiosis 1 and secondary spermatocytes are observed . luminal surface of the seminiferous epithelium. A cytoplas- more elongated than at step 8 spermatids. mic lobe of step 15 spermatids was small, but very densely Stage X stained. In late stage VII, spermiation occurred. Leptotene The nucleus underwent flattening and elongation at step spermatocytes appeared in this stage. A sex vesicle of 10 spermatids. Chromatin condensation began at the apex of pachytene spermatocytes was obviously identified. The the nucleus. The nucleus of the pachytene spermatocyte was nucleolus and Golgi complex of pachytene spermatocytes round and stained lightly in a distinct region. became more prominent. Stage XI Stage VIII The nucleus of step 11 spermatids continued its flatten- Mature spermatids completely disappeared from the sem- ing, elongation and condensation of chromatin. Diplotene iniferous epithelium. One kind of spermatids was recognized spermatocytes differentiated from pachytene spermatocytes in stages VIII to XII. The shape of step 8 spermatids was in this stage. slightly elongated. The nucleus came intact with the cell sur- Stage XII face. Zygotene spermatocytes were seen in this stage. The nucleus still continued its flattening and condensation Stage IX of chromatin. The spermatid head decreased in length at step Remodeling of the nuclear shape began at step 9 sperma- 12 spermatids. Meiotic anaphase or telophase of meiosis 1 tids. The ovoid nucleus of spermatids became flattened and and secondary spermatocytes were observed. 34 T. MIZUKAMI ET AL.

Fig. 3. Schematic drawing of the cycle of the seminiferous epithelium in the greater Japanese shrew mole. Roman numerals represent each stage. Arabic numerals beneath the Roman numerals show the relative frequency of each stage as a percentage. Arabic numerals beneath each spermatid show each step. A, In, B, differentiated spermatogonia (A, B, Intermediate) ; PL, L, Z, P, D, primary spermatocytes; PL, preleptotene phase; L, leptotene phase; Z, zygotene phase; P, pachytene phase; D, diplotene phase; II, secondary spermatocyte; M, mei- otic figure :RB, residual body.

DISCUSSION matid nucleus migrate towards the basement membrane dur- ing acrosomal formation, whereas in the musk shrew, the As in most of other mammals, one cross section of the spermatid nucleus remains in the middle region of the semi- seminiferous epithelium in the greater Japanese shrew mole niferous epithelium and only the acrosome extends towards showed a single stage of the cycle. Although spermatogene- the basement membrane. The spermatid nucleus of the sis in the greater Japanese shrew mole fundamentally resem- greater Japanese shrew mole also remained in the middle bles that in the mouse, musk shrew, Watase’s shrew and region of the seminiferous epithelium and only the acrosome other mammalian species, some slight differences were extended towards the basement membrane, though the elon- detected. While the cycle of the seminiferous epithelium of gation of the acrosome was not prominent. Similar to the the greater Japanese shrew mole was divided into 12 stages greater Japanese shrew mole, in the Spanish mole [27] and and spermatids were subdivided into 15 steps, the cycle of European common shrew [22], the spermatids’ nucleus the seminiferous epithelium of the musk shrew was divided remained in the middle region of seminiferous epithelium into 13 stages and developing spermatids were subdivided and only the acrosome extended towards the base to a lesser into 13 steps [16]. That of the Watase’s shrew was divided degree. At this point, the acrosomal formation in the greater into 12 stages and spermatids were subdivided into 13 steps Japanese shrew mole was more similar to that in the Spanish [1]. That of the Spanish mole was divided into 10 stages and mole ( occidentalis) [27] and European common shrew spermatids were subdivided into 13 steps [27]. In the greater ( araneus ) [22] than in the musk shrew and Watase’s Japanese shrew mole, the extension of the acrosome towards shrew. It is well known that insectivores have a large the basement membrane took a shorter period (2 steps) than acrosome in comparison with other mammalian species [22]. in the musk shrew (3 steps), and Spanish mole (3 steps) and The development of the large acrosome should have a strong almost the same as that in the Watase’s shrew, which showed relation with the peculiar acrosomal formation in insecti- differences in number of stages among these four kinds of vores. It seems likely that the prominent extension of insectivores. In most mammals, both the acrosome and sper- acrosome induces enlargement of acrosome. CYCLE OF SEMINIFEROUS EPITHELIUM IN SHREW MOLE 35

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