山階鳥研報 (J. Yamashina Inst. Ornith.), 19: 45-55, 1987

The Male Sex Accessories in the Annual Reproductive Cycle of the Pied contra contra

S. K. Gupta* and B. R. Maiti**

Abstract. The male sex accessories were studied from gravimetry, histology and biochem- ical standpoint during the annual testicular cycle and nesting cycle in a sub-tropical , the Pied Myna (Sturnus contra contra). The findings revealed that all the sex accessories showed cubodial to low columnar, non-ciliated and non-secretory cells, with low seminal glomus weight and sialic acid content during the non-breeding phase (August to January). The epithelial cells were moderately enlarged with a moderate increase in seminal glomus weight and sialic acid concentration during the pre-breeding phase (February and March); greatly enlarged with differentiation of cells and secretory activity followed by a further increase in weight and of sialic levels of the seminal glomus during the breeding phase (April and May); and regressed with reverse changes in the seminal glomus during the post-breeding phase (June and July). During the nesting cycle (i.e. the breeding phase) particularly the early nest-building period, the values of all the above parameters in the sex accessories increased. They were highest in the late nest-building period and decreased gradually in the egg-laying, incubation and nestling periods. It is suggested that in the Pied Myna, sex accessory activity varies simultaneously with the annual gonadal cycle, being low during the non-breeding phase, at a moderately increased in the pre-breeding phase, at a maximum during breeding and declining thereafter. But during the nesting cycle sex accessory activity increased in the early nest-building period, attained a peak in the late nest-building period and declined gradually in the egg-laying, incubation and nestling periods. Changes in the sex accessories are considered to be the result of changes in ovarian steroid activity during the gonadal cycle of this bird.

Introduction

Detailed study of the male genital tract has been carried out only in domestic (Lake 1957, 1979, Tingari 1971, 1972, Budras & Sauer 1975, Hess et al. 1976, Hess & Thurston 1977, Aire 1979 a, b, 1980, 1982). Information available on the genital tract in wild birds which constitute about 99% of total avian , is very inadequate (Lafts & Murton 1973). In few wild birds investigated so far, the male genital tract has been reported to be well-developed only during the breeding phase of seasonal reproductive cycle (Bullough 1942, Bailey 1953, Mehrotra 1962, Sarkar & Ghose 1964, Middleton 1972, Silverin 1975). However, the detailed cytomorphological study of the changes in all the sex accessories during the annual testicular cycle has been carried out only in some fringillid birds (Bailey 1953). The latter author found that cyto-differentiation and enlarge- ment of the sex accessories were most pronounced the breeding phase. In other birds, the Received 17 December 1986 * Present Address: Department of Zoology, H. L. N. College, Yamuna Nagar 135001, Haryana, . ** Address for reprints: Histophysiology Laboratory , Department of Zoology, University of Calcutta (Ballygunge Campus), 35 Ballygunge Circular Road, Calcutta 700019, India.

45 46 S. K. Gupta & B. R. Maiti epithelial height and the seminal glomus weight also increased during this phase (Middleton 1972, Silverin 1975). Histological changes that might occur simultaneously in different sex accessory structures during the nesting cycle of wild birds are not known as yet. Moreover, sialic acid content of the sex accessory organ (seminal glomus), which is known to be androgen dependent in a wild avian species (Gupta et al. 1984), has not been studied during the annual testicular cycle in any wild bird so far. In the current investigation, we have examined the changes in all the sex accessories of a sub-tropical passerine bird, the Indian Pied Myna both during the annual testicular cycle as well as in relation to the nesting cycle.

Materials and Methods

The adult male specimens of Indian Pied Myna, Sturnus contra contra L, Were captured from their natural populations near Calcutta. The birds were killed by cervical dislocation. The accessory sexual organs (epididymal region with the testis, vas deferens and seminal glomus) were quickly dissected out. The seminal glomus was weighed on a torsion balance (Roller-Smith, U. S. A.). The tissues were fixed in Bouin's fixative and processed for routine microtomy. 5μm thick sections were stained with Masson's trichrome technique. In addition, serial sections were also taken for the epididymal region and for the seminal glomus in each month and in each stage of the nesting cycle. The epididymal region was serially cut for location of the tubulus rectus, rete testis, vasa efferentia, and epididymal tubules. The seminal glomus was also serially cut for ascertaining regional differences, if any. Heights of the epithelium in the epididymal tubules, vas deferens and seminal glomus (peripheral and central tubules) were measured with an ocular micrometer from five different locations of each section (from ten random sections of each specimen). Sialic acid content of the seminal glomus was estimated by thiobarbituric acid method of Warren (1959). O. D. was read at 532nm and 549nm in Perkin-Elmer Spectrophotometer (model 550S).

Results

Gross anatomy In Indian Pied Myna, the male sex accesory system comprised of (i) tubuli recti, (ii) rete testis, (iii) vasa efferentia, (iv) epididymal tubules, (v) vas deferens and (vi) seminal glomus. A precise anatomical organization of these organs was studied from serial histological sections. The tubuli recti located mostly inside the tunica albugenia, were connected broximally with the seminiferous tubules and distally with the rete testis (Fig. 1). The rete testis was a network of cavernous channels, present mostly within the testicular capsule (intra-capsular), while a very small part of it was located outside the capsule (extra-capsular) (Fig. 2). The rete testis opened abruptly in the vasa efferentia which showed epithelial folds. The epididymal tubules were more numerous than those of the vasa efferentia and were without epithelial folds. However, the transition between these two regions was gradual. The vas deferens extended from caudal end of of the testis to the distal part The Male Sex Accessories in the Annual Reproductive Cycle of the Pied Myna 47 of the abdominal cavity where it opened into a mass of highly convoluted tubules, the seminal glomus.

Changes during the annual reproductive cycle During the annual testicular cycle, the activity of the sex accessory organs passed through four distrinct phases, viz., (i) the non-breeding phase (August to January), (ii) the pre-breeding phase (February and March), (iii) the breeding phase (April and May), and (iv) the post-breeding phase (June and July).

Non-breeding phase All the accessory sexual organs were inconspicuous and showed a narrow lumen lined by a cuboidal to low columnar epithelium of non-ciliated and non-secretory cells (Figs. 5 and 7). The epithelial height in the epididymal tubules, vas deferens and seminal glomus was low. The seminal glomus weighed less with low sialic acid content (Table 1).

Pre-breeding phase All the accessory sexual organs were slightly enlarged during this phase and showed wider lumen. The epithelial height increased marginally in various regions (Table 1). Weight and sialic acid content of the seminal glomus also increased to some extent (Table 1). There was, however, no secretory activity or cell differentiation in any region of the accessory sex organs.

Breeding phase During this phase, the accessory sexual organs looked conspicuously enlarged. The lumen was relatively much wider. Values for height of the epithelium in the epididymal tubules, vas deferens and seminal glomus, and for the weight and sialic acid content of the seminal glomus increased significantly in April and became maximum in May (Table 1). Remarkable histological changes were noticeable in all the accessory sexual organs except the rete testis. In the tubulus rectus, the lining epithelium contained two types of cells: (i) those with large round nuclei located towards the basement membrane, and (ii) those with small oval nuclei located towards the lumen (Fig. 1). The lumen contained cytoplasmic residual bodies, immature germ cells and few sperms. Examination of serial sections revealed the occurrence of two types of tubules (proximal and distal) in the vasa efferentia, with both types lined by a folded epithelium of tall columnar cells. (i) Proximal efferent tubules were short in length and showed predominantly non-ciliated cells with the nuclei located away from the basement membrane. Infranuclear and supranuclear "clear cytoplasmic areas" were usually present in these cells. The lumen was very small (Fig. 3), (ii) Distal efferent tubules were long and contained predominantly ciliated cells with the nuclei located towards the basement membrane. "Clear cytoplasmic areas" were absent. Instead, cytoplasm looked granulated. The lumen was relatively much larger (Fig. 4). The transition between the proximal and distal tubules was gradual and both types appeared to be highly secretory. Sperms were rarely 48 S. K. Gupta & B. R. Maiti The Male Sex Accessories in the Annual Reproductive Cycle of the Pied Myna 49

Table 1. Annuai changes in the sex accessories of the male Pied Myna

*Mean±standard error**central tubules

seen in their lumen. In the epididymal tubules, two types of cells, viz., ciliated and non-ciliated, could be recognized in the lining epithelium (Fig. 6). In both types of cells, the nuclei were large and round, and located generally towards the basement membrane. The cell cytoplasm was densely granular. Fuchsino- philic secretory droplets were encountered at the apical part of the cell. The lumen was usually empty but occasionally showed immature germ cells with a few sperms. There was a conspicuous increase in the length and diameter of the vas deferens and it assumed a convoluted appearance. The lining epithelium became folded and both ciliated and non-ciliated tall columnar cells were present. Sperms were rare. In the serial sections, there was no conspicuous histological difference in the proximal, middle

EXPLANATION OF FIGURES

(Photomicrographs. Mason's trichrome stain, ×400)

Fig. 1. Intra-testicular tubulus rectus (ITR) connecting the seminiferous tubule with the (intra-capsular) rete testis (IRT) in the Pied Myna in May. Fig. 2. The opening of the rete testis into the vasa efferentia. A small extra-capsular rete testis (ERT) is seen to join with the main intra-capsular rete testis (IRT) with the proximal efferent tubule (PET) in May. Fig. 3. Cross section of the proximal efferent tubule (in May) showing narrow lumen lined by nonciliated tall columnar cells. The nuclei are located away from the basement membrane. Infranuclear and supra-nuclear "clear cytoplasmic areas" are seen. Fig. 4. Distal efferent tubule (in May) with large lumen lined mostly with cicliated tall columnar cells. The nuclei are located near the basement membrane. "Clear cytoplasmic areas" are absent. 50 S. K. Gupta & B. R. Maiti The Male Sex Accessories in the Annual Reproductive Cycle of the Pied Myna 51 or distal regions of the seminal glomus. However, two types of tubules were clearly distinguishable: (i) Peripheral tubules and (ii) Central tubules. In the peripheral tubules (Fig. 8), the epithelium was much thicker and folded. Cell cytoplasm was poorly granulated and the nuclei were generally elongated. Numerous sperms , mostly in bundles, were seen in the lumen with their heads directed towards the lining epithelium . In the central tubules (Fig. 9), the epithelium was not folded. Cell cytoplasm was densely granular and the nuclei were round. Also, sperms present in the lumen were neither in bundles nor pointed towards the epithelium. However, in both types of tubules, the epithelium consisted mostly of non-ciliated secretory cells and the lumen contained abundant materials including innumerable sperms . How- ever, apical blebs were distinctly seen in the peripheral tubules but were absent in the central tubules. The sialic acid level of the seminal glomus increased to a maximum extent (Table 1).

Post-breeding phase

Regressive changes were noticed in all the accessory sexual organs during this phase . The secretory activity declined. The epithelial height in the epididymal tubules, vas deferens and seminal glomus was significantly reduced. Ciliated cells were lost. Abundant cellular debris was seen specially in the tubulus rectus and rete testis. The distinction between proximal and distal efferent tubules in the vase efferentia, and between central and peripheral tubules in the seminal glomus was gradually lost. There was a significant fall in the seminal glomus weight and sialic acid content, and sperms with heads directed towards the epithelium were not observed.

Changes during the nesting cycle Breeding activity was noticed during the months of April and May. It could be divided into five stages, viz., (i) early nest-building, (ii) late nest-building, (iii) egg-laying, (iv) incubation and (v) nestling stage. Salient changs in the accessory sexual organs seen during different stages of the nesting cycle are given below. The accessory sexual organs gradually developed during the early nest-building stage, maxi-

EXPLANATION OF FIGURES

(Mason's trichrome stain, ×400)

Fig. 5. The epididymal tubules (in January) with very narrow lumen lined by non-ciliated low columnar cells containing scanty cytoplasm (non-breeding phase). Fig. 6. The enlarged epididymal tubule in May (breeding phase) showing wide lumen. Ciliated and non-ciliated columnar cells with granular cytoplasm are seen in the epithelium. Fig. 7. The seminal glomus in January (non-breeding phase) containing narrow tubules with very narrow lumen and thick muscle layers. The epithelium is a single layer of non-ciliated cuboidal cells with scanty cytoplasm. Fig. 8. The peripheral tubule in the seminal glomus in May (breeding phase) showing much thicker and folded epithelium. Note numerous sperms with their heads directed towards the epithelium. Fig. 9. The central tubule in the seminal glomus in May (breeding phase) showing unfolded , less thick epithelium with densely granulated cells as compared to those of peripheral tubule. Numerous sperms are present in the lumen but their heads are not directed towards the epithelium. 52 S. K. Gupta & B. R. Maiti mally developed during the late nest-building stage and gradually declined thereafter. The histological features were essentially similar to those described for the breeding phase. In the vasa efferentia, proximal and distal efferent tubules could not be distinguished in the nestling period. The tubular size, epithelial cell differentiation and the secretory activity of the vasa efferentia, epididymal tubules, vas deferens and seminal glomus increased from early nest-building to late nest-building stage and declined gradually in subsequent stages. Changes in the seminal glomus were more conspicuous than in other organs. The weight of the seminal glomus increased significantly from early nest-building (25.97±2.34) to late nest-building (44.65±3.21) and declined gradually thereafter (egg laying: 35.28±3.06, incubation: 29.70±3.21 and nestling: 24.53±2.87). Fluctuation in epithelial height also followed a similar pattern. Frequency of sperms with their heads pointing towards the lining epithelium was more upto egg-laying stage and noticeably decreased in incubation and nestling stages. Abundance of sperms were seen in the lumen of the seminal glomus in all the stages of the nesting cycle, but immature germ cells were rarely seen in any stage of the nesting cycle.

Discussion

The present investigation clearly indicates that all the accessory sexual organs, except the rete testis, undergo distinct changes during the annual reproductive cycle in the Indian Pied Myna, since they remain ill-developed during the non-breeding phase (August to January), become slightly enlarged during the pre-breeding phase (February and March), develop markedly during the breeding phase (April and May) and regress rapidly during the post-breeding phase (June and July). Annual cyclic changes in some of the accessory organs have been reported earlier in some wild birds (Middleton 1972, Silverin 1975). In addition, the current study also reveals existence of close relationship between the activity of the accessory sexual organs and the nesting cycle in the Pied Myna. As the nest-building activity begins in April, active development of the whole genital tract involving rapid increase in size of the tubules with differentiation and secretory activity of the epithelial cells occurs. This development continues throughout the nest-building stage and attains peak when nest-construction is nearing completion. Regression of the accessory sexual organs has been found to synchronize generally with the onset of egg-laying, incubation and nestling stages of the nesting cycle in this avian species. The present study reveals certain important histological features marked in different accessory sexual organs of the Indian Pied Myna, hitherto unreported for any wild avian species. This has been apparent from the study of serial sections of these organs in the Indian Pied Myna. These structures are: (i) an intra-testicular tubulus rectus, (ii) a short extra-capsular rete testis, (iii) two different types of tubules in the vase efferentia, viz., proximal and distal, and (iv) two distinct types of tubules in the seminal glomus, viz., central and peripheral. The failure of previous authors to recognize the existence of these structures may in part be due to the study of only few representative sections of these organs. In addition, structural organization of the accessory sexual organs in the breeding Indian Pied Myna differs markedly from what is commonly known for the poultry birds. The Male Sex Accessories in the Annual Reproductive Cycle of the Pied Myna 53

The conspicuous differences are: (i) the seminal glomus is present in the Pied Myna but absent in the poultry birds, (ii) the epididymal tubules are numerous as compared to that of the vasa efferentia in the Pied Myna but reverse is true for the poultry birds, (iii) the vasa efferentia have small epithelial folds in the Pied Myna as compared to the long irregular epithelial folds in the poultry birds and (iv) few sperms are seen in the vasa efferentia, epididymal tubules and vas deferens of the Pied Myna but numerous sperms are seen in these organs in the poultry birds. Consistent availability of sperms in huge number in the seminal glomus of the breeding Indian Pied Myna and scanty spermatozoa in other accessory sexual organs even during the breeding period indicate that in this avian species, sperms are stored perhaps in the seminal glomus as reported for other passerine birds (see Middleton 1972). This is in contrast with the probable storage of sperms in the vas deferens of the domestic fowl and Japanese Quail (Lake 1975, de Reviers 1971, Amir et al. 1973, Clulow & Jones 1982). Also, in the latter birds, maturation of sperms takes place in the epididymal regions (Munro 1938, Clulow & Jones 1982). But in the Indian Pied Myna, retention of spermatozoa mainly in the seminal glomus and its high secretory activity suggest that seminal glomus may be the probable site of sperm maturation in this species. The suggestion is strengthened by the fact that sialic acid which has been involved in sperm maturation process in mammals (Prasad & Rajalakshmi 1976), is also found in the seminal glomus of the Pied Myna. Wolfson (1954) and Middleton (1972) have also assigned a sperm maturation role of the seminal glomus in other avian species. It is known that in birds, sex accessory structures depend largely upon testicular androgen (Lofts & Murton 1973, Lake 1979). Moreover, sialic acid content of the seminal glomus has been reported to depend largely upon testicular androgen studied in the present avian species, the Tree Pie, because sialic acid level of the seminal glomus declined following castration and was restored by testosterone treatment in this bird (Gupta et al. 1984). In the present study, sialic acid content of the seminal glomus also altered during the annual testicular cycle. In the same avian species, testicular steroidogenic activity is also known to alter during the breeding cycle (Gupta 1983). Thus, changes in sex accessory activity during the annual testicular cycle including nesting cycle could be due to an alteration in testicular androgen in the Tree Pie.

Acknowledgements

This work was supported partly by grants from the special Assistance Programme of the University Grants Commission, Government of India , to the Department of Zoology, University of Calcutta and partly from the award of Teacher Fellowship scheme of the University Grants Commission to SKG (UGC/3139/Fellow/T).

References

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Aire, T. A. 1980. The ductuli efferences of the epididymal region of birds. J. Anat. 130: 707-723. Aire, T. A.1982. The surface morphology of the ducts of the epididymal region of the drake (Anas platyrhynchos) as revealed by scanning and transmission electron microscopy. J. Anat. 135: 513-520. Amir, D., Braun-Eilon, B. & Schindler, H. 1973. Passage and disappearance of labelled spermatozoa in the genital tract of the male Japanese quail in segregation or cohabitation. Annls. Biol. Anim. Biophys. 13: 321-328. Bailey, R. E. 1953. Accessory reproductive organs of male fringillid birds-seasonal variations and response to various sex hormones. Anat. Rec. 115: 1-20. Budras, K. D. & Sauer, T. 1975. Morphology of the epididymis of the cock (Gallus domesticus) and its effect upon the steroid sex hormone synthesis. I. Ontogenesis, morphology and distribution of the epididymis. Anat. Embryol. 148: 175-196. Bullough, W. S. 1942. The reproductive cycle of the British and continental races of the . Phil. Trans. Roy. Soc. London ser B 251: 165-246. Clulow, J. & Jones, R. C. 1982. Production, transport, maturation, storage and survival of spermatozoa in the male Japanese quail. Coturnix coturnix. J. Reprod. Fert. 64: 259-266. de Reviers, M. 1971. Le development testicularie chez le coq. II. Morphologie de l'epithelium seminifere et establisement de la spermatogenese. Annls. Biol. Anim. Biochem. Biophys. 11: 531-546. Gupta, S. K. 1983. Reproductive biology of Indian pied myna, Sturnus contra contra. Ph. D. Thesis, University of Calcutta. Gupta, S.K., Bhat, G. & Maiti, B. R. 1984. Gonadal hormone-dependent changes in the concentrations of sialic acid and fructose in the seminal glomus and of glycogen in the oviduct of the Indian pied myna. Gen. Comp. Endocrinol. 54: 389-391. Hess, R. A. & Thurston, R. J. 1977. Ultrastructure of the epididymal region of the turkey (Meleagris gallopavo). J. Anat. 124: 765-778. Hess, R. A., Thurston, R. J. & Biellier, H. V. 1976. Morphology of the epididymal region and ductus deferens of the turkey (Meleagris gallopavo). J. Anat. 122: 241-252. Lake, P. E. 1957. The male reproductive tract of the fowl. J. Anat. 91: 116-129. Lake, P. E. 1979. Male genital organs. In Form and Function in Birds 2: 1-61. King, A. S. & Mclleland, J. (Eds). London: Academic Press. Lofts, B, & Murton, R. K. 1973. Reproduction in birds. In Avian Biology 3: 1-108. Farner, D. S. & King, J. R. (Eds.) New York: Academic Press. Mehrotra, P. N. 1962. Cyclical changes in the epididymis of the goose, Anser melanotus. Quart. J. Micros. Sci. 103: 377-383. Middleton, A. L. A. 1972. The structure and possible function of the seminal sac. Condor 74: 185-190. Munro, S. S. 1938. Fuctional changes in fowl sperm during their passage through the excurrent ducts of the male. J. Exp. Zool. 79: 71-92. Prasad, M. R. N. & Rajalakshmi, M. 1976. Comparative physiology of mammalian epididymis. Gen. Comp. Endocrinol. 28: 530-537. Sarkar, A. & Ghosh, A. 1964. Cytological and cytochemical studies on the reproductive cycle of the subtropical male house-sparrow, La Cellule 65: 111-126. Silverin, B. 1975. Reproductive organs and breeding behaviour of the male pied flycatcher Ficedula hypoleuca (Pallas). Ornis. Scand. 6: 15-26. Tingari, M. D. 1971. On the structure of the epididymal region and ductus deferens of the domestic fowl (Gallus domesticus). J. Anat. 109: 423-435. Tingari, M. D. 1972. The fine structure of the epithelial lining of the excurrent duct system of the testis of the domestic fowl (Gallus domesticus). Quart. J. Exp. Physiol. 57: 271-295. Warren, L. 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234: 1971-1975. Wolfson, A. 1954. Sperm storage at lower than body temperature outside the body cavity in some passerine birds. Science 120: 68-71. The Male Sex Accessories in the Annual Reproductive Cycle of the Pied Myna 55

ホオ ジロム ク ドリSturnus c. contraの オスの生殖付属腺 の年周期変化

亜 熱 帯 地 域 の ス ズ メ 目鳥 類 で あ る ホ オ ジ ロム ク ドリSturs c. contraの オ ス の生 殖 付 属 腺 の年 変 化 を,重 量 測 定 に よ り,ま た組 織学 的,生 化学 的 観点 か ら調 査 した。 そ の結 果,オ ス の生 殖 付 属 腺 の 消長 は,生 殖 腺 の 年 周 期 と同 時 に起 こ る もの と思 われ た。 す な わ ち,非 繁 殖 期 に は活 性 が 低 く,繁 殖 期 前 に活 性 が高 ま り,

繁 殖 期 間 中 に最 も活性 が 高 くな っ た。更 に詳 し く述べ る と,そ の活 性 は造 巣 前 期 に増 加 しは じめ,造 巣 後 期 に ピー クを む か え,産 卵 ・抱卵 ・育雛 期 に は徐 々 に減 少 して い く。オス の生 殖 付 属 腺 の この変 化 は,メ ス の 巣 卵 ホ ル モ ンの活 性 の変 化 に よ る もの で あ ろ うと思 わ れ た。

S. K. Gupta & B. R. Maiti: カルカッタ大学動物学部組織生理学教室