Cytological Survey of the Walleye (Stizostedion- vitreum, M. ) Pituitary Gland In Relation to Annual Temperature Cycle and Its ~eproduktive Cycle

Transcription

1 Cytological Survey of the Walleye (Stizostedion- vitreum, M. ) Pituitary Gland In Relation to Annual Temperature Cycle and Its ~eproduktive Cycle A Thesis Submitted To the Faculty University of Wisconsin La Crosse Mack G. Cook In Partial Fulfillment of the Requirements for the Degree of Master of Science August 1974

2 UNIVERSITY OF WISCONSIN La Crosse Wisconsin COLLEGE OF ARTS, LETTERS AND SCIENCES Candidate: Mack G. Cook CY. We recommend acceptance of this thesis to the College of Arts,Letters, and Sciences in partial fulfillment of this candidate's requirements for the degree Master of Science in Biology. The candidate has completed his oral defense of the thesis. dn&- Thesis Committee Chairman ' Date This thesis is approved for the College of Arts, Letters, and Sciences

3 ABSTRACT The value of the walleye (Stizostedion vi treum, Mitchill ) as a resource and as a member of the biotic community necessitates an accurate des- cription of its reproductive endocrinology and a means of monitoring its reproductive status. This has been accomplished by a cytological survey of the pituitary of 100 walleyes from navigational pools 6 and 7 4 C of t e upper Mississippi River. Gonadal tissue was also removed and analyzed to determine the specific stage of the fish's reproductive cycle. Pituitaries were removed through a dorsal flap in the cranium during 1972 and The average diameter of gonadotrop cells was u during March. This decreased to 8.79 u in July and a gradual increase occurred following the July value. Eta, MSH and thyrotrops followed this trend. In March eta cell diameters were 8.99 u, MSH cells were 8.34 u and, in April thyrotrops measured 8.04 u in diameter. These cell diameters decreased to 6.79 u for eta cells, 7.06 u for MSH cells and 6.7 u for thyrotrops. The gonadal cells increased in size and maturity from u in March to u in May. The optimum temperatures for spawning of 6.1' C to 17.2' C occurred from the beginning of March until the end of April. This corresponds with time of decreasing cell diameters seen in the pituitary. Presently the appearance of the pituitary cells, expecially gonadotrops, suggests a normal, unaltered condition. Unintentional or accidental disturbance of the habitat, most probably the parameter of temperature, would produce a change in the cycle of gonadotrop size changes,

4 .....,....., TABLE OF CONTENTS...,... LIST OF TABLES,,,.,,,,.,.,,,.,.,..,.... i v LIST OF FIGURES...,;....,... v cage L I g OF PLATES... vi INTRODUCTION... 1 MATERIALS AND METHODS... 6 "RE6ULTS DISCUSSION BIBLIOGRAPHY APPENDIX iii

5 LIST OF TABLES 1. Number of mature walleyes of each sex collected during each month of 1972 to 1973 from navigational pools 6 and 7 of the upper Mississippi River Monthly measurements of the average cell diameters in microns of four pituitary cell types found in each sex of walleye collected from navigational pools 6 and 7 of the upper Mississippi River during 1972 and Appendix. Table -, 3. ight - length data for 100 walleyes from pool 6 and 7 of the upper Mississippi River Scale length - total length data for 100 walleyes from pool 6 and 7 of the upper Mississippi River Mean calculated total lengths at each annulus for the 1968 to 1972 year classes of 103 walleyes collected from pools 6 and 7 of the upper Mississippi River from 1972 to Calculated growth increments of each age class of walleye collected in the upper Mississippi River during its respective year of growth Page

6 Figure LIST OF FIGURES 1. Monthly averages of the diameters of four pituitary cell types from walleyes collected in navigational pools 6 and 7 of the upper Mississippi River in 1972 and Annual water temperature range in pools 6 and 7 of the upper Mississippi River calculated from temperatures recorded during collections, from a local office for the Department of Natural Resources and from a local nuclear power plant. A1 so designated are temperatures at which walleye spawning normal and optimally occurs Average monthly diameters of ova and stage of spermatogenesis in collected walleyes from pools 6 and 7 of the upper Mississippi River. ermatogenesis in male fish was described in?!i ur stages each with an arbitrary value of 150 to facilitate application to the same scale used to plot ova sizes Walleye gonadotrop sizes compared to gonadal cell sizes from fish collected during March, April and May of Appendix Figure 5. Weight and length relationship of 100 walleyes from pool 6 and 7 of the upper Mississippi River during 1972 and Total length - scale length relationship of 100 walleyes from Mississippi River (navigational pools 6 and 7) during 1972 and Page

7 Plates LIST OF PLATES 1. Cross section of entire pituitary, Matsuo's stain, (pars intermedia to left, rostral pars distalis to right) Eta (prolactin) cells in the rostral pars distalis, Matsuo's stain, 100x 12 Page Eta cells, Matsuo's stain, 16x Cross section of entire pituitary, Matsuo's stain, 6.3x, pars intermedia to the right and green gonadotrops are found in the caudal pars distalis Cross section of entire pituitary, Matsuo's stain, 6.3x, caudal pars distalis in lower right Cross section of pituitary, PAS methyl blue stain, 6.3x, caudal pars distalis extends to left under pars intermedia i 7. ~onadotro~ follicle, Matsuo's stain, loox, vessels in upper right and lower left Peninsula of adenohypophysis, PAS methyl blue stain, 1OOx Peninsula of adenohypophysis,. Matsuo's, 16x Thyrotrops lining nerve tissue at left center, Mallory's anilin blue collagen stain, loox Gonadotrop follicles, PAS methyl blue,.l6x Gonadotrop follicles, PAS methyl blue, loox Gonadotrop follicles, Matsuo's, loox Granules and general stain reaction in gonadotrops, PAS methyl blue, loox Gonadotrops staining reactions,pas methyl blue, loox Sphere cells with orange globules, Mallory's anilin blue collagen stain, loox Sphere cells, Mallory's anilin blue collagen stain, loox... 26

8 Plates 18. MSH cells with PAS + reaction at basement membrane. PAS methyl blue. loox MSH cells, Matsuo's stain, loox Gonadotrops surrounded by thyrotrops. Matsuo's. loox Walleye ova, lead hematoxylin stain, 6.3~ Page

9 INTRODUCTION The walleye (Stizostedion vitreum, Mitchill), a member of the family percidae, is an important game fish in the northern Mississippi River drainage (Niemuth et al., 1969). The welfare of this natural resource rests on its capacity to reproduce or successfully spawn year after year. Success depends on the synchrony of the fish's behavior and its environment (Hoar and Randall,vol.III, 1969),and the ultimate physiological control of this activity resides in the pituitary. A histological survey of the wall eye pituitary throughout the year provided a comparison between existing environmental conditions and the tinctorial appearance of specific cells. Treated histologically, these cells take on separate identitges, and in the wake of recent investigations, functions have been confidently ascribed in most cases to each cell type. In this way the walleye pituitary can provide a unique monitor of the fish's reproductive condition (Ball and 01 ivereau, 1966; Hoar and Randal 1,vol 111, 1969; Lagios, 1965; Pickfordand Atz, 1957; Sage 1967; Sage and Bromage, 1970 ; Schreibman et a1., 1973; Sokol, 1961). Housed in a depression of bone, the teleost pituitary rests anteriorly and medially to the inferior lobe of the infundibulum (Hoar and Randall, vol.11, 1969; Kerr, 1941; Lagler et al., 1962). The pituitary is composed of both neural and epi the1 ial tissue, the former originating from the diencephalon and the latter derived from the buccal epithelium of Rathke's pouch (Schreibman et al., 1973). A delicate infundibular stalk connects the gland to the floor of the diencephalon. As in the majority of teleosts, there is no hypothalamo - hypophyseal portal

10 system (Lagios, 1965), although a common blood supply usually does first bathe the neural tissue and then proceeds to the adenohypophysis. This suggests a primitive form of the median eminence found in higher tetrapods (Kerr, 1942; Sokol, 1961). In this early stage of pituitary investigation the three lobes of the adenohypophysis have received several assorted names. The terminology divised by Schreibman et al. (1973) considering function and location, will be used here. Several authors referred to in this investigation use terms identifying the pituitary lobes which differ from those of Schreibman et al. (1973). The table below is provided as an easy reference for comparing these names that apply to th, same areas. Schreibman's terms Others (3C' Rostra1 pars distalis ---- proadenohypophysi s partie folliculaire Caudal pars distalis proximal pars distal is mesoadenohypophysis uebergangsteil Pars intermedia meta - adenohypophysis neuro - intermediate The anteriormost region, termed rostra1 pars distalis, was relatively avascular and had little contact with the neural hypophysis (Lagios, 1965; Kerr,1942). Its small, closely packed erythrosinic cells are the source of prolactin which is responsible for the fish's adaptability to freshwater (Kerr, 1942; Li and Warner, 1972; Sokol, 1961). The next region, moving posterior, was the caudal pars distalis. This lobe was the largest of tfe three. Its cells (thyrotrops, somatotrops and gonadotrops) were variable in size, shape, and staining reaction. The gonadotrops 2

11 represent the major source of endocrine control over the reproductive physiology of the fish (Lagios, 1965; Li and Warner, 1972; Robertson and Wexler, 1962a,b; Schreibman et al., 1973; Scruggs, 1951; Sokol, 1961) The most posterior lobe, the pars intermedia, had extensive contact with interdigitating peninsulas of the neurohypophysis. The acidophilic cells here may function in the control of melanocytes (Lagios, 1965). The pituitary exists as a mediator between the fish's nervous and endocrine systems or external and internal environment (Pickford and Atz, 1957). The structural method of communication between the hypothalamus and the adenohypophysis remains a controversial subject. As mentioned, the presence of a median eminence is questionable, and Lagios (1965) suggested the common blood supply between neural tissue and genohypophysis. Pickford and Atz (1957) stated that a plexus of.vessels existed between the neural hypophysis and the pars distalis. Sokol (1961) observed neurosecretions accumulating and moving into the caudal pars distalis, and Dalange (as mentioned in Sokol 1961) described net - like terminations of nerve fibers on gonadotrops in the sea horse (Hippocampus). Kerr (1942) also described a plexus of vessels between neural and gonadotropic tissue. Although the method of transfer is still in question, evidence supporting endocrine activity in response to neural stimulation by environmental factors does exist. Marshall (1967) altered the external environment of anabantoids (Trichopsis vittatus and T. pumilus) by subjecting them to a controlled light regime and in this way identified their spawning time as a few hours before darkness. If the light cycle was altered, the spawning time also changed. The brook trout (Salvelinus fontinalis) and the brown trout (m trutta)have been 3

12 raised under controlled light conditions and their spawning times have been advanced by as much as four months or delayed six weeks (Allison, 1951; Pickford and Atz, 1957). In all cases, excluding the species of blind fish, light will affect the reproductive condition of fish as influenced by the pituitary (Pickford and Atz, 1957). In poikilothermic animals temperature must be considered as an influential environmental element. Evidence suggested that temperature may work in harmony with light or, in some cases, it could be more important than light (Pickford and Atz, 1957). The eggs of the seiffertilizing hermaphroditic fish Rivul us marmoratus (F. cyprinodontidae) produced a higher percentage of male young when raised at low temperature (Harrington, 1967). In the Killifish (Fundulus, F. cyprinodontidae) spermat@anial multiplications occurred at 10' C but spermatogenesis did not proceed until higher temperature were reached (Pickford and Atz, 1957). In the goldfish (Carassius auratus, F. cyprinodontidae), injections of gonadotropins elicited spawning conditions in fish kept at temperatures too low to cause normal ripening. Apparently a lack of pituitary secretion could be the obstacle to gametogenesis at these lower than normal temperatures. In this case, the effect of temperature on the gonads alone can not be overlooked although the evidence of increased neurosecretions of some freshwater fish in spring and decreased secretion in autumn suggested the more sensitive temperature control would be via the pituitary (Pickford and Atz, 1957). Limited information regarding other environmental factors such as rate flow, turbulence, turbidity, and water chemistry exists for some tropical fish. With regard to the temperate freshwater fish, I have found no significant evidence of the fish's response to these parameters. 4

13 To successfully spawn a fish must contend with these environmental conditions. This has given rise to reproductive cycles which, in fish, closely match the seasons and adjust to their subtle fluctuations. The adult fishes' ability to sense and react to their surroundings provides eggs and young fish with the best possible chance of survival. Imediately after spawning, the walleyes gonads began production of the next year's gametes. By late fall the gametes were well developed and they entered a period of slow growth through the colder and less active winter months. The spring rise in temperature stimulated the fish into a rapid spurt of growth and final maturation of gonad cells. When the correct light and temperature conditions dictated, the fully mature eggs and milt were released completing another annual cycle (Hoar and Randall, 1969). The gona@trops confined to the caudal pars distalis of the pituitary were the cells that can be expected to reflect the most dramatic changes in morphology which correspond to this gonadal development and the overt seasonal changes in walleye behavior. This survey of the pituitary focusing on these cells served as a monitor of the reproductive condition as mediated by environmental stimuli. Any change in stimuli would be first noticed in these cells.

14 METHODS AND MATERIALS The pituitary glands and gonads used in this study were taken from 100 walleyes collected from 1972 to 1973 in navigational pools 6 and 7 of the upper Mississippi River. This section of the river borders the Wisconsin counties of Trempealeau, Monroe and Vernon. In this portion of the river no unnatural effluents flow into the river that could alter its natural condition. Methods used to obtain specimens included electro-fishing, gill nets of 7.5 cm. (3 inch) bar mesh, and hook and line. Despite the use of all three methods, the collecting success on mature individuals was greatly affected by the time of year and the weather. The walleyes were most vulnerable during their spawning period of February to May (Iable 1). Total lengths and weights of all fish were recorded Samples of,4-.10,scal-eawere,removed from.below +he lateral lihe where the margin of C the pectoral fin intersects the lateral line. Scale impressions were read using a 32 nun lens and condenser on an Eberback microprojector. combination produced a scale magnification of 40x and made possible the actual aging of the fish by counting annuli. These were distinguished most clearly in the lateral field of the scale where crossing over of circuli and crowding were obvious clues. Also, erosion of circuli This usually in the radii, spacing variations between circuli, and irregularities in circuli suggested the presence of an annulus. irregularities were not considered. Scales showing obvious Each scale was measured from its focus to its posterior margin along the largest and most directly anterior- posterior radii. Gonadal tissue was removed from each fish through a short incision 6

15 Table 1 Number of mature walleyes of each sex collected during each month of 1972 to 1973 from navigational (2~1 pools 6 and 7 of the upper Mississippi River.

16 Femal es 'Males Total Jan Feb. 25 Mar. 20 Apr June 0 July Aug. % 7 Sept. Oct. Nov. Dec.

17 made from the anus and directed anteriorly. In each case tissue from the posterior tip of the gonad was taken and fixed in Bouin-Hollande fluid. 0 This tissue was imbedded in paraplast medium (melting point C) and sectioned at 8-10 u. avoid damage caused by the coarse egg membranes. Sectioning at this thickness was necessary to Slides of the gonad tissue were stained with hematoxylin and eosin and a lead hematoxylin procedure. A modification using fast green and lead hematoxylin was also employed for greater contrast. positive verification of the sex. These slides made possible a The cellular measurements of the gonad tissue provided a method of assessing the stage of development and activity. A micrometer reticle with 10 mm divisions into 100 units was used for all measurements. Random sections of ovary material were placed under the objective and the first te%midsaggital sections of ova encountered were measured and averaged. Four criteria were used to judge testicular material. The sample was immature if only spermatogonial cells were present, underdeveloped if primary and secondary spermatocytes were seen, prespawning if secondary spermatocytes and spermatozoa were present and mature and ready for spawning when only spermatozoa were observed- Removal of the pituitary gland involved cutting the cranium between the orbits, then cutting posteriorly along each side of the head producing a flap in the roof of the cranium. When pryed up, the brain was exposed.lifting the medulla exposed the pituitary which was immediately taken out and fixed in Bouin-Hollande fluid. This operation was performed as soon after the fish had left the water as possible, a factor especially critical during the winter months. 8

18 If allowed to freeze, the tissue was difficult to handle and stained material assured rapid and efficient fixation. The tissues were then imbedded in paraplast medium and sectioned at 5-6 u. Every second slide of pituitary tissue was stained with a PAS - methyl blue method (Wilson and Ezrin, 1954). The alternate slides were subjected to "Procedure A-Walker" (Emmart et al. 1966) and a modification of Matsuo's staining method for bird pituitary glands (Matsuo, 1954). The latter staining method proved most effective when used in the fol 1 owing procedure: 1. Xylene and dehydrate to water 2. Orange G soln., 15 min. orange G (Chroma-Gesellschaft and Schmid CO.).5 gm. 'glacial acetic acid. distilled water 3. Rinse 4. Acid Fuchsin soln., 2 min. acid fuchsin glacial acetic acid distilled water.5 gm, 3 ml. 300 ml. 5. Rinse 6. 2 % Phosphomalybdic acid, 2 min. 7. Rinse 1 % methyl green 1 % acid violet 50 % absolute alcohol 7 parts 2.5 parts 5 parts

19 9. Rinse 10. Blot, 95 % absolute alcohol, 5 min. 11. Clove oil, 5 sec. clove oil 3 parts absolute alcohol. 1 part 12. Clear and mount This produced purple to red acidophi 1 s, green basophi ls, violet ampophi 1 s and negative chromophobes. A midsaggital section of the pituitary was used to measure gonadotrops. The caudal pars distalis in this section was scanned under a low power magnification. When a new objective producing a magnification of 400x was switched into place, the first 20 cells seen were measured and averag size were recorded. '?h ' Total cell diameter, nuclear diameter, and granule Water temperatures were recorded during the coll ection of samples. Temperature records were also 'obtained from alocal office of the..... Department of Natural ~esources 1 ocated' in. ~a~rosse, Wisconsin. This data probably indicated only surface temperature and a more accurate measurement of the overall water temperature was desired. Two local nuclear electric power plants also recorded the temperature of their inflow water. These plants are located in Genoa,Wisconsin, and water used for their operation is pumped from below the water surface of pool 8 in the Mississippi River. The temperatures are recorded daily throughout the year. These records complement each other and provided a yearly picture of both surface and total water temperatures. 10

20 RESULTS Histological preparation and analysis was made on a total of 100 walleyes, 69 females and 31 males. During certain periods of the year, some individuals were scarce; for example, few males were captured from June to January (Table 1). Several immature fish were captured and the condition of cells and development of some lobes in these pituitaries was not consistent enough to be useful in a descriptive study. These individuals were not considered in the total number of walleyes collected. The shape of the walleye pituitary gland was typically oval (Plate 1). A small cleft ran from beneath the gland up the front widening anteriorly to allow for the bulb shaped lobe situitated there. 9 ~orsal-7$', only the delicate infundi bular stalk composed of neural fibers connected the gland to the floor of the brain. These nerve fibers occupy most of the dorsal area. A large mass of the nerve tissue moved down and into the posterior aspect of the gland occupying as much as one third of the total pituitary area. Some bundles of neurohypophyseal fibers interdigitated with the area in the center of the gland and few bundles of fibers reached out toward the anterior part of the pituitary. This extensive interdigitation of the neurohypophysis into the adenohypophysis of the walleye pituitary was an outstanding feature. It served as a landmark providing a means of rapid orientation. The adenohypophysis was divided into three areas distinguished by the differences in cell types. The rostra1 pars distalis was the smallest lobe (Plate 1). It formed a crescent in the anteriormost portion of the gland. The top point of the crescent ended in the entering neurohypophyseal 11

21 Plate 1 - Cross section of entire pituitary, Matsuohs Stain, (pars intermedia to left, rostral pars distalis to right). Plate 2 - Eta (prolactin) cells in the rostral pars distalis, Matsuo's stain, 100x.

22 fibers and the bottom point extended under the middle area of the gland. The body of this lobe was solidly packed with acid fuchsin staining (eta)cells. Vascular tissue or nerve fibers were rarely seen in this lobe. The closely packed eta cells were small and accept a polygonal shape due to the close contact with neighboring cells. Their nuclei were eccentric and the nucleoli are indistinctjplates 2,3). These acidophil ic cells accepted the acid fuchsin of Matsuo's procedure producing a lavender color throughout; in the PAS - methyl blue procedure, the cells appeared blue. The size of the eta cells of the walleye averaged 7.81 u in diameter in the female fish and 7.89 u in males (Table 2). Over the year these cells showed a slight rise and fall in size. The maximum s5ze occurred in March (8.99 u) followed by a steady decline in size until Sasember (6.79 u). This progression of size fluctuation is shown on Figure 1. A peak noticed during October reflected data based on information from only one individual captured during that time and consequently cannot be considered representative of all the walleyes at that time. The heavily granulated cytoplasm and sparsely staining nuclei changed little during the year in structure or affinity for the acid stains in Matsuo's procedure. The caudal pars distalis was the largest lobe in the pituitary. Its boundaries were defined by the rostral pars distalis to the anterior, the pars intermedia posteriorly, and the neurohypophysis covering the dorsal surfacs. This lobe itself formed the ventral surface of the pituitary (Plates 4,5). Laterally the caudal pars distalis extended under the rostral pars distalis and posteriorly it extended far under the pars 13

23 Table 2 Monthly measurements of the average cell diameter '%I in microns of four pituitary cell types found in each sex of walleye collected from navigational pools 6 and 7 of the upper Mississippi River during 1972 and 1973.

24 I Plate 3 - Eta cells, Matsuo's stain, 16x Plate 4 - Cross section of entire pituitary, Matsuo's stain, 6.3x, pars intermedia to the right and green gonadotrops are found in the caudal pars distalis. 14

25 Plate 5 - Cross section of entire pituitary, Matsuo's stain, 6.3x, caudal pars distalis in lower right. Plate 6 - Cross section of pituitary, PAS methyl blue stain, 6.3x, caudal pars distalis extends to left under pars intermedia.

26 "u Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Average Thyrotrops (female) (male) Gonadotrops (female) c-. 01 (male) Eta cells (female) (ma1 e) MSH cells (female) (male)

27 Figure 1 <\I i' Monthly averages of the diameters of four pituitary cell types from wall eyes collected in navigational pools 6 and 7 of the upper Mississippi River in 1972 and 1973.

28

29 intermedia (Plates 1,6). The caudal pars distal is was highly vascularized with several vessels often seen in close proximity to gonadotrop follicles (Plate 7). The remaining and most distinctive characteristic of this lobe was the extensive interdigitation of the neurohypophysi s. This interdigitation resulted in the formation of several fingerlike peninsula of adenohypophysis tissue; in some cases these were only five cells thick (Plates 1,8 and 9). Thyrotrops lined the adenohypophysis peninsula forming a border where the caudal pars distal is met the neurohypophysis. The thyrotrops were variable in shape but most often were columnar with their axes perpendicular to the basement membrane lining the lobe (Plates 8,lO). The nuclei were oval and usually in the distal end of the cell. The average width of the thyrotrops was 7.22 u in females and 7.56 u in males (Table 2). 'The length appeared to be 2 to 3 times the width of the cell. The size fluctuation during the year again showed steady decline from the maximum of 8.03 u in width in April to the minmum of 6.4 u in November (Figure 1). T@ thyrotrops were basophilic and also accept PAS, although the c. bright red color associated with a positive PAS reaction was usually confined to the end of the cell directed toward the neurohypophysis (Plate 8). Using Matsuo's tetrachrome, the thyrotrops displayed a red (violet) color. The nuclear chromatin material was present in large amounts and stained darker. Gonadotrops were found inside the adenohypophysis peninsula interior to the thyrotrops. These cells became more dominant moving ventrally until at the ventral edge only gonadotrops were found surrounded by 18

30 Plate 7 - Gonadotrop follicle, Matsuo's stain, 100x, vessels in upper right and lower left. Plate 8 - Peninsula of adenohypophysis, PAS methyl blue stain, 100x. 19

31 Plate 9 - Peninsula of adenohypophysis, Matsuo's, 16x. Plate 10 - Thyrotrops lining nerve tissue at left center, Mallory's anilin blue collagen stain, 100x. 20

32 occasional chromophobes or thyrotrops. Gonadotrops were commonly found in clusters or oval folliclelike structures. These were also seen most often at the ventral edge of the, gland (Plates 7, 11,12 and 13), The gonadotrops obtained the largest cellular diameters and were the most variable in shape and size. In females, the gonadotrops averaged 9.47 u in diameter and in males the average was 9.72 u (Table 2), although cells as small as 6 u in diameter and as large as 15 u were found. When these cellular size, fluctuations were compared to the time of year, a regular cycle was again apparent. The maximum size for gonadotrop diameters was seen during March (10.56 u). and a progressive decrease followed which lasted until August when the minimum average cell diameter was recorded (7.75 u). The cell diameters steadily increased following the August low. This increase continued until diameter me.asurements reached approximateljl 9.5 u and here the cell size remained constant until the next spawning period (Figure 1). The cyclic trend of decreasing cell sizes from March to August followed &I by an increase was mirrored by nuclear sizes during March to May. During the rest of the year nuclear sizes were between 4.59 u and 4.97 u in diametey. At the extremes of the cycle, cytological changes became evident. During March and April, when cells were large, granulation was very heavy and stained brightly; also, nuclei stained dark and were very distinct. In July and August (the months of minimum cell sizes), the cell boundaries were difficult to distinguish and cytoplasm appeared vacuolated (Plate 7). These basophilic gonadotrops gave a strongly positive PAS reaction producing the typical bright red color. This color was restrictied to

33 Plate 11 - Gonadotrop follicles, PAS methyl blue, 16x. Plate 12 - Gonadotrop follicles, PAS rnethly blue, 100x. 22

34 Plate 13 - Gonadotrop follicles, Matsuo's, 100x. Plate 14 - Granules and general stain reaction in gonadotrops, PAS methyl blue, 100x.

35 \ thickly packed granules rather than the cytoplasm in general, nuclei accepted the counter stain methyl blue and showed dispersed The dark staining chromatin and prominent nucleoli (Plates 14,15). The same cells, when stained with Matsuo's procedure, appeared green with dark green nuclei. Some sections were stained alternately with Matsuo's procedure and with the PAS test. This provided a comparison of cell reactions to a tinctorial stain and a standard biochemical test In some cases only 5 u separated the cells stained under different procedures. The cells accepting the methyl green of the Matsuo's stain also reacted positively to the PAS confirming their identity, under these two procedures, as gonadotrops. The cells Kerr (1942) refers to as sphere cells were also found in the adenohypophysis peninsula of the caudal pars distalis of the walleye when Mallory's anilin blue collagen stain was applied. The bright orange globules in these cells varied greatly in size and number. They seemed to coalesce as they developed, often to the extreme of occupying the entire space of the cell's cytoplasm (Plates 16,17). Cells containing the largest globules always appeared near an extension of the neurohypophysis. The pars intermedia occupied the posterior portion of the gland except for the thin ventral extension of the caudal pars distalis (Plates 1,4). This lobe supported the largest concentration of nerve fibers and often seems to be a layer only two or three cells thick covering the large lobe of the neurohypophysis. Cells in the pars intermedia were cuboidal to slightly columnar. There was often a positive PAS reaction along their basement membrane (Plate 18).

36 Plate Plate 16 - Sphere cells with orange globules, Mallory's anilin blue collagen stain, 100x. 2 5

37 Plate 17 - Sphere cells, Mallory's anilin blue collagen stain, 100x. Plate 18 - MSH cells with PAS+ reaction at basement membrane, PAS methyl blue, 100x.

38 With Matsuo's stain they appeared dark blue or purple with red nuclei (Plate 19). The MSH cells of this lobe also decreased in total cell diameter from March to May, and they increased gradually the rest of the year. This cycle of size change was indicative of the other cells of the adenohypophysis although the MSH cell size variation was less radical. Determination of sex of the walleye specimens was difficult at best, except during prespawning months when gonads were visually swollen with roe or milt. At other times of the year anything less than a microscopic check of gonad material was not sufficient. Depending on the time of year, the translucent outer membrane of the gonads was collapsed on itself like an empty sack. From late fall (November)to spring (March-May), the male gonads appeared creamy white and swoll en and the female gonads were extremely 1 arge, often displacing other organs in the body cavity. Tha large ova inside the gonad gave the whole organ an amber color. In place of seminiferous tubules found in mammal male gonads* separate packets of gamete cells were seen in the walleye gonad. These packets matured as a unit during the year. The process of spermatogenesis in these packets began immediately following spawning with the appearance of spermatogonia in the testes packets. By October, cells of Sertoli were found surrounding primary spermatocytes filling the packets. During February and March a rapid transition from secondary spermatocytes to spermatozoa occurred. The ova of the female walleyes began development immediately foll owing spawning. Their cytoplasm was homogeneous, and the nuclei 27

39 Plate 19 - MSH cells, Matsuo's stain, 100x.

40 were clear'with dark material at the edges (see Hoar and Randall, vol. 111, 1969 for a review of gamete cytology in fish). Size increases and appearance of egg membranes provided the means of detecting change in the ova (Figure 3). During March ova size averaged u in diameter, this increased to u in May, and release of the eggs followed. Development was evident again in July with the appearance of ova u in diameter. These increased in size to u in diameter during September. A gradual decline to a constant size followed in the months from October to February. The eggs remained in this condition until the following year's spawning season when the rapid increase in size began again. September. All visible egg membranes were present by August or early The size of the gametes from March to May were inversely related to the size changes of gonadotrops during the same spring months (Figure 4). The line for this relationship is described by the following formula: The weight of the collected walleyes varied from 110 grams to 3600 grams. The range of total lengths was 250 to 680 cm. This data was grouped in 12 intervals and recorded in the Appendix, Table 3. Calculation of a weight-length relationship (below) from this information produced an equation describing a linear relationship. Appendix, Figure 5 displays this data as applied to a linear graph. w = a (TL)" log w = log TL 29 w = weight TL = total length a = intercept n = slope

41 Further information describing the normal condition of the collected fish was found using the scalemeasurements, measured from focus to margin of radii. Eleven intervals were prepared and recorded with their respective total lengths intheappendix, Table 4. Using the equations below a scale-total length relationship was developed. The line produced from these caiculations described the data as plotted in Appendix, Figure 6. TL = a + b (SL) a = b = TL = total length a = intercept on ordinate b = slope SL = scale length Scale measurements also made possible aging of walleyes collected and back calculation of growth. two years old to six years old. were.recorded in $he.appendix, Table 5. Walleyes collected ranged in age from Mean total lengths at each annulus Growth increments o.f each age class during its respective year of growth were calculated by subtracting successivefigures on Table 5 in the age class row and recording them in the respective year class column in Appendix, Table 6. From this information we could deduce that the fish sampled had an acceptably normal growth pattern during the sampling period. These calculations correspond favorably with the age and growth information on walleyes compiled by Carlander and Whitney in Clear lake, Iowa 1935 to Discrepancies that do exist in this data are explanable, such as, the variation in the ponderal index reflecting gravid conditions in female fish, and the curvilinear line in Appendix, Figure 5 suggesting Lee's phenomenon, and the unexpect growth of the 1968 year class, figured using only two individuals. The fish used may be accepted as representative of the population.

42 The water temperature records from personal data, the Department of Natural Resources and the two power plants on the Mississippi River showed a rapid rise in temperature occurred during February, March and April which corresponded to the prespawning and spawning periods. August, September, and October were marked by a comparatively rapid decline in temperature (Figure 2).

43 Figure 2 Annual water temperature range in pools 6 and 7 of the upper Mississippi River calculated from temperatures recorded during collections, from a local office for the Department of Natural Resources and from a local nuclear power plant. Also designated are temperatures at which walleye spawning normal and optimally occurs.

44

45 Figure 3 Average monthly diameters of ova and stage of spermatogenesis in collected walleyes from pools 6 and 7 of the upper Mississippi River. Spermatogenesis in male fish was described in four stages each with an arbitrary value of 150 to facilitate application to the same scale used to plot ova sizes.

46

47 Figure 4 Walleye gonadotrop sizes compared to gonadal cell sizes from fish collected during March, April and May of 1973.

48

49 DISCUSSION Kerr (1942) described the yellow perch (Perca fluviatilis, F. Percidae) pituitary as relatively primitive in some respects. Characteristics of the walleye pituitary that confirm this primitive state were the short and easily broken infundibular stalk and all three adenohypophyseal lobes that were'contacted to some extent by the neurohypophysis. A more advanced characteristic was the lack of any hypophyseal recess. The arrangement of the three adenohypophyseal lobes of the walleye pituitary resembled that of the kill ifish (Fundulus heterocl i tus, f. Cyprinidae), (Scruggs,1936), black surfperch (Embiotica jacksoni, F. hbiotocidae)(lagios, 1964), and the top minnow (Poecilia reticulata, F. Poecil iidae) (Sage and Bromage, 1970). In this arrange- ment the long axis of the gland ran anteriorposteriorly, the rostral pars distalis covered the anterior end, the caudal pars distalis occupied the inid-ventral area, and the pars intermedia made up the posterior portion. The lobes of other teleost pituitaries varyed greatly making anatomical generalities dangerous. goldfish (Carassius auratus,f. For example, the Cyprinidae)adenohypophyseal lobes are layered with the caudal pars distalis sandwiched between the rostral pars distalis above and the pars intermedia below. The Salmonidae pituitaries are also oval but usually oriented with a dorsal-ventral long axis. In the case of the channel catfish (Ictalures punctatus, F. ~Ictaluridae), the infundibular stalk enters anteriorly and holds the gland firmly to the floor of the brain (Scroggs, 1936). neurohypophyseal fibers entered dorsally on the walleye pituitary and interdigitated extensively with the caudal pars distalis, less with the The

50 pars intermedia and little or not at all with the rostral pars distalis., Contact between neural tissue and adenohypophysis is common to all teleosts, only the extent of interdigitation and the point of attachment of the infundibular stalk vary. The shape of the caudal pars distalis varied subtly from the description presented by Kerr (1942) for the yellow perch (Perca -- fluviatilis, F. Percidae). It should be emphasized that the rostra1 pars distalis did appear to be embedded in the anterior of the, gland separate from the caudal pars distalis. When sectioned, this was less apparent although the boundary between the rostral and caudal pars distalis was made very distinct using Matsuo's stain. In the walleye, the caudal pars distalis did not extend under the rostral lobe midsaggitally (Plate 3). The extension of the cells of the caudal..pars,di.s+xlis under,the.pars ;itviermedia was observed over the entire width of the gland. As the source of gonad stimulating hormones, the gonadotrops in the caudal pars distalis of the walleye reacted positively to the " periodic acid schiff (PAS ) reagent. The teleost gonadotrops were described as mucoid (basophil ic) with PAS + granules. These granules contain FSH, a glycoprotein, and LH, also a glycoprotein. Both were selectively oxidized and combined with the Schiffs reagent to produce the observed PAS t or red colored compound. These cells were confined to the caudal pars distalis of the walleye pituitary although they did spread to the rostral pars distalis in the freshwater eel, (Anquil la anquilla, F. Anquilldae), salmon (Oncorhynchus kisutch, F. Salmonidae), and trout (Salmo trutta, F. Salmonidae)

51 (Hoar and Randall, vol. 11, 1969). Thyrotrops in the same lobe also reacted positively to PAS. Contrary to work on some species such as Poecilia (Sage and Bromage, I i 1970,b) the thyrotrops of the walleye were PAS + only along their basement membrane. These cells did change during the year, usually in time with the reproductive cycle, and this could affect the amount of granulation and staining reaction seen, although in the walleye thyrotrops this reaction was consistent throughout the year. To erase any doubt about their identity as thyrotrops, the morphological characteristics as described by Lagios (1964), Li and Warner (1972), Pickford and Atz (1957), Sage and Bromage(1970), Schreibman et al., (1973), and Sokol (1961) were used to verify the identification using the tinctorial reaction. Matsuo's staining procedure provided a second confirming method of cell identification. When sections were stained alternately with PAS and Matsuo's procedure, a comparison of specific areas and cells was possible. Using the Schiffs reagent all PAS cells could be located. On the next section sliced from the sample, Matsuo's stain segregated the PAS cells into two groups, those a-ppearing green to blue (gonadotrops)and the red or violet cells (thyrotrops)(plates 1,20). The gonadotrop cells showed a progressive decline in cell diameter during March, April and May(Figure 1). In previous investigations, the detail of decreasing dismeters was given only cursory attention. The gonadotrops were said to increase in size until spawning, the size then decreased, and vaculoated cells were seen. Only Li and Warner (1972) mention that the cycle of gonadotrop size variation in

52 Plate 20 - Gonadotrops surrounded by thyrotrops, Ratsuo's, 100x.

53 the alewife (Alosa pseudoharengus, F, C1upeidae)may have preceeded the reproduction cycle. Figures 1 and 4 illustrated the gonadotrops reaching their maximum size in March and they decreased in size for two months preceeding spawning or the maximum gamete cell development. This regression was described by the line y = x X The statistical significance of the data described by this line is represented by a chi squared value of.994. indicating that the results obtained were different than those produced by chance alone. This gonadotrop decrease was opposite to the trend seen in the gametes (Figure 3). Here the cells increased in size from March through May. Again on Figure 4, the maximum size of gamete cells was not reached until gonadotrop sizes had decreased sufficiently. As mentioned by Turner (1968). the main function of one of the gonadotropins was the stimulation of the gamete cell development. This seemed to be occurring under the direct exhaustion of gonadotropins originating from the gonadotrops in the pituitary as indicated by their decrease in size. Also during May, several cells had a foamy or vaculoated appearance suggesting an emptying of their contents. This latter observation agreed with work done by Lagios in 1969 on the black surfperch (Embiotica jacksoni, F. Embiotocidae), by Li and Warner in 1960 on the alewife (Alosa pseudoharengus, F. Clupeidae), by Roberts and Wexler in 1962 on chinook salmon (Oncorhyncus tschawytscha, F. Salmonidae), by Sokol in 1961 on the common guppy (Lebistes reticulatus, F. Poeciliidae) and the killifish (Fundulus heteroclitus, F. Cyprinidae).

54 .!! Following spawning, an increase occurred in the gonadotrops' diameter (Figure 1). The size of these cells leveled off at approximately 9 u and remained there until the prespawning activity previously mentioned. This autumn rise in cell size corresponded to an increased ova size and spermatogenesis activity (Figure 3). This comparative activity could be coincidental ; the gonadotrops could have been recovering after their spent condition resulting from spawning. The gonads were recently emptied, so they were also beginning a new cycle with newly developing cells. A second explanation for the gonadotrop size increase could have been the production and secretion of FSH to stimulate the redevelopment in the gonads which would explain the second rise in activity in that area. In this case the sudden increase in ova diameter from u in March to the year's maximum of u in May, and the decrease.of gonadotrop diameters from u in March to 9.37 u in May could have been attributed to LH. In the gonads, or target organs of these gonadotropic hormones, it was noticed that the maturing ova developed a granulosa layer of covering cells in late August and early September (Plate 21). Although questionable, most investigators consider this granulosa to be the source of estrogen. Consequently, during autumn this increased blood content of estrogen would cause the pituitary to diminish its production of FSH and increase its output of LH (Turner, 1968; Hoar and Randal1,vol ). The presence of LH. therefore, would be possible after the fall of the year. The leydig cells of lobule boundary cells (Marshall and Lofts, 1956) were not seen until six months prior to spawning. Lagios (1964) also failed to find Leydig cells in the black surfperch until

55 Plate 21 - Walleye ova, lead heniatoxylin stain, 6.3~.

56 December and January when prespawning activity began in that fish. This evidence suggested that the March-May gonadotrop activity occurred at a time when the blood content of LH would be high. This presupposed the existence of separate gonadotropic hormones although reports do exist refuting the possibility of two separate hormones or separate cell types (Lagios, 1964; Pickford and Atz, 1957). Alternately, the separation of gametogenic and androgenic function was suggested by work done by Schreibman and Kallman (1967) as mentioned in Schreibman et al. (1973). Also in some teleost, freshwater eel (Anquil la anquilla, F. Anquilldae), coho salmon (Oncorhynchus kisutch, F. Salmonidae), and the minnow (Cyprinus auratum, F. Cyprinidae), specifically the coho salmon, two different gonadotrops and gonadotropins have been identified (Chestnut, 1970 in Schreibman et a1.,1973; Hoar and Randall, 1969) These pituitary cellular activities and resulting gonad change were integral to the spawning activity of the walleye. In Wisconsin, this activity was expected when water temperatures were between 6.1' and 17.2' C., the peak occurred between 8.8' and 10' C.. Males were the first to move to the spawning grounds at temperatures as low as 3.3' C. (Niemuth, 1969). These points were labled on the annual temperature curve (Figure 2) recording temperatures taken during the collecting period of this investigation. Niemuth mentioned that I spawning normally takes place between mid-april and May first. Figure 2 1 showed the temperatures at these times to be in.complete accordance with those expected. Both products of oogenesis and spermatogenesis 0 were being released as of May and the gonadotrops were also approaching

57 a spent condition in May. This information suggested that despite alteration of some physical parameters of the river, the variation in temperature was slight and the effect on gonadotrop histology or activity was negligible. This information indicated that the gonadotrops of the walleye decrease in size while the gonadal cells were increasing in size and maturity. Both events began a few months prior to spawning and continued i 1.. until the end of April or until the fish spawned during which time the 1 I : temperature steadily increased., This summary suggested a number of criteria useful in describing the event of spawning. First the gonadotrops swell and shrink periodically and these cell measurements can be used to predict the reproductive condition of the walleye. Several investigators suggested these size changes occur in harmony with water temperatures. temparatures can also be useful in describing spawning. Consequent1 y water Finally, measurements of ova and development of spermatozoa provided good indicators on the reproductive timetable. Knowledge of these criteria would be of advantage to a commercial hatchery because they would provide a means of predicting the occurrence of spawning and this would minimize losses. method of control or a means of scheduling spawning. Even better would be a Presently the most popular method for this type of control is the use of pituitary injections. the action of the gonadotrop cells. These injections compare closely with Normally the gonadotrops swell to their maximum size (10.56 u in March for walleyes) and this would be I I analogous to the drawing in of pituitary extract with a syringe. During the months prior to spawning these cells decrease in size as they

58 Slo~ly inject their contents into the blood (Figure 1) as in the decreased space in the syringe. The inaccuracy in this analogy rests in the time factor. The gonadotrops exert their influence slowly over a period of three to four months before spawning, the artificial injections introduce large doses of gonadotropic hormones at once. Results were normally expected within 24 hours (Fontenele, 1955). 1 More interesting was the source of hormones used for artificial injections. For a commercial operation the best source would be one that produces the desired effect for the least cost. For this, fish that were mature and ripe or within two or three months of spawning were most sought after (Pickford and Atz, 1957). This period corresponded with the time at which gonadotrops were at their maximum size. This provides another method of describing the spawning event, when gonadorops were big enough and the temperature was right, a highlyeffective spawn stimulating extract was obtainable. This method of control is feasible in the commercial hatchery but in the natural habitat the most influential factor is temperature. In the case of poikilotherms a massive rise in temperature or an unseasonal temperature rise would also alter the reproductive clockwork. In this case the dose is critical since in poikilotherms a change of one degree centigrade will alter the animals metabolism by ten percent. The gonadotrops of the walleye would be expected to reflect such a change. The gonadotropic activity of walleyes collected from the Mississippi River in has now been monitored cytelogically under what appears to be normal temperatures for this area. Reference to this type of information could detect a change in the reproductive pattern that the walleye has spent eons of years adapting to.