Systematic Fish Pathology Part 13. Pathology of gonads and early life stages

1. Systematic Fish Pathology Part 13. Pathology of gonads and early life stages Prepared by Judith Handlinger With the support of The Fish Health Unit, Animal Health Laboratory, Department Of Primary Industries, Parks, Water and Environment, Tasmania, for Australian Animal Pathology Standards program(AAPSP)

2. Course Outline A. Systematic Fish Pathology 1.Consider the Fish: An evolutionary perspective on comparative anatomy and physiology 2. Pathology of the kidney I – interstitial tissue Part A 3. Pathology of the kidney II – interstitial tissue Part B 4. Pathology of the kidney III – the nephron 5. Pathophysiology of the spleen 6. Fish haematology 7. Fish immunology – evolutionary & practical aspects 8. Pathology of the digestive system I – the oesophagus, stomach, & intestines. 9. Pathology of digestive system II – the liver and pancreas, swim bladder, peritoneum. 10. Pathology of fish skin 11. Pathology and diseases of circulatory / respiratory system – heart, gills and vessels 12. Pathology of the musculoskeletal system and nervous systems 13. Pathology of gonads and early life stages (this module) A: Gonads B: Early life stages

3. Acknowledgments & Introduction. • This is the thirteenth and final module of the systematic examination of fish pathology, which aims to convey an approach to diagnosis and cover fish reactions of each organ system rather than to cover all fish diseases. Like previous modules, it is based largely on representative pathology found in the diagnostic laboratory of the Tasmanian Department of Primary Industries, Parks, Water & Environment (DPIPWE). • It was funded by the Australian Animal Pathology Standards program with in-kind support of DPIPWE as acknowledged previously. • Photos for this series, especially those of gross pathology, are generally also from DPIPWE archives and were generated by multiple contributors within DPIPWE Fish Health Unit. • Contributors of cases from other laboratories have been acknowledged wherever possible and specific material and photographs used with permission where the origin is known. Any inadvertent omissions in this regard are unintended. • References quoted are listed at the end.

4. GONAD ANATOMY & MICROANATOMY Although fish gonads are in general very similar to those of other vertebrates, there are species differences in: SEX DIFFERENTIATION – Some fish (e.g. barramundi) can change sex, usually from a smaller male (as sperm production is less energy demanding than egg production), to older larger females(protandroushermaphroditism). BREEDING PATTERN: – Species of fish may breed continuously; have several egg batches per year; spawn once a year; or have a single spawning. NUMBER OF GAMETES – Numbers do vary with above factors, but may be markedly reduced with internal fertilization & incubation, which occurs in the ~3% of fish that are live-bearers (e.g. the guppy). Syngnathid (seahorses etc) males incubate the eggs, and also produce very low sperm levels. ANATOMICAL DIFFERENCES - The ovary of the majority of fish species is a typical sac with multiple internal folds. Eggs are released into an oviduct, and thence to the environment (cystovarian). Salmonids, (regarded as a primitive fish group), have a semi-open ovarian system, with the eggs being released into the abdominal cavity and expressed at spawning via a V-shaped vent (technically an oviduct) near the anus (secondary gymnovarian type). The importance of understanding normal structure is enhanced by the increased frequency of abnormal gonads due to deliberate manipulation (production of sterile triploids, hybrids) & environmental contamination by endocrine disruptors.

5. Ovarian development & maturation (salmonids) : Most fish have paired gonads that lie ventro-lateral to the swim bladder, though these can be partially or fully fused in some species. Most fish are either male or female – though some change sex throughout their life, and a very few are functional hermaphrodites (e.g some Killifish) This immature salmonid shows one of a pair of orange immature ovaries lying ventro-lateral to the swim bladder (arrow).

6. The ovary in this salmonid is undergoing maturation, with vitellogenesis of eggs. Vitellogenesis is a hormonally initiated (oestrogenic) process of production of vitellogenin which begins in the liver, and leads to deposition of yolk (consisting of vitellogenin and other yolk proteins) into oocytes as part of their maturation. Ovary Corpuscle of Stannius Kidney

7. Mature salmonid ovary, showing large ova covered by a thin ovarian wall. (Ignore the granulomatous liver.)

8. Ripe (spawning) rainbow trout ovary. Note that the ovarian wall overlying the mature eggs has broken down, releasing the ova into the abdominal cavity, along with ovarian fluid which then helps propel the ova from the V-shaped vent near the anus(arrow), during spawning. Release of sperm by the selected male occurs promptly, following this propulsion of released eggs from the abdomen.

9. Ova being expressed by gentle manual pressure (artificial stripping).

10. Gonad development: Early immature gonad (G) lying ventral to the deflated swim bladder (S) and the kidney (K) of a very young Atlantic salmon (Objective magnification x 10). K * S * Detail of this gonad. At this stage both sexes look similar & cannot be differentiated with histology. Large endodermally derived gonadocytes or germ cells (*) (often just referred to as oocytes) are present in both sexes. Multiplication at this stage is mitotic, as the fish grows (in this section one mitotic figure is visible). In the maturing ovary, the oogonia are eventually transformed into nonyolky primary oocytes that go through the first stages of meiosis. They remain arrested at the prophase of the first meiotic division during addition of yolk (vitellogenesis), and cannot be fertilized until the mature eggs undergo appropriate hormonal stimulation. G

11. Two more gonads from this fish cohort, showing both the large oogonia, plus support & endocrine cells (theca cells in the follicular envelope of each oocyte which produce the steroid sex hormones – see slide 15).

12. Ovarian development: Atlantic salmon female (2 gram), with gonad (G) showing more differentiation as an ovary, with the characteristic internal folding. (Objective magnification x 4). … with small primary oocytes (*). (Objective magnification x 20). G *

13. A 850 g Atlantic salmon female, showing a larger ovary with more differentiated primary oocytes, and the characteristic thin capsule & internal folding, cut in cross section. Compare this to the thicker outer wall and definite lumen of a closed-type ovary (donated slide).

14. Ovary of a 13 month old Atlantic salmon female, showing uniformity in the population of most developed oocytes (to become eggs of the first spawning), organized into cords within a thin capsule. Developing oocytes (still in the pre-vitellinogenesis stage) make up the majority of the tissue. Cells lining the follicles (granulosa and thecal cells, both involved in estradiol production), are also important, the outer thecal cells producing testosterone, which is converted to estrogen (mainly E2) by the granulosa cells. These & following salmonid gonad slides courtesy Nicole Ruff, SALTAS, used with permission.

15. Detail of two developing ova, abutting the liver (below). Each oocyte is lined by a developing dense egg membrane (zonaradiata) and 2 layers of follicular cells (an inner layer of granulosa cells (white arrows) which are flattened in early previtellogenicoocytes and plump during vitellogenesis, inside a more diffuse layer of thecal layer of stromal cells (yellow arrows) that is continuous over adjacent ova. At this stage the first polar body (from the first meiotic division) is extruded and the second meiotic division occurs. This also generates a polar body (that is, one half of the chromosomes acquires the nucleus & yolk, the other is discarded, eventually). This second polar body is retained and released only after fertilization. Ova maturation: Examples of normal salmonid vitellogenesis are not available as the ovary of maturing salmonids are too large for inclusion in routine diagnostic sections (unless specifically indicated). This Danio species ovary does show vitellogenesis, at various stages as these species may have multiple small batches of eggs throughout the season, rather than synchronous development of one large batch. Globules of the egg protein vitellin within the ooplasmimpart the oeosinophiliato the larger ova above. Note the primary oocytes that are not undergoing maturation remain largely basophilic. The abundentvitellin is produced by the liver – which is therefore also more basophilic, reflecting the large amount of RNA etc for its production. The retention of the second polar body until after fertilization is utilized for the production of triploid fish, as subtle treatment of the egg at that stage (short heat, cold, or pressure shock regimes) can induce the 2nd polar body to be retained and re-incorporated into the nucleus.

16. Trout ovary showing nearly fully developed oocytes, plus several earlier stages that are not undergoing maturation this year. Because the mature ova are smaller (typical of many deeper water marine fish), the 2 distinct populations of ova (this years eggs & undeveloped eggs), can be better seen in this mature trevalla ovary.

17. Detail of the egg membrane of a salmonid egg, showing why this has been called the zonaradiata(as least during development). The developing membrane has also been called the zonapellucida, chorion, vitelline envelope or vitelline membrane. The outer layers are no longer present in this spawned egg. The membrane is composed of specific proteins which are highly conserved in higher vertebrates & a variable proportion of muco-polysaccharides. It gains water and hardens rapidly after exposure to water, and as a hardened shell can support up to 100 times more weight than oviduct / unspawned eggs. It is penetrated by the fine pore channels as shown, and one small opening (the micropyle) for sperm access. The micropyle is generally sufficiently narrow to permit the passage of a single sperm. This also closes within minutes of eggs being activated by water at spawning. Review references re egg shell & yolk production, liver functions that produced them, hormonal control & the implications of endocrine disruptors: Arukwe & Goksøyr, 2003; Kagawa, 2013.

18. A pregnant female Guppy (Poeciliareticulata). Obviously relatively few sperm are necessary for fertilization internally, especially as few eggs at a time can be incubated. Although remaining internal, developing larvae are fully dependent on yolk, as for other fish. Note batches of ooocytes with differing levels of maturation..

19. Sexual maturation in males: Maturation can also be a high energy-demand stage for males, often largely due to the high stress levels associated with maturation & mating competition. This, and the reduced immunity believed to be related to the competition stress, often lead to skin lesions or other mild conditions (see Part 10, Skin). Mature testes may also reach considerable size, particularly in once-a-year spawners, such as this mature male greenback flounder (testes causing the abdominal distention).

20. The development of secondary sexual characteristics, such as the jaw hook on the male rainbow trout (top), are also energy demanding. Note the leakage of ova from the mature female (bottom) As well as the development of secondary sexual characteristics, energy balance in mature salmonids is markedly affected by cessation of feeding, which occurs during migration back to freshwater in wild populations.

21. Testes development: testes of a young immature (9 month 0ld) male Atlantic salmon. Developing has progressed well beyond primary gonocyte stage, but spermatogenesis has not commenced. The testes structure of anastomosingseminiferous tubules is discernable at this magnification, though the thin interstitial areas and as yet the lack of a lumen, make this sometimes hard to see. Tubule outlines, and even an occasional lumen (*), are better seen at higher magnification. *

22. The testes structure is better seen in more mature testes, such as this precocious (9 months old & described as almost spermiating) male salmon. [Precocious males, maturing in their first year, are relatively common in salmon populations. Their occasional success in achieving egg fertilization under the fins of older males sometimes still in combat ensures that there is genetic cross-over between what may be largely separated year-classes, normally maturing at 3 years age.] Tubules outlined by connective tissues, with small clusters (circle) of germ cells which multiply & undergo a similar transformation through 1o & 2o spermatogonia & spermatocytes, then divide by meiosis into spermatids which become sperm. • Fish sperm are small, having sufficient energy reserves for ½ - 1 minute free swimming activity, which commences on activation by ovarian fluid or water. [After that time, it is unlikely the sperm will still be in the vicinity of the ova anyway!] • The content of ATP declines in old sperm towards the end of the breeding season, though some fish species have sperm that can generate additional ATP. Measures of sperm quality in fish that are non-histological, including techniques to measure the energy (ATP) content are given by Fauvel et al, 2010.

23. Another 9 month old precocious male salmon … Interstitial Leydig cells are the major source of testicular steroids, together with Sertoli cells. The large cells within the tubule are spermatogonia. With probably more immature cells, showing the progression of spermatogenesis. Note that the apparent depth of the seminiferous epithelium can appear (erroneously) greater in tangential sections of the tubule. Note also the larger cells, upper tubule. Summary references for a description of all development stages of both sexes, plus the hormones controlling these processes, see Van derVen, & Wester, 2014 (a or b); Sundararaj, 1981.

24. This greenback flounder testes (as above) shows similar synchronized production of a large number of sperm, many now spawned so tubules appear nearly empty. Mature males of species with a single annual spawning have more synchronized sperm production of a large number of sperm, as shown by this Danio sp. male. Basal cells and stem cells are present, but no intermediate development stages (mostly a bag of sperm).

25. Less developed male of this or another Danio sp., showing a mixture of development stages of sperm.

26. High sperm numbers, but continuous production, is shown in this Blenny testis. Within each tubule, small clusters of cells (as shown above, sometimes called cysts of cells), are at the same level of maturation, so both mature & immature spermatocytes are present in all tubules.

27. In contrast, this male Platy (Xiphophorus sp.) has continuous production of fewer sperm as multiple batches of internally incubated eggs are produced (live bearers). Mature sperm occupy areas nearer the sperm duct, aligned (in their still inactive state) so that tails project into the tubule lumen.

28. Another Platy male, showing areas with different levels of spermatogenesis, plus interstitial hormone producing cells. … and more clearly showing the whorled sperm tails (arrow).

29. Also well demonstrated in a Guppy (Poeciliareticulata), another live-bearer of the same fish family.

30. The extreme in low normal sperm production is the Syngnathid group (seahorses), producing a testis like that of this male big bellied seahorse (Hippocampus abdominalis) that may not be immediately recognized as such. (x4 objective magnification.) Tufts of spermatogenic cells lining testes tubules. (x 20 objective magnification.) The groups of germ cells are similar to other male fish, but very few more developed sperm can be seen, and fully mature sperm sufficiently rare to be missed. In this group, the female transfers eggs to the pouch of the male prior to fertilization. It is generally said that sperm are released directly into the pouch, but this has been shown to be anatomically impossible for at least some species of seahorse – which still have very low sperm production, despite sperm having further to swim to reach the eggs. (Van Look et al, 2007). (One might expect that the transfer process still provides more control of the fertilization process and for direction of the ejaculate directly onto the eggs, but a mystery still remains.)

31. GONAD PATHOLOGY Though gonads may be involved in infectious disease, especially systemic diseases, Much of the pathology is associated with failure of normal development, or alteration of normal function by external influences such as hormone disruptors. Stress (particularly severe stress such as capture stress) can also disrupt normal development – a major cause of initial failure of many attempted culture attempts. To study these processes, a knowledge of normal gamete turnover is required.

32. E DEGENERATION & RESORPTION OF OVA Much of gamete associated pathology is associated with failure to spawn, and resorption of the mature gametes (especially ova, which represent a major energy investment). Devitalized ova and eggs (i.e., once spawned), turn white with coagulation of the vitellin & associated proteins. The white eggs shown here were not fertilized, and subsequently died. Eggs retained internally will eventually also turn white, but most vitellin is first resorbed.

33. Section of an ovary of a group of rainbow trout that were not stripped during the appropriate time (no opportunity to do so, due to a farm flood). Fish were very sensitive to stress, some succumbing to infections, though most eventually recovered. Most fish has eggs free in the abdominal cavity. This fish shows one still in the ovary (top). There is little evidence yet of resorbtion of this large ovum, but resorption plus a light inflammatory reaction is occurring in 3 oocytes that commenced vitellogenesis. Eosinophilicvitellogenin derived proteins and lipoprotein are present, but no longer evenly distributed. As the contents are resorbed, these oocytes will eventually appear similar to the collapsed oocytes (arrowed) where vitellin has been completely resorbed.

34. Showing elevated vitellin in liver, kidney (blood and tubules), and spleen. The effect of elevated blood vitellin levels from resorption of oocytes on other organs has been covered in Parts 4, 9A & 10A (kidney, liver, skin). Very high levels have similar physiological imposts as elevated levels of other proteins. Vitellin levels of liver can also be elevated by an increase over normal production due to hormone disruptors (see below).

35. The end stage of resorption of salmonid eggs retained within the abdomen is shown here (above, left) as collapsed egg capsules, surrounded by a light peritoneal reaction.

36. Vitellinresorption can occur in any species, induced by failure to spawn or prior to that stage due to abortion of vitellogenesis following stress. This slide shows eggs of a goldfish showing immature oocytes (2 stages); one mature oocyte (top centre); and several degenerating ova (*) showing a collapsing capsule & withdrawal of vitellin; and one older fully resorbed egg remnant (arrow). * * * *

37. Retained eggs within the ovary of a goldfish (closed type ovary). E

38. Gonad abnormalities Is this Danio sp. gonad normal? Ova are present but none are undergoing vitellogenesis (as shown above), & several oocytes appear to be degenerating. Possibilities that need to be considered in this group includes the common sale of sterile inter-species hybrids (not all X-s sterile). Danio species are also well studied for effects of hormone disruptors. Gonad of a Danio sp. aquarium fish, cohort of above Danio examples. Some had bacterial infections – possibly stress associated.

39. Is this testis normal? (Zebrafish, Daniorerio)? Both ova & developing sperm present. Yes, it is normal! The zebrafish testis starts as a testis-ovary. After about 40 days post hatch (though timing varies with strain), zebrafish are sexually differentiated (either male or female). [Some strains are reported to retain a few ova into later stages.] The number may increase under the influence of hormone-disrupting pollutants (see below). Photo by Leo van derVen http://en.wikibooks.org/wiki/The_Zebrafish_in_Toxicology/Testis_Development Reference: Van derVen et al, 2014 a or b, for a full description.

40. Hormone disruptors & fish gonads There is increasing concern at the effect of hormone disruptor pollutants on fish reproduction. One Danio species (the Zebra fish, Daniorerio) is one of several species in which these effects are well studied, with reports freely available. Others include the fathead minnow & Japanese Medaka. Listed findings in summary (to illustrate complexity only) • Estrogenic effects: • in females: atresia of mature oocytes • in males: larger cysts of spermatogonia; interstitial compartment more prominent partly due to vitellogenin; occasional ova-testes (foci of mature ovary as well as normal testes) • Anti-estrogenic effects: • in females: ovarian regression - oviducts filled with debris, increased macrophages & debris; invagination of oocyte membrane; hypertrophy of granulosa cells • in males: asynchrony of spermatogenesis; large clusters of Leydig cells • Androgen effects: • in females: apparent inhibitory effect on ovulation. • in males: expansion of the interstitial component; imbalance of spermatogonia size & ratios • Anti-androgenic effects: • In males: intersex fish with testis-ova (scattered ova); enlarged Sertoli cells; enlarged clusters of Leydig cells. References: Van derVen et al, 2014b (with interactive photos); Wester et al, 2003. Broader hormone disruptor issues : Anon, 2014.

41. Abnormal gonad development due to triploidy: Gonad of a young (13 months old) salmon, recognisable by its structure as ovary, but with abnormal cells. Primary oogonia are not developing normally, and there is an increased proportion of the gonad composed of fibrous and interstitial tissues. This abnormal ovary is the result of deliberate human intervention: the treatment of eggs during the fertilization process to prevent extrusion of the second polar body of the ovum, to produce a triploid fish, which are generally sterile. The level of gonad disruption varies, and there is always the possibility of some fertile fish. (Methods for producing triploids vary: both temperature and pressure shocks have been used, with selected methods varying between species. ) These & following triploidy slides courtesy Nicole Ruff.

42. This vestigial ovary (same series as above), shows the variability that can be obtained with sub-optimal triploidy inducing treatments. A few well developed oocysts remain. The other ovary was normal, filled with oocysts. Another vestigial ovary, with one focus of developing oocytes within a fibrous-like matrix.

43. Abnormal testes - triploid male salmon. Some primary undifferentiated gonocytes are present, but the organ is disorganized and underdeveloped. The advantages of triploidy for production fish are: Such fish normally grow more rapidly. (This does, however, result in increased vulnerability to marginal nutritional imbalances – see, for example, Part 12A, muscluloskeletal section.) The resulting sterility is desirable firstly because it avoids the drop in growth and flesh quality of maturing fish, and therefore extends the effective growing season. Under some circumstances, aquaculture regulators have requested sterile animals to prevent resulting self-sustaining wild populations. However the reliability of sterility with such manipulations is generally insufficient to make this a reliable method of control.

44. Abnormal gonad products can also induce a reaction, particularly when they degenerate as shown in this triploid.

45. Testes degeneration & mineralization: Sub-gross view of a section of Atlantic salmon testes showing variable areas of palor. Some areas contain mature sperm (plus some cell debris – no developing sperm). This pattern of sperm production, followed by marked degeneration, is likely to be the result of yet another human manipulation: the treatment of female eggs with a short testosterone treatment to produce pseudomales (the development of gonads as testes in genotypic (XX) females). This is to provide all-female (X) sperm. The progeny of normal females fertilized by such sperm is always female – all female stock being another way to avoid the quality decline of maturing males. The problem is that although sperm are produced, there is no oviduct in female salmon to transform into a sperm duct. Therefore sperm from pseudomales are recovered by surgical removal of the testes. Any fish missed have effectively a sperm duct blockage. This leads to pathology resembling spermatic granulomas in terrestrial animals, in that sperm released from blocked, ruptured tubules are antigenic when they interacts with the host immune system. Note: doses of testosterone are small & applied only to the egg stage to produce a parent fish. Thus treatment is a generation away from any production fish. Others show more degeneration of sperm mass, plus markedly abnormal areas with mineralization.

46. GONADS & INFECTION Gonads may be involved in generalized infections (for example, bacterial kidney disease; mycobacteriosis), but with pathogens specific to gonads being few and mostly restricted to parasites. Nevertheless trans-ovarian transmission of pathogens (viruses & bacteria), is the main concern underlying much pre-spawning hatchery testing, especially for salmonids. Parasites in the gonad: Flathead ovary with Philometra infection. Incidental finding. These nematodes can migrate through many tissues, eventually usually reaching the subcutis.

47. Salmon anatomy & post-spawning peritonitis: The semi-open ovarian anatomy of salmonids makes them vulnerable to retrograde infections. This is increased with poor hatchery handling or hygiene, or excessive anaesthesia for sedation during hand stripping which leaves fish with an extended period of poor vent sphincter control. Post-spawning Vagococcussalmoninaruminfection, rainbow trout. Other gram positive bacteria such as Carnobacteriumpiscicola are also common.

48. Infection in this rainbow trout is chronic, probably a post-spawning infection the previous year as pale collapsed (mostly resorbed) ova from the previous year are adhered to surfaces,while this year’s near-mature ova are still within the ovary. Mixed Carnobacterium& Vagococcus infection, with a purulent reaction. 93/2788 Ovary Spleen

49. GONAD NEOPLASIA A number of gonad tumours have been described, but our only available example is a goldfish with a suspected ovarian cystadenoma. Epizootics of gonad tumours in golfish/carp hybrids have been reported from the Great Lakes, USA, and attributed to hormonal imbalances. These tumours arise in the Sertoli cell. They cause sterility in males & prevent females spawning. Gonad tumours have also been seen in koi.

50. References (gonad) • Arukwe, A & Goksøyr, A. 2003. Review. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology. http://www.comparative-hepatology.com/content/2/1/4 • Kagawa, H. 2013. Oogenesis in Teleost Fish. Aqua-BioScience Monographs, 6 (4), 99–127. http://www.terrapub.co.jp/onlinemonographs/absm/pdf/06/0604.pdf • Fauvel, C., Suquet, M., and Cosson, J. 2010. Evaluation of fish sperm quality. Journal of Applied Ichthyology, 26 (5), 636–643. http://archimer.ifremer.fr/doc/00015/12642/13921.pdf • Van derVen, L & Wester, Piet. The Zebrafish in Toxicology. 2014a http://en.wikibooks.org/wiki/The_Zebrafish_in_Toxicology • Van derVen, L & Wester, Piet. The Zebrafish in Toxicology. 2014b Histology and Histopathology Atlas of the Zebrafish V2.01. http://zfishtoxpat.comoj.com/index.html • Wester, P.W., van den Brandhof, E.J., Vos, J.H., van derVen, L.T.M. 2003. RIVM report 640920001/2003. Identification of Endocrine Disruptive Effects in the Aquatic Environment - a Partial Life Cycle Assay in Zebrafish. http://www.rivm.nl/bibliotheek/rapporten/640920001.pdf • Sundararaj, B.I. 1981. ADCP/REP/81/16 - Reproductive Physiology of Teleost Fishes: A Review of Present Knowledge and Needs for Future Research. FAO Corporate Document Repository. http://www.fao.org/docrep/x5742e/x5742e00.htm#Contents • Van Look, K. J. W., Dzyuba, B., Cliffe, A., Koldewey, H.J., & Holt, W.V. 2007. Dimorphic sperm and the unlikely route to fertilisation in the yellow seahorse. The Journal of Experimental Biology 210, 432-437. • Anon, 2014. Endocrine (Hormone) Disruptors. U.S. Fish & Wildlife Service, Environmental Quality. http://www.fws.gov/contaminants/issues/endocrinedisruptors.cfm