A seasonal study of male reproduction and sperm physiology of three coldwater flatfish

Seasonal patterns of sperm release and changes in sperm quality were studied in winter flounder and Atlantic halibut. While more than half of male winter flounder began producing milt by January, only small amounts of milt continued to be detectable during the prespawning period lasting from January to April. Milt production increased, sperm quality improved and spermatocrit declined during the summer spawning period in May and June, briefly coupled with peak plasma levels of testosterone (T) and 11 ketotestosterone (11-KT). In halibut, the period of milt production in males also began in January and continued into May. Unlike winter flounder, sperm motility in halibut was highest during the earliest stages of the spawning season and a remarkable reduction in sperm motility occurred by the end of the season in association with marked increases in the density of the milt. -- Light and electron microscopy observations revealed that primitive features characterize the structure of both the winter flounder and halibut spermatozoa consisting of an unmodified head and a short midpiece enclosing a single unmodified mitochondria ring. The basal body, accommodated in the caudal nuclear fossa, is made up of the distal centriole and a centriolar collar consisting of 9 pericentriolar columns, 9 bridges and an outer ring. Serial microscopic sampling and examination of sperm morphology demonstrated the existence of abnormal spermatozoa in both species throughout the reproductive season. These abnormalities involved the head, tail and cell membrane damage. In both species, the lowest proportion of abnormal sperm was found during the period that sperm motility was the highest. -- The impact of hormonal manipulation of reproduction in mature male winter flounder was examined by means of hypophysectomy (hypex), pituitary replacement therapy (PRT) and the administration of gonadotropin releasing hormone analogue (GnRH-A). During the spawning season, while GnRH-A treatment increased the production of milt and advanced sperm production, milt was diluted and sperm output for the entire reproductive season was not augmented. In hypexed male flounder, by contrast, milt became more concentrated and sperm production declined along with reduced plasma androgen levels. A restoration of milt hydration in hypexed males followed PRT in December and January. Moreover, PRT increased plasma 11-KT levels in intact males at this early seasonal stage of spermiation and appeared to be responsible for advancement of spermiation in a large number of prespawning males. Finally, none of these treatments altering the hormone levels of mature males resulted in detectable changes in either sperm motility or egg fertilization rates. -- Studies of the genital tract in male winter flounder indicated that the testicular efferent duct system (TEDS) is composed of a testicular primary duct (PD) and the sperm duct (SD) lined primarily by columnar and cuboidal epithelial cells. Secretion in these cells appeared to be most active during the period of vigorous sperm release in the spawning season and relatively quiescent at other periods, parallelling seasonal changes in milt production and sperm motility. Observations in vivo indicated that the pH of milt increased and milt density decreased as one proceeded distally along the male genital tract (from PD to SD), since sperm motility was increased perhaps after storage for several weeks in the TEDS in May. This suggests that the testicular efferent duct system plays a role in the mediation and maintenance of sperm function. -- Milt properties in the winter flounder were characterized, and showed that seminal fluid osmolality ranged from 356-377 mOsm kg⁻¹ with a pH of 8.0. Although protein electrophoretic profiles were very similar between the seminal fluid and blood plasma, the protein, phospholipid (PL), cholesterol (Chl) and some ion (Na⁺ and Cl⁻) concentrations were lower in the seminal fluid. Of all these substances, only PL and Chl concentrations were found to be negatively correlated with sperm motility. -- Like other teleosts, the testicular development in male yellowtail flounder can be classified into 5 progressive stages, i.e. 1) early testis development, 2) rapid testis development, 3) limited spermiation, 4) full spermiation and 5) spent. Despite this apparent progression of development in testes, the seasonal rate of mature and spermiating yellowtail flounder never fell below 40% even during September and December when the lowest gonad-somatic index values were observed. Frequent observations of spermatocytes in the testis of spermiating cultured males and males from the wild suggest that overlapping cycles of spermatogenesis occur in yellowtail flounder, representing a somewhat distinctive mode of male flatfish reproduction in this species