Publisher Summary It has long been recognized that the control of gonadal function by pituitary hormones (gonadotropins) is a general feature of vertebrate reproductive physiology. Numerous studies on the hormones of eutherian mammals have established the existence of two chemically distinct types of gonadotropin molecules in the pituitary-luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Recent biochemical studies on these molecules have revealed that each is a glycoprotein consisting of two chemically non-identical subunits, designated α and β. The physiological actions of the two gonadotropins in mammals are still subject to intensive investigation; however, it is agreed that FSH and LH have somewhat different roles in the regulation of gonadal function. This chapter describes a flow diagram of the protocol for the fractionation of pituitary hormones, with particular reference to gonadotropins and growth hormone (GH), from various nonmammalian species. The strong resemblance in chromatographic behavior among the fractions identified as FSH and LH from most nonmammalian species and their mammalian counterparts provided preliminary evidence of chemical similarity among the different species of FSH and of LH. Biochemical analyses on the six highly purified species of LH and FSH (chicken, turkey, alligator, snapping turtle, sea turtle, and bullfrog) yielded additional evidence for the homologies between mammalian and nonmammalian hormones.

Evolution of gonadotropin structure and function.

The gonadotropins of lower vertebrates

The process by which spermatid cytoplasmic volume is reduced and cytoplasm eliminated during spermiogenesis was investigated in the bullfrog Rana catesbeiana. At early phases of spermiogenesis, newly formed, rounded spermatids were found within spermatocysts. As acrosomal development, nuclear elongation, and chromatin condensation occurred, spermatid nuclei became eccentric within the cell. A cytoplasmic lobe formed from the caudal spermatid head and flagellum and extended toward the seminiferous tubule lumen. The cytoplasmic lobe underwent progressive condensation whereby most of its cytoplasm became extremely electron dense and contrasted sharply with numerous electron-translucent vesicles contained therein. At the completion of spermiogenesis, many spermatids with their highly condensed cytoplasm still attached were released from their Sertoli cell into the lumen of the seminiferous tubule. There was no evidence of the phagocytosis of residual bodies by Sertoli cells. Because spermatozoa are normally retained in the testis in winter and are not released until the following breeding season, sperm were induced to traverse the duct system with a single injection of hCG. Some spermatids remained attached to their cytoplasm during the sojourn through the testicular and kidney ducts; however, by the time the sperm reached the Wolffian duct, separation had occurred. The discarded cytoplasmic lobe (residual body) appeared to be degraded within the epithelium of the Wolffian duct. It was determined that the volume of the spermatid was reduced by 87% during spermiogenesis through a nuclear volume decrease of 76% and cytoplasmic volume decrease of 95.3%.

Spermiogenesis in the bullfrog (Rana catesbeiana): A study of cytoplasmic events including cell volume changes and cytoplasmic elimination

The ultrastructure of the main cellular components of testicular interstitial spaces in three Anuran species was studied during two short periods of the seasonal cycle and after treatment with 5- and 6-hydroxydopamine (5- and 6-OHDA). In early December when seminiferous tubules are completely filled with resting spermatozoa in Rana, Leydig cells display a well developed smooth- and rough-surfaced ER, numerous granular vesicles being untypical of Mammalian Leydig cells, mitochondria with tubular cristae and only few lipid droplets. In late April shortly after spermiation has occurred in most animals studied Leydig cells exhibit a low degree of activity as shown by the presence of numerous large lipid inclusions, reduced amounts of ER and granular vesicles, but increased numbers of dense bodies. In addition, a few rather undifferentiated cells are observed. In principle, the same differences between winter and late spring Leydig cells are seen in Bufo, although the smooth-surfaced ER which is partly arranged in whorls is much more pronounced in this species whereas granular vesicles are lacking. In December intertubular spaces display a few mitotic figures of Leydig cells. In Xenopus no obvious seasonal changes are observed in Leydig cells. As in Rana, there are numerous granular vesicles. The agranular ER is moderately developed. Intratesticular seminal excretory ducts consist in all three species studied of very low differentiated cells being almost completely filled with filaments (∼100 A). Seasonal changes do not seem to occur. Morphological equivalents for secretory or resorptive processes are neither observed in early December nor in late April around the time of spermiation (in Rana and Bufo). At the connection site of seminiferous tubules and excretory ducts a basal laminal labyrinth is commonly observed. In Xenopus smooth muscle cells form an incomplete sheath around seminiferous tubules and are also uniformly distributed in the intertubular spaces without any obvious relationships to vessels. Rana and Bufo lack a peritubular contractile cell layer. After application of 5- and 6-OHDA (5-OHDA: three injections of 200mg/kg at 12 hr intervals; 6-OHDA: three doses of 100–150 mg/kg on three consecutive days) cytoplasmic inclusion bodies with lamellar and crystalloid internal patterns develop in Leydig cells from lysosome-like structures. In addition, formation of dense bodies from mitochondria is seen in a few instances. The significance of these alterations is discussed.

https://link.springer.com/content/pdf/10.1007/BF00219962.pdf

Fine structure of the male genital tract and kidney in the anura Xenopus laevis Daudin, Rana temporaria L. and Bufo bufo L. under normal and experimental conditions

Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians. The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined. Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials. Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.

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J. Russo

Papers

Effect of HCG on the enzymic activity of toad testes.