Induction Of Spermiation Research Articles

Applied reproductive technologies and genetic resource banking for amphibian conservation

As amphibian populations continue to decline, both government and non-government organisations are establishing captive assurance colonies to secure populations deemed at risk of extinction if left in the wild. For the most part, little is known about the nutritional ecology, reproductive biology or husbandry needs of the animals placed into captive breeding programs. Because of this lack of knowledge, conservation biologists are currently facing the difficult task of maintaining and reproducing these species. Academic and zoo scientists are beginning to examine different technologies for maintaining the genetic diversity of founder populations brought out of the wild before the animals become extinct from rapidly spreading epizootic diseases. One such technology is genetic resource banking and applied reproductive technologies for species that are difficult to reproduce reliably in captivity. Significant advances have been made in the last decade for amphibian assisted reproduction including the use of exogenous hormones for induction of spermiation and ovulation, in vitro fertilisation, short-term cold storage of gametes and long-term cryopreservation of spermatozoa. These scientific breakthroughs for a select few species will no doubt serve as models for future assisted breeding protocols and the increasing number of amphibians requiring conservation intervention. However, the development of specialised assisted breeding protocols that can be applied to many different families of amphibians will likely require species-specific modifications considering their wide range of reproductive modes. The purpose of this review is to summarise the current state of knowledge in the area of assisted reproduction technologies and gene banking for the conservation of amphibians.

Existence of APGWamide in the testis and its induction of spermiation in Haliotis asinina Linnaeus

Immunolocalization by using rabbit polyclonal antibody against APGWamide demonstrated that there was APGWamide immunoreactivity (APGW-ir) in the varicosed nerve fibers in the capsule, connective tissue and trabeculae of the testis whereas there was no APGW-ir in the ovary of sexually mature broodstocks of Haliotis asinina. In vivo bioassay, by injecting APGWamide into foot muscle of mature males and females (age over 24 months), showed that most males (87.50%) injected with 200 μl of 10 − 3 M APGWamide (170 μg APGWamide/animal) could be induced to spermiate whereas fewer males (43.75%) injected with 200 μl of 10 − 6 M APGWamide (0.17 μg APGWamide/animal) could spermiate. Following injection with 10 − 3 M, 53% of males spawned within 3 to 4 h, while the remaining males spawned within 2 to 3 h. The former tended to release more sperm at the range of 1.04 to 2 × 10 9, and the latter at the range 3.62 × 10 7 to 2.07 × 10 8 cells, respectively, and that the sperm of the former group appeared to be more motile. Following the injection of 10 − 6 M, all inducible males spawned within 2–3 h, and 43% of spawned males have sperm around 2 × 10 9, while 57% have fewer sperm number ranging from 2.75 × 10 7 to 9.94 × 10 8 cells. In contrast to males, none of the mature females with ripen ovary could be induced to ovulate. These data suggest that APGWamide is an important signaling molecule that plays a role in the male reproductive process that could be used to induce spermiation.

Sexually mediated shedding of Myxobolus fallax spores during spermiation of Litoria fallax (Anura)

Myxobolus fallax (Myxosporea) infects the testes of the dwarf green tree frog Litoria fallax without apparently affecting the host's health, behavior, or testicular sperm numbers or quality. We investigated the shedding M. fallax spores and the relationship between M. fallax infection and fertility in L. fallax. During natural spawning, comparisons were made between the prevalence and intensity of M. fallax infection, spore shedding, and fertilization rates. During the hormonal induction of spermiation, comparisons were made between the prevalence and intensity of M. fallax infection, and the number of sperm and spores shed. During natural spawning, the prevalence or intensity of infection or spore shedding did not affect fertility. Spermiation and spore shedding was induced in 10 males by the administration of human chorionic gonadotrophin, with 10 controls. Histology showed that all 10 males were infected by M. fallax. The controls shed no sperm or spores. Nine infected males shed sperm 3 h post-administration (PA), and 3 of these also shed spores. Only the 3 males that shed sperm and spores at 3 h PA also shed sperm at 6 h PA, at which time they also shed spores. In total, the 3 males that shed spores gave higher sperm numbers than the 6 males that did not shed spores. Overall, these results show that the shedding of M. fallax spores is sexually mediated through reproductive hormones without affecting fertility.

Effect of different methods for the induction of spermiation on semen quality in European eel

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g−1 fish (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer-assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium-velocity spermatozoa, as well as different motility parameters. Sperm samples from A-D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E-treated males showed 0% motile cells. No significant differences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A–D groups.

Induction of spermiation in common carp after enhanced intestinal uptake of sGnRH-A and Pimozide

The inherent ability of the carp Cyprinus carpio gut to absorb intact salmon gonadotropin hormone-releasing hormone analogue (sGnRH-a)+Pimozide (Pim), without loss of their bioactivity is very limited. Artificial intestinal absorption enhancement boosted sGnRH-a and Pim induced GtH II release and milt production significantly. The intraperitoneal route of administration was more effective than the oral one. A potential application of the oral route for peptidergic drug delivery and the possibility of developing a dietary delivery system is discussed.

Induction of spermiation in common carp after enhanced intestinal uptake of sGnRH‐A and Pimozide

The inherent ability of the carp Cyprinus carpio gut to absorb intact salmon gonadotropin hormone‐releasing hormone analogue (sGnRH‐a)+Pimozide (Pim), without loss of their bioactivity is very limited. Artificial intestinal absorption enhancement boosted sGnRH‐a and Pim induced GtH II release and milt production significantly. The intraperitoneal route of administration was more effective than the oral one. A potential application of the oral route for peptidergic drug delivery and the possibility of developing a dietary delivery system is discussed.

Comparative effects of avian and piscine gonadotrophins on gonadal steroidogenesis, and of avian and piscine pituitaries on induction of spermiation and ovulation in the loach and white silver carp

This study reports the effects of avian gonadotrophins (GTH) on gonadal steroidogenesis in vitro, and of avian pituitaries on induction of ovulation and/or spermiation in the loach ( Paramisgumus dabryanus and Misgurnus anguillicaudutus) and white silver carp ( Hypophthalmichthys molitrix). Minced testes or isolated ovarian follicles obtained from sexually-mature loach and silver carp, were incubated in Medium 199 at 25 °C for 4 or 12 h, with avian (chicken, duck, turkey, ostrich or goose) luteinizing hormones (LH), follicle stimulating hormones (FSH), or piscine (common carp, grass carp, black silver carp or loach) GTH, under continuous aeration of 95% oxygen and 5% carbon dioxide and shaken at 100 cycles per min. All avian LH and piscine GTH assayed were capable of evoking dose-related responses of testicular testosterone formation and ovarian estradiol-17β formation by the two fish. Avian LH, however, were generally more potent than piscine GTH. All avian FSH tested were inactive. In the loach ovulation experiment, fish were kept under a lighting regime of 12L and 12D at two temperatures (22 °C and 16 °C). Each fish received one intramuscular (i.m.) injection of a dose of one pituitary equivalent of chicken, duck, common carp or grass carp pituitaries. Ovulation rates in the loach treated with avian or piscine pituitaries were over 90%. Most of the loach kept at 22 °C and 16 °C ovulated, 10–14 h and 22–26 h, respectively, after the injections. Controls did not ovulate. In the white silver carp experiment, fish were kept in open ponds. Each fish received two i.m. injections, 6 h apart, of a dose of six pituitary equivalents per injection. The milt production in the fish treated with either chicken or common carp pituitaries was greatly increased. The abdomens of treated female fish became more distended 15 h after the first injection relative to their treatment condition and the controls; induction of ovulation was only partially successful in terms of the number of eggs ovulated. The effects of avian and piscine pituitaries on induction of ovulation and/or spermiation in the two species of fish were indistinguishable. This study indicates that avian LH are highly acceptable by both testicular and ovarian receptors of the loach and white silver carp. Clearly, avian LH are very potent in stimulating gonadal steroidogenesis in a number of teleostean fish. These findings also suggest that avian LH or pituitaries have a potential use in the center of reproduction in aquaculture.

17 alpha,20 alpha-Dihydroxy-4-pregnen-3-one is the naturally occurring spermiation-inducing hormone in the testis of a frog, Rana nigromaculata.

Spermiation, the process in which vertebrate spermatozoa are detached from investing Sertoli cells into the lumen of the seminiferous tubule, is a prerequisite for the successful fertilization. Using an in vitro Rana nigromaculata spermiation bioassay, we have shown that gonadotropin initiates spermiation by inducing the synthesis of delta 4-steroids by testis fragments. Among all of the delta 4-steroid metabolites produced by R. nigromaculata testis fragments, spermiation-inducing activity was confined to only one metabolite; this metabolite was identified as 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17 alpha,20 alpha-DP). Induction of spermiation by gonadotropin in vitro was accompanied by marked elevations in 17 alpha,20 alpha-DP concentrations in incubation media. These findings provide evidence that 17 alpha,20 alpha-DP is the nautrally occurring spermiation-inducing hormone in R. nigromaculata.

Induction of spermatogenesis and spermiation by a single injection of human chorionic gonadotropin in intact and hypophysectomized immature European eel ( Anguilla anguilla L.)

Intact and hypophysectomized male silver eels ( Anguilla anguilla) in fresh water received a single injection of human chorionic gonadotropin (hCG) (250 C) or solvent (0.15 M NaCl). No effect of solvent was observed. Spermatogonia proliferated in testis of hCG-treated intact or hypophysectomized eels. One month after the injection, primary and secondary spermatocytes were found. After 3 months, numerous spermatozoa were present. In hypophysectomized eels, hCG was also effective even though maturing germ cells were less numerous and spermiation was less frequent than in intact animals. Within 1 week after hCG injection, plasma levels of free and glucuroconjugated androgens (testosterone and 11-oxotestosterone) rose significantly in intact and hypophysectomized fish. The highest values were observed within 1 month, and then plasma levels decreased to pretreatment values. The most important changes were observed in the case of free 11-oxotestosterone. The long-term effects of hCG can be explained partly by the long half-life of this hormone. The effects of hypophysectomy on the response of testis to hCG caused us to think that some endogenous pituitary secretions must interfere in the intact fish so that maximal effects of hCG, especially on the induction of spermiation, are obtained.

Induction of spermiation in anurans by mammalian pituitary gonadotropins and their subunits

Tests with highly purified ovine FSH and LH in three species of anurans show that the spermiation response in these amphibia does not discriminate between the two pituitary gonadotropins: FSH and LH are essentially equipotent. Similar results are obtained with both hypophysectomized and intact frogs. Less purified gonadotropin preparations (NIH) give more variable results: LH is considerably more potent than FSH in Rana pipiens, but FSH is about twice as potent as LH in Hyla regilla and Eleutherodactylus coqui. There is a slight synergism between FSH and LH. However, the use of trypsin to inactivate LH and the use of antiserum to block FSH confirmed that the activity of each purified gonadotropin is an intrinsic property of the molecule. The relative potencies of the highly purified gonadotropin preparations in the anuran spermiation test differed markedly from those observed with the same preparations in mammalian and reptilian assays. In anurans, the relative potency of purified LH was about 20 times greater than in other systems, whereas, the relative potency of the highly purified FSH was only about 1 5 that observed in the other tests. The two individual subunits of LH had little activity in the frogs (ca. 2.5% of LH) and there was slight difference in their potencies. In contrast the β subunit of FSH was relatively potent (about 25% of native FSH), but the α subunit was inactive (<3%). In all three species of anuran, a crude extract of anuran (bullfrog, R. catesbeiana) pituitary glands was as potent as the most active purified mammalian hormone.

Involvement of gonadotropin and steroid hormones in the spermiation of the goldfish ( Carassius auratus)

3β-hydroxysteroid dehydrogenase activity has been located in the interstitial cells of the goldfish testis. This activity falls rapidly after hypophysectomy reaching a very low or undetectable level within 25 postoperative days. Replacement therapy using salmon gonadotropin showed that this enzymic activity and spermiation are dependent on pituitary gonadotropin. However, the enzymic activity did not increase significantly at spermiation and spermiation could be induced even when there is a very low or undetectable level of 3 β-ol dehydrogenase. A single injection of salmon gonadotropin induces spermiation and hypertrophy of the interstitial cells. Androgen was also found to be effective for the induction of spermiation. These facts suggest that despite the lack of a direct correlation between 3 β-ol dehydrogenase and spermiation, the pituitary gonadotropin may act on the interstitial cells and induce the secretion of androgens which in turn bring about spermiation.