Methyl farnesoate stimulates testicular growth in the

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Methyl farnesoate stimulates testicular growth in the freshwater crab ... eter in MF injected crabs and pro- ... fluid, dehydrated through an alcohol series, cleared ...

Naturwissenschaften 86, 394–395 (1999)

Springer-Verlag 1999

Methyl farnesoate stimulates testicular growth in the freshwater crab Oziotelphusa senex senex fabricius Y. Kalavathy, P. Mamatha, P. Sreenivasula Reddy Department of Biotechnology, Sri Venkateswara University, Tirupati 517 502, India Received: 25 August 1998 / Accepted: 18 February 1999

Abstract The influence of methyl farnesoate (MF) on testicular growth in the fresh water crab Oziotelplusa senex senex was studied. MF stimulated testicular growth as evidenced by increased testicular weight, testicular index and testicular follicle diameter in MF injected crabs and provides evidence that MF acts as a male reproductive hormone in crustacea. Since the first description of the mandibular organs by Le Roux (1968), there has been considerable speculation regarding the role(s) of this tissue in crustacea. The mandibular organs found in several decapod crustaceans, including crabs, lobsters, crayfishes and in prawns, produce the terpene methyl farnesoate (MF; Laufer et al. 1987). MF is the unepoxidated form of insect juvenile hormone III and may have roles in crustaceans similar to those of juvenile hormone in insects. In insects juvenile hormone plays several regulatory roles including gonadotropic (Herman and Bennet 1975) and morphogenetic factor (Wigglesworth 1970). In crustaceans MF is involved in the secretion of ecdysone from Y organs (Tamone and Chang 1993), reproduction (Laufer et al. 1986, 1998; Borst et al. 1987; Liu and Laufer 1996) and morphogenesis (Laufer et al. 1997a,b). We recently reported stimulation of ovarian maturation in MF injected fresh water crabs (Reddy and Ramamurthi 1998). In the present study we demonstrate Correspondence to: P. Sreenivasula Reddy e-mail: pcjobs6mail.com, Tel.: c91-8574-50666 ext 361, Fax: c91–8574–20119 394

the stimulation of testicular growth by MF in the fresh water crab, Oziotelphusa senex senex. Oziotelphusa senex senex Fabricius, the Indian field crab, is a decapod crustacean normally inhabiting burrows in the paddy fields and irrigation canals in South India. Animals were collected from rice fields in and around Tirupati (Andhra Pradesh, India) and acclimated to laboratory conditions for 10 days before being used for experimentation. Only male crabs weighing 30–32 g in the intermolt (C4) stage were used in the study. During the experimental period the crabs fed on sheep flesh ad libitum. Trans,trans-MF obtained gratis from Prof. R. Ramamurthi, Department of Zoology, S.V. University, Tirupati, was dissolved in 95% ethanol, which was the solvent vehicle. The quantity of 10 ml ethanol solution was mixed with crustacean saline to obtain a final concentration of 16 ng/100 ml. The crabs divided into three groups of 30 each. The first group, which served as initial control, received no treatment and was killed on the first day of experiment. The testes were isolated and weighed from these crabs after determining their weights. The second group received 100 ml injections of crustacean saline containing 10 ml 95% ethanol, through the arthrodial membrane of the coxa of third pair of walking legs, and served as concurrent control. The third group were injected with MF at a dose of 16 ng per crab in 100 ml volume. Earlier we reported that the hemolymph volume (ml) of the crab Oziotelphusa is 27% of body mass

(Reddy and Ramamurthi 1998). The calculated hemolymph volume for 30 g per crab used was found to be 8.0 ml, then the final concentration of MF is 2 ng/ml, which is the physiological concentration of MF in Scylla serrata (Tobe et al. 1989). Injections were given on the 1st, 7th and 14th days. These two groups of crabs were killed on the 21st day and, as in the first group, their body weights and testicular weights were determined. No deaths occurred in control or in experiment groups. The testicular index was calculated using the formula: testicular indexp(wet weight of testis/wet weight of crabs)!100. For microscopic study, the testes were fixed in Bouin’s fluid, dehydrated through an alcohol series, cleared in xylene, embedded in paraffin (mp. 56–587 C), sectioned at 7-mm thickness and stained with hematoxylin and eosin. The diameters of testicular follicle in each testis were determined with the aid of a compound microscope and ocular micrometer. The data were analyzed by one-way analysis of variance. MF administration resulted a significant (P~0.001) increase in testicular weight, testicular index and testicular follicle diameter than was found for the control and concurrent control crabs (Table 1). The increase in testicular weight (7.69%), testicular index (6.98%) and testicular follicle diameter (8.62%) of the concurrent control crabs was not significantly different from the initial control crabs, indicating that not significant variations had occurred during the experimental period. The present results demonstrate that MF accelerates testicular growth in crabs. Although the sexual differentiation, development of secondary sexual characters of males and function of male reproductive system are under the control of the androgenic gland, two peptide hormones affect the reproduction in male crustaceans: gonad-inhibiting hormone (GIH) from the medulla terminalis ganglionic Xorgan-sinus gland complex in the eyestalks and gonad-stimulating hormone (GSH) from the brain and thoracic ganglia (Otsu 1960, 1963). In addition to these, the above results reveal that MF also acts as one of the

Naturwissenschaften 86 (1999) Q Springer-Verlag 1999

Table 1. Testicular weight, testicular index and testicular follicle diameter of the crab, Oziotelphusa senex senex, after 21 days of various experimental conditions Group

Testis weight (mg)

Testicular index

Testicular follicle diameter (ml)

Initial control (np30) Concurrent control (np30) MF-injected (np30) F value

0.13B0.016 a 0.14B0.014 a (7.69) 0.37B0.02 (184.62) 62.36*

0.43B0.003 a 0.46B0.003 a (6.98) 1.24B0.007 (188.37) 7629*

74.25B0.233 a 80.65 B0.306 a (8.62) 243.19B1.035 (227.53) 22535*

*P~0.001 Values are meanBSEM of 30 individual crabs. Values in the parenthesis are the percentage change from control a Means with the same superscript are not significant (Student-Newman-Keuls multiple comparison test)

GSH. The stimulatory action of MF on testicular growth could have been due to stimulation of GSH synthesis and release or inhibition of release of GIH or direct action of MF on testes. It may be that all the three hormones (GSH, GIH and MF) play a very important in regulating the reproduction in crustaceans. We thank Prof. Ernest S. Chang, Bodega Marine laboratory, United States, for his encouragement and advice. P.S.R. thanks the management of Pondicherry University, Pondicherry, for granting extra-ordinary leave, during which the present work was carried out and the Staff of Biotechnology Department, S.V. University, Tirupati for support. Portions of this work are a result of research suported by D.S.T. (SP/SO/CO4/96), New Delhi.

Borst DW, Laufer H, Landau M, Chang ES, Hertz WA, Baker FC, Schooley DA (1987) Methyl farnesoate and its role in crustacean reproduction and development. Insect Biochem 17 : 1123–1127

Herman WS, Bennet DC (1975) Regulation of oogenesis, female specific protein production, and male and female reproductive gonad development by juvenile hormone in the butterfly, Nymphalis antiopa. J Comp Physiol 99 : 321–338 Laufer H, Landau M, Homola E (1986) The synthesis and regulation of methyl farnesoate, a new juvenile hormone for crustacean reproduction. Adv Invertebr Reprod 4 : 135–143 Laufer H, Borst D, Baker FC, Carrasco C, Sinkus M, Reuter CC, Tsai LW, Schooley DA (1987) Identification of a juvenile hormone-like compound in a crustacean. Science 235 : 202–205 Laufer H, Paddon J, Paddon M (1997a) A hormone enhancing larva production in the pacific white shrimp Penaeus vannamei. In: Alston DE, Green BW, Clifford HC (eds) IV Symposium on Aquaculture in Central America: Focussing on Shrimp and Tilapia. Tegucigalpa, Honduras, pp 161–162, Laufer H, Ahl J, Takac P, Rotllant G (1997b) The role of methyl farnesoate in crustacean morphogenesis. In: Kawashima S, Kikuyama S (eds) Advances in comparative endocrinology, XIII International Congress on Comparative Endocrinology, pp 43–50, Moduzzi, Bologna Laufer H, Biggers WJ, Ahl JSB (1998) Stimulation of ovarian maturation in the crayfish

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Procambarus clarkii by methyl farnesoate. Gen Comp Endocrinol 111 : 113–118 Le Roux A (1968) Description d’organes mandibulaires nouveaux chez les Crustaces Decapodes. CR Acad Sci Paris 266 : 1414–1417 Liu L, Laufer H (1996) Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata. Arch Insect Biochem Physiol 32 : 375–385 Otsu T (1960) Precocious development of the ovaries in the crab, Potamon dehaani, followed implantation of the thoracic ganglion. Annot Zool Jpn 33 : 90–96 Otsu T (1963) Bihormonal control of sexual cycle in the freshwater crab, Potamon dehaani. Embryologia 8 : 1–20 Reddy PS, Ramamurthi R (1998) Methyl farnesoate stimulates ovarian maturation in the freshwater crab Oziotelphusa senex senex Fabricius. Curr Sci 74 : 68–70 Tamone SL, Chang ES (1993) Methyl farnesoate stimulates ecdysterioid secretion from Y-organs in vitro. Gen Comp Endocrinol 89 : 425–432 Tobe SS, Young DA, Khoo HW (1989) Production of methyl farnesoate by the mandibular organs of the mud crab, Scylla serrata. Gen Comp Endocrinol 73 : 342–353 Wigglesworth VB (1970) Insect hormones, Freeman, San Francisco

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