Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17α-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-α) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17α-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17α-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function

Efficient \u3cem\u3eIn Vitro\u3c/em\u3e Regeneration System From Seed Derived Callus of Perennial Ryegrass (\u3cem\u3eLolium Perenne\u3c/em\u3e) And Annual Ryegrass (\u3cem\u3eLolium Multiflorum\u3c/em\u3e)

The commercially important ryegrasses in cool temperate climates throughout the world are annual ryegrass (Lolium multiflorum L.) and perennial ryegrass (Lolium perenne L). Improvements through conventional breeding have been slow as they are usually heterozygous and highly self-infertile. Hence, there is a need to use modern biotechnological tools to the development of improved rye grass cultivars for incorporating value added traits. Successful transformation of rye grasses has been done using suspension cells, which is time consuming and laborious (Spangenberg et al., 1995, 1998). We report here a rapid and highly efficient in vitro plant regeneration system from seed derived callus in annual and perennial rye grasses

A Novel Genotype Independent Protocol for In Vitro Plant Regeneration from Mature Seed Derived Callus of Tall Fescue (\u3ci\u3eFestuca Arundinacea\u3c/i\u3e Schreb.)

Tall fescues (Festuca arundinacea Schreb.) are cool season forage and turf grasses of significant agricultural importance in different grassland countries. Genetic improvement of tall fescues by conventional selection procedures is slow, since these are predominantly, cross-pollinated, hexaploid and generally infertile (Jauhar, 1993). Genetic Engineering approaches for incorporation of agronomically useful traits may contribute to the development of improved tall fescue cultivars (Spangenberg et al., 1998). However for any genetic engineering studies, it is essential to develop a genotype-independent, reproducible and efficient in vitro plant regeneration protocol. In the present study, we analyzed the effects of different sterilization procedures for in vitro seed germination and studied the effects of different concentrations and combinations of 2,4-D and BAP on callus induction, growth and regeneration potential of two cultivars of tall fescue

Androgenic Response of Cultured Anthers and Microspores of Sorghum

Sorghum anthers with uninucleate microspores were dissected out of spikelets and plated on MS, B5 and N6 media with kinetin, NAA and 2,4-D. The callus induction frequencies (percentage of anthers plated) for the 3 hybrids tested were 60, 20-30 and 15-20%, for CSH 9, CSH 5 and CSH 12R, respectively. Uninucleate microspores showed the most cell division, and cultures of anthers at earlier (small cells without vacuoles) or later (binucleate microspores) stages did not show any androgenic response. The highest response was obtained at 26°C. After 12 d the anthers on all media, especially B5 and N6 started to turn black. Sucrose was more effective at inducing divisions (50-60% of microspores) than maltose (15-20%). Combined treatment with NAA and 2,4-D at 2.0 mg/litre induced divisions. When NAA and 2,4-D (2 mg/litre) were combined with kinetin at 0.05, 0.1, 0.15 and 0.2 mg/litre, the response increased, with a maximum number of multicellular microspores observed at 0.2 mg/litre kinetin. Microcalli that were growing within the anthers after 20-30 d of culture were transferred to MS medium; irregular masses of calli were observed after 30 d. It is concluded that sorghum microspore cultures may be less dependent on genotype or culture conditions than are anther cultures

Immunobiology of a synthetic luteinizing hormone receptor peptide 21-41

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function

Regeneration of sorghum from shoot tip cultures and field performance of the progeny

A system for rapid plant regeneration through somatic embryogenesis from shoot tip explants of sorghum [Sorghum bicolor (L.) Moench] is described. Somatic embryogenesis was observed after incubation of explants in dark for 6–7 weeks through a friable embryogenic callus phase. Linsmaier and Skoog medium supplemented with 2,4- dichlorophenoxyacetic acid (2 mg

Alterations in sperm characteristics of follicle-stimulating hormone (FSH)-immunized men are similar to those of FSH-deprived infertile male bonnet monkeys

The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle-stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)-induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields alphat values, which is another index of chromatin packaging. Higher alphat values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA-FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross-reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate

SCREENING OF MUNGBEAN [VIGNA RADIATA (L.) WILCZEK] GENOTYPES FOR SALT TOLERANCE

ABSTRACT: Thirty nine mungbean genotypes exhibiting distinct and significant response during screening for salt tolerance at early seedling growth stage were screened at vegetative, flowering and pod-filling growth stages up to the harvest under two salinity stress levels i.e. 50 mM and 75 mM NaCl along with their respective control treatment. The experiment was conducted in earthen pots lined with polythene bags in complete randomized block design with reliable growth and physiological characteristics along with yield attributes. The results illustrated significant variations and adaptability among all the genotypes under salt stress. The tolerant genotypes were observed for less reduction in RWC, MSI, total chlorophyll and carotenoid contents, plant length, survival, K + /Na + ratio, and grain yield even under high salinity level (75mM NaCl) with respect to their non-stressed plants. However, the susceptible genotypes showed greater reduction in the measured parameters under salinity stress. On the basis of low and best performance of each genotypes under high salinity levels, total eleven genotypes TCR86, PLM380, PLM562, WGG37, IC615, PLM891, IC2056, IC10492, PLM32, K851, and BB92R were selected from this study which will be screened further for salt tolerance for the identification of most salt tolerant and susceptible genotypes to be used in breeding for the genetic improvement of mungbean for saline soils. The study indicated that selection of genotypes according to their performance under saline condition is very important for the selection of salt tolerant genotypes

Culture and regeneration of mesophyll-derived protoplasts of sorghum [Sorghum bicolor (L.) Moench]

A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l–1 kinetin and 2 mg l–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity