Effect of Neutralization of Endogenous Follicle Stimulating Hormone (FSH) or Luteinizing Hormone (LH) on Ovarian Lipids in the Hamster: A Histochemical and Biochemical Evaluation

The effect of neutralizing FSH or LH on ovarian lipids in the cycling hamster was studied. In the normal cycling hamster on the day of proestrus, histochemical examination revealed the presence of sudanophilic lipids in the granulosa cells of the follicles and in the interstitium. A clear reduction in the intensity of lipid staining was observed on proestrus in the ovary of hamsters treated with FSH antiserum on the previous proestrus. Similar treatment with antiserum to LH, on the other hand, caused an accumulation of lipids in these structures. Estimation of the free and esterified fractions of cholesterol and triglycerides in the nonluteal tissue of the ovary of hamsters on proestrus following treatment with FSH antiserum on the previous proestrus revealed a significant reduction in all 3 lipid components. Even a short term deprivation of FSH caused a similar reduction in these lipids in the ovary. In contrast, treatment with LH antiserum either on the previous proestrus or on the previous day (diestrus-2) resulted in an enhancement in esterified cholesterol and triglycerides, while it caused a reduction in the free cholesterol fraction of the ovary on proestrus.It is suggested that though treatment with antisera to either FSH or LH causes a disruption in follicular maturation, their effect on lipid metabolism is different. A positive role for FSH and LH in maintaining normal sterol and triglyceride levels in the nonluteal ovarian tissue of cycling hamster is indicated.

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

A Rapid Bioassay for Measuring Inhibin Activity

A sensitive and rapid bioassay of inhibin is described. Swiss albino female mice, 27 days old, weighing 25-30 g, were primed with 20 IU hCG, given in 2 equal doses, administered at 0900 h and 1800 h. An increment of 200% in uterine weight could be observed when the animals were autopsied the next day at 1000 h. The validity of the endpoint chosen, as a function dependent on FSH, was shown by the fact that neutralization of endogenous FSH by FSH antiserum led to an inhibition of uterine weight increase. Further injection of the inhibin material caused an actual reduction in the heightened levels of FSH (ng/ml) brought about by hCG: Control, 207 ± 13.6; hCG, 365 ± 28.8 and hCG inhibin, below the detection level of radioimmunoassay. Inhibin preparation from ovine testicular extract when tested at 3 dose levels showed a dose dependent and significant suppression in uterine weight increase. The assay was statistically validε = 2.5; slope = 23.9; λ = 0.104 and intra- and interassay variation = 8.3% and 11.7%, respectively. Since the mouse uterine weight assay is specific, has greater sensitivity and is uniformly reproducible, it is suggested that this assay procedure is ideally suited to assess the activity of different inhibin test preparations as well as to follow the purification of inhibin activity from crude material.

Luteal-phase defect induced by deprivation of FSH at a specific period of the follicular phase prevents pregnancy in the bonnet monkey (Macaca radiata)

Female bonnet monkeys were injected i.v. with 25 µl antiserum to FSH on Days 5, 6 or 7 of the cycle: the length of the luteal phase was shortened but there was no alteration in cycle length. Proven fertile females (N = 6) were caged throughout the period of the experiment (6 cycles) with proven fertile males and treated with 25 µl FSH antiserum on Day 7 of each of 3 successive cycles. Out of 18 cycle exposures during the treatment phase, 17 were ovulatory, but no pregnancies occurred. In the post-treatment phase, 5 monkeys became pregnant within 3 cycle exposures. These results show that it is possible to render female monkeys infertile by creating luteal insufficiency and this can be achieved repeatedly in a reproducible manner by depriving the cyclic females of FSH support on Day 7 of consecutive cycles.

Effect of a specific estrogen antibody on pregnancy establishment in the bonnet monkey (Macaca radiata)

The requirement for estrogen for pregnancy establishment has not been conclusively demonstrated in primates. Selective neutralization of estrogens was achieved in mated female monkeys during preimplantation and postimplantation periods by injecting characterized estrogen antiserum from either day 14 to 18 or day 28 to 32 of cycle. While estrogen deprivation during preimplantation period in 5 animals exposed to 14 ovulatory cycles resulted in only one pregnancy, only 3 of 13 monkeys treated during postimplantation period continued pregnancy to term. In comparison with controls (4 of 5 monkeys becoming pregnant), the percent protection against pregnancy in animals treated during preimplantation period was 93. The pregnancy termination in 10 of 13 monkeys treated during postimplantation period when compared with normal postimplantation pregnancy wastage in our colony (2%) is also highly significant (P less than 0.01). The present study demonstrates a critical need for estrogen during the peri-implantation period for a successful pregnancy establishment in primates.

5-Fluorouracil induces structural changes in rat liver tRNA

5-fluorouracil (FUra) has been shown to modulate the aminoacylation function of rat liver tRNA. The present study was aimed at studying the structure-function relationship of FUra-substituted tRNA. Male Wistar rats (2-3 month old) were given a single i.p. injection of FUra at 50, 250, or 500 mg/kg body wt. and FUra-substituted total liver tRNA, i.e. tRNA(FUra50, 250, and 500, respectively, were isolated 3 h later. Normal tRNA (tRNA(N)) was isolated from saline-treated control rats. Thermal denaturation studies showed higher melting temperatures for tRNA(FUra) compared to tRNA(N). Heat denaturation followed by renaturation of total tRNA did not affect the activity of tRNA(N) and tRNA(FUra50), where as tRNA(FUra250 and 500) lost 35% and 72% of activity, respectively, compared to the corresponding group of non-denatured tRNA. Antibodies specific to rat liver tRNA recognized normal and FUra-substituted tRNA in the order of tRNA(N) > tRNA(FUra50) > or = tRNA(FUra250) > tRNA(FUra500) in an avidin-biotin micro-enzyme linked immunosorbant assay. tRNA(N) or tRNA(FUra50) preincubated with tRNA antiserum showed 74% and 59% of aminoacylation activity, respectively, compared to that of corresponding tRNA preincubated with normal rabbit IgG. However, activities of similarly treated tRNA(FUra250 and 500) were not affected. The observations of possible changes in the secondary structure of rat liver tRNA upon incorporation of FUra are discussed.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for follicle-stimulating hormone mediation in the hemiorchidectomy-induced compensatory increase in the function of the remaining testis of the adult male bonnet monkey (Macaca radiata)

Hemiorchidectomy (HO) in the adult male bonnet monkey results in a selective increase in circulating concentrations of FSH and testosterone, and this is accompanied by compensatory increase in sperm production by the remaining testis. We investigated the possible role of increased FSH concentration that occurs after HO in the compensatory increase in the activity of the remaining testis. Of eight adult male bonnet monkeys that underwent HO, four received i.v. injections every other day for 30 days of a well-characterized ovine FSH antiserum (a/s) that cross-reacts with monkey FSH. The remaining four males received normal monkey serum (NMS) as control treatment in a protocol similar to that employed for ais-treated males. Blood samples were collected between 2100 and 2200 h before and 1/2, 1, 3, 5, 7, 14, 22, and 29 days after HO. Testicular weight, number of 3 beta-hydroxy steroid dehydrogenase-positive (3 beta-HSD+) cells, and DNA flow cytometric analysis of germ cell populations were obtained for testes collected before and at the termination of NMS or ais treatment. In NMS-treated males, circulating serum FSH concentrations progressively increased to reach a maximal level by Day 7 after HO (1.95 +/- 0.3 vs. 5.6 +/- 0.7 ng/ml on Days -1 and 7, respectively). Within 30 min of ais injection, FSH antibodies were detected in circulation, and the antibody level was maintained at a constant level between Day 7 and end of treatment (exhibiting 50-60% binding to I-125-hFSH). Although circulating mean nocturnal serum testosterone concentration showed an initial decrease, it rose gradually to pre-HO concentrations by Day 7 in NMS-treated males. In contrast, nocturnal mat serum testosterone concentrations in a/s-treated males remained lower than in NMS-treated controls (p < 0.05) up to Day 22 and thereafter only marginally increased. Testicular weights increased (p < 0.05) over the pre-HO weight in NMS- but not in ais-treated males. After HO, the number of 3 beta-HSD+ cells (Leydig cells) was markedly increased but was significantly (p < 0.05) higher in NMS-treated males compared to a/s-treated males. A significant (p < 0.05) reduction in the primary spermatocyte population of germ cells was observed in ais-treated compared to NMS-treated males. These results suggest that the increased FSH occurring after HO could be intimately involved in increasing the compensatory functional activity of the remaining testis in the male bonnet monkey.

Selective immunoneutralization of luteinizing hormone results in the apoptotic cell death of pachytene spermatocytes and spermatids in the rat testis

The selective withdrawal of pituitary gonadotropins through specific antibodies is known to cause disruption of spermatogenesis. The cellular mechanism responsible for the degenerative changes under isolated effect of luteinizing hormone (LH) deprivation is not clear. Using antibodies specific to LH we have investigated the effect of immunoneutralization of LH on apoptotic cell death in the testicular cells of the immature and the adult rats. Specific neutralization of LH resulted in apoptotic cell death of germ cells, both in the immature and the adult rats. The germ cells from control animals showed predominantly high molecular weight DNA, while the antiserum treated group showed DNA cleavage into low molecular weight DNA ladder characteristic of apoptosis. This pattern could be observed within 24 h of a/s administration and the effect could be reversed by testosterone. The germ cells were purified by centrifugal elutriation and the vulnerability of germ cell types to undergo apoptosis under LH deprivation was investigated. The round spermatids and the pachytene spermatocytes were found to be the most sensitive germ cells to lack of LH and underwent apoptosis. Interestingly, spermatogonial cells were found to be the least sensitive germ cells to the lack of LH in terms of apoptotic cell death. Results show that LH, in addition to being involved in the germ cell differentiation, is also involved in cell survival and prevent degeneration of germ cells during spermatogenesis. Apoptotic DNA fragmentation may serve as a useful marker for the study of hormonal regulation of spermatogenesis and the specific neutralization of gonadotropic hormones can be a reliable model for the study of the molecular mechanism of apoptosis.

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: Results of a pilot study

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar, The volunteers responded only to the first two immunizations, The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively, During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable, A 50 mu l aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of I-125-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively, The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded, Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging, Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles, There was no production of antibodies capable of interacting with non-specific tissues, It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen, This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype `O' produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Studies of bacteriophage T4 tail fibers and tail fiber genes

The distal half of the bacteriophage T4 tail fiber interacts with the surface of the bacterium during adsorption. The largest polypeptide in this half fiber is the product of gene 37 (P37). During assembly of the tail fiber, P37 interacts with the product of gene 38 (P38). These two gene products are incompatible with the corresponding gene products from the related phage T2. T2 P37 does not interact with T4 P38 and T2 P38 does not interact with T4 P37. Crosses between T2 and T4 phages mutant in genes 37 and 38 have shown that the carboxyl end of P37 interacts with P38 and with the bacterial surface. In the corresponding region of gene 37 and in gene 38 there is no recombination between T2 and T4. In the rest of gene 37 there are two small regions with relatively high recombination and a region of low recombination.

When T2/T4 heteroduplex DNA molecules are examined in the electron microscope four nonhomologous loops appear in the region of genes 37 and 38. Heteroduplexes between hybrid phages which have part of gene 37 from T4 and part from T2 have roughly located gene 37 mutations in the heteroduplex pattern. For a more precise location of the , mutations a physical map of gene 37 was constructed by determining the molecular weights of amber polypeptide fragments on polyacrylamide gels in the presence of sodium dodecyl sulfate. When the physical and heteroduplex maps are aligned, the regions of low recombination correspond to regions of nonhomology between T2 and T4. Regions with relatively high recombination are homologous.

The molecular weight of T2 P37 is about 13,000 greater than that of T4 P37. Analysis of hybrid phage has shown that this molecular weight difference is all at the carboxyl end of P37.

An antiserum has been prepared which is specific for the distal half fiber of T4. Tests of the ability of gene 37 hybrids to block this antiserum show that there are at least 4 subclasses of antigen specified by different parts of P37.

Observations in the electron microscope of the tailfiber - anti- body complexes formed by the gene 37 hybrids and the specific anti- serum have shown that P37 is oriented linearly in the distal half fiber with its N-terminus near the joint between the two half fibers and its C-terminus near the tip of the fiber. These observations lead to a simple model for the structure of the distal half fiber.

The high recombination in T4 gene 34 was also investigated. A comparison of genetic and physical maps of gene 34 showed that there is a gradient of increasing recombination near one end of the gene.

Electrophoretic and immunotaxonomic studies of three species of marine gastropods from Portonovo coast with reference to population management

Electrophoretic and serological studies of foot muscle protein of three species of Cerithiacea (Telescopium telescopium, Cerithidea fluviatilis and C. obtusum) were carried out to understand their relationships. Living specimens were collected from mud flats and mangrove swamps off Portonovo. Polyacrylamide electrophoresis of proteins from foot muscle extract of T. telescopium, C. fluviatilis and C. obtusum showed that the former had a different densitometric profile as well as more number of protein bands; but the later two species showed a closer related pattern as well as lesser number of protein bands. At the same time these two species are distinguished from each other in their total number of bands and Rf values. Immunological studies using micro-Ouchterlony double diffusion tests which absorbed antiserum indicated that C. fluviatilis and C. obtusum were more closely related as revealed by an identity reactions than T. telesopium as shown by non-identity reactions. Results are discussed in relation to ecological and morphological adaptations