In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.     

Organ-specific antigens of Clonorchis sinensis

This study was carried out to find out specific proteins from different organs of Clonorchis sinensis. Crude extract, organ-specific and excretory-secretory (ES) proteins were analyzed by immunoblot with infected human sera. The bands of 7- and 17-kDa were main component of intestinal fluid and ES protein and commonly found in all organspecific proteins. The 17-kDa protein was observed from ES antigen, intestinal fluid, eggs and sperms, 26- and 28-kDa proteins were from the uterus, vitellaria, and ovary, and 34-, 37-, 43- and 50-kDa proteins were mainly from the testis and sperms. Serum of mice immunized with sperms reacted to the 50-kDa protein by immunoblotting and immunohistochemical staining showed a positive reaction at the seminal receptacle and seminiferous tubule. The present results show that the 7-kDa protein is a common antigen of every part or organ of C. sinensis, but different organs express their specific antigenic protein bands.     

Protein profiles in pin and thrum floral organs of distylous Averrhoa carambola L.

Floral organs (tepal, stamen, style, including stigma, and ovary) from immature and mature (1 day prior to anthesis) flower buds of pin and thrum morphs of Averrhoa carambola were subjected to one and two-dimensional IEF/SDS-PAGE gel electrophoresis and visualized by silver staining. One-dimensional gel electrophoresis of organ extracts from mature floral buds showed a number of protein bands common to all organs in both pin and thrum morphs. In the stamen and style, these bands differed in intensity between the two morphs. Under two-dimensional gels, the differences in protein profiles between the two morphs were more distinct in these organs. When compared with polypeptide spots from leaflets, a total of 14 floral organ-specific polypeptides was detected, the majority appearing in the stamen, followed by the style but none in the ovary. In the stamen, most of these polypeptides were detected in both the pin and thrum morphs. However, in the style, a 72-kDa polypeptide was detected exclusively in the pin morph, and this was also the most abundant floral organ-specific polypeptide. Floral organ-specific polypeptides of 45 kDa (detected in stamens of the thrum morph) and 70 kDa (detected in stamen and style of both morphs) were found to bind concanavalin A.     

Anovulation in non-reproductive female Damaraland mole-rats (Cryptomys damarensis)

Within colonies of Damaraland mole-rats (Cryptomys damarensis), anovulation in non-reproductive females is thought to play an important role in maintaining reproductive skew. Pituitary sensitivity and ovarian structure were examined in three groups of females that differed with respect to their social environment and breeding status to determine whether anovulation is due to inhibitory social cues or is merely the result of a lack of copulatory stimulation. The contribution of gonadal steroid negative feedback to neuroendocrine differences in the reproductive systems of the respective groups was also investigated. LH secretion after a 0.5 micrograms GnRH challenge in females that had been removed from the presence of the breeding individuals for at least 6 months (removed non-reproductive females) was significantly higher than in non-reproductive females in the colony, but significantly lower than in reproductive females. In both removed non-reproductive females and reproductive females, corpora lutea were observed in ovaries of seven of eight females, indicating that ovulation occurs spontaneously in subordinate females on removal from the breeding pair. Circulating progesterone concentrations in removed non-reproductive females were significantly higher than in non-reproductive females, indicating that circulating progesterone is not responsible for infertility in non-reproductive females. Indeed, after hystero-ovariectomy, reproductive females continued to show significantly greater GnRH-stimulated LH secretion than non-reproductive females. Thus, differential inhibition of gonadotrophin secretion in breeding and non-breeding females occurs independently of gonadal steroids. It is concluded that female Damaraland mole-rats are spontaneous ovulators and that anovulation results from inhibitory social cues within the colony, not a lack of copulatory stimulation. Since non-reproductive females are infertile, inhibition of the hypothalamo-pituitary-gonadal axis has the potential to play a causal role in maintaining reproductive skew in colonies of C. damarensis.     

The Freshwater Mussel (Elliptio complanata) as a Sentinel Species: Vitellogenin and Steroid Receptors

Freshwater mussels, Elliptio complanata were collected froma reference and pollutant-impacted pond on Cape Cod, MA. Glutathione-S-transferase(GST) activity was measured in gill, hepatopancreas and foot.In addition, content of seven heavy metals were measured inwhole bodies. GST activity was significantly elevated in hepatopancreasand foot, as was whole body cadmium level in animals from thecontaminated site suggesting that these animals have been exposedto organic and inorganic contaminants. Sodium dodecyl acrylamidegel electrophoresis (SDS-PAGE) analysis showed putative vitellogeninswith molecular weight 180 and 205 kDa bands only in the ovary.In non-denatured gel electrophoresis ovarian extracts revealedtwo higher molecular weight bands at 550 and 700 kDa, whichwere reproductive stage specific. Western blotting of SDS-PAGEand non-denatured gels using the anti-scallop yolk-protein antibodyconfirmed the presence of cross-reacting bands of the same molecularweights in the ovary but not other tissues. Although severalexperiments involving steroid hormone exposure were done, nosignificant changes in vitellogenin protein levels were observed.However, using an anti-human ERß antibody, ERßpositive bands were observed both in female foot, and the ovary.No cross reactivity with the antibody was observed in hepatopancreas.Additional studies are required to resolve questions of vitellogeninregulation and the role of (xeno)estrogens in bivalve molluscs.     

Aspects of reproduction in female Ross seals (Ommatophoca rossii)

Ovaries were collected from 26 Ross seals shot in the King Haakon VII Sea south of the 60 degrees latitude during 3 periods, between 12 January and 2 February 1980-1982. The structure of the ovaries resembled that of other pinnipeds. A corpus luteum was found in 18 of 26 females, which therefore were regarded as reproductive. However, no females were lactating or visibly pregnant (presence of fetus in the uterus). Of the remaining 8 females (non-reproductive), 3 had neither a corpus luteum nor a corpus albicans. Ovulation did not alternate between ovaries in 4 of 10 females. Ovarian weight was greater in reproductive females than in non-reproductive females, and was also correlated with presence of a corpus luteum. Follicular development was more advanced in reproductive females than in non-reproductive females, and it was also more pronounced in the ovary containing a corpus luteum. The finding of a high percentage of females with a corpus luteum, but with no fetus in the uterus, together with histological characteristics of the ovarian structures in the present study, and earlier published data on mating, pupping and moulting, provide circumstantial evidence for delayed implantation in Ross seals.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

The Floral Specific Proteins of Cucumber

Soluble proteins extracted from various organs of cucumber (Cucurnis sativus L. ) were analyzed using the technique of SDS one dimensional gel electrophoresis. The majority of the proteins were expressed in more than one organ, while those specific to only one organ were comparatively rare. Only two of all the proteins analized appeared to be truely organspecific. A 40.0 kD protein and a 30.8 kD protein were observed only in extracts of reproductive organs stamen and ovary. Some proteins were specific only to certain reproductive organ. They are 35.0 kD and 34.0 kD in sepal, 18. 8 kD, 28.5 kD, 31.0 kD, 37.0 kD and 39.0 kD in stamen, 45.0 kD in style with stigma and 32.5 kD in ovary. IEF-SDS two-dimensional'electrophoresis showed that many of the one-dimensional gel bands represented several proteins dots in the 2-D and that some of them were unique to an organ-type of the flower. Protein analysis of the various organs showed that stamens contained the greatest amount and petals the least amount of protein.     

黄瓜器官特异蛋白的研究

利用 SDS单向电泳对黄瓜 ( Cucumissativus L .)的根、茎、叶、花萼、花冠、雄蕊、花柱和子房的可溶性蛋白进行了分析和比较。检测到花冠中的 2 3.5 k D和 33.0 k D,雄蕊中的 1 8.8k D、2 8.5 k D、31 .0 k D、37.0 k D和 39.0 k D,花柱中的 4 5 .0 k D及子房中的 32 .5 k D蛋白 ,分别为各自器官中的器官特异蛋白质。对花冠、雄蕊、花柱和子房的可溶性蛋白的 IEF- SDS双向电泳分析也确定了相应于 SDS单向电泳上特异蛋白带的蛋白质斑点。而且相应于 SDS单向电泳上的一条带 ,在 IEF- SDS双向电泳上可能是一个以上的分子量相同而等电点不同的几个蛋白质斑点。各种器官的蛋白质含量以雄蕊为最高、花萼为最低。     

Immunochemical studies on a phenobarbital-inducible esterase in rat liver microsomes

Detergent extracts of liver microsomes from drug-treated rats were analyzed semiquantitatively in crossed immunoelectrophoresis in combination with a zymogram technique for nonspecific esterase activity. One esterase-active antigen was quantitatively induced by phenobarbital as demonstrated with both antimicrosomal antiserum and a monospecific antiserum prepared against this antigen. No similar inducation of this or any other esterase-active antigen was detected after 3-methylcholanthrene treatment. With the monospecific antiserum, the antigen was also detected in other organs, capable of carrying out hydroxylation reactions, such as lung and kidney. It was, however, not induced by phenobarbital in these organs. Quantitative enzyme assays with acetanilide as substrate indicated that the phenobarbital-inducible esterase could also function as an amidase. Furthermore, from absorption studies, it was concluded that this antigen is located on the cytoplasmic side of the microsomal vesicles. Crossed immunoelectrofocusing experiments revealed that this esterase-active antigen consists of five subcomponents with different charge properties.     

Role of brain hormone in oogenesis of the Indian freshwater leech,Poecilobdella viridis (Blanchard) during the annual reproductive cycle

The possible role of a brain hormone in oogenesis of Poecilobdella viridis has been investigated by brain extirpation and brain extract injections during non-reproductive (November to January) and reproductive (March to May) periods. Brain extirpation during the non-reproductive period ceased maturation of the ovary. It is inferred that brain secretion bears possibly a gonadotropic principle which governs and regulates the oogenesis during the annual reproductive cycle.     

The effect of long non-reproductive periods on the genital health in captive female white rhinoceroses (Ceratotherium simum simum, C.s. cottoni)

White rhinoceroses suffer from a low reproductive rate in captivity. Intensive efforts to propagate specifically the northern white rhinoceros have been very limited. The dismal outlook for this subspecies in the wild makes successful ex situ breeding programs paramount. In this context, this study examined 48 southern and 6 northern white rhinoceroses using ultrasound and faecal hormone analysis to elucidate causes for female reproductive failure and to determine whether long non-reproductive periods have a detrimental impact on genital health. Results showed that 76% of the nulliparous females had intact hymenal membrane indicating these females had never been bred, at an age when their wild counterparts have delivered multiple offspring. Fifty-six percent of the studied population had various reproductive pathology. Cystic endometrial hyperplasia; leiomyomas of the cervix, uterus and ovary, adenoma; para-ovarian cysts and hydromucometra represent the scope of lesions identified. The stages of the lesions in nulliparous females correlated with age (r = 0.4, P < 0.05). Due to the severity of the lesions, 28% of the study population was considered post-reproductive. Therefore, the reproductive life span in some individuals was 10-20 years shorter than expected. However, in parous females the incidence of pathological lesions was significantly lower (P < 0.0001). Seventy-eight percent females studied had erratic or absent luteal activity. The hormone data corresponded with two ultrasonographic levels of ovarian activity, active and inactive, occurring within an age range of 3-19 years and 15-38 years, respectively. This suggests the lack of ovarian activity by reproductive mid-life in non-reproducing females. The accuracy of the ultrasound findings was validated by necropsy in nine animals showing a strong positive correlation (r2 = 0.9, p < 0.001). Our data suggests that the development of reproductive pathology and ovarian inactivity in white rhinoceros is an age-related consequence of long non-reproductive periods. This asymmetric ageing process of the genital organs can be prevented with the achievement of at least one pregnancy.     

Physicochemical properties of kidney-specific antigen

Using specific antirenal sera obtained from rabbits and absorbed with a mixture of extracts from heterologous organs, a specific antigen was detected in human and CBA mouse renal extracts. Its molecular weight was found to amount to about 100 000 dalton. It is salted out with ammonium sulfate at 50-70% saturation of renal extract and is destroyed on extract heating for 30 min at 75 degrees C. This antigen is sensitive to trypsin and papain but resistant to hyaluronidase. It is partially destroyed by DNase and RNase, provided the latter ones are used in comparatively high doses (1 mg per 0.3 ml extract) and exposure lasts one day. Based on the study of the physicochemical properties it is suggested that the kidney-specific antigen may be a ribonucleoprotein or a deoxyribonucleoprotein but cannot be attributed to glycoproteins.     

Allocation of resources to reproduction in Styrax obassia in a masting year

An analysis is presented of three possible pathways of reproductive allocation, namely, allocation of resources to reproductive organs from reproductive shoots, from non-reproductive shoots and from the main trunk. These pathways were examined by comparing the amount of storage starch in reproductive shoots, non-reproductive shoots and the main trunk in Styrax obassia, a typical masting tree species, during a year of little flowering (1999) and in a mass-flowering year (2000). In addition, we measured rates of light-saturated photosynthesis in leaves of reproductive and non-reproductive shoots to examine the contribution of photosynthetic production to reproductive costs. In both the main trunk and non-reproductive shoots the pattern of seasonal variation in the amount of starch did not differ between 1999 and 2000. However, in the mass-flowering year, the amount of starch in the reproductive shoots was less than that in non-reproductive shoots during the growing season. Thus, reproductive shoots bore most of the cost of reproduction, although non-reproductive shoots and the main trunk also bore some of the cost. Mass-based rates of light-saturated photosynthesis of the leaves of reproductive shoots were significantly higher than those of non-reproductive shoots during both the flowering and the fruiting period. However, leaves of reproductive shoots had a significantly smaller area, a lower mass per area, and lower concentrations of nitrogen than leaves of non-reproductive shoots, although the number of leaves did not differ between the two types of shoots. Therefore, the amount of photosynthate per shoot was significantly lower in reproductive shoots than in non-reproductive shoots. These results suggest that the cost of reproduction depends predominantly on storage starch in reproductive shoots, although it is still unclear how much photosynthate is allocated to reproductive organs from non-reproductive shoots.     

A histological investigation of the occurrence of non-reproductive female bluefin tuna Thunnus thynnus in the Mediterranean Sea

The presence of non-reproductive Atlantic bluefin tuna Thunnus thynnus females in the Mediterranean Sea was investigated through histological analysis of the gonads. Three hundred and twenty-six ovary samples were collected from adults captured at different locations in the Mediterranean Sea during the reproductive seasons between 1998 and 2008. Only three specimens were considered to be in a non-reproductive state: two of them were in a reabsorbing state showing ovaries with early vitellogenic oocytes and extensive α and β atresia of vitellogenic follicles; the third showed gonads with perinucleolar oocytes and was considered to be in a resting state. The low occurrence of non-reproductive individuals found in this study makes it unlikely that non-reproductive individuals aggregate with reproductive ones during their migration towards spawning grounds. Further research is suggested in order to investigate the potential presence of non-reproductive individuals on non-spawning grounds during the reproductive season.     

Virus-augmented delayed hypersensitivity skin tests in gynecological malignancies

Summary Cultured human tumor cells of various histologic origins were infected with PR8/A/34 influenza virus. Nonviable crude membrane extracts were derived from the infected and uninfected cells. The extracts were coded and tested for their ability to produce delayed hypersensitivity skin reactions (DHSR) in allogeneic patients with squamous uterine cervical carcinoma, epithelial ovarian carcinoma, and malignant melanoma. Augmented antigen sensitivity to the virus-modified extracts compared with virus alone or to the unmodified extracts was observed in all patient groups. There was insufficient specificity to delineate a response by individual tumor type and related tumor extract, but some of the observed responses suggested tumor or organ site associations. Cervical carcinoma patients reacted more frequently to the virus-modified cervix extract, which also produced a high frequency of response in patients with ovarian carcinoma and melanoma. Ovarian carcinoma patients demonstrated increased sensitivity to both virus-modified ovarian carcinoma extracts, although 14 of 21 patients also showed responsiveness to one of the unmodified ovarian extracts. Malignant melanoma patients showed increased sensitivity to all virus-modified extracts except one of two derived from the ovarian carcinoma, and demonstrated a significantly augmented response to the virus-modified melanoma extract when the response to this extract was compared with that in ovarian carcinoma patients.The augmented reactions appear to be due to an association of the PR8 virus and as yet undetermined cellular components rather than to the virus alone. The possible involvement of tumor-associated determinants and the clinical significance of this phenomenon require further investigation.     

Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.     

An immunologic study of the secretory products of rat Clara cells

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.     

Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.     

Serological Comparison of the Three Morphological Phases of Coccidioides immitis by the Agar Gel Diffusion Method

Hyperimmune sera against spherules and against arthrospores of Coccidioides immitis were prepared by inoculation of rabbits. The antibody content of these sera was studied by the agar gel diffusion method. It was observed that antispherule pooled sera formed multiple precipitin bands with extracts of spherules and of arthrospores. The antiarthrospore pooled serum, however, failed to precipitate with the spherule extract, and formed a single band in the presence of an arthrospore solution. When the spherule and the arthrospore extracts were tested with a variety of different antisera, it was observed that the spherule preparation formed bands only in combination with anti-purified spherule pooled serum, whereas the arthrospore extract precipitated with anti-purified spherule, antiarthrospore, and anti-Histoplasma capsulatum pooled sera. It was also observed that a spherule culture supernatant solution formed five precipitin bands in combination with anti-spherule pooled sera, formed one band with pooled antiserum from rabbits with coccidioidomycosis, and did not precipitate in the presence of antiarthrospore pooled serum. Coccidioidin, however, formed two bands in the presence of any of these antisera. It was therefore concluded that extracts from the spherule phase of C. immitis differed from solutions obtained from the arthrospore and mycelial phases.