Search

Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplemented D-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17 beta-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3 beta hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.

An antibody against basic fibroblasts growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30-33 and 46 kDa in immunoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.

Carbonyl reductase activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The enzyme activity increased after the preovulatory surge of luteinizing hormone (LH) on proestrus, and the enzyme content began to increase prior to the LH surge. Although the enzyme content reached the highest level at 2000 h and remained at a plateau for 8 h, the enzyme activity increased linearly until it reached the highest level at 0800 h on the morning of estrus. At their maximum, enzyme activity and content were approximately 1.5-fold and 2-fold greater, respectively, then basal diestrus values. The enzyme protein amounted to 1-4% of the ovarian cytosolic protein. An immunohistochemical study revealed that the enzyme was primarily localized in interstitial gland cells and theca interna cells of secondary and Graafian follicles as well as atretic follicles.

Previous studies from this laboratory have demonstrated immunostainable beta-endorphin-like material (beta-EP) in Leydig cells and epithelia of the epididymis, seminal vesicle and vas deferens of the rat. These observations would be strengthened if it could be demonstrated that they were not a peculiarity of the rat. Accordingly, we now present immunocytochemical evidence for the presence of beta-EP in the Leydig cells of mouse, hamster, guinea pig and rabbit. No immunoreactive material was identified in Sertoli, myoid, endothelial or germ cells of any of the species examined. Immunostainable beta-EP was also demonstrated in the epididymides of mouse, guinea pig, rabbit, and rat, but not hamster. Immunostainable material was also present in the epithelia of the vas deferens and seminal vesicles of mouse and rat, the only two species thus far examined. Since beta-EP was present in Leydig cells, we wondered whether this peptide could be identified in other steroid-producing tissues. When rat ovaries and adrenals were reacted with anti-beta endorphin, staining was demonstrated in corpus luteum and adrenal cortex. No staining was observed in the adrenal medulla or other portions of the ovary. In order to determine whether the beta-EP detected in the testis and epididymis was derived from a pituitary source, animals were hypophysectomized and tissues examined 2 weeks later. Both the Leydig cells and the epididymal epithelium remained immunostainable. In summary, immunostainable beta-EP has been identified in Leydig cells of five species. Stainable material is also present in the epithelium of other portions of the male reproductive tract and in steroid-secreting cells of the ovary and the adrenal. Such beta-EP may have a paracrine function in the testis and other portions of the male reproductive tract.

Soluble extracts of Bufo ovaries agglutinate sialidase-treated rabbit erythrocytes. Unlike other amphibian lectins this agglutination activity does not require the presence of calcium ions. It is specifically inhibited by D-galactose and its derivatives. Thiodi-D-galactoside is the most potent saccharide inhibitor followed by lactose and methyl-beta-D-galactoside, respectively. D-Fucose, D-glucose and D-mannose do not inhibit the activity at concentrations at or above 100 mM. The lectin has been purified 500-fold to apparent homogeneity from the ovaries by salt extraction and affinity chromatography on lactose-aminophenyl-agarose, with a yield of about 0.2%. The molecular mass determined by gel filtration under native conditions was 30 kDa; polyacrylamide gel electrophoresis in SDS gave a molecular mass of 15 kDa, suggesting that the lectin is a dimer. The lectin has an isoelectric point of 40 and contains a high proportion of acidic amino acids.

Mouse follicular oocytes cultured for more than 72 h in modified Krebs-Ringer solution in vitro undergo spontaneous segmentation to form "blastomeres" with or without nuclear fragments or chromatin. Glycosaminoglycans (GAGS) isolated from bovine follicular fluid (bFF) and added to the suspending medium at concentrations of 0.25 mg/ml or greater prevented the occurrence of spontaneous segmentation in isolated cumulus-free mouse oocytes in a dose-dependent manner. The active material was isolated from bFF by chromatography on Dowex 1-X2. The fraction eluted with 0.5 M NaCl possessed the greatest biological activity. Preparations of purified bFF-GAGs and hyaluronic acid and chondroitin sulfate from a commercial source prevented the occurrence of segmentation. Hyaluronic acid was more potent than chondroitin sulfate. The present results show that GAGs prevent segmentation of oocytes in vitro.

Germinal vesicles of oocytes from Pleurodeles waltlii were used for immunization of BALB/c mice to obtain hybridomas secreting monoclonal antibodies. The hybridomas were screened for reactivity of their antibodies against lampbrush chromosomes of oocytes, as revealed by indirect immunostaining. Antibodies labelling the lampbrush chromosomes were also tested on histological sections of oocytes, embryos, and larvae of Pleurodeles. Characterization of the antigens was accomplished through immunoblotting of two-dimensional electrophoretic gels of germinal vesicle proteins. The ten monoclonal antibodies giving a positive reaction were classed into five groups. Group 1, exemplified by antibody A33, recognizes all the lampbrush chromosome transcribing sites (loops). Moreover, it differentially labels the cell nuclei during embryonic and larval development. Group 2, antibody B71, also stains all the loops of the lampbrush chromosomes, but does not react with cell nuclei of embryos and larvae. Group 3, antibody A1, labels specific loops, some of which are heterozygous in the strain of P. waltlii used. These heterozygosities have allowed us to localize and to characterize a chromosomal segment on bivalent IV which is heteromorphic in the two partners of the bivalent. We suggest that this heteromorphism represents a morphological distinction between Z and W heterochromosomes. Moreover, this antibody reacts with only one transcription unit along a loop that contains several units. Group 4, antibody B24, stains the only two structures in the lampbrush chromosomes of P. waltlii that do not have a loop organization, the mass "M" and the spheres. Group 5, antibody A35, reacts with the chromomeres. The antigens corresponding to antibodies A33 and B24 have been identified as proteins, which have apparent molecular weights of 80 and 104 kilodaltons, respectively. They correspond to proteins abundant in the germinal vesicles. All the antibodies described here cross-react with the lampbrush chromosomes of five other species of Urodeles.

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.

The 5'-terminal nucleotide sequences of ribosomal 18-S and 28-S RNA of seven species of eukaryotes including three mammals, one bird, one amphibian, one echinoderm and one slime mold, were analyzed either by means of terminal phosphorylation of RNA with polynucleotide kinase of by fingerprint analysis of uniformly labeled RNA. The following conclusions were obtained. 1. The 5'-terminal sequences of the 18-S RNA of the mouse, chicken and Dictyostelium discoideum were pUpApCp(Cp,Up)Gp---, suggesting strongly that all the eukaryotes had this same sequence at the 5'-terminus. Preliminary analysis of the 5'-termini of the 18-S RNA from human, rat, Xenopus and sea urchin cells revealed the same pUp(Np)Gp--- type 5'-terminal structure, supporting the above hypothesis. 2. The 5'-terminal sequences of the 28-S RNA of the human, rat, mouse and chicken cells were all pCpGp---, whereas those of the lower animals such as Xenopus, sea urchin and Dictyostelium were different.

To understand the possible structures of testicular inhibin, we have isolated cDNAs coding for inhibin subunits from human testicular cDNA libraries. In this study we report that the nucleotide and predicted amino acid sequences for human testicular inhibin beta-B-subunit are similar to those of human ovary. In rat testis two species of beta-B-subunit mRNA [4.4 and 3.3 kilobases (kb)] appeared to be present in equal concentration, as opposed to rat ovary where a predominant band of 4.4 kb and a minor band of 3.3 kb were observed. One major species of beta-A-subunit mRNA (6.5 kb) was identified in both testis and ovary. The concentration of beta-A-subunit mRNA in the testis was very low, representing only 0.5% of that in rat ovary. The accumulation of beta-B-subunit mRNA peaked at 20 days of age and declined thereafter in a pattern similar to that of the alpha-subunit gene. Hypophysectomy caused a marked increase in the concentration as well as the total content of beta-B-subunit but no change in beta-A-subunit mRNA in rat testis. We have previously reported that FSH markedly increased alpha-subunit mRNA levels both in vivo and in vitro. By contrast, neither FSH nor testosterone has any significant effect on the accumulation of beta-A- or beta-B-subunit mRNAs in hypophysectomized animals or Sertoli cell primary cultures. We conclude that 1) the mRNAs for both beta-subunits are not regulated by FSH; and 2) hypophysectomy does not change and increases, respectively, the mRNAs for the beta-A- and beta-B-subunits. We conclude that the inhibin subunit mRNAs are differentially regulated in rat testis.

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.

Zollinger-Ellison syndrome is usually caused by a gastrin-secreting tumor in or near the pancreas. We describe a patient in whom an ovarian cystadenocarcinoma was the cause of the syndrome. The patient presented with a short history of peptic ulceration and development of a large pelvic mass. Investigations demonstrated a basal acid output of 37.8 mEq/h and a maximal acid output of 36.0 mEq/h, and the plasma concentration of gastrin was 830 pg/ml (normal less than 100). Secretin and calcium infusion tests were positive, and a meal test was compatible with Zollinger-Ellison syndrome. Imaging studies demonstrated a normal liver and pancreas but a large cystic right ovarian mass. Resection of the mass resulted in a marked reduction in gastric acid output, a fall in plasma gastrin concentration to normal, negative calcium and secretin tests, and a normal (positive) meal test. Histology of the mass showed it to be a mucinous cystadenocarcinoma. The tumor stained with immunoperoxidase technique was positive for gastrin, and the cyst fluid contained high concentrations of gastrin and calcitonin. One year later, the patient has no biochemical or imaging evidence of tumor. Ovarian, gastrin-producing tumors and pancreatic gastrinomas cannot be distinguished by provocative tests, and negative imaging studies do not exclude a pancreatic tumor. Patients with an ovarian mass and Zollinger-Ellison syndrome should have a bilateral oophorectomy and a careful exploration of the pancreatic area.

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.

Techniques employed for the recovery from biological tissues of noncombustible fine particles such as asbestos, talc, kaolin and diatomaceous material were assessed by electron microscope x-ray microanalysis. Recovery procedures which have been proven successful for lung tissue were found to be impracticable for more solid types of tissues. Digestion techniques employing acids, alkalis or enzymes and standard incineration procedures were found to be unsatisfactory for human adrenal, cervix, liver and ovarian tissues when the resultant residues were examined by electron microscope microanalysis. The recovered particles were often completely masked with residues which were shown to be composed of organic elements. The use of oxygen during the incineration process completely removed this contaminating material in nearly all cases studied. When such procedures were used, clearly defined particles were recovered from the tissue, thereby permitting x-ray analysis. A quantitative analysis could then be made to estimate the particle content in a variety of tissues.

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent-solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000.

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.

Immunocytochemical staining for relaxin in ovarian sections of pregnant mice from day 11 through day 18 of gestation revealed that only corpora lutea (CL) of pregnancy are stained. Evaluation of serial sections of ovaries from a day 16 pregnant mouse revealed that the only luteal structures present are CL of pregnancy. The number of CL present in each ovary equaled the number of implantation sites in each related horn (7 on the right side and 8 on the left side). These large CL varied in shape, being round in some profiles to very elongate in others. All CL were immunochemically stained for relaxin using the peroxidase-antiperoxidase method of L. Sternberger (Immunocytochemistry, 2nd ed. Wiley, New York, 1979). The intensity of the strain varied from cell to cell within each CL. Small luteal structures that were observed to be immunochemically stained for relaxin were demonstrated to represent the periphery of CL of pregnancy. No luteinized follicles were observed and interstitial cells and follicles were not immunochemically stained in any of the day 16 serial ovarian sections or in any of the ovarian sections from pregnant mice on the other days of gestation studied. CL of previous cycles were not observed to be present in the ovaries at days 15, 16, or 18 of gestation. However on day 14 and before, CL of previous cycles were observed and they did not exhibit any relaxin immunostaining. Immunocytochemical studies using the biotin-avidin system revealed that no relaxin immunostaining could be demonstrated in the ovaries of cycling mice at any stage of the estrous cycle. In conclusion, this study revealed that the only ovarian structures demonstrating relaxin immunocytochemical staining in the mouse were CL of pregnancy.

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.

The immunohistochemical localization of inhibin in porcine and bovine ovaries was studied, using monoclonal antibodies to bovine follicular fluid 32K inhibin (bFF 32K inhibin) and a polyclonal antiserum to porcine follicular fluid 32K inhibin (pFF 32K inhibin). In order to obtain a precise immunohistochemical staining, various fixations were tested with an immunoblotting procedure. Acetone fixation followed by celloidin embedding proved to be most appropriate for the immunohistochemical study of inhibin. A consistent pattern emerged in all sections prepared from porcine and bovine ovaries. Granulosa cells were specifically stained by the antibodies, and especially the cells constituting the inner layer were more intensely stained than the cells close to the theca.

A gonadotropin-releasing hormone (GnRH)-binding inhibitor (GnRH-BI) was purified from bovine ovaries and identified as histone H2A. In the present studies, the biological effects of partially purified and purified ovarian GnRH-BI, as well as calf thymus histone H2A, were examined in rat ovarian cells. Since GnRH has direct antigonadotropic actions on these cells, the effects on luteinizing hormone-stimulated cAMP accumulation in luteal cells and follicle stimulating hormone-induced cAMP and progesterone production in granulosal cells were evaluated. Antigonadotropic activity in both luteal and granulosal cells coeluted directly with GnRH-BI activity during purification from bovine ovaries, and the antigonadotropic effects were dose dependent and reversible. In contrast to GnRH, GnRH-BI maximally inhibited gonadotropin responses and the effects of GnRH-BI were not blocked by a GnRH antagonist. The purified ovarian GnRH-BI and calf thymus histone H2A had identical antigonadotropic properties, and the half-maximal concentrations for inhibiting the gonadotropin responses of granulosal and luteal cells was 2 and 5 microM, respectively. In conclusion, the ovarian GnRH-binding inhibitor, identified as histone H2A, not only inhibits the high affinity binding of GnRH to rat ovarian membranes but also evokes GnRH-like antigonadotropic responses in rat ovarian cells that do not appear to be mediated by binding to GnRH receptors.

We have identified a 110-kDa pI 5.6 phosphoprotein with DNA binding properties in the rat pachytene spermatocyte nuclear matrix. By immunoblotting and indirect immunofluorescence assays using polyclonal antibodies against the 110-kDa protein, we observed that it was germ cell nuclear matrix specific, more prominent in pachytene spermatocytes compared to premeiotic spermatogonia or postmeiotic round spermatids, and present in rat oocytes and in germ cells of mouse and monkey. We propose that this protein could play an important role in the meiotic process.

Developmental comparisons of silkmoth ribosomal proteins were performed by parallel two-dimensional electrophoretic analyses of proteins derived from developing adult wings and from ovarian follicles at the prechoriogenic and choriogenic stages of development (before and during secretion of the chorion, respectively). Proteins of 40S and 60S subunits were prepared and analyzed separately. A single major developmental difference was observed, exclusively in choriogenic follicles: the majority of a 40S subunit protein, S6, was shifted to a more acidic form, possibly as a result of phosphorylation. A less prominent change was apparently due to quantitative variation between two forms of one large subunit protein. The developmental comparisons were performed in two species of the genus Antheraea, with consistent results. Comparisons between the two species revealed remarkable evolutionary conservation of the ribosomal protein patterns, sharply contrasting with the evolutionary diversification of chorion structural proteins in the same two species. The only detectable interspecies differences in ribosomal components were (a) slightly more acidic behaviour of one small-subunit protein and more basic behaviour of one large-subunit protein in Antheraea polyphemus as compared to Antheraea pernyi, and (b) presence of an apparently species-specific 60 S subunit protein in A. pernyi, which in A. polyphemus probably co-migrates with a neighbouring protein.

Beta-endorphin (beta-EP) immunostainable cells were demonstrated in human ovarian tissue using a non-cross-reacting anti-beta-EP serum and the avidin-biotin-peroxidase detection technique. In ovaries from ovulating and premenopausal women, beta-EP immunoreactivity was localized in the luteinized cells of theca interna of maturing follicles with almost negligible staining in granulosa cells; cells of primary follicles did not stain. In corpora lutea, luteinized cells in both theca interna and granulosa, layers were equally positive. In postmenopausal ovaries, staining was detectable only in scattered luteinized stromal cells. This is the first report on the presence of immunoreactive beta-EP in human ovaries, in which beta-EP seems to be produced by the same sex cord cells engaged in active steroidogenesis and may be under gonadotropin central regulation. The significance of this finding is discussed.

Ovarian steroid contents and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured during the days after first ovulation in rats unilaterally ovariectomized in late prepuberty. In addition, follicle counts were made at second estrus and second metestrus. During the cycle following first ovulation, ovarian estradiol contents in unilaterally ovariectomized (ULO) rats were significantly increased as compared to intact rats on the day of metestrus, on diestrus 1 and on second estrus. Ovarian progesterone was significantly increased on the days of metestrus, on diestrus 1, second proestrus and second estrus, but no differences were seen in ovarian androgen contents. After ULO there was an indication of an augmented FSH surge at the first and the second ovulation. Follicle counts revealed that the total number of healthy as well as of atretic antral follicles on the day of second estrus was significantly increased after ULO, due to increased numbers of the smallest antral follicles. At second metestrus the number of larger antral follicles (350-500 micron 3) and the total number of healthy antral follicles was higher after ULO. It is concluded that the compensatory process after ULO involved increased recruitment of small antral follicles. Activities in the remaining ovary were not simply doubled but a new hormonal balance was established.

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

A relaxin-like molecule has been isolated from the ovaries of the spiny dogfish (Squalus acanthias) which consists, like porcine relaxin, of two chains linked by the insulin-type disulfide bonds. The total number of amino acids is 54 of which 24 are in the A chain and 30 in the B chain. The molecular masses, calculated from the amino acid compositions, are 2510 Da for the A chain and 3370 Da for the B chain, making a total of 5880 Da. The N-terminus of the B chain is protected by a 5-oxoproline (pyrrolidone carboxylic acid) residue which is also found in the same position in the relaxins of sand tiger shark, pig, and man, whereas the relaxin of the rat has its 5-oxoproline residue at the N-terminal of the A chain. By all available criteria, S. acanthias relaxin is a typical member of the relaxin family although the sequence homology to mammalian relaxins is limited to about 45% of its amino acid residues. In contrast, the dogfish relaxin shows about 80% homology with sand tiger shark relaxin (the first such interspecies similarity to be observed) and has about twice the biological activity (mouse pubic symphysis test) when compared to sand tiger relaxin.

An enzymic method for recovering primordial follicles from the pig ovary consists of incubating cortical slices for 2 h with 0.025% collagenase 1A. An average of 185,000 or 419,000 primordial follicles per ovary were recovered from ovaries collected in Cambridge and Kansas, respectively. Following a discontinuous Percoll gradient, primordial follicles can be separated from contaminating somatic cells by mouth pipette or a micromanipulator to collect 100-1500 follicles but for large scale recovery of approximately 30,000 follicles flow cytometry is recommended. Two types of primordial follicles can be distinguished by electron microscopy: peripheral clusters of small oocytes with an incomplete investment of pregranulosa cells and a deeper region of individual oocytes surrounded by a complete layer of pregranulosa cells. The viability of the purified primordial follicles is attested by their ability to synthesize proteins for at least 12 h after incubation with [35S]methionine. Moreover, the primordial follicles showed several polypeptide bands in common with mature oocytes especially with Mr of 60,000-90,000 but with considerable differences from somatic cells.

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.

A monoclonal antibody (IgG1) has been prepared that specifically detects Chinese hamster ovary cells expressing a multidrug-resistant phenotype. This antibody recognizes the membrane P-glycoprotein (Mr 170,000) associated with drug resistance as determined by enzyme-linked immunosorbent assay with purified P-glycoprotein and by Western blot analysis of cell extracts from drug-resistant and drug-sensitive cells. By immunofluorescence methods, the antibody also reacts strongly with viable and ether:ethanol-fixed resistant cells but does not react with the parent drug-sensitive cell line. Thus, this antibody can bind with live cells allowing discrimination by immunohistochemistry between drug-resistant and drug-sensitive Chinese hamster ovary cells.

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

Measurement of receptor-bound unlabelled physiologically active lutropin (luteinizing hormone, LH) was possible by a modified radioimmunoassay. The conventional radioimmunoassay conducted at 4 degrees C was inadequate, whereas the modified assay performed at 37 degrees C could measure receptor-bound lutropin. The radioimmunoassay at 37 degrees C takes only 36h for completion compared with 5-7 days at 4 degreesC. The sensitivity and range of dose-response curves are, however, unaltered. The validity of the technique was established by a number of criteria.

The content of 5-methylcytidylic acid in nuclear DNA and mito chondrial DNA of Xenopus laevis and HeLa cells has been determined. Both nuclear DNA's contain 5-methylcytidylic acid. The 5-methylcytidylic acid content of X. laevis DNA is 1.7 mole percent of total nucleotides, and that of HeLc cell DNA is 0.7 mole percent. In neither mitochondrial DNA could any 5-methylcytidylic acid be detected; the limit of sensitivity was judged at below 0.1 mole percent for X. laevis DNA and below 0.05 mole percent for HeLa cell DNA.

The major high mannose-type glycopeptides present in Chinese hamster ovary cells have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, and (Man)6(GlcNAc)2-Asn. The structures of these glycopeptides were determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Their complete structures are: manalpha1 leads to 2Manaalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, Manalpha1 leads to 2Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, and Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn. These structures are compared with the structures of the peptide-bound oligosaccharide intermediates that are processed to form complex-type oligosaccharides. From these results, it is proposed that the high mannose-type oligosaccharides are a product of "incomplete" processing of the protein-bound oligosaccharide along the same pathway which leads ultimately to the formation of a complex-type oligosaccharide.

The influence that extracts of different T. infestans organs possess to induce Trypanosoma cruzi differentiation from epimastigotes to metacyclic forms in Grace's and modified Grace's media was analyzed. Growth of epimastigotes was adequate in Grace's medium when homogenates of stomach, testes, and ovaries from recently fed insects were added. Growth of epimastigotes was adequate when testes or ovaries obtained from recently fed triatomes were added to modified Grace's medium. Similar results were obtained in Grace's or modified Grace's media supplemented with stomach or intestinal homogenates obtained from "starved" triatomes. None of the above culture conditions induced differentiation of epimastigotes to the metacyclic stage. When extracts of intestine or stomach obtained from triatomes fed 24 to 48 hr previously were used to supplement the modified Grace's medium, growth and differentiation to the metacyclic stage was induced in a high percentage of the population. When extracts of intestine obtained from recently fed insects were used to supplement Grace's medium, differentiation and growth was an in modified Grace's medium. No important differences were observed if cultures were prepared at pH 6.4 or 7.0. The triatome food source did not seem to be important for the differentiation. The parasite population after differentiation to metacyclic type forms showed a higher degree of infectivity than the original population constituted mostly by epimastigotes. These studies suggest the importance of factors from the insect vector in differentiation of T. cruzi.

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.