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The present investigation provides three lines of evidence for the presence of a pro-form of neuropeptide Y (NPY) in plasma and follicular fluid. First, by the demonstration of NPY-immunoreactive material of a size corresponding to the estimated mol wt of pro-NPY. Second, an antiserum specific for the C-terminal tyrosine amide of NPY and peptide YY does not react with this material. Third, it was possible to convert the pro-NPY extracted from plasma and follicular fluid using the protease, Endoproteinase-Lys C, to a NPY-immunoreactive form eluting slightly before NPY on a G-50 column. The size of the digested product was consistent with a cleavage of pro-NPY resulting in an immunoreactive species, NPY-Gly-Lys. Pro-NPY was also found in tissue culture media from the human neuroendocrine cell line SH-SY5Y. As in the case of plasma and follicular fluid, another NPY immunoreactive species eluted from a G-50 gel filtration column slightly before synthetic human NPY. Analysis of this material with an antibody directed against the tyrosine amide of NPY in combination with isoelectric focusing revealed that this peak consisted of at least two immunoreactive forms of NPY. In conclusion, at least three different forms of NPY immunoreactivity are likely to be present in plasma, follicular fluid, and cell tissue culture media; pro-NPY, a degradation form of pro-NPY, or a biosynthetic intermediate and NPY.

A peptide that inhibits egg development in mosquitoes (oostatic factor) has been purified from the ovaries of female Aedes aegypti. The factor is a decapeptide with a molecular mass of 1047.6. The primary sequence has been determined as NH2-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-COOH from mass spectra recorded on a quadrupole Fourier transform instrument. The amino acid sequence exhibits sequence correlation to mammalian, plant, and several viral proteins. Injection of synthetic analogs into mosquitoes, biting midges, flies, and fleas inhibited proteolytic enzyme biosynthesis in the midgut. Binding studies with [3H]oostatic factor indicated that the midgut epithelial cells have a factor-specific receptor.

The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 1 crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31/32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

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.

Ovaries from a female plaice (Pleuronectes platessa) that had been injected with human chorionic gonadotrophin were incubated in vitro with 17 alpha-hydroxy[1,2,6,7-3H]progesterone. The major steroids produced by the ovaries were tentatively identified as 17 alpha,21-dihydroxy-4-pregnene-3,20di-one (11-deoxycortisol; 17,21-P), 17 alpha,21-dihydroxy-5 beta-pregnane-3,20-dione (3 alpha, 17,21-P-5 beta). A high proportion of these steroids was found in a conjugated form (sulphates or glucuronides). Radioimmunoassays were developed for 11-deoxycortisol and for 3 alpha,17,21-P-5 beta and were applied to fractions of mature male and female plaice plasmas and plaice ovarian incubates that had been separated on thin-layer chromatography. The presence of all three steroids, in vivo and in vitro, was confirmed. Particularly high amounts of conjugated 3 alpha,17,21-P-5 beta were found in the plasma of mature females (200-400 ng ml-1). The 3 alpha,17,21-P-5 beta radioimmunoassay also identified 3 alpha,17 alpha-dihydroxy-5 beta-pregnane-20-dione in all three fluids, despite the fact that this steroid was not among the radioactive incubation products of the ovary. These findings are compared with those from another flatfish, the dab (Limanda limanda), where the major gonadal steroids have been shown to be 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and its 5 beta-pregnane (3-keto and 3 beta-hydroxyl) metabolites.

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.

In order to determine whether leukotrienes, products of the lipoxygenase pathway, are involved in ovulation, pairs of rabbit ovaries were treated with the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) while being perfused in vitro. The control ovaries from each rabbit received luteinizing hormone (LH) (1.5-2.25 micrograms ml-1) while the contralateral ovaries were treated with LH + NDGA (100 microM) or LH + CA (100 microM). The numbers of ovulations from both the LH + NDGA- and LH + CA-treated ovaries were significantly higher (P less than 0.05) than from their respective LH-stimulated controls. Treatment with NDGA alone in the perfusate did not cause any ovulation, while CA alone caused one ovulation from one of six ovaries perfused. Ovarian tissue levels of prostaglandins after 7 h of perfusion with LH + NDGA or with LH alone showed that, in five of the six ovaries perfused in this group, the tissue levels of PGE2, 6-keto-PGF1 alpha and PGF2 alpha were higher in the presence of NDGA. The mean differences were significant (P less than 0.05) for prostacyclin but not significant (P greater than 0.05) for PGE2 and PGF2 alpha. Our interpretation of the findings is that, when used for blocking the lipoxygenase pathway, NDGA and CA increase the substrate availability for the cyclo-oxygenase pathway of arachidonic acid metabolism, resulting in a net increase in prostaglandins. The increased ovarian levels of prostaglandins, especially prostacyclin, may cause the observed increase in ovulation rate. Consequently, although the leukotrienes may be involved in the mechanism of ovulation in the rabbit, their effects appear to be less pronounced than those of prostaglandins.

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.

The control of FSH and LH secretion by the pituitary gland is clearly multifactorial. The evidence that LHRH influences the release and stores of both FSH and LH is considerable, and equally well documented is the fact that testosterone and oestradiol will suppress the secretion not only of LH but also of FSH. Recently evidence points to the existence of inhibin which will suppress FSH secretion but which in higher doses will also decrease LH levels. It is therefore likely that the ratio of FSH to LH secreted by the gonadotrope is determined by the summation of the action of these factors on cellular mechanisms that remain to be determined.

Ovarian poly (A) + RNA from Xenopus laevis and Xenopus borealis was used to construct two cDNA libraries which were screened for histone sequences. cDNA clones to H4 mRNA were obtained from both species and an H3 cDNA clone from Xenopus laevis. The complete DNA sequences of these clones have been determined and are presented. These new sequences are compared with other H3 and H4 DNA sequences both in the coding and 3' noncoding regions. We find that there is considerable non-random codon usage in ten H4 genes. In addition there are some sequence similarities in the 3' noncoding regions of H3 and H4 genes.

Fundamental and clinical studies on T-1982 (cefbuperazone), a new cephamycin antibiotic, were carried out, and the following results were obtained. When T-1982 was administered at a dose of 1 g by intravenous drip infusion for 30 minutes or 1 hour, the concentration in serum showed as high as 23.0 micrograms/ml or 25.0 micrograms/ml even 2 hours after administration. The concentrations in the genital tissues about 5 hours after administration ranged 1.2-45.6 micrograms/g for 30 minutes drip infusion and 0.9-26.8 micrograms/g for 1 hour drip infusion. From these results, T-1982 was supposed to maintain the in vivo concentration to inhibit 80-100% the growth of bacteria such as S. aureus, E. coli, Klebsiella, Proteus, S. marcescens and Gram-negative anaerobic bacteria, B. fragilis which were often isolated clinically in the field of obstetrics and gynecology. When T-1982 was administered at a dose of 1-2 g twice a day to 14 patients with female genital infection; 2 intrauterine infection, 2 pyometra, 7 pelveoperitonitis, 1 adnexitis, 1 adnexal abscess and 1 vaginal cuff abscess, the clinical results were excellent in 9, effective in 4 and poor in 1. The efficacy rate was 92.9%. No side effects nor abnormalities in laboratory findings were observed in any of the 14 cases. These results suggest that T-1982 has efficacy for the treatment of obstetrical and gynecological infections.

The presence of the blood group substances A, B, AB, Lea, and Leb and of CEA was demonstrated by peroxidase-antiperoxidase or fluorescein immunohistochemistry in histological sections of normal ovarian tissue and ovarian tumors of varying differentiation. In surface cells of normal ovaries and of benign tumors, blood group substances (BGS) were found in patterns corresponding to the serum blood group type of the patients. BGS were localized exclusively at the apical border of normal cells. CEA was almost always absent. In borderline lesions the presence of BGS was reduced by approximately 13%. The polar differentiation was often lost, and the BGS were unevenly distributed in the cytoplasm. The percentage of CEA-specific staining increased from less than 5% in normal tissue and benign tumors to 15% in mucinous and 10% in serous borderline tumors. Malignant tumors contained altered distribution patterns of BGS and CEA. In contrast to normal ovarian epithelia, BGS positivity was diminished distinctly; the decrease was particularly evident for Leb presence by reduction from 75 to 10%. CEA was observed in nearly 50% of the carcinomas (more often in mucinous than in serous carcinomas). Our study revealed that expression of blood group antigens was related to benign lesions, whereas its loss was associated with a greater malignant potential. Inverse correlations were found for CEA. Thus, immunohistological determination of these marker substances may be applied to tumor prognosis.

All the steroids produced by Atlantic croaker ovaries during final oocyte maturation (FOM) were assayed for their ability to induce germinal vesicle breakdown (GVBD) of croaker oocytes in vitro. Ovarian tissue in the process of FOM was removed from Atlantic croaker (Micropogonias undulatus) and incubated with human chorionic gonadotropin (hCG) and pregnenolone in tissue culture medium for 8 hr. Steroids were extracted from the medium and fractionated by HPLC and TLC. Fractions were bioassayed for their potency to induce GVBD of Atlantic croaker oocytes in vitro. 17 alpha, 20 beta,21-Trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) was identified as the predominant steroid product and the major maturation-inducing steroid produced by the ovary of Atlantic croaker in vitro. A small amount of another steroid with equal potency to induce GVBD was tentatively identified as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P4); however, over ten times more 20 beta-S than 17 alpha,20 beta-P4 accumulated in the incubation medium. Trace amounts of testosterone and estradiol-17 beta were also isolated. Ovarian tissue incubated under more physiological conditions, without the addition of excess pregnenolone, failed to produce 17 alpha,20 beta-P4. These results provide further evidence that 20 beta-S is a major maturation-inducing steroid in Atlantic croaker.

Human Chorionic Gonadotropin (HCG) is major physiological luteotropic factors for the human corpus luteum. The observations strongly suggest that the human ovary possesses a gonadotropin receptor in the cell membrane. We studied the HCG receptor in normal human ovary and ovarian tumors. Twenty-three human ovarian specimens and 16 ovarian tumor specimens were obtained from women patients having gynecological surgery. Ovaries were homogenized and sonicated. The homogenates were centrifuged at 2000 g for 15 min. After sucrose density gradient ultracentrifugation (78,000 g, 4 h), two fractions were collected from layer of 33% and interface between 33% and 37%. Thirty micrograms of ovarian protein, 8 ng 125I-HCG and unlabeled HCG in a final volume of 0.5 ml of 0.05 mol/L Tris buffer were incubated at 30 degrees C for 2 h. The results were shown in the table.

A subclone of Chinese hamster ovary cells, R40F, has been selected for its unusually high resistance to lethal concentrations of Cd and Zn. Although there is a 33% loss in Cd resistance when R40F cells are cultured in the absence of exogenous metals, the Zn resistance remains unaltered. These cells are 80% tetraploid and demonstrate an increased capacity for metallothionein protein synthesis. When compared to wild type cells cultured in the absence of exogenous metals, R40F cells exposed to 200 M Cd for 48 h exhibited an approximate 200-fold increase in metallothionein-I (MT-I) protein. A 32P-labeled mouse MT-I cDNA was employed in solution hybridization studies to measure the level of MT-I mRNA in wild type and R40F cells. Cd (0.5 M) induces MT-I mRNA about 2.5- and 5-fold in wild type and resistant Chinese hamster ovary cells, respectively. When optimally induced, the resistant cells have about 80-fold more MT-I mRNA than the sensitive cells. Southern blot analysis of HincII-cleaved DNA indicates that the MT-I gene is amplified approximately 60- to 75-fold in R40F cells.

The abilities of the two different mol wt forms of rat placental lactogen (rPL) to maintain luteal steroidogenesis in pregnant rats in the absence of PRL were determined. The two forms of rPL present in day 12 pregnant rat serum were separated by gel chromatography, lyophilized, and reconstituted in a volume of saline equal to the original volume of serum. Rats were injected with 0.4 mg ergocryptine (ECO) on day 6 of pregnancy to suppress PRL and were then treated with a preparation of the large mol wt (LMW) hormone, the small mol wt (SMW) hormone, or both molecules (2 ml/day). Control animals received saline. Twice daily injections of the amount of LMW hormone contained in 1 ml day 12 pregnant rat serum reversed the abortifacient effect of ECO. In contrast, administration of the SMW hormone did not maintain either pregnancy or progesterone levels. Administration of both mol wt forms of rPL was also capable of maintaining luteal function. Sera obtained from day 18 pregnant rats containing only the SMW hormone had no luteotropic activity when administered, yet treatment with sera of day 12 pregnant rats sustained progesterone synthesis and fetal survival after ECO treatment. rPL were measured in the peripheral circulation and in the uterine vein throughout pregnancy by radioreceptor assay (RRA) using particulate membranes from either rabbit mammary gland or rat ovaries. Two peaks of activity were observed in the peripheral circulation by the two RRAs: one between days 11-14 and another between days 17-21, with a decline in activity between days 14-16. In contrast, levels of rPL in the uterine vein remained elevated throughout pregnancy. Concentrations of the LMW placental luteotropin were 5-10 times higher by ovarian RRA (O-RRA) than by mammary gland RRA (MG-RRA) between days 11-13, but concentrations of the SMW placental lactogen were found by the two RRAs to be similar in the later stages of pregnancy. Gel filtration of day 12 pregnant rat serum revealed two peaks of PRL-like activity. The O-RRA detected 19 times more LMW placental luteotropin than did the MG-RRA in the first peak of activity, yet measured equivalent amounts of the SMW placental lactogen in the second peak. Similar to results found in the peripheral circulation, levels of LMW placental luetotropin in the uterine vein measured by MG-RRA were significantly lower than those determined by O-RRA until day 14 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

A peptide with relaxin activity in guinea pigs but not in mice has been extracted from the ovaries of pregnant sand tiger sharks (Odontaspis taurus). The structural similarity of this peptide to porcine relaxin includes molecular size approximately 6000 daltons), number of chains, and possibly, disulfide cross-links. The relaxin-type peptide isolated from shark ovaries contains the amino acid residues tyrosine, proline, and histidine, which are absent in the porcine hormone. The amino acid composition of shark relaxin, therefore, resembles that of porcine insulin to a greater extent than does the amino acid composition of porcine relaxin. This finding supports the idea that shark relaxin may be a primitive relaxin that has undergone fewer mutations than porcine relaxin since the putative duplication of the insulin gene. The data presented here suggest that the putative duplication of the insulin gene, which might have given rise to relaxin, has occurred much earlier than the separation of sharks from the general branch of animals that eventually gave rise to mammals.

Carotene is stored as a tissue reserve in the corpus luteum. With different carotene supplementations in heifers (0, 100, 200 and 300 mg/animal and day) it could be clearly seen that the carotene concentration in the corpus luteum (2.3, 27, 50 and 81 micrograms/g) was directly dependent on it. On the other hand, the weight of corpus luteum (3.8 to 4.6 g) was not influenced (P greater than 0.05) by carotene supplementation. Apart from corpus luteum carotene was also stored in orange coloured pigment corpuscles of ovary. The carotene concentration of the pigments was 6 to 70 times higher (158, 150, 334 and 487 micrograms/g) than in the corpus luteum. Weights of the pigments rose with increased carotene doses. However, the weights amounted to merely 4 to 11% of the corpus luteum's mass. The amount of carotene in corpus lutea of animals added with carotene contained was higher than that of pigments. Further investigations are necessary to characterize the physiological importance of the pigment corpuscles.

The occurrence of endogeneous lectins in the ovaries of four fish species has been studied by indirect immunofluorescence staining with antibodies against individual lectins. Paraffin sections of the ovary of perch (Perca fluviatilis L.) were treated with an antibody against perch lectin. In cryostat sections of the tench (Tinca tinca L.) ovary, the L-rhamnose-specific lectin "I" was detected with a specific antibody. In cryostat sections of both roach (Rutilus rutilus L.) and rudd (Scardinius erythrophthalmus L.) ovaries, lectins were localized using a single antibody against roach lectin. The isolation of tench lectins is briefly described. In the fish species employed for this study, lectins are associated exclusively with the content and surrounding membrane of cortical vesicles situated within the cytoplasm of maturing oocytes. The positive reaction with lectin antibody was observed almost immediately after the formation of the first cortical vesicles in the peripheral cytoplasm of early previtellogenic oocytes. Their lectin content increases during the later stages when cortical granules fill the whole cytoplasm before moving towards the cell periphery, as the oocyte starts to accumulate yolk. The presence of lectins within cortical vesicles is significant also in view of the polysaccharide content of these structures. In the vitellogenic oocytes lectins seem to move towards the cell periphery and accumulate beneath the plasma membrane. Our observations are discussed in view of the present ideas on the intracellular function of lectins, and with respect to the role of cortical vesicles in fertilization and in post-fertilization modifications of the egg envelopes.

A pharmacokinetics and platinum concentration in the gynecologic organs were measured following the intravenous drip infusion of 375 mg/m2 of CBDCA. Total platinum (TP) in plasma decreased biphasically and free platinum (FP) was almost monophasically eliminated. The t 1/2 of FP was about 51 min. and the percentage of FP out of TP at four hours after administration was more than 92%, which was much higher proportion compared with that of cisplatin (CDDP). A cumulative excretion of platinum in the urine within 24 hours was 69%. The platinum concentration in the benign part of portio and endocervix were higher than that of malignant portion, although there were no significant differences in other pelvic organs such as ovary and tubes. However, pelvic lymph-nodes showed less platinum concentration both in metastatic and non-metastatic tissues. Platinum concentration in the tissue did not change significantly with lapse of time. Bone marrow suppression was observed especially in the patients who had received chemotherapy or radiotherapy. No renal toxicity was observed even without any hydration and gastro-intestinal toxicity was much milder than that of CDDP.

A histological and histochemical study of the ovary of Dysdercus koenigii has been carried out to ascertain the origin of protein, lipid and carbohydrate components of the yolk during vitellogenesis. Germaria of the telotrophic ovarioles of this insect comprise mononucleate and binucleate trophocytes, prefollicular cells and oogonia ensheathed in two membranes. The follicular epithelium derived from the prefollicular cells is multilayered in the beginning of the oocyte development but is progressively reduced to one layer in later stages. Chromatin material in the binucleate trophocytes condenses to form DNA-positive blobs. RNA-positive material flowing from the nutritive cords can be seen at the nutritive cord-oocyte junction. Protein bodies appear first in the intercellular spaces of follicular epithelium during the early vitellogenic stages indicating the supply of yolk protein from the haemolymph. Lipids originate as L1 bodies made up of unsaturated phospholipids in the nucleus (germinal vesicle). These bodies by addition of saturated triglycerides from haemolymph change to L2 bodies, which in turn, knock off phospholipid part and change of L3 bodies. Polysaccharides come as extrusions from the germinal vesicle while glycogen is first seen in the intercellular spaces of follicular epithelium and then passes into the oocyte indicating its origin in the haemolymph.

Oxytetracycline (OTC) was administered by constant IV infusion to 3 healthy postpartum cows at rates predicted to approach a steady-state plasma concentration of 5 micrograms/ml. After 8 hours of constant IV infusion, genital tissues were surgically removed. The mean plasma-to-tissue ratios of concentrations of OTC were 0.95, 1.33, 1.88, and 1.04 (2 cows only) for caruncles, endometrium, uterine wall, and ovaries, respectively. Differences between the ratios of any 2 of the uterine tissues were statistically significant (P less than 0.05). Intrauterine (IU) infusions of 5.5 mg of OTC/kg were administered to 3 healthy postpartum cows and 3 postpartum cows with metritis. The mean values of the fraction of the drug absorbed from the uteri of cows given IU infusions of OTC were 0.50 and 0.23 for healthy postpartum cows and postpartum cows with metritis, respectively. Concentrations of OTC were high in the caruncles and endometrium of all cows at 24 hours after IU infusions of the drug. Concentrations in the plasma, uterine wall, and ovaries were low, with mean concentrations of OTC in these tissues in postpartum cows with metritis being lower than those in the same tissues of healthy postpartum cows. Computer-simulated genital tissue concentrations of OTC after twice daily IV doses of 11 mg/kg indicated that this dosage regimen would provide postpartum uterine tissue concentrations greater than 5 micrograms/g throughout the dosage interval in all tissues, except the uterine wall.

This study demonstrates the reasonable reliability of fine needle aspiration cytology in ovarian tumors: 91% of benign and 84.5% of malignant tumors were diagnosed correctly by aspiration cytology. The concentration of estradiol-17 beta in cyst fluid may be helpful in distinguishing follicle cysts from neoplastic cysts. Cytologic differentiation of aspirated ovarian tumors was reasonably accurate. With respect to the approximately 1.6% of severe pelvic inflammations in our patients, we suggest that there are few indications for the use of aspiration cytology in ovarian tumors. In spite of a worldwide trend towards cytologic diagnoses by means of aspiration biopsy, there are still few studies on the aspiration biopsy of ovarian tumors. This is surprising because fine needle puncture of ovarian tumors is neither more trouble-some nor more complicated than the well-established cytologic biopsy of the prostate. This paper reports on the reliability of this method and its capability in the differentiation of various types of ovarian tumors. The differentiation of ovarian cysts is of particular interest.

The presence of binding sites for nonecdysteroid steroids was investigated in the cytosol of several tissues of the migratory locust, Locusta migratoria migratorioides. Binding of androgens was not observed. Most tissues, however, showed nonsaturable binding of estrogens and in some tissues saturable progestin binding could be demonstrated. A pregnenolone binder, that was found to be present in the male copulatory organ, was further studied. It showed a dissociation constant of 4.4 (+/- 1.6) X 10(-8) M. This is the first report of a nonecdysteroid steroid-binding factor in an insect tissue.

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed intermolecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.

A single dose of cadmium chloride (2.2 mumol/100 g of body weight) brought about in the sensitive KP strain alterations of the ovarian structure and disturbances in the ovarian cycle manifested by a prolongation of diestrus. Following cadmium administration and increase in the amount of atretic follicles, especially those showing stages 1I and 2II of degeneration, was observed in the ovaries of KP mice. The corpora lutea contained numerous degenerated cells and were infiltrated by abundant connective tissue cells. The used dose of cadmium chloride showed no influences upon progesterone level. The absence of changes in the ovarian morphology and in the duration of the oestrus cycle in CBA females suggest that this strain is resistant to cadmium chloride.

Paraffin sections of ovaries from 24 goats stained by Acetone Sudan Black B method for bound lipids and Copper-Phthalocyanin method for phospholipids revealed at least two categories of interstitial cells containing sudanophilic lipids with phospholipids. First category were those which were assumed to be formed from theca interna cells of atretic follicles. Second category included those originating probably from the granulosa cells of primordial follicles and form a continuous zone under the tunica albuginea. Another zone of interstitial gland cells presumed to be originated from the germinal epithelium has also been recognized under the latter. Appearance of sudanophilic material in relation to atresia of follicles in goat ovary has been reported.

Electron probe analysis by x-ray spectrometry is used in biology for simultaneous determination of the concentrations of any elements with a higher atomic number than that of carbon in single samples with volumes of 0.01 to 0.5 nl. In this technique, deposits prepared from identical volumes of biological fluids and standard solutions are totally covered by the electron beam, and the measured x-ray intensities for each element directly compared. The possibility of intensity quantification depends on the thinness of the dried deposits obtainable by various preparatory techniques. Factors affecting the accuracy of the results include droplet stability under the electron beam, the identity of the degree of oxidation of the elements in biological fluids and standards, sample mass thickness, beam voltage, and matrix effects. Minimum detectable concentrations in the 0.05 mmol.l-1 range are now achievable. This technique is the only one applicable in cases where available volumes are too small to determine the concentrations of several elements on the same sample (for instance of Na, Mg, S, P, Cl, K, Ca, Fe and Co), or even to determine the concentration of a single element (e.g. Mg). Although the droplet technique has so far mainly been used in renal physiology, it has also been applied in reproductive and digestive physiology. Isolated cells are analyzed according to the same principle of totally covering the cell by the electron beam. During the last decade, the vast increase in the relevant literature has testified to the contribution of the microdroplet technique to various fields of biology.

Relaxin was measured by radioimmunoassay in follicular fluid from sows which were of different reproductive states. In non-pregnant animals, follicular fluid relaxin values ranged from 0.23 to 666 ng/ml. For pregnant animals the range was 172.6-32 707 ng/ml and for sows with polycystic ovaries this range was 5.0-2800 ng/ml. For polycystic ovaries only there was a correlation (r = 0.74) between the diameter of the follicle and relaxin concentration in the respective fluid. Gel filtration species of relaxin, of molecular weights 27500, 19000 and 6000. Relaxin was extracted from 1 kg of ovaries collected from prepubertal pigs and gel filtration studies indicated the presence of large molecular weight species in addition to a molecule of approximately 6000. These experiments suggest that the corpus luteum is not the sole source of relaxin in the pig.

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2'-dAMP to inhibit the 3' leads to 5' exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.

New methods have been developed for the measurement of ATP, GTP, and UTP pools of microscopic cell colonies of 50-1000 cells. These methods are based on stoichiometric generation of ATP from ADP with GTP and UTP serving as phosphate donors, followed by measurement of ATP with firefly luciferase. This technique has been used to generate dose-response curves describing the effects of mycophenolic acid and pyrazofurin on individual colonies of Chinese hamster ovary cells.

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.

Rats were continuously exposed to constant activity of tritium in drinking water (HTO group) or to tritium organically bound in food (T-food group) in the period from conception of F1 generation through maturity. Female offspring were killed at the age of 21 and 71 days and the oocytes in their ovaries were counted. Mean dose rates absorbed in the ovaries were for the HTO groups 7.25 +/- 0.37 and 14.73 +/- 0.79 mGy/day and for the T-food group 4.84 +/- 0.25 mGy/day. Reduction in the oocyte number in the ovaries of females exposed to tritiated food was bigger than in the ovaries of females exposed to tritiated water. The dependence of the survival of small oocytes on the dose rate and the corresponding total accumulated dose had an exponential character. The damaging effect of tritium was for the period from conception to 21 days of age bigger than from 21 to 71 days of age. Of all stages of oocyte development, the highest sensitivity to tritium irradiation was observed in small oocytes and oocytes with one complete layer of follicle cells. As a result, relative number of growing and large oocytes increased.

The levels of ascorbic acid were measured in the tissue of 53 ovarian carcinomas and compared to concentrations in normal human ovarian tissue. The mean ascorbic acid concentrations per gram of tissue was 218 microgram in carcinoma tissue and 382 microgram in intact ovarian tissue. The difference was significant, with p less than 0.001. A relationship was found to exist between ascorbic acid concentrations in tumor tissue, on the one hand, stages of the disease, on the other. The importance of ascorbic acid to tumour growth is discussed.

Microvitellogenin, a female specific protein found in hemolymph and eggs of adult female Manduca sexta (tobacco hornworm moth) has been purified to homogeneity. It is a 31,000 dalton protein lacking covalently bound carbohydrate. It appears in the hemolymph near the time of adult eclosion. The appearance of microvitellogenin in adult hemolymph is not dependent upon the presence of juvenile hormone. Uptake of both vitellogenin and microvitellogenin are greatly reduced in the absence of juvenile hormone.

The ovarian ruptured follicles larger than 0.2 g. of wet weight were collected from laying hens and an ovarian oviposition-inducing factor (OOIF) was extracted from the follicles. The extract was chromatographed on a column of Sephadex G-75. Material from the active region for inducing premature oviposition was subjected to thioglycollate or trypsin treatment. It was found that oxytocic activity of this material was completely destroyed by thioglycollate buy not by trypsin, when tested on the contractility of the isolated hen's uterus. The OOIF was found to be diffusible through a cellophane bag. It is suggested that the OOIF may be a relatively small molecule have a disulfied bridge and it seems to be a hormone or hormone-like substance resembling the posterior pituitary hormones in the chemical nature.

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).