Christoph Kirchner

Untersuchungen an uterusspezifischen Glykoproteinen während der frühen Gravidität des KaninchensOryctolagus cuniculus

SummaryBetween the 3rd and 6th day post coitum the dry substance of uterine fluid contains nearly 60% peptide chains, on the 6th day 19 h p.c. only 35%. The remaining 40 and 65% resp. consist mainly of carbohydrates (cf. III.1). By means of disc electrophoresis, thin layer chromatography following hydrolysis and molecular sieve chromatography, we were able to characterize 4 uterine specific proteins, 2 of which are carbohydrate rich glycoproteins. Their relative concentrations were determined between 0 and 8 day p.c. (of. III.2): Glycoprotein I can already be found during oestrus. Its concentration increases slightly from the 3rd to the 5th day p.c., more rapidly up to the 6th day 6 h p.c., and decreases later (of.III.2b). The molecular weight is estimated to be around 50,000 (cf.III.4). Postalbumin as well as Uteroglobin can be found shortly after ovulation. Its concentration stays nearly constant between 3rd and 6th day p.c., and decreases during the 6th day p.c. (of.III.2b). Its molecular weight is in the same range as that of Uteroglobin, nearly 30,000 (of.III.4. Uteroglobin is most probably free of carbohydrates. Its concentration increases rapidly between 16 and 40 h p.c., slightly until the 5th day p.c., and decreases later (cf.III.2b). At 6th day p.c. about 50% of the peptide chains of uterine fluid are Uteroglobin (cf.III.2.g). Glycoprotein II is not secreted before the 6th day p.c. It has a comparativly high content of galactose (cf.III.3.d). Its concentration increases rapidly during the 6th day p.c. (cf.III.2.b). The moleculare weight is higher than 70,000 (cf.III. 4).These proteins and glycoproteins have also been found inblastocoelic fluid after the 5th day p.c. (cf.III.2.c).Aftersuperovulation induced by hormones, and natural mating, the pregnancy specific protein pattern is not fully developed (cf.III.2.d). Inpseudopregnancy, triggered by mating with vasectomized males, the same proteins as in normal pregnancy were found from the 6th day 6 h to the 6th day 19 h p.c. If no blastocysts are implanted, the protein pattern of the 6th day 19 h p.c. persists nearly unchanged until the 9th day p.c. (cf.III.2.e).Duringimplantation uterus and blastocyst lumens are filled with serum proteins, which tend to cover up the secretion proteins (cf.III.2.f).9 constituents of carbohydrates could be identified in uterine fluid. There was no evidence for freemonohexoses, particularly free glucose. Theoligomere carbohydrates were free or bound to proteins (cf.III.3.). We were able to separate Glycoprotein I, Glycoprotein II, Albumin + Uteroglobin with Sephadex G 150 and G 75 SF (cf.III.4).21 freeamino acids were found in uterine and blastocoelic fluid by means of thin layer chromatography. The concentrations of amino acids in uterine fluid were comparable to those of serum. The dry substance of uterine fluid contains more glycine, alanine, glutamic acid and aspartic acid than that of serum (cf.III.5).The possible role of amino acids in thenutrition of the blastocyst and the possible influence of uterine specific proteins and glycoproteins onnutrition and morphogenesis of the rabbit embryo were discussed.ZusammenfassungVom 3.–6. Tage post coitum enthält dieTrockensubstanz von Uterusflüssigkeit rd. 60% Peptidketten, am 6. Tage 19 Std p.c. nur noch 35%. Der Rest besteht vor allem aus Kohlenhydraten (s.III.1). Mittels Disc-Elektrophorese, Dünnschichtchromatographie nach Hydrolyse und Molekularsiebchromatographie ließen sich 4uterusspezifische Proteine — 2 davon sind kohlenhydratreiche Glykoproteine — physikalisch und chemisch charakterisieren und ihre relativen Konzentrationen vom 0.–8. Tage p.c. bestimmen (s.III.2): Glykoprotein I ist schon während des Oestrus zu finden. Seine Konzentration steigt vom 3.–5. Tage p.c. wenig, bis zum 6. Tage 6 Std. p.c. stärker an und nimmt später wieder ab (s.III.2.b). Sein Molekulargewicht liegt bei 50 000 (s. III.4). Postalbumin läßt sich ebenso wie Uteroglobin schon kurz nach der Ovulation nachweisen. Seine Konzentration ist vom 3.–6. Tage p.c. annähernd konstant und sinkt im Verlaufe des 6. Tages p.c. ab (s.III.2.b). Es hat ein Molekulargewicht um 30 000, ähnlich wie Uteroglobin (s.III.4). Uteroglobin ist vermutlich kohlenhydratfrei. Seine Konzentration steigt von 16–40 Std p.c. stark, bis zum 5. Tage p.c. gering an und sinkt danach wieder ab (s.III.2.b). Am 6. Tage p.c. beträgt sie rd. 50% (s. III.2.g). Glykoprotein II wird erst am 6. Tage p.c. sezerniert. Es ist gekennzeichnet durch einen hohen Galaktosegehalt (s. III.3.d). Seine Konzentration steigt im Verlaufe des 6. Tages p.c. stark an (s.III.2.b). Sein Molekulargewicht liegt über 70000 (s. III.4).Diese Proteine und Glykoproteine wurden ebenfalls in derBlastozystenflüssigkeit vom 5. Tage p.c. an nachgewiesen (s. III.2.c).Nach hormoninduzierterSuperovulation und natürlichem Deckakt bildet sich das uterusspezifische Proteinmuster nicht voll aus (s. III.2.d). BeiPseudogravidität, induziert durch den Deckakt mit vasektomierten Männchen, sind im Uterus in der Zeit vom 6. Tage 6 Std — 6. Tage 19 Std p.c. die gleichen Proteine vorhanden wie bei normaler Gravidität. Finden Implantationen von Blastozysten nicht statt, so bleibt das Proteinmuster des 6. Tages 19 Std p.c. bis zum 9. Tage p.c. hin fast unverändert erhalten (s. III.2.e). Im Verlaufe derImplantation werden Uterus- und Blastozystenlumen von Serumproteinen überflutet, welche die Sekretproteine weitgehend überdecken (s. III.2.f).In der Uterusflüssigkeit wurden 9 Kohlenhydratbestandteile identifiziert. FreieMonohexosen, insbesondere freie Glukose, konnten nicht nachgewiesen werden. Dieoligomeren Kohlenhydrate sind frei oder an Peptide gebunden (s. III.3). Mit Sephadex G 150 und G 75 SP ließen sich Glykoprotein I, II, Albumin und Uteroglobin + Postalbumin voneinander trennen (s. III.4).21freie Aminosäuren wurden mit der Dünnschichtchromatographie in Uterus- und Blastozystenflüssigkeit identifiziert und die der Uterusflüssigkeit im Vergleich zu Serum semiquantitativ bestimmt. Bezogen auf gleiche Mengen Trockensubstanz sind in der Uterusflüssigkeit vor allem höhere Anteile Glycin, Alanin, Glutaminsäure und Asparaginsäure als im Serum vorhanden (s. III.5).Die mögliche Bedeutung der Aminosäuren für dieErnährung der Blastozyste, wird diskutiert; ebenso der mögliche Einfluß der uterusspezifischen Proteine und Glykoproteine aufErnährung und Morphogenese des Kaninchenkeimes.Between the 3rd and 6th day post coitum the dry substance of uterine fluid contains nearly 60% peptide chains, on the 6th day 19 h p.c. only 35%. The remaining 40 and 65% resp. consist mainly of carbohydrates (cf. III.1). By means of disc electrophoresis, thin layer chromatography following hydrolysis and molecular sieve chromatography, we were able to characterize 4 uterine specific proteins, 2 of which are carbohydrate rich glycoproteins. Their relative concentrations were determined between 0 and 8 day p.c. (of. III.2): Glycoprotein I can already be found during oestrus. Its concentration increases slightly from the 3rd to the 5th day p.c., more rapidly up to the 6th day 6 h p.c., and decreases later (of.III.2b). The molecular weight is estimated to be around 50,000 (cf.III.4). Postalbumin as well as Uteroglobin can be found shortly after ovulation. Its concentration stays nearly constant between 3rd and 6th day p.c., and decreases during the 6th day p.c. (of.III.2b). Its molecular weight is in the same range as that of Uteroglobin, nearly 30,000 (of.III.4. Uteroglobin is most probably free of carbohydrates. Its concentration increases rapidly between 16 and 40 h p.c., slightly until the 5th day p.c., and decreases later (cf.III.2b). At 6th day p.c. about 50% of the peptide chains of uterine fluid are Uteroglobin (cf.III.2.g). Glycoprotein II is not secreted before the 6th day p.c. It has a comparativly high content of galactose (cf.III.3.d). Its concentration increases rapidly during the 6th day p.c. (cf.III.2.b). The moleculare weight is higher than 70,000 (cf.III. 4).These proteins and glycoproteins have also been found inblastocoelic fluid after the 5th day p.c. (cf.III.2.c).Aftersuperovulation induced by hormones, and natural mating, the pregnancy specific protein pattern is not fully developed (cf.III.2.d). Inpseudopregnancy, triggered by mating with vasectomized males, the same proteins as in normal pregnancy were found from the 6th day 6 h to the 6th day 19 h p.c. If no blastocysts are implanted, the protein pattern of the 6th day 19 h p.c. persists nearly unchanged until the 9th day p.c. (cf.III.2.e).Duringimplantation uterus and blastocyst lumens are filled with serum proteins, which tend to cover up the secretion proteins (cf.III.2.f).9 constituents of carbohydrates could be identified in uterine fluid. There was no evidence for freemonohexoses, particularly free glucose. Theoligomere carbohydrates were free or bound to proteins (cf.III.3.). We were able to separate Glycoprotein I, Glycoprotein II, Albumin + Uteroglobin with Sephadex G 150 and G 75 SF (cf.III.4).21 freeamino acids were found in uterine and blastocoelic fluid by means of thin layer chromatography. The concentrations of amino acids in uterine fluid were comparable to those of serum. The dry substance of uterine fluid contains more glycine, alanine, glutamic acid and aspartic acid than that of serum (cf.III.5).The possible role of amino acids in thenutrition of the blastocyst and the possible influence of uterine specific proteins and glycoproteins onnutrition and morphogenesis of the rabbit embryo were discussed.

Uteroglobin in the rabbit

SummaryThe localization and release of uteroglobin (UGL) were investigated immunohistologically in the oviducts and uteri of female rabbits from oestrus through the 7th day post coitum and the blastocyst on the 7th day post coitum.UGL was detected within Fallopian tube cells even during oestrus. Granules of UGL appeared toward the bases of these cells. Subsequently, the cells became almost entirely filled with UGL. Drop-like protrusions of the apical cytoplasm suggest a mechanism of apocrine extrusion. All stages of filling and extrusion were visible during the entire preimplantation period.During oestrus, synthesis of UGL within uterine cells becomes sufficiently advanced so that extrusion has either already begun or is about to begin. UGL positive material first appears in the supranuclear regions. Later the entire cytoplasm shows a positive reaction. An uneven distribution of UGL cells is observed in the endometrium. Since only the glands adjacent to the myometrium and cells of the cavum epithelium contain UGL, a striking mosaic of UGL positive and negative cells results.The present report is the first detecting UGL in single cells of the blastocyst. Both entodermal and ectodermal cells proved to be UGL positive. The synthesis and section of UGL in the oviduct and uterus and the possible origins of UGL in the blastocyst are discussed.

Uteroglobin and the accumulation of progesterone in the uterine lumen of the rabbit

SummaryThis study was undertaken to determine whether the influx of progesterone into the uterine lumen of the rabbit, in the preimplantation period, is dependent onuteroglobin (UGL). Rabbits were ovariectomized and, three months later, treated with two defferent doses of progesterone. Purified UGL was injected into one uterine horn and, as a control,immunoglobulin G (IgG) was injected into the other. After four days, the animals were sacrificed their uteri flushed, and the progesterone content of the washes was determined by radioimmunoassay.Animals with the lower serum progesterone level (2.8 ng/ml) had a significantly different uterine horn progesterone content. The hormone accumulation in the horn containing UGL was 2.3 to 7.5 times higher than in the horn containing IgG. Animals with a higher serum progesterone level (7.2 ng/ml) showed no differences. The hormone content was equally high in both horns, presumably due to the synthesis of endogenous UGL being reactivated by the hormone treatment.The validity of these experiments as models for the events during early pregnancy and the physiological role of progesterone available inside the uterus are discussed.

Progesterone dependent uptake of uteroglobin by rabbit endometrium

SummaryUnlabeled or3H-labeled UGL, isolated from rabbit lungs, was injected intraluminally into the uteri of ovariectomized low dose progesterone substituted and unsubstituted animals as well as into mated animals on the fifth day of pregnancy. It was offered to the endometrium, in this last case, in competition with endogenously synthesized UGL.3H-labeled IgG served as a control. The animals were killed 24 h after the experiment.Immunohistograms and autoradiograms showed that UGL was taken up by the whole endometrium of the ovariectomized animals but only when progesterone was present. In the preimplantation uterus, uptake was limited to clusters of smaller cells accumulated in the lumen adjacent to the epithelium, whereas IgG remained within the uterine lumen, forming a dense layer on the endometrial surface.

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

SummaryProteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.

Purification and immunohistology of a glycoprotein secreted from the rabbit uterus before implantation

SummaryTwo antigens of the β-glycoprotein fraction from rabbit uterine secretion of the seventh day post coitum were purified firstly by gel filtration on Sephadex G 150, then either by ion exchange chromatography on DEAE Sephacel or chromatofocusing on PBE 94. By the use of a specific antiserum, raised in female sheep, two antigens with α2- and β2-mobility in agar gel electrophoresis could be demonstrated. Immunohistochemical staining of the uterine epithelium at the seventh and eighth day post coitum showed the antigens to be localized in a ciliated cell type of conspicuous shape, which is supposed to be the site of synthesis. Stain accumulated mainly in the apical part of the cell, but there were also small deposits around the nucleus.

Elektronenmikroskopische Untersuchungen über die Blastozyste des Kaninchens vor der Implantation in bezug auf ihre Wechselbeziehung zur uterinen Umgebung

SummaryUltrastructure of the preimplantation rabbit blastocyst was studied in view of its ability to resorbe substances from the uterine environment resp. to transport them to the blastocoelic cave. The architecture of the blastocyst covering, the cell-to-cell contacts of the trophoblast and entoderm, and the unusual protuberance vesicles in the trophoblast are discussed concerning their possibly function.ZusammenfassungDie Ultrastruktur der Kaninchenblastozyste vor der Implantation wurde untersucht im Hinblick auf ihre Fähigkeit, Substanzen aus der uterinen Umgebung zu resorbieren bzw. sie in die Blastozystenhöhle zu transportieren. Der Aufbau der Keimhülle, die Zell-zu-Zell Kontakte im Trophoblast und Entoderm und die ungewöhnlichen Protuberanzvesikel des Trophoblasten werden unter dem Gesichtspunkt ihrer möglichen Funktion diskutiert.Ultrastructure of the preimplantation rabbit blastocyst was studied in view of its ability to resorbe substances from the uterine environment resp. to transport them to the blastocoelic cave. The architecture of the blastocyst covering, the cell-to-cell contacts of the trophoblast and entoderm, and the unusual protuberance vesicles in the trophoblast are discussed concerning their possibly function.

Immunohistological studies on the distribution of yolk proteins in the stick insect (Carausius morosus)

Abstract The path of yolk proteins through stick insect embryos at two developmental levels was studied immunologically. In 18-day-old embryos, all the blastema takes up the yolk proteins directly. In 30-day-old embryos, two transport systems are developed: embryonic contraction moves the yolk particles throughout the embryonic cavities, and amoeboid cells complete the yolk transport to its destination.

Untersuchungen über die Auflösung der Keimhüllen beim Kaninchen: Der Einfluß des uterinen Milieus

The effect of uterine environment on lysis of the blastocyst coverings in the rabbit was studied in vivo and in vitro. After transplantation of 4, 5, and 6 days post coitum old blastocysts in foster does, the stage of pregnancy of which differed ± 2 days from the corresponding age of the blastocysts and after in vitro culture of 4 day old blastocysts, we investigated histologically the protease activity and the structure of the blastocyst coverings. The experiments revealed that only an exact synchronous in vivo medium is capable of initiating lysis or of support started lysis, although growth of the blastocysts seemed to be normal in foster uteri as well as in culture dishes. A series of figures on structural changes of the blastocyst between the 4 and the 8 day post coitum is compiled.SummaryThe effect of uterine environment on lysis of the blastocyst coverings in the rabbit was studied in vivo and in vitro. After transplantation of 4, 5, and 6 days post coitum old blastocysts in foster does, the stage of pregnancy of which differed ± 2 days from the corresponding age of the blastocysts and after in vitro culture of 4 day old blastocysts, we investigated histologically the protease activity and the structure of the blastocyst coverings. The experiments revealed that only an exact synchronous in vivo medium is capable of initiating lysis or of support started lysis, although growth of the blastocysts seemed to be normal in foster uteri as well as in culture dishes. A series of figures on structural changes of the blastocyst between the 4 and the 8 day post coitum is compiled.