ウズラ卵管における卵外皮形成の組織学的研究

鳥類卵の卵外皮形成機構を明らかにする目的で,ウズラ卵管の峡部,峡子宮部,子宮部,および脱灰したウズラ卵卵外皮の組織学研究を行なった. 1. 峡子宮部には固有の粘液細胞が存在する.本細胞には脂蛋白が濃密に存在する. 2. ウズラ卵卵外皮はニワトリのそれと類似の層形成ならびに各層の構造を有する.卵殻乳頭は中心核と周辺部とに分けられる. 3. 中性粘液多糖類が卵殼膜,卵殼乳頭中心核,クチクラに,酸性粘液多糖類が卵殼乳頭,卵殼乳頭,卵殼基質にみとめられる. 4. 卵殼乳頭中心核には脂蛋白も豊富である. 5. 既報および本論文の成績に基づいて,卵管の分必物と卵外皮構成物質との相互関係を検討した.To clarify the formation of egg-coverings in birds, the isthmus, isthmouterine region and uterus of quail oviducts and their egg-coverings were investigated histologically. The obtained results are as follows. 1. Mucous cells proper to the isthmouterine region are termed as isthmouterine mucous cells. Their features are different from the isthmal and uterine mucous cells. The isthmouterine mucous cells are characterized by high stainability for lipoprotein reaction. 2. Stratification of decalcified quail egg-coverings and structures of each layer of them are similar to those of hen's eggs. The mammilla is divided into central core and superficial portion. 3. Neutral mucopolysaccharides are present in the shell membranes, central core of the mammilla and cuticle; acid mucopolysaccharides in the superficial portion of the mammilla and shell matrix. 4. The central core contains a certain amount of concentrated lipoprotein. 5. The author discussed the interelationship between the oviducal secretions and the constituents of the egg-coverings, on the basis of findings reported in previous and present papers

鶏の唾液腺ムチンの組織化学

　鶏の口腔に開口するいわゆる唾液腺はいずれも粘液性腺であるといわれている. しかしこの唾液腺粘液物質の性状についての報告は少ない.そこで鶏の唾液線ムチンを組織化学的に観察した. 　調べた9 種の唾液腺はすべて粘液性腺であり，粘液物質は酸性粘液多糖類の性状を示した.しかし腺により多少酸性粘液の性状が異なっていた.詳しくは，単口上顎腺，前舌腺，口角腺のムチンは主として非硫酸性粘液多糖類の性質を示し，口蓋腺，前および後顎下腺，蝶形翼状腺，後舌腺，輪状披裂腺のそれは主として硫酸性粘液多糖類の性質を示した.The mucin of the salivary glands of the chiken was examined histochemically. The results obtained are as follows. (1) In all the salivary glands, the contents of the glandular cell and the secretion in the glandular lumen were strongly periodic acid-Schiff reactive, azure A-metachromatic at a low value of pH, and alcianophilic, and showed affinity to various acid mucin stains. Methylation completely deprived the glandular cell of such properties as alcianophilia and metachromasia. Saponification after methylation almost completely restored those properties blocked by methylation. Digestion with such an enzyme as diastase, hyaluronidase, or sialidase failed to modify the staining properties of the glandular cell. The cell was almost negative for ninhydrin-Schiff and mercuric bromphenol blue reactions for protein. From the results of these histochemical reactions, it was concluded that the salivary glands of the chiken were all mucin-secreting ones, and that the mucosubstance contained in the glands was acid mucopolysaccharide in nature. (2) The acid mucin of the salivary glands was considered as compounds of nonsulfated and sulfated acid mucopolysaccharide. It was different in composition from one gland to another. The salivary glands were divided into two groups on the basis of this difference. The maxillary, anterior lingual, and angulus oris glands were composed mainly of nonsulfated acid mucopolysaccharide and classified into one group. The palatine, anterior and posterior submandibular, spheno-pterygoid, posterior lingual, and crico-arytenoid glands consisted mainly of sulfated acid mucopolysaccharide and formed the other group

鶏の卵管内の精子の分布と局在，特に膣腺における精子の貯蔵について

　鳥類は崎乳類と異なり交尾後かなりの期間卵管内に精子を保持していることはよく知られている.この研究は鶏の交尾後の卵管内の精子の分布と局在ならびに滞溜期間を卵管の切片標本によって組織学的に調べた.観察は受精後一定間隔毎の自然交尾および各種の方法による人工受精鶏の卵管によって行われた. 　調べた受精後約4週間以内の卵管30例はすべて精子を含んでいた.卵管内に多量の精子が長期間認められるところは卵管上部のカラザ部と卵管下部の腫起始部(腔関口より下方約1cmの間)の2部分であった.詳しい精子の位置はカラザ部では主に粘膜ヒダの間隙やカラザ分泌腺の腺腔であり，腫起始部ではこの部に特徴的に存在する大型の管状腺(股腺)の腺腔内であった. この所見は従来の卵管の内谷物からの塗抹標本による精子の分布の所見と一致するのみならず，さらに卵管の組織学的機造の面から精子の局在を明かにしたことになる. 　一般に腫腺がカラザ部より，より多量に且つ長期lζ 精子を保持していた. このことは従来の精子の貯溜郎と考えられていたカラザ部に対し，臆腺が一次的貯溜所の役割lを果しているように見える.The distribution and location of sperms in the fertile oviduct of the domestic fowl was observed. For this purpose, section preparations were made from the oviduct at varying intervals of time after the last mating or artificial insemination. The results obtained are summarized as follows. 1. Sperms were distributed constantly in abundance and observed for a long time after insemination both in the chalaziferous portion and at the beginning of the vagina. The sperms were actually located in the grooves of the mucosal folds and the chalaziferous glands of the chalaziferous portion and in the lumina of the vaginal glands at the beginning of the vagina. The chalaziferous portion had been described by VAN ORIMMELEN as location of sperms, but the vaginal glands were found by the present author as such for the first time. 2. The vaginal glands were large simple tubular ones, about 1 cm long, situated in the lamina propria of the mucosal folds, far from the vagi nal orifice. They see med to be special glands characterized by the presence of cholesterin-ester-like lipid. 3. More sperms were stored in the vaginal glands for a longer time than in the chalaziferous portion. Sperms were found in the chalaziferous portion 2 or 3 weeks after insemination and in the vaginal gland on the 27th day after insemination, although it was not clear whether they were active or dead. 4. The perms stored in the crypts of the chalaziferous portion were agglutinated in the depths of the grooves, forming bundle-like clump , shortly after insemination. They became scattered with the lapse of time after insemination. The sperms stored in the vaginal glands formed packed masses, which were not disintegrated for a Long time. 5. The sperms forming masses had generally a normal structure, but those distributed independently had an abnormal one, lacking tail or acrosome. 6. The migration of sperms into the vaginal glands was voluntary. This was confirmed by using various experimental methods of insemination. In other words, the vaginal glands seemed to have some factor which favored the survival of sperms. From these results, it is concluded that the vaginal glands may act as a preliminary reservoir for sperms until the sperms reach the infundibulum, where they are finally stored

鶏卵の卵殼形成の走査電子鏡検

鶏卵の卵殻形成の機構が,走査電子顕微鏡下で形態的に観察された. 卵管子宮部に下降した直後の卵は,卵殼膜のみに包まれた状態であった.卵殼の形成が始まると,まず卵殼膜表面に微細な粒子が沈着した.ついで卵殻膜の表面の所々に凝塊様隆起が現われた.この隆起は主として有機性物質より成り,石灰沈着の核のような作用をした.後にこの有機性基質の回りに石灰沈着が進み,乳頭状の突起となった.これが卵殼乳頭層の乳頭突起である.乳頭突起は次第に丘状に発達し,最後にはお亙いにゆ合して一層の石灰層,すなわち乳頭層となった. 乳頭層の形成後は,石灰沈着が乳頭突起の上に,卵殼表面に放射状に進み,卵殼海綿層が形成された.卵殼海綿層の形成は徐々に行なわれた.The morphological mechanism of the shell formation of hen's eggs was observed in developing eggs in the uterus by scanning electron microscopy. The shell formation was started in the egg just after the egg had arrived at the uterus. The first indication of the shell formation was the deposition of small sand-like granules on the shell surface. The subsequent indication of the shell formation was the appearance of small-sized organic protrusions on the shell surface. These protrusions increased in size as the shell formation advanced and formed nuclei of calcification. The deposition of calcium salts progressed around the organic protrusions, and mammillary knobs were formed. Previous organic concretions were encrusted by the deposition of calcium and became a central organic core, which was located at the center of the mammillary knob. Mammillary knobs were first small in size and later increased in size progressively. At last, they fused with one another to form a mammillary layer. After the formation of the mammillary layer, a spongy layer developed successively on the mammillary knob. It became thick with additional deposition of calcium salts in a radial direction

ニワトリとウズラの卵管子宮部の螢光色素

　Porphyrin が広く鳥類の卵の外皮に分布することは明らかにされているが，卵管における形成と卵への沈着の機序は明らかでない.別報で著者らは，卵の外皮の本色素の分布を示したが，今回は卵管の本色素の分布を検索した.材料としてウズラ，ニワトリ(ロード・アイランド・レッド，白色レグホーン)を用いて，次の結果を得た. 　肉眼的には，各卵管の子宮部粘膜に特異的な禍色の帯色をみとめ，それと一致して赤色~桃色螢光がみとめられた.これらの性質から， porphyrin 色素が分布していることは明らかである.その色調と螢光はウズラで著名であるが，ニワトリでは淡く弱い. 　子宮部組織の凍結切片の観察では， 3種の烏の各組織で粘膜上皮に限って赤色~桃色螢光がみとめられるが，ウズラで最も著名である.該上皮には，卵殻形成時のウズラで褐色色素穎粒が容易にみとめられるが，他のものでは検出し難い.何れの卵管でも，子宮腺には色素穎粒はみとめられない. 　子宮部のパラフイン切片での詳細な観察では，ウズラとロード・アイランド・レッドで粘膜上皮に色素穎粒がみとめられるが，白色レグホーンではみとめられない.子宮腺lこは，何れでも色素穎粒がみとめられない.Macroscopical and microscopical observations were made on the oviducts of the Rhode Island Red, White Leghorn and quail, with special reference to porphyrin pigments. The results obtained are summarized as follows. Macroscopically, the mucous membrane of the uterus in each bird was colored in various brown and fluoresced red to pink. The brightness of fluorescences was related with the depth of the colorations, and they were distinct in the quail uteri, showing dark brown coloration and red fluorescence, and were moderate, showing light brown and pinkish fluorescence, in the Rhode Island Red and White Leghorn uteri. In frozen sections, distinct fluorescence and collected brown pigment granules were observed in the mucous membrane of the uterus in some cases of the quails. In the uteri of the others, weak fluorescences were observed, but the pigment granule was unobservable. Through the careful searching in the paraffin sections, the pigment granules were revealed in the mucous epithelium of the quail and Rhode Island Red uteri. Neither the fluorescence nor the pigment granule was found in the uterine gland in each material. From these findings, it was concluded that fluorescent porphyrin pigments were distributed in the mucous epithelium of the uterus in the quail, as well as the Rhode Island Red and the White Leghorn, although "pigment granule" was invisible in the sections of the White Leghorn uteri

うずら卵管の組織学的研究 : 子宮部の粘膜上皮の分泌物について

　前報(1965) では，うずら卵管子宮部の粘膜上皮の繊毛細胞(apical cell) の色素(porphyrin) について 報じたが，子宮部粘膜上皮と卵形成との関係を明らかにするために，その粘膜上皮を構成している繊毛 細胞(apical cell) と粘液細胞(basal cell) のそれぞれの分泌物について，組織化学的に観察し，またこれ らの分泌像が卵の形成の各時期でどのように変化するかについて検索した. 　該上皮の分泌物は， apical cell における色素顆粒(porphyrin) およびPAS 陽性顆粒，basal cell におけ る塩基性粘液穎粒である.色素顆粒は蛋白反応にのみ陽性で，色素蛋白であること， PAS 陽性顆粒は中 性粘液多糖類一蛋白複合体であること，粘液頼粒は酸性粘液多糖類一蛋白複合体であることがそれぞれ 示された. 　一方各時期における分泌像の変化については， basal cell は，峡部に卵殻膜で包まれた軟卵がある時期 に多量の粘液を充し，ついでその卵が子宮部に至ると，卵殻形成以前に粘液を放出する. apical cell の 両頼粒は常に相平行した関係を示し，卵が子宮部にあって卵殻が形成されている問に顆粒は著しく増加 し，卵殻完成に伴なって放出される. 　これらのことから， basal cellの粘液顆粒は卵殻の有機基質の形成に，apical cell の色素顆粒および PAS 陽性顆粒はクチクラ形成に関係するものと考えられる.In this investigation, the observations were made on the secretion materials in the surface epithelium of the quail uterus, with reference to their histochemical properties and their quantitative changes related to the egg formation. These results are summarized as follows: 1. The mucous surface epithelium in the uterus of the quail consisted of the apical ciliated cells and the basal mucous cells as described in the domestic fowl by RICHARDSON (1935) and JOHNSTON et al. (1963). 2. Three types of secretion materials were found in the epithelium; the pigment granules (porphyrin) and the PAS-positive granules in the apical cells and the basophilic mucin granules in the basal cells. 3. Histochemically, the pigment granules were presented as pigment protein, the PAS-positive granules as neutral mucopolysaccharide-protein complex and the mucin granules as acid mucopolysaccharide-protein complex. 4. The PAS-positive granules and the pigment granules showed parallel appearances in their quantitative changes in the cells; they were maximum in the stage of the shell formation, then released into the uterine lumen to form their mingled structures, which were considered as the cuticular material. 5. The mucin granules in the basal cells were maximum in the stage of shell-membrane formation, and then released into the lumen, prior to the pigment and the PAS-positive granules. They were considered as related to the formation of the organic matrix of the shell

ニワトリとウズラの卵の外皮における螢光色素(Porphyrin)

　Porphyrin色素が広く烏類卵の外皮に分布することは，化学的には明らかにされているが，それらの外皮各層(クチクラ・卵殻・卵殻膜)における分布は，形態学的には明らかにされていない.また，本色素の卵管での形成ならびに卵への沈着の機序も明らかにされていない. これらの解明の手がかりとして，ウズラとニワトリ(ロード・アイランド・レッド，白色レグホーン)の卵を用い，外皮各層の本色素分布を検索した. 　各卵は，クチクラと卵殻に本色素分布を示したが，その量と分布様式には種類によって差異がみとめられた.すなわち: 　ウズラ卵では，自然状態で，クチクラにおける色素分布が著明で，卵殻はほぼ白色ないし微青色にすぎない. しかし脱灰することによって，かなりの色素が卵殻に分布していることが示された. 　ロード・アイランド・レッド卵では，自然状態で，クチクラと卵殻にそれぞれ中等度の着色がみとめられる.脱灰した卵殻では，その色調はウズラ卵殻より濃い. 　白色レグホーン卵では，外皮が極めて淡く着色するか，あるいは白色である.自然状態では，前者はクチクラと卵殻に淡い若色を示し，後者では， クチクラは透明に近く，卵殻は白色である. しかし詳細に検査して，両者ともクチクラと卵殻に微量ながら螢光色素の分布がみとめられた. 　3種の卵の卵殻膜には，各卵についての検査では，色素の分布は明らかでない.The distribution of fluorescent pigments in the coverings of the Rhode Island Red, White Leghorn and quail eggs were morphologically examined. Fluorescences and coloration were observed in the cuticles and shells of these eggs. The strength of fluorescences was in parallel with the depth of colorations. The pigmentation was conspicuous in the quail eggs, moderate in the Rhode Island Red eggs and scarce in the White Leghorn eggs. Differences in the modes of pigmentation were observed according to the eggs of the different kinds of birds; as to the quail eggs, it was more prominent in the cuticle than in the shell, as to the Rhode Island Red eggs, almost the same and medial in the above both, and as to the White Leghorn eggs, scarce in the above both. Pigmentation of the shell-membranes was not clearly observed. It was concluded that, porphyrin pigments characterized by red fluorescences were distributed, at least, in the shells and cuticles of these eggs

Scanning electron microscopy of the hen's egg shell

The structure of the hen's egg shel1 has been examined by many researchors under the optical microscope, polarization microscope, and electron microscope. In the present study, the structure of the egg shell of the White Leghorn hen was examined under the scanning electron microscope. Completed eggs just after oviposition were used in this study. They were broken mechanical1 y with a knife, and the inside was washed shortly with a physiological saline solution. The shells were fixed in a 10% formalin solution. After fixation, they were calcified by 3% glacial acetic acid. They were also dipped into 30~35 % sodium hydroxide to remove organic substances from them. The resulting materials were coated with gold or aluminum, and examined under the scanning electron microscope, Type JSM-2 (Japan Electron Optics Laboratory, Ltd.), at 25 KV accelerating voltage. As is already known, the avian egg shell, from inside to outside, consists of the inner shell membrane, the outer shell membrane, the mammillary layer, the spongy layer, and the cuticle. By scanning electron microscopy, the inner shell membrane was divided into two layers, an inner and an outer layer. The inner layer of the inner shell membrane had an amorphous granular appearance and contained fine fibers. The outer layer of the inner shell membrane was formed by a meshwork of fibers. These fibers were 1~2μ in width and run parallel to the surface.They were surrounded by dusty substances that made their demarcation somewhat unclear. The outer shell membrane was formed by a relatively dense meshwork of fibers. These fibers were 3~4μ in width and looked like tapes. They anastomosed with one another in such manner as to form knots. The outer surface of the outer shell membrane was not so sharply defined. Some of the fibers entered the calcified part of the mammillary knobs. The mammillary layer was made of numerous conical mammillary knobs (mammillae) arranged regularly with their base on the spongy layer. The calculous mammillary knobs from which organic substances had been removed were 50~60μ in diameter at their base, and the diameter diminished toward the top. The top of each knob was somewhat flattened, and the center was crateriform (the central depression). Several small canals were open at the bottom of the central depression. The border of the central depression and the slope of the protrusions were indented sharply with many sulci. The individual knob had a large cavity, whose long axis was perpendicular to the surface. The cavity was filled with organic matrix of the natural egg. The mammillary layer changed gradually toward the spongy layer. The spongy layer was densely calcified, yet the calcification was denser near the outer surface than in the deeper portion of the layer. On the other hand, the shell layer was constructed with dense and coarse calculous columns, which were arranged radially to the surface at regular intervals. In the clacified shell, these matrix fibers were 1~2μ in width and gave off fine branches. There was anastomosis among neighboring branches. The outer surface of the shell was not smooth, and had a strikingly convexo-concave appearance. The concave area of the surface had contained air pores. Some air pores were open in the convex area. The air pores penetrated the spongy layer and opened at the interspace between the mammillary knobs. The cuticle was a thin structureless membrane about 10μ thick

うずら卵管の組織学的研究 : 腺および粘液細胞の分泌物の組織化学的研究

　鳥類卵管における卵の形成に関しては多くの研究があり，卵管各部と卵の各構成要素との関係はほぼ明らかにされているが，細部については不明な点が残されている. したがって，卵の形成に関係すると考えられる腺と粘液細胞の分泌物について組織化学的観察をおこない， より詳細に卵の形成について明らかにしようと試みた.本報ではそれらの基本的所見を記載した. 　うずら卵管における腺および粘液細胞の分布は，にわとりのそれらに類している. 腺で分泌顆粒のみとめられるのは，漏斗部後位，膨大部，峡部前位の各部のものであった.粘液細胞の発達と分布から，粘液の放出量は，膨大部，峡部，漏斗部，子宮部，腟部の順と考えられる. 　粘液は，漏斗部，膨大部，子宮部では硫酸基を含む酸性粘液多糖類，峡部，腟部では，中性粘液多糖類の性質を示した. これらの粘液物質の蛋白は，漏斗部，膨大部では乏しく，峡部，子宮部ではかなり多いことが示されたが，各部の粘液には，シスチンが合まれていることが示された. 　各部の腺の分泌顆粒は，いずれも中性粘液多糖類ー蛋白複合体であることを示したが，特異的に峡部前位の腺では，その顆粒にシスチンがみとめられた.Histochemical observations were made on the secretion materials of the mucous cells in the surface epithelium and the glands of the oviduct of the quail. The results obtained are summarized as follows: 1. The histological features were almost similar to the findings on the domestic fowl described by BRADLEY (1928), RICHARDSON (1935) and FUJII et al. (1965). However, the secretion granules were observed in the glands in the posterior infundibulum, magnum and the anterior isthmus. In other glands including so-called chalaziferous gland (RICHARDSON, 1955), secretion granules were not found. 2. From the view of the reactions for the mucopolysaccharides, it was concluded that the mucous cells in the infundibulum, magnum and the uterus contained sulfated, acid mucopolysaccharides, and on the contrary to these, in the isthmus and the vagina neutral mucopolysaccharides. 3. The protein content in the mucosubstances of the mucous cells were observed scanty in the infundibulum and the magnum, and somewhat rich in the isthmus and the uterus. As a specific amino-group, the positive reaction for cystine or cystine-compounds was showed in the mucosubstances in every region. 4. The secretion granules of the glands in the posterior infundibulum, magnum and the anterior isthmus showed several common characters of polysaccharideprotein complex. Specifically, only the secretion granules of the gland in the anterior isthmus presented positive reactions for cystine or cystine-compounds

Scanning Electron Microscopy on the Structure of Abnormal Hen's Eggshell

本研究は数種の異常卵殻の微細構造を走査電子顕微鏡下で調べた。観察は10％硫化ソーダで卵殻から有機質を除いた試料で行った。主な所見は以下のとおりであった。 (1) 皺状卵殻(corrugated shell)の卵殻皺の隆起部の構造は正常であったが,隆起部の間の溝状部では乳頭層の異常が顕著であり,同時に海綿層の形成不全を伴っていた。 (2) 疣状卵殻(pebbly-textured shell)は2型に大別された。一つは外卵殻膜の上に有機性凝塊が沈着し,これによって卵殻石灰質層が隆起したものであった。卵殻乳頭層に有機性凝塊に対応する大きさの陥没部が形成されており,この部の乳頭突起は異常であった。他の一つは卵殻海綿層に有機物凝塊が沈着し,これによって海綿質が隆起したものであった。海綿質層に有機物凝塊に対応する空洞が形成されていた。両者いずれの場合にも隆起部の卵殻質は粗雑であった。 (3) 斑状卵殻(mottled shell)は卵殻構造は正常であったが、卵殻質は卵殻表層と海綿層が散在的に粗雑であった。 (4) ヒビ割れ卵殻(checked shell)は乳頭層に裂け目を生じていた。裂け目は海綿層の深層に達していたが,上層では完全に修復されていた。 (5) 薄殻(thin shell)は2型に大別された。一つは海綿層の形成過程に産出されたものであり,粗雑な卵殻質を有するものであった。他の一つは卵殻質は緻密であるが,乳頭層に欠陥があって海綿層が正常の厚さに達し得ないものであった。 (6) 軟卵殻(soft shell)は乳頭層の形成過程において放出されたものであり,発達中の乳頭突起だけからなっていた。 (7) 外観的異常を伴う卵殼は共通的に乳頭層が異常であった。乳頭層の異常は乳頭突起の乳頭核の異常に起因していた。The fine structure of various abnormal eggshells of hens were examined under scanning electron microscope. The findings are as follows. 1. Corrugated shells had remarkable and specific abnormalities in their structure. The protruded area of wrinkles was normal as for its structure and shell texture, whereas the concaved area between the wrinkles was particularly thin due to the deficiency of the spongy layer. The mammillary layer just beneath the protruding wrinkles was almost normal in structure, whereas the one of the concaved area showed no complete formation. 2. Rough and pebbly-textured shells were classified into two types. One had prominences which were made by the deposition of foreign organic masses on the shell membrane in the early stage of the formation of the mammillary knobs. In this case, various sizes of craters, in which the formation of the mammillary knobs had been extensively disturbed, were formed in the mammillary layer. Other prominences were made by the deposition of similar objects on the fairly developed spongy layer. In this case, no abnormalities of the mammillary layer were detected. 3. Mottled shells had a highly rough and very porous shell texture, particularly in the superficial layer of the shell and the spongy layer around the air pores. The remainder of the shell was normal. 4. Checked shells had internally streak-like clefts in the mammillary layer, these extended into the deep layer of the mammillary layer. These cracks had not been sealed by the addition of calcified material, so that they must have been formed in the early stage of the shell formation. 5. Thin-shelled eggs were divided into two types. One was the thin shell with large air pores during its formation and with a porous shell texture. They were expelled before the full formation of the spongy layer. Another type was the thin shell which did not develop to normal thickness due to its incomplete mammillary layer. 6. Soft-shelled eggs had only the developing mammillary knobs on the surface of the shell membrane. They were expelled prematurely in the course of the formation of the mammillary layer. 7. From the above results, it is considered that the abnormality of eggshells is caused mostly by the incomplicity of the mammillary knobs, and that the formation of the mammillary knobs is influenced by the degree of normality of the shell membrane formation