The dynamic structure of rabbit blastocyst coverings

Summary The neozona is the innermost layer of the complex blastocyst coverings of the rabbit and is located between the mucoprotein layer and the trophoblast. As shown previously the neozona begins to replace the zona pellucida from the 4th day post coitum (d p.c.) on (Denker and Gerdes 1979). In the present study, rabbit blastocyst coverings were checked for regional differences in their composition, comparing the embryonic and the abembryonic pole of the blastocyst, at 5 and 6 d p.c. These two stages were chosen because at 5 d p.c. a complete trophoblast layer is still present at both the embryonic disc (Rauber's layer) and the extraembryonic regions (mural and abembryonic pole trophoblast), whereas at 6 d p.c. Rauber's layer has largely degenerated. Correlation of regional differences in blastocyst coverings structure with presence or absence of an intact trophoblast is taken as suggestive evidence for a role of the trophoblast in the formation or the structural modification of blastocyst coverings components.Blastocysts of both stages were fixed in glutaraldehyde with and without ruthenium red and processed for TEM. The neozona was found to be almost equally well developed in all regions at 5 d p.c. On contrast, at 6 d p.c. (Rauber's layer defective) the neozona is consistently found to be much thinner at the embryonic disc than in the extraembryonic regions where the trophoblast is still intact. This is the first report on regional differences of the structural composition of blastocyst coverings within the same blastocyst. It is interpreted as evidence for a physiological role of the trophoblast in the formation of the neozona. The biochemical nature of the putative trophoblastic factors involved is still to be identified.     

The dynamic structure of rabbit blastocyst coverings

Summary The extracellular coverings which surround rabbit blastocysts are far more complex structures than the zona pellucida of other species. Since previously published views of their composition, structure and identification of the various layers are highly controversial, a detailed investigation of the stages between morula and implantation has been undertaken using both electron microscopical and histochemical methods.Rabbit blastocyst coverings undergo considerable structural and chemical transformation from the early until the late blastocyst stages. Morulae are surrounded by zona pellucida and mucoprotein layer (a highly sulfated, sialic acid-free mucosubstance which is derived from the tubal secretion). In the early blastocyst (i.e. from 31/2 d p.c. on), the zona loses its high content of periodate-accessible vicinal hydroxyl groups (PAS reaction) and protein, and there is morphological evidence for an erosion (particularly from the inside), suggesting enzymatic lysis in addition to mechanical stretching due to the expansion of the blastocyst. The zona pellucida disappears completely at 41/2 d p.c. At the same time, deposition of new material begins in its place. This newly formed layer will be called neozona. Until implantation, it increases considerably in thickness, finally representing between 2/5 and nearly 1/2 of the total thickness of the blastocyst coverings. Histochemically, the neozona is characterized as a moderately acid mucosubstance, rich in protein and in periodate-accessible vicinal hydroxyl groups, containing sulfate esters as well as sialic acid. Its chemical composition is in many respects comparable to that of the zona pellucida. At least part of the neozona material may be derived from the trophoblast. This new aspect of the physiology of the preimplantation trophoblast, i.e. its secretory activity, is discussed. The possibility that uterine secretion components are also involved in formation of the neozona (as well as in dissolution of the zona pellucida) is envisaged. Observations suggesting a chemical modification of the inner parts of the mucoprotein layer and impregnation of this layer with sialic acid-containing glycoproteins are also discussed.An additional layer, the gloiolemma, which derives from the uterine secretion, is deposited at the outer surface of the mucoprotein layer after 6 d p.c. At the onset of implantation, i.e. around 7 d p.c., rabbit blastocyst coverings are, therefore, composed of three layers of different origin: neozona, mucoprotein layer and gloiolemma.Abbrevations d p.c. days post coitum - EM electron micrograph - G gloiolemma - HgBPB Hg-biomophenol blue staining - HNA hydroxynaphthaldehyde reaction - M mucoprotein layer - N neozona - P perivitelline space - PAS periodic acid — Schiff reaction - T trophoblast - Z zona pellucida     

Shedding of the zona pellucida in normal pregnancy and in various hormonal states in the mouse

Summary Blastocysts were rinsed out of the uterus by glutaraldehyde on Day 4 and 5 in normally pregnant and in ovariectomized, hormone-treated mice and subsequently prepared for and examined by scanning electron microscopy (SEM).The zona pellucida surface showed no differences in early and late stages, either in normal pregnancy or after ovariectomy and administration of ovarian hormones. In all types of treatment, hatching of the blastocyst from the zona was observed.The shedding process was oestrogen-dependent and the zona-shedding rate of normal pregnancy could be provoked by 0.01 or 0.1 g oestradiol-17 given either late on Day 3 or early on Day 4 but not early on Day 3. A subimplantation dose of oestrogen (0.001 g) did not seem to influence the shedding process, nor did 0.1 g if pretreatment with progesterone was omitted after ovariectomy.The zona-free blastocyst in normal pregnancy usually showed signs of abembryonic proliferation and sometimes detachment injuries in the abembryonic pole, indicating incipient implantation. Similar appearances were noted in blastocysts from progesterone-maintained, ovariectomized mice given 0.01 or 0.1 g oestradiol-17 early on Day 4 but not on Day 3. In progesterone-maintained, ovariectomized mice given the above oestrogen doses on Day 3, the zona-free blastocysts were similar to those observed in corresponding animals not treated with oestrogen, i. e. without signs of incipient implantation at any stage. A subimplantation dose of oestrogen (0.001 g) had no visible effect on the surface of the zona-free blastocyst.It is concluded that hatching occurs under normal conditions and can be provoked by various hormonal treatments. Further, the location or maturity of the morulae may account for the lack of oestrogen response in zygotes early on Day 3. Since a zona-shedding curve close to the normal one was obtained after oestrogen treatment that did not induce implantations, it is inferred that the oestrogen surge may not constitute a discrete peak.     

Ultrastructural studies of the temporal relationship between loss of zona pellucida and appearance of blastocyst-induced stromal changes during normal pregnancy in rats

Summary Ultrastructural studies were undertaken to investigate the temporal relationship between loss of the zona pellucida around the blastocyst and the appearance of decidual changes in the endometrial stroma during normal implantation in rats. Blastocyst-free and blastocyst-containing sites of pregnant uterine horns were studied and compared with control sites from contralateral salpingectomized horns and horns of pseudopregnant animals from 24.00 h on Day 4 and onwards. There were no membrane contacts between the blastocyst and the uterine epithelium at 10.00 h on Day 5 and earlier because of an intervening zona pellucida. From 14.00 h onwards, however, such contacts were present and at 18.00 h, the zona pellucida had disappeared and the blastocyst had attached onto the uterine epithelium.The stromal cells of pregnant and control horns were indistinguishable from each other at 24.00 h on Day 4, but from 06.00 h on Day 5 onwards specific changes were noted in the stromal cell nucleoli of the pregnant horns. The results therefore suggest that the first morphological sign of decidualization occurs about 12 h before the Pontamine Blue reaction and is initiated by the blastocyst early on Day 5 while it is still encased by the zona pellucida.Supported by grants from the Swedish Medical Research Council, Project No 12X-70 to Prof. O. Nilsson     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

囊胚序贯培养在体外受精-胚胎移植中的应用

目的 探讨囊胚序贯培养系统在体外受精-胚胎移植技术（IVF—ET）中的临床应用。方法 进行了17个周期的囊胚序贯培养，将胚胎体外培养至第5—6天，达囊胚期后移植。结果 共取卵250枚，受精率68％，囊胚形成率为52％，囊胚种植率为28％，获8例临床妊娠。结论 序贯培养囊胚移植更适合卵子和胚胎的体外发育，对提高临床妊娠率，降低多胎率有良好的应用前景。     

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium

Summary Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of ³⁵S-methionine and ³H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.     

Uteroglobin in the developing rabbit conceptus in vivo and in vitro

Summary Uteroglobin (UGL) was measured in day- 4 to day-10 rabbit conceptuses by a competitive ELISA. Levels in blastocyst fluid, tissues, coverings and in the early fetus were determined separately. The total amount of UGL increased from 18.4 ng to 6.8 g per conceptus. The UGL content of individual day-6 blastocysts was studied in vitro. Culturing was carried out up to 60 h in Ham's F10 medium with polyvinylpyrrolidone as macromolecular component, with and without progesterone, and with progesterone plus estradiol. UGL was determined in the blastocyst fluids, tissues with coverings and in the culture media. After labelling with [³⁵S]-methionine, protein patterns of total blastocysts and of culture media were analysed by two-dimensional gel electrophoresis and fluorography. The morphology of cultured blastocysts was examined by electron microscopy. During 60 h of culture, the blastocysts expanded in diameter by 84%, and released 19% of their initial UGL content into the medium, independent of the hormonal substitution. Neither de novo synthesis, nor degradation of UGL was found: the protein remained unlabelled in fluorography, and its total quantity was not significantly different from that of non-cultured controls. Trophoblast, endoderm and embryoblast cells showed well preserved cell organelles and intercellular junctions, while the morphological differentiation of the germ layer was inhibited.     

CpG ODN对ZP~(121-140)表位合成肽诱导的免疫应答和免疫避孕效应影响

目的:探讨CpG ODN对透明带(四)抗原表位ZP~(121-140)合成肽诱导的免疫应答和免疫避孕效应影响.方法:应用人工合成ZP2~(121-140)表位肽,与20μg CpG ODN或等量完全弗氏佐剂(CFA)混悬后左胫前肌免疫雌性BALB/c小鼠,初次免疫后2、4、6周各加强免疫一次,共免疫四次.在每次加强免疫前和末次免疫后两周断尾取血,分离血清,分析特异性lgG抗体及非特异性细胞因子IFN-γ、TNF-Ⅱ,IL-10水平;收集小鼠阴道冲洗液,离心取上清,分析特异性IgA抗体水平.免疫小鼠生育实验结束后,取卵巢组织行病理学分析.结果:CpG ODN组诱导的特异性IgG和IgA水平较CFA组有增高趋势,但无显著性差异(P＞O.05).CpG ODN组诱导的非特异性IFN-γ和TNF-水平明显高于CFA组(P＜0.05);而CpG ODN组诱导的IL-10水平显著低于CFA 组(P＜0.05).两种佐剂对小鼠的受孕率影响没有显著性差异,但CpG ODN组孕鼠每胎产仔数明显低于CFA组(P＜O.05).病理学分析显示实验小鼠卵巢组织无病理性变化.结论:CpG ODN对Zp~(121-140)合成肽诱导的免疫应答和免疫避孕效应略优于CFA,更适合用于避孕疫苗佐剂研究.     

激光辅助在囊胚玻璃化冷冻及复苏过程中的合理应用

目的探讨激光辅助在囊胚玻璃化冷冻和复苏过程中的合理应用及其在囊胚玻璃化冷冻效果和移植后妊娠率提升中所起的作用。方法囊胚玻璃化冷冻前行激光辅助皱缩使囊胚腔脱水;囊胚复苏后于内细胞团扩张前行激光辅助孵化。统计囊胚复苏后存活率、2h完全扩张率、孵出率及移植妊娠率。结果囊胚复苏238例,移植237例,平均移植囊胚1.87个,妊娠145例,临床妊娠率61.18%;共解冻囊胚445个,存活率99.78%,培养2h完全扩张率99.78%,孵出率68.1%。结论冷冻前行激光辅助皱缩对囊胚玻璃化冷冻的成功是必要的,可以避免内细胞团损伤,保证复苏后高存活率;囊胚复苏后在内细胞团扩张前行激光辅助孵化安全、高效,克服了囊胚孵出障碍,对提升妊娠率有帮助。     

D5和D6冻融囊胚移植的比较

目的分析D5和D6冻融囊胚移植的结局。方法 D5和D6囊胚利用玻璃化冷冻法分别冷冻,待患者子宫内膜达到7～8mm时,以第5天为移植窗,复苏2h后移植。结果 D5冷冻囊胚复苏后的存活率（99.02%）与D6冷冻囊胚复苏后的存活率（98.51%）没有差异,D5冻融囊胚移植的临床妊娠率（62.20%）和种植率（40.69%）略高于D6冻融囊胚移植的临床妊娠率（57.40%）和种植率（36.87%）,但没有显著差异,而D6冻融囊胚移植后的流产率（18.75%）显著高于D5（10.77%）（P〈0.05）。结论 D5冻融囊胚移植能够提高新生儿出生率。     

Human blastocyst culture and derivation of embryonic stem cell lines

Human embryonic stem cell (hESC) biology is expected to revolutionize the future of medicine by the provision of cell-based therapies for the treatment of a variety of deliberatig diseases. The tremendous versatility of hESCs has reinforced this hope. To understand the biology of these my sterious cells and attempt to differentiate them into desirable tissues, bona fide hESCs that maintain their stability with time are required for research and clinical application. This review discusses the various protocols to derive and propagate hESCs from high quality embryos. The nature and properties of hESCs are also described together with unanswered questions that need to be addressed if this science is to be taken to the bedside.     

Infertility observed in female rats treated with N-acetyl-L-cysteine: Histopathological examination of ovarian follicles and recovery of fertility

ABSTRACT  We previously reported infertility in female rats that received N-acetyl-L-cysteine (NAC) intravenously at a dosage of 1000 mg/kg/day. Unfertilized oocytes and gestation day 1 and 2 embryos were assessed morphologically, and the results suggested that absence or thinning of the zona pellucida (ZP) is related to infertility. However, the morphological characteristics of oocytes before ovulation and recovery from the effects of NAC were not clarified. In the present study, the ovarian follicles were histopathologi-cally examined and the recovery of reproductive function was evaluated to investigate the effects of NAC. Female Sprague-Dawley rats at 10 weeks of age received NAC intravenously at 1000 mg/kg/day for more than 1week. Thinning of the ZP was observed in the ovarian follicles in all stages of growth by light microscopy. Outflow of the components of the ZP between the corona radiata and disarrangement of the corona radiata were more pronounced in growing follicles than in large secondary follicles. Similar findings were observed by electron microscopy, and the effects of NAC were limited to the ZP. Infertility and thinning of the ZP were observed in the no-recovery NAC group, but not in the recovery NAC group, in which animals recovered within four estrous cycles after NAC administration. It has been reported that the ZP is expressed by oocytes or by both oocytes and granulosa cells, but no changes were noted in these cells. The present findings suggest that NAC affects the ZP directly and that reproductive function may recover from the effects of NAC.     

The axis of polarity of the mouse blastocyst is specified before blastulation and independently of the zona pellucida

BACKGROUND: Rather than being prepatterned, orientation of the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living conceptuses. METHODS: Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for recording the position along the axis of the centres of the patches of labelled cells. RESULTS: These centres showed significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae. CONCLUSIONS: These findings further support the view that the Em-Ab axis of the mouse blastocyst is normally prepatterned and provide no evidence of a role for the ZP in its specification.     

An electron microscopic study of sperm penetration into the human egg investments

Summary The ultrastructure of human spermatozoa located in the cumulus cells and the zona pellucida of a pronuclear egg, and in the zona pellucida of a two-cell egg, both fertilized in-vivo, has been analysed in order to understand how the human spermatozoon penetrates the investing coats of the oocyte. Among the 36 spermatozoa found in the cumulus cells, 31 were phagocytosed by cumulus cells and 5 were wedged in the matrix between the cells. These spermatozoa were acrosome-reacted and their equatorial segment was intact. Six of the seven spermatozoa found in the zona pellucida (four spermatozoa in the pronuclear egg and three in the two-cell egg) had lost the equatorial segment, while the other one was partially reacted. The sperm heads were located in slits with sharp edges. From these findings it was concluded that in the human (1) only few and normal spermatozoa seem to reach the cumulus cells after natural insemination, (2) the acrosome reaction probably occurs sometime before the spermatozoa reach the vicinity of the corona cells, (3) the reaction of the equatorial segment seems to occur during or before the initial phase of zona penetration, since the spermatozoa located in the matrix of the zona pellucida had no equatorial segment. No evidence of the presence of spermatozoa with an intact acrosome in the matrix of cumulus cells or with an intact equatorial segment in the zona pellucida were found.     

内毒素对去卵透明带2-细胞小鼠胚胎体外发育的影响

目的 观察不同剂量浓度内毒素对去卵透明带2—细胞小鼠胚胎体外发育的影响。方法 获取小鼠2—细胞胚胎，用胰蛋白酶法去除卵透明带后，分别置于含有0，1pg/ml，5pg/ml和10pg/ml内毒素的CZB液滴中培养，此外，用蛋白酶法去除卵透明带，在不含内毒素的CZB液滴中培养，观察胚胎体外发育情况。结果 用胰蛋白酶法去卵透明带的2—细胞小鼠胚胎，其囊胚率随着培养液中内毒素剂量的增加而降低，且5pg/ml和10pg/ml时与对照组(不含内毒素)相比差异显著(P＜0．001)。在有内毒素存在时，停滞在2—细胞或4细胞期的胚胎彼此分离排成列，不再呈球形。部分卵裂球出现空泡或碎裂。另外，用蛋白酶法去除透明带的2—细胞鼠胚的囊胚率为61．7％，显著低于胰蛋白酶法的73．8％(P＜0．001)。结论 微量内毒素即明显抑制去卵透明带2—细胞小鼠胚胎的体外发育，并表现出剂量效应；去卵透明带2—细胞小鼠胚胎试验可应用于培养液的微量内毒素检测；胰酶法去除卵透明带后胚胎的囊胚率优于蛋白酶法。     

Uterine fibroblast growth factor-2 and embryonic fibroblast growth factor receptor-1 at the beginning of gastrulation in the rabbit

Fibroblast growth factor-2 (FGF-2) induces gastrulation of rabbit blastocysts in vitro and is present in the uterine secretion at day 6 after mating. The following study was made in order to show if changes in the uterine FGF-2 concentration or in the FGF receptor concentration of the embryonic tissues point to a regulation of this event. By the use of the ELISA technique and immunohistochemistry, FGF-2 concentration was determined in the endometrical tissue, uterine secretion and blastocyst between day 4 and day 8 of pregnancy, in the uterine secretion after induction of pseudopregnancy, in day 6 blastocysts after in vitro culture, and FGF immunoreactivity was localized in the endometrial tissue. FGF receptor-1 (FGFR-1) concentration was examined correspondingly in the blastocyst. Cross-linking experiments using ¹²⁵I-FGF-2 were done to identify binding proteins in the blastocyst. In the uterine secretion, FGF-2 was constantly high up to day 6.5 but showed an increase thereafter. Similar values in pseudopregnant uterine secretions indicated that the growth factor was of uterine origin. It was probably synthesized by the uterine epithelium as shown by immunohistochemistry. Under culturing conditions, the blastocyst produced small amounts of FGF-2. In the blastocyst, FGFR-1 as well as binding of ¹²⁵I-FGF-2 showed a dramatic increase from day 6.0 to day 6.5, coinciding with the onset of gastrulation. Receptor antigenicity was located in the embryonic disc at day 6.5 and day 7.0. Two binding proteins of about 200 and 130 kDa were found by cross-linking. The results indicate that a regulation of growth factor influence on embryonic differentiation is more probable via expression of the embryonic receptor than via differential release of the uterine growth factor.     

The structure of rabbit retinal Müller (glial) cells is adapted to the surrounding retinal layers

Summary Radial glial (Müller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Müller cells within the different topographical regions of the retina, and of the Müller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Müller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Müller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Müller cell processes (and somata) extend thin cytoplasmic bubbles ensheathing the neuronal somata, as do the velate astrocytes in the brain. In the plexiform layers, Müller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Müller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.     

人类辅助生殖技术中DAY3胚胎质量与囊胚形成相关性分析

目的探讨人类辅助生殖技术中,day3胚胎质量与囊胚形成的相关性。方法将符合纳入标准的130例患者day3胚胎移植后剩余胚胎进行囊胚培养至day6,分析患者年龄、day3胚胎分级、day3胚胎细胞数及培养时间与囊胚形成的关系。结果患者年龄与囊胚形成无明显相关;囊胚形成率与day3胚胎质量评分和细胞数呈显著正相关,day3评分为Ⅰ-Ⅱ级胚胎囊胚形成率明显高于Ⅲ-Ⅳ级胚胎,day3细胞数≥6的胚胎囊胚形成率明显高于≤5的胚胎囊胚形成率。综合分析day3胚胎分级、细胞数、培养时间与囊胚形成率的关系,囊胚形成率最高的是≥6细胞Ⅰ-Ⅱ级胚胎,其次是≥6细胞Ⅲ-Ⅳ级胚胎,≤5细胞的其他两组囊胚形成很低,各组胚胎day6囊胚形成率均明显高于day5。结论 day3胚胎的细胞数、质量分级及囊胚的培养天数都能够影响囊胚的形成,其中day3胚胎的细胞数更为重要,培养至day6使day3胚胎质量较差和发育迟缓的胚胎有机会形成囊胚,有效地筛选和保存了有一定发育潜能的胚胎。     

The ontogeny of dendrite bundles in rabbit visual cortex

Summary Time schedule and mechanisms of the appearance of dendrite bundles in laminae IV and II/III of the visual cortex have been investigated in the rabbit from the first appearance of the cortical plate during fetal development up to adult stages. Sections cut either perpendicularly or tangentially to the cortical surface were used for light-and electron-microscopic analysis. Dendrite bundles appear during the late fetal period and the first postnatal days in a biphasic process. The first step takes place during late fetal development. Due to migration of neuroblasts along radial processes of glial cells, column-like compartments of neuropil are formed in laminae II–IV which contain the apical processes of the nerve cells situated in deeper layers. They represent the units of origin for the later dendrite bundles. The second step is initiated immediately after birth when axons arrive and small oblique and horizontal dendrites start to sprout from perikarya as well as from the apical dendrites. These new cell processes proliferate and grow rapidly. However, during this growth process groups of apical dendrites of pyramidal cells remain together, and they later form the bundles. In the beginning of this process many apical dendrites are connected with each other by punctae adhaerentiae. The basic pattern of dendrite bundles is present before the eyes open and before the majority of spines and synaptic contacts are formed.