The effect of Norethisterone acetate on the uterus of albino rats: histological, histochemical and ultrastructure study.

BACKGROUND: Norethisterone acetate (NETA), also known as norethindrone acetate is a progestogens medication that is widely used in birth control pills, menopausal hormone therapy, and for the treatment of gynecological disorders as abnormal uterine bleeding and endometriosis. There is a lack of detailed histological information regarding the effects of NETA on the uterine structure. So, the present study focuses on the uterine histological, histochemical and ultrastructure changes following the exposure to NETA in the albino rats. To do this aim, fourteen adult female albino rats were used. They were randomly divided into two equally groups: Control group and NETA treated group. Albino rats of control group were administered daily food, water and orally distilled water only, while rats of NETA treated group were administered daily orally 20 µg of NETA dissolved in 2 ml distilled water, food, and water. The experiment was continued for three weeks. RESULTS: The findings of the present work indicated that the use of NETA has negative effects on the endometrial epithelium (proliferation, autophagy and apoptosis), glands (necrotic, apoptotic or pseudosecretory glands) and stromal and myometrial reactions (granulocytes, connective tissue remodeling, apoptosis, myocytes hypertrophy). CONCLUSION: This work revealed that NETA has desynchronized progestogenic effect on the uterine tissues of the albino rat and thereby prevent implantation and pregnancy.

Suppression of uterine and placental ferroptosis by N-acetylcysteine in a rat model of polycystic ovary syndrome.

The mechanisms that link hyperandrogenism and insulin (INS) resistance (HAIR) to the increased miscarriage rate in women with polycystic ovary syndrome (PCOS) remain elusive. Previous studies demonstrate that increased uterine and placental ferroptosis is associated with oxidative stress-induced fetal loss in a pre-clinical PCOS-like rat model. Here, we investigated the efficacy and molecular mechanism of action of the antioxidant N-acetylcysteine (NAC) in reversing gravid uterine and placental ferroptosis in pregnant rats exposed to 5α-dihydrotestosterone (DHT) and INS. Molecular and histological analyses showed that NAC attenuated DHT and INS-induced uterine ferroptosis, including dose-dependent increases in anti-ferroptosis gene content. Changes in other molecular factors after NAC treatment were also observed in the placenta exposed to DHT and INS, such as increased glutathione peroxidase 4 protein level. Furthermore, increased apoptosis-inducing factor mitochondria-associated 2 mRNA expression was seen in the placenta but not in the uterus. Additionally, NAC was not sufficient to rescue DHT + INS-induced mitochondria-morphological abnormalities in the uterus, whereas the same treatment partially reversed such abnormalities in the placenta. Finally, we demonstrated that NAC selectively normalized uterine leukemia inhibitory factor, osteopontin/secreted phosphoprotein 1, progesterone receptor, homeobox A11 mRNA expression and placental estrogen-related receptor beta and trophoblast-specific protein alpha mRNA expression. Collectively, our data provide insight into how NAC exerts beneficial effects on differentially attenuating gravid uterine and placental ferroptosis in a PCOS-like rat model with fetal loss. These results indicate that exogenous administration of NAC represents a potential therapeutic strategy in the treatment of HAIR-induced uterine and placental dysfunction.

The Use of Atomic Force Microscopy in Comprehensive Assessment of the "Mother-Placenta-Fetus" System in Obstetric and Endocrine Pathology during Pregnancy.

Atomic force microscopy is not very popular in practical health care, therefore, its potential is not studied enough, for example, in obstetrics when studying the "mother-placenta-fetus" system. Our study summarizes the possibilities of using atomic force microscopy for detection of various circulatory disorders and vascular changes at the microscopic level in the uterus (endometrium and myometrium), placenta, and umbilical cord in the main variants of obstetric and endocrine pathology. For instance, in the case of endocrine pathologies, changes in the form of stasis, sludge, diapedesis, ischemia, destruction and separation of endotheliocytes in villous blood vessels were found in the mother. The oxygen content in erythrocytes also naturally decreased in pathologies; poikilo- and anisocytosis were observed.

Ultrastructure of the uterus, embryonic envelopes and the coracidium of the enigmatic tapeworm Tetracampos ciliotheca (Cestoda: Bothriocephalidea) from African sharptooth catfish (Clarias gariepinus).

Transmission electron microscopy (TEM) was used to study the ultrastructure of the uterus and egg morphology in the enigmatic bothriocephalidean tapeworm Tetracampos ciliotheca. The uterine wall, underlain by well-developed muscle bundles, consists of a syncytial epithelium which is characterized by the abundance of free ribosomes, mitochondria and cisternae of granular endoplasmic reticulum (GER). On the apical surface of the uterine epithelium, there is an abundant network of cytoplasmic microlamellae projecting into the uterine lumen. The lumen is filled with freely lying eggs which are located close to the uterine wall but do not contact with the microlamellae of the uterine epithelium. The developed eggs possess an oncosphere surrounded by four envelopes: (1) a thin egg shell; (2) an outer envelope; (3) a syncytial, ciliated inner envelope; and (4) the oncospheral membrane. The mature hexacanth is armed with three pairs of oncospheral hooks, as well as somatic and hook muscles and five types of cells (1) binucleated subtegumental cell, (2) somatic cells, (3) penetration gland cells, (4) nerve cells and (5) germinative cells. Considering the relative scarcity of descriptive and comparative studies on the ultrastructure of the uterus and egg morphology in the order Bothriocephalidea, we concluded that Tet. ciliotheca displays a unique type of egg development. Based on these results, we discuss plausible ideas relating to the function of these structures for consideration in future studies.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2-0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Preservation of Mesenchymal Stem Cell-Derived Extracellular Vesicles after Abdominal Delivery in the Experiment.

Light luminescent microscopy was used to study the distribution of extracellular microvesicles with PKH26-stained membranes secreted by placenta-derived mesenchymal stromal cells in the uterine tissues at different terms after injections to intact rats and after abdominal delivery (a model of cesarian section). Microvesicles migrated through the uterine tissues and were detected for at least 8 days after injection. In some cases, microvesicles were more numerous in the uterus after cesarian section modeling, which can be related to blockade of microcirculation and lymph flow due to inflammation accompanying surgical intervention. The content of microvesicles in the uterine tissues gradually declined due to macrophage phagocytosis and, probably, due to their migration into the vascular bed. Despite their size, properly stained extracellular microvesicles can be detected by light microscopy in tissues after injections.

Insights from imaging the implanting embryo and the uterine environment in three dimensions.

Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo.

The orientation of a decellularized uterine scaffold determines the tissue topology and architecture of the regenerated uterus in rats†.

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

Ultrastructural markers of tissue endometrial receptivity in patients with recurrent implantation failure.

The scanning electron microscopy of the endometrial surface epithelium during the 'implantation window' was performed in 119 patients with uterine factor of infertility or recurrent miscarriage due to endometrial hypoplasia. Ultramorphological picture of the surface endometrial epithelium was characterized by aplasia and hypoplasia of pinopodes (67.39%), dense cell - cell contacts (69.53%), heteromorphy of secretory cells (15.22%) in combination with atypia of microenvironment cells (50%) in patients with infertility. The asynchronous development of pinopodes (46.67%) and the absence of intercellular contacts separation during the 'implantation window' (84.44%) was observed in patients with recurrent miscarriage. The revealed disturbance determines the mechanisms of the blastocyst adhesion violation and trophoblast invasion in the different stages of implantation in patients with uterine factor of infertility and recurrent miscarriage.

Uterine ALK3 is essential during the window of implantation.

The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-ß family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3(flox) (/flox)-Pgr-cre-positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor ß (TGF-ß) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity.

Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations.

PURPOSE: Using a comprehensive flow cytometric panel, do endometrial immune profiles in adverse reproductive outcomes such as repeat implantation failure (RIF) and repeat pregnancy loss (RPL) differ from each other and male-factor controls? METHODS: Six-hundred and twelve patients had an endometrial biopsy to assess the immunophenotype. History on presentation was used to subdivide the population into recurrent implantation failure (RIF) [n = 178], recurrent pregnancy loss (RPL) [n = 155], primary infertility [n = 130] and secondary infertility [n = 114]. A control group was utilised for comparative purposes [n = 35] and lymphocyte subpopulations were described. RESULTS: Distinct lymphocyte percentage differences were noted across the populations. Relative to controls and RPL, patients with a history of RIF had significantly raised uterine NKs (53.2 vs 45.2 & 42.9%, p < 0.0001). All sub-fertile populations had increased percentage peripheral type NKs (p = 0.001), and exhibited increased CD69+ activation (p = 0.005), higher levels of B cells (p < 0.001), elevated CD4:CD8 ratio (p < 0.0001), lower T-regs (p = 0.034) and a higher proportion of Th1+ CD4s (p = 0.001). Patient aetiology confers some distinct findings, RPL; pNK, Bcells and CD4 elevated; RIF; uNK and CD56 raised while CD-8 and NK-T lowered. CONCLUSIONS: Flow cytometric endometrial evaluation has the ability to provide a rapid and objective analysis of lymphocyte subpopulations. The findings show significant variations in cellular proportions of immune cells across the patient categories relative to control tissue. The cell types involved suggest that a potential differential pro-inflammatory bias may exist in patients with a history of adverse reproductive outcomes. Immunological assessment in appropriate populations may provide insight into the underlying aetiology of some cases of reproductive failure.

Endometrial pinopode biomarkers: Molecules and microRNAs.

Ultrastructural changes on the apical surface of the luminal epithelium of the uterus are known as pinopodes. Their morphology in species and in special species is associated with different results about size, duration, and percentage of surface area covered by pinopodes. The content of pinopodes is different in rodents and humans. In mice and rats pinopodes have many vacuoles and no organelle that extends to the actin stalk above the microvilli. Human pinopodes do not have a large vacuole and contain the golgi complex, a rough endoplasmic reticulum, secretory vesicles, and mitochondria that extend from the entire cell surface. It has been suggested that pinopodes are good markers of endometrial receptivity and implantation window. There are several molecular markers related to the presence of pinopodes, including integrins, leukemia inhibiting factor (LIF), l-selectin, HOXA10, glutaredoxin, glycodelinA, heparin-binding epidermal growth factor, mucins, and microRNAs (miRNAs). Multiple lines of evidence have indicated that miRNAs could affect the expression of LIF and pinopodes in the endometrium and these molecules play key roles in implantation window processes. Here, we have summarized the morphology and function of pinopodes. Moreover, we have highlighted several molecules in relation to pinopodes that could be used as biomarkers.

Intrauterine exosomes are required for bovine conceptus implantation.

Exosomes, extracellular vesicles, are present in uterine flushing fluids (UFs), which are involved in conceptus-endometrial interactions during peri-implantation periods. Despite several studies on intrauterine exosomes conducted, the roles conceptus and endometrial exosomes play during peri-implantation periods have not been well characterized. To investigate the effect of bovine intrauterine exosomes on conceptus implantation, exosomes isolated from bovine UFs during peri-implantation periods were subjected to global protein analysis. The analysis detected 596 exosomal proteins, including ruminants' pregnancy recognition factor IFNT, and 172 differentially expressed proteins with more than 1.5-fold changes in UFs on days 17, 20 and 22 pregnancy (day of conceptus implantation is initiated on days 19-19.5). Treatment of primary bovine endometrial epithelial cells with exosomes from day 17 UFs up-regulated the expression of apoptosis-related genes, and treatment with exosomes from day 20 and 22 UFs up-regulated the expression of adhesion molecule. Based on these findings, intrauterine exosomes should be considered as an essential constituent for successful implantation.

The first ultrastructural observations of the egg-forming complex of Calicotyle affinis, an endoparasitic monocotylid monogenean which lacks a uterus.

This ultrastructural study of the female reproductive system of Calicotyle affinis, a monogenean without a uterus, demonstrates, for the first time in a monogenean, the presence of accessory cells located in the peripheral region of the ovary and a single large cell whose surface is penetrated by deep invaginations which restrict the passage of oocytes through the lumen of the distal extremity of the ovary. The cytoarchitecture of the epithelial lining of the two vaginae is a syncytium formed by an anucleate epithelial lining with sunken epithelial perykaria. The wall of the seminal receptacle is an enlarged prolongation of the vaginal epithelial lining. Fertilization occurs in the fertilization chamber and fertilized oocytes retain cortical granules within their cytoplasm, a characteristic known to occur in free-living flatworms. Our study also highlights concentrations of two distinct groups of Mehlis' gland cell ducts on either side of the proximal end of the ootype distinguished by ultrastructural characteristics of their secretory granules. The epithelial wall of the ootype is formed by a single layer of regular, columnar, glandular epithelial cells; these cells are closely adjacent, conjoined towards their apical region by septate junctions and produce rounded, electron-dense granules which are discharged into the ootype lumen via a merocrine or holocrine mechanism. Released granules concentrate around the eggshell and form an additional fibrous coat. The morphological diversity observed in the female reproductive system of the Monogenea is commented on it relation to clarifying patterns in monogenean evolution and for understanding the phylogeny of the Neodermata.

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae).

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non-invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non-invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.

The unique uterine structure of the basal monogenean Chimaericola leptogaster (Monogenea: Polyopisthocotylea), an ectoparasite of the relictual holocephalan fish Chimaera monstrosa.

Ultrastructural studies of the monogenean uterus are few in number and no non-polystomatid polyopisthocotyleans have been investigated. The uterus of Chimaericola leptogaster, a basal polyopisthocotylean monogenean, has several unusual features, including six reflexed loops comprising four ascending and three descending, longitudinally oriented, linear sections. At the ultrastructural level, three readily distinguishable uterine regions and other distinctive characteristics are apparent. One novel feature occurs in the proximal uterus, where the lining forms a so-called 'single-layered multi-rowed cellular epithelium', which includes two types of cells, tall (ca. 14-19 µm in height) and short (ca. 6-9 µm in height) cells, both lying on the basement membrane. Although known from other bilaterian groups, this is the first record of this type of epithelium in the Neodermata. The lining of the middle uterine region comprises a single regular layer of columnar glandular epithelial cells, which produce numerous rounded, electron-dense granules associated with Golgi complexes. The presence of the uterine glands in the middle region of the uterus is an unusual feature for a monogenean, having previously been described only for basal orders of the Cestoda, i.e. the Gyrocotylidea, Caryophyllidea and Spathebothriidea. Seen in cross-section, the epithelium of the distal uterus contains three areas of tall single-layered columnar epithelium (ca. 30 µm deep) interspersed by three areas of flattened epithelium (ca. 0.2-0.9 µm deep). Such a pattern is quite different from those reported for other monogeneans and, indeed, other neodermatan groups. The investigation has shown that the outer layer of the fully formed eggshell is assembled from epithelial secretions in the middle uterine lumen, but is modified in terms of its shape in the distal uterus. Possible phylogenetic implications arising from the unusual features described are discussed in relation to other neodermatan groups and recent molecular phylogenies of the Bilateria.

New species of Aberrapex Jensen, 2001 (Cestoda: Lecanicephalidea) from eagle rays of the genus Myliobatis Cuvier (Myliobatiformes: Myliobatidae) from off Argentina.

Three new species of Aberrapex Jensen, 2001 (Cestoda: Lecanicephalidea) have been collected from species of Myliobatis Cuvier along the coast of Argentina. Aberrapex ludmilae sp. n. parasitises M. goodei Garman in the San Matías Gulf. This species is unique in a combination of features including the shape and extent of the uterus, scolex size, testis distribution, and by lacking an external seminal vesicle and postovarian vitelline follicles. Aberrapex sanmartini sp. n. from M. goodei in San Blas Bay and A. vitalemuttiorum sp. n. from M. ridens Ruocco, Lucifora, Díaz de Astarloa, Mabragaña et Delpiani in coastal waters off Buenos Aires Province, are distinguished from all other congeners by the microthrix pattern on the scolex surface, uterus shape and extension, position of the connection of the uterine duct to the uterus, presence and extension of an external seminal vesicle, and its overall size and number of proglottids. The specimens of M. goodei and M. ridens show locally distinct cestode faunas, which are correlated with well-defined biogeographic regions. Some of these areas correspond with mating and nursery zones for species of Myliobatis.

Immunohistochemistry of Telocytes in the Uterus and Fallopian Tubes.

The seminal work of Popescu and colleagues first demonstrated the existence of a new cell type - the telocytes. We were among the first who reported the presence of such cells in the female genital tract and performed TEM examinations, as well as immunohistochemical staining in the attempt to find a specific marker. Telocytes from rat and from the human uterus and from human fallopian tube were extensively investigated initially by comparison with interstitial cells of Cajal. Progress in telocyte research led to the identification of different subtypes suggestive for a heterogeneous telocyte population which can even coexist in the same location. As a consequence, the functions of TCs are still elusive and can be considered a versatile phenomenon that depends on a variety of conditions, including signal reception and transmission of information via extracellular vesicles or by direct intercellular contact.

Fine structure of uterus and non-functioning paruterine organ in Orthoskrjabinia junlanae (Cestoda, Cyclophyllidea).

Some cyclophyllidean cestodes provide protection for their eggs in the external environment by providing them with additional protective layers around the egg membranes. In attempting to examine such adaptations, the microanatomy and fine structure of the uterus of pregravid and gravid proglottids of the cyclophyllidean cestode Orthoskrjabinia junlanae, a parasite of mammals that inhabit a terrestrial but moist environment, were studied. In the initial stages of uterine development, developing embryos locate freely in the lumen of a saccate uterus that later partitions into chambers. Each chamber that forms encloses several embryos. The chambers are surrounded by muscle cells that synthesize extracellular matrix actively. The paruterine organs consist of stacks of flattened long outgrowths of muscular cells, interspersed with small lipid droplets. In the gravid proglottids, the size of paruterine organ increases and consists of flattened basal and small rounded apical parts separated by constrictions. The fine structure of the organ wall remains the same: sparse nuclei and stacks of flattened cytoplasmic outgrowths but internal invaginations or lumen in the paruterine organ are absent. Completely developed eggs remain localized in the uterus. Based on the comparative morpho-functional analysis of uterine and paruterine organs and uterine capsules in cestodes, we conclude that these non-functioning paruterine organ in O. junlanae is an example of an atavism. We postulate that the life cycle of the parasite, which infects mammals living in wet habitats, where threats of desiccation of parasite ova is reduced, has favoured a reversion to a more ancestral form of uterine development.

Functional ultrastructure of the parenchymatic capsules of the cestode Thysanotaenia congolensis (Cyclophyllidea, Anoplocephalidae, Inermicapsiferinae).

Proglottids from adult Thysanotaenia congolensis from naturally infected black rats Rattus rattus from Santiago Island, Cape Verde, were examined by transmission electron microscopy (TEM). The uterus in mature proglottids is composed of an ephemeral transverse tube or sac that breaks down, releasing eggs into the parenchyma where they are sequestered in groups and encapsulated by parenchymatous layers. In gravid proglottids, eggs accumulate in groups of 6-12 at the distal end of sac-like uterine ducts. As eggs accumulate, the end of the uterine ducts expands until it pinches off, releasing groups of eggs into the parenchyma surrounded by remnants of uterine epithelium. These epithelium-bound groups of eggs remain in the parenchyma until they are encapsulated with several parenchymatous layers, forming parenchymatic egg capsules, typical for mature and gravid proglottids of Inermicapsiferinae. The parenchymatic capsules originate from the medullary parenchyma of immature proglottids, which undergoes differentiation into the three layers of gravid proglottids: (1) an outer connective tissue layer composed of long delicate filaments of unknown chemical nature embedded in a granular extracellular matrix; (2) a middle layer appearing as an accumulation of large closely packed PAS-positive mucous goblets that are intensely metachromatic after toluidine blue staining and (3) an inner compact layer composed of lipid-containing cells, muscle cells with elongated muscle fibres and cells of various sizes and shapes forming or containing calcareous corpuscles. The mature hexacanths of T. congolensis are surrounded by reduced oncospheral envelopes consisting of remnants of a very thin membranous layer of degenerating embryophore with long, irregularly shaped cytoplasmic processes and by remnants of uterine epithelium extending as numerous apical microlamellae into the parenchymatic capsule lumen.