Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P<0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P<0.05), whereas the numerical density of cells in the stroma did not change (P>0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P<0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.

Morphological classification of bovine ovarian follicles.

Follicle classification is an important aid to the understanding of follicular development and atresia. Some bovine primordial follicles have the classical primordial shape, but ellipsoidal shaped follicles with some cuboidal granulosa cells at the poles are far more common. Preantral follicles have one of two basal lamina phenotypes, either a single aligned layer or one with additional layers. In antral follicles <5 mm diameter, half of the healthy follicles have columnar shaped basal granulosa cells and additional layers of basal lamina, which appear as loops in cross section ('loopy'). The remainder have aligned single-layered follicular basal laminas with rounded basal cells, and contain better quality oocytes than the loopy/columnar follicles. In sizes >5 mm, only aligned/rounded phenotypes are present. Dominant and subordinate follicles can be identified by ultrasound and/or histological examination of pairs of ovaries. Atretic follicles <5 mm are either basal atretic or antral atretic, named on the basis of the location in the membrana granulosa where cells die first. Basal atretic follicles have considerable biological differences to antral atretic follicles. In follicles >5 mm, only antral atresia is observed. The concentrations of follicular fluid steroid hormones can be used to classify atresia and distinguish some of the different types of atresia; however, this method is unlikely to identify follicles early in atresia, and hence misclassify them as healthy. Other biochemical and histological methods can be used, but since cell death is a part of normal homoeostatis, deciding when a follicle has entered atresia remains somewhat subjective.

Studies of granulosa cell maturation in dominant and subordinate bovine follicles: novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1.

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0+/-S.E.M. 0.4 mm; n=16), partially dominant (12.0+/-0.6 mm; n=18) and fully dominant (15.0+/-0.4 mm; n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3beta-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-alpha and beta-B were observed as expected for follicle sizes examined. After adjusting for size differences, only CYP11A1 was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV alpha-1 (COL4A1), laminin beta-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations, CYP11A1 mRNA levels were highly correlated with the focimatrix genes COL4A1, NID1 and -2 and HSPG2. Thus, focimatrix may potentially regulate CYP11A1 expression, and the regulation of both could be important in follicular dominance.

Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei.

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with alpha-ketohexanoic acid and NAD(+) grew under these conditions. Crystals of form I diffracted to beyond 3.0 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 A, beta = 96.3 degrees . Crystals of form II diffracted to beyond 2.0 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 A, alpha = 109.1, beta = 107.5, gamma = 95.9 degrees. The calculated values for V(M) and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry.

Dietary manipulation of Bos indicus x heifers during gestation affects the reproductive development of their heifer calves.

The effect of nutrition during the first and second trimesters of pregnancy in composite beef heifers on reproductive parameters of their female calves was determined in the present study. At artificial insemination, heifers were assigned to one of four treatment groups (i.e. HH, HL, LowH and LL) depending on the level of crude protein intake (H = high; L = low) for first and second trimesters of pregnancy. Gonadotrophin concentrations and ovarian parameters were measured in their female calves at 5 and 23 months of age. Crude protein intake was positively associated with dam plasma urea (P < 0.001). The density of healthy follicles in heifers at the time of death was negatively correlated with dam plasma urea at Day 179 (P = 0.009). Heifers from LowH dams had a smaller-sized prepubertal largest ovarian follicle (P = 0.03) and lower densities of primordial and primary follicles (P = 0.02) and healthy antral follicles (P = 0.009) when they were killed. There was a positive correlation between plasma FSH concentrations at 5 and 23 months of age (P = 0.02), as well as between the sizes of the largest ovarian follicles at 6 and 23 months of age (P = 0.01). In conclusion, the reproductive development of heifers may be affected by prenatal nutrition during early and mid-gestation.

Uptake of the 35 kDa major surfactant apoprotein (SP-A) by neonatal rabbit lung tissue.

Secreted surfactant is made up of both phospholipid and protein components. Therefore, we investigated the possibility that surfactant apoproteins might be taken up by the alveolar type II cell in a manner similar to the uptake of surfactant phosphatidylcholines. Day 2 neonatal rabbits were infused via the trachea with a solution of carrier surfactant and 125I-labelled surfactant apoprotein (SP-A, Mr approx. 35,000). Most of the 125I-SP-A remained within the alveolus; however, a fraction of the 125I-SP-A was taken up by the lung tissue from the alveolus in a time-dependent manner. The small amount of radiolabeled material detected in blood, liver or kidney tissues of 125I-SP-A-infused animals was not trichloroacetic acid (TCA) precipitable, i.e., probably represented degradation products. In contrast, the proportion of TCA-precipitable 125I-SP-A in lung tissue or lavage samples did not change as function of time after tracheal administration. Two-dimensional gel electrophoresis of the 125I-SP-A present in the lavage samples or associated with lung tissue was used to show that a small proportion of the 125I-SP-A was partially degraded in the lung tissue and alveolus. These data are suggestive that the SP-A is taken up by lung tissue, perhaps in a manner similar to the uptake of surfactant phospholipid by the alveolar type II cell.

Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum. Evidence for a ligand-induced conformational change.

A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion. The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate. The crystals diffract well and X-ray photographs have established that they are in the space group R32. Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit. Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell. Such a conformational rearrangement may represent an important step in the catalytic cycle.

Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli.

Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme. Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively. Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits.

Synaptic organisation of lumbar sympathetic ganglia of guinea pigs: serial section ultrastructural analysis of dye-filled sympathetic final motor neurons.

The authors serially sectioned seven dye-filled neuronal somata and more than 1.6 mm of their dendrites from the lumbar sympathetic ganglia of guinea pigs and examined them ultrastructurally to determine the distribution of preganglionic synaptic inputs to their dendrites and cell bodies. Most of the surface of the neurons was covered with Schwann cells. Apposing boutons were rare, with an average density of one axosomatic bouton per 125 microm2 of somatic membrane and one axodendritic bouton per 25 microm of dendrite. Many dendritic segments that were more than 50 microm long completely lacked any apposing boutons. Although the average density of apposing boutons was low, local densities could be high, so that clusters of up to four adjacent boutons occurred on cell bodies and dendrites alike. The spatial arrangement of the apposing boutons for each of the cells examined here was not significantly different from a random distribution. Consequently, the number of apposing boutons observed for any neuron was simply proportional to the amount of neuronal surface sampled in the serial section run. About 50% of boutons directly apposing the neurons lacked any detectable presynaptic specialisations. When they were present, the presynaptic densities had a mean length of about 220 nm, with no difference between boutons that made axosomatic or axodendritic appositions. By applying these data to complete reconstructions of the dendritic trees of dye-filled sympathetic neurons at the light microscopic level, the authors estimated that few neurons in the lumbar sympathetic chain of guinea pigs would receive more than 200 synapses or apposing boutons and that many of them would receive less than 100 synapses. Up to 50% of these boutons would be predicted to make axosomatic contacts. These new observations provide a strong morphological framework for a better understanding of how sympathetic final motor neurons process their preganglionic synaptic inputs.

Synaptic organisation of neuropeptide-containing preganglionic boutons in lumbar sympathetic ganglia of guinea pigs.

Within the lumbar sympathetic ganglia of guinea pigs, the endings of different populations of neuropeptide-containing preganglionic neurons form well-defined pericellular baskets of boutons around target neurons in specific functional pathways. We have used multiple-labelling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to investigate synaptic organisation within pericellular baskets labelled for immunoreactivity to calcitonin gene-related peptide (CGRP), substance P (SP), or the pro-enkephalin-derived peptide, met-enkephalin-arg-gly-leu (MERGL) in relation to their target neurons. Different functional populations of neurons, identified by their neurochemical profile, showed a significant degree of spatial clustering and predicted well the distribution of specific classes of pericellular baskets. Most of the boutons in a basket were completely surrounded by Schwann cell processes and did not form synapses. The synapses that were present were made mostly onto dendrites enclosed by the Schwann cell sheath surrounding the neuron within the basket. These dendrites probably originated from neurochemically similar neighbouring neurons. Nevertheless, some of the boutons in the baskets did form synapses with the cell body or proximal dendrites of the neuron they surrounded. Occasionally, cell bodies received a relatively high number of synapses and close appositions from boutons in a pericellular basket. Synaptic convergence of two immunohistochemically distinct types of preganglionic inputs was found in baskets of SP-immunoreactive or MERGL-immunoreactive, but not CGRP-immunoreactive, boutons. Taken together, our results show that the appearance of pericellular baskets is primarily due to the packing of the target neurons. The grouping of functionally similar classes of neurons with their pathway-specific projections of peptide-containing preganglionic neurons suggests that peptides could exert their effects in relatively well-defined zones within the ganglia.

Extracellular matrix of the bovine ovarian membrana granulosa.

Much is known about the control of the development of ovarian follicles by growth factors and hormones. The study of extracellular matrix in the ovary, though, is a relatively new area. To date much research has focused on identifying the matrix components present, and more recently, its production and the physiological roles. In this review we focus on the changes that occur in the follicular basal lamina from primordial follicles through to ovulation and formation of the corpus luteum, the changes that occur during follicular atresia, and we discuss our observations of a novel matrix which forms in the membrana granulosa. The follicular basal lamina changes considerably during follicular development in its expression pattern of type IV collagens. Of the laminin chains examined, there appears only to be an increase in amount, except for laminin alpha2. It is expressed only in a small proportion of healthy antral follicles and in the majority of atretic antral follicles. Call-Exner bodies have the same composition as the basal lamina, except they do not contain laminin alpha2, even when the follicular basal lamina does. The novel matrix that develops within the membrana granulosa is similar in composition to Call-Exner bodies which occur predominantly in preantral follicles, except that it is far more common in large antral follicles, does not induce polarization of the surrounding granulosa cells, and does not contain follicular fluid-like material as the Call-Exner bodies of some species do. The expression of this matrix occurs prior to and during the time when granulosa cells express steroidogenic enzymes. It does not exist in corpora lutea. In addition large luteal cells, derived from granulosa cells, do not appear to have a basal lamina. These findings suggest that the maturational changes in the membrana granulosa are accompanied by changes in the matrix.

Extracellular matrix in ovarian follicles.

A lot is known about the control of the development of ovarian follicles by growth factors and hormones, but less is known about the roles of extracellular matrix in the control of follicular growth and development. In this review we focus on the specialized extracellular matrix of the basal laminas that are present in ovarian follicles. These include the follicular basal lamina itself, the Call-Exner bodies of the membrana granulosa, the subendothelial and arteriole smooth muscle basal laminas in the theca, and the basal lamina-like material of the thecal matrix. We discuss the evidence that during follicle development the follicular basal lamina changes in composition, that many of its components are produced by the granulosa cells, and that the follicular basal laminas of different follicles have different ultrastructural appearances, linked to the shape of the aligning granulosa cells. All these studies suggest that the follicular basal lamina is extremely dynamic during follicular development.

Development of the ovarian follicular epithelium.

A lot is known about the endocrine control of the development of ovarian follicles, but a key question now facing researchers is which molecular and cellular processes take part in control of follicular growth and development. The growth and development of ovarian follicles occurs postnatally and throughout adult life. In this review, we focus on the follicular epithelium (membrana granulosa) and its basal lamina. We discuss a model of how granulosa cells arise from a population of stem cells and then enter different lineages before differentiation. The structure of the epithelium at the antral stage of development is presented, and the effects that follicle growth has on the behavior of the granulosa cells are discussed. Finally, we discuss the evidence that during follicle development the follicular basal lamina changes in composition. This would be expected if the behavior of the granulosa cells changes, or if the permeability of the basal lamina changes. It will be evident that the follicular epithelium has similarities to other epithelia in the body, but that it is more dynamic, as gross changes occur during the course of follicle development. This basic information will be important for the development of future reproductive technologies in both humans and animals, and possibly for understanding polycystic ovarian syndrome in women.

Dynamics of the membrana granulosa during expansion of the ovarian follicular antrum.

As an endocrine organ, the ovary has some unique characteristics. The formation, the maturation and the regression of the hormone producing cells really determine the timing, the amount and the type of hormone secreted. Here, we focus on the granulosa cells of ovarian follicles which express 17beta-hydroxysteroid dehydrogenase type 1 and cytochrome P450 aromatase. Follicles only produce estradiol late in follicular development before either ovulation or atresia ensues. We discuss the evidence that the membrana granulosa has many characteristics in common with other epithelia, including that it arises from stem cells. The corollary of this is that individual cells within the membrana granulosa are of different ages or stages of specialization. This is evident as regional differences across the membrana granulosa in terms of cell ages, shapes, gene expression, and even behaviour on cell death. We discuss theoretical considerations of the effects of antrum formation on the behavior of the membrana granulosa, and show evidence for differences between follicles in cell shapes, basal lamina phenotypes and location of younger cells, which we speculate is due to different rates of antrum expansion. Clearly, the membrana granulosa is dynamic, and this could explain much about the differences in the behaviors of cells from within the membrana granulosa, and between ovarian follicles.

The physiology of the ovary: maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum.

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3-7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate beta-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with beta-carotene reduced the amount of cross-linking. We conclude that the high levels of beta-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.

Production of extracellular matrix, fibronectin and steroidogenic enzymes, and growth of bovine granulosa cells in anchorage-independent culture.

A proportion of the granulosa cells from bovine antral follicles will survive, like stem cells, in anchorage-independent culture. To study these cells, bovine granulosa cells were isolated from medium-sized follicles (3-5 mm), plated out (in aliquots of 2.5 x 10(4) viable cells) onto a 1 mL agar base, and overlaid with 1 mL of methycellulose solution in culture medium (control). The cells were cultured (14 days) and then processed for histology (n = 14) or Western immunoblotting (n = 5). Under control conditions or after treatment with basic fibroblast growth factor (bFGF; 50 ng mL-1), a proportion of the granulosa cells divided to produce colonies; individual cells remained small. bFGF increased the number of cells harvested (15.8 +/- 7.3-fold, as measured indirectly by the relative amount of the nuclear La antigen), increased the average diameter of the colonies from 88.9 +/- 13.5 microns to 136.5 +/- 4.9 microns and stimulated the production of fibronectin 5.7 +/- 1.5-fold (P < 0.05). An extracellular matrix, which has previously been shown to be a basal lamina, was observed in 19.1% of the colonies (total of 350 colonies examined; n = 8 experiments). Cells treated with dibutyryl cAMP (1 mM) hypertrophied and had 50 +/- 28.7-fold and 102.6 +/- 55.8-fold higher levels of cholesterol side-chain cleavage cytochrome P450 (P < 0.001) and 3 beta-hydroxysteroid dehydrogenase (P < 0.01) respectively (n = 5). Thus, granulosa cells with characteristics of stem cells can divide and produce extracellular matrix, or be induced to differentiate when in culture without anchorage.

Insulin-like growth factor binding proteins in follicular fluid from morphologically distinct healthy and atretic bovine antral follicles.

In bovine follicles 2-5 mm in diameter, two morphologically distinct types of healthy follicles and two types of atretic follicles have been described recently. Healthy follicles either have columnar basal granulosa cells with follicular basal lamina composed of many layers or 'loops' or they have rounded basal cells with a conventional single-layered, aligned follicular basal lamina. In atretic follicles, cell death either commences at the basal layer and progresses to the antrum (basal atresia) with macrophage penetration of the membrana granulosa or death progresses from the antrum in a basal direction (antral atresia). Little is known about how these different phenotypes develop. To determine whether insulin-like growth factor binding protein (IGFBP) levels in follicular fluid differ between these different types of follicles, we measured IGFBP levels in fluids from these follicles. A total of 61 follicles were assessed by light microscopy and characterized by morphological analysis as either healthy, with columnar or rounded basal granulosa cells, or as undergoing antral or basal atresia. The IGFBP concentration in the follicular fluid of individual follicles from the four groups (n = 12-20 per group) was identified by Western ligand blots using (125)I-insulin-like growth factor (IGF)-II as a probe. Insulin-like growth factor binding proteins 2, 3 (44 and 40 kDa), 4 (glycosylated and non-glycosylated) and 5 were observed. The levels (per volume of fluid) of IGFBPs 2, 4 and 5 were greater in atretic follicles than in healthy follicles. However, there were no statistical differences in levels of each IGFBP between either the two types of healthy follicle or between the two types of atretic follicles. Thus, IGFBP levels are not related to the different types of healthy or atretic follicles.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: impact for allograft rejection.

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet ß-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores ß-cell-matrix attachment, a recognized requirement for ß-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

The roles of the ovarian extracellular matrix in fertility.

In the mammalian ovary there is considerable and continuous remodelling of tissue during both fetal and adult life, necessitating changes in extracellular matrix. Matrix is a diverse group of molecules varying in its composition and roles, which include regulation of growth factor activity and cell behaviour. Here we discuss four topical aspects of matrices in ovaries. (1) Our current state of knowledge of latent TGFFbeta binding proteins that can bind the extracellular matrix fibrillins. Fibrillins and latent TGFbeta binding proteins may be very important given the genetic linkage data implicating a role for fibrillin 3 in polycystic ovarian syndrome. They will almost certainly be important in the stromal compartments of the ovary by regulating TGFbeta bioactivity. (2) Follicles which have an unusual ultrastructural follicular basal lamina and poor quality oocytes. The results suggest that the use of oocytes from these follicles should be avoided in assisted reproductive technologies. (3) Evidence that expression of components of focimatrix correlates with expression of aromatase and cholesterol side-chain cleavage in granulosa cells. Focimatrix is a novel type of basal lamina associated with granulosa cells with expression beginning before deviation and continuing until ovulation. It may be involved in maturation of granulosa cells and selection of the dominant follicle. (4) Evidence is presented in support of a hypothesis that follicular fluid accumulates in follicles due to the osmotic potential of hyaluronan and versican, which are matrices produced by granulosa cells and too large to traverse the follicular antrum. These examples illustrate the diversity of matrix and foreshadow potential important discoveries involving extracellular matrix in ovaries.

Molecular composition of the peri-islet basement membrane in NOD mice: a barrier against destructive insulitis.

AIMS/HYPOTHESIS: This study examined whether the capsule which encases islets of Langerhans in the NOD mouse pancreas represents a specialised extracellular matrix (ECM) or basement membrane that protects islets from autoimmune attack. METHODS: Immunofluorescence microscopy using a panel of antibodies to collagens type IV, laminins, nidogens and perlecan was performed to localise matrix components in NOD mouse pancreas before diabetes onset, at onset of diabetes and after clinical diabetes was established (2-8.5 weeks post-onset). RESULTS: Perlecan, a heparan sulphate proteoglycan that is characteristic of basement membranes and has not previously been investigated in islets, was localised in the peri-islet capsule and surrounding intra-islet capillaries. Other components present in the peri-islet capsule included laminin chains alpha2, beta1 and gamma1, collagen type IV alpha1 and alpha2, and nidogen 1 and 2. Collagen type IV alpha3-alpha6 were not detected. These findings confirm that the peri-islet capsule represents a specialised ECM or conventional basement membrane. The islet basement membrane was destroyed in islets where intra-islet infiltration of leucocytes marked the progression from non-destructive to destructive insulitis. No changes in basement membrane composition were observed before leucocyte infiltration. CONCLUSIONS/INTERPRETATION: These findings suggest that the islet basement membrane functions as a physical barrier to leucocyte migration into islets and that degradation of the islet basement membrane marks the onset of destructive autoimmune insulitis and diabetes development in NOD mice. The components of the islet basement membrane that we identified predict that specialised degradative enzymes are likely to function in autoimmune islet damage.