Apoptosis does not affect the vasculature of the corpus luteum of pregnancy in the rat.

There has been much research into the mechanics of angiogenesis and many studies have demonstrated that newly formed vessels regress during angiogenesis. This vascular involution has been shown to involve basement membrane dissolution and endothelial cell apoptosis. The corpus luteum provides an ideal in vivo model to study physiologic angiogenesis and studies have shown that involution of newly formed vessels occurs during corpus luteum regression. However, few studies to date have investigated the role of apoptosis on the vasculature which develops during pregnancy. By the use of the TUNEL technique to detect apoptotic cells and immunohistochemistry to distinguish between endothelial cells and pericytes, this present study demonstrated that the vasculature of the corpus luteum of pregnancy in the rat does not undergo apoptosis.

Endocrine and testicular changes in a short-day seasonally breeding bird, the emu (Dromaius novaehollandiae), in southwestern Australia.

Seasonal changes in testicular morphology and blood plasma concentrations of LH, testosterone, and prolactin are described for captive male emus in southwestern Australia. Testicular mass and testicular testosterone did not differ between the non-breeding (spring-summer) and the breeding (autumn-winter) seasons. Nevertheless, the testes obtained in the breeding season (May and August) were nearly two fold greater in mass than those collected in the non-breeding season (October and February). The highest testicular concentrations of testosterone were observed in February and lowest in October, while the values during the breeding season were intermediate. The patterns of histological changes in the testes also indicate that emus breed over the autumn-winter months. Tubule diameter was larger in the breeding season than in the non-breeding season, whereas the relative volume of the interstitium was larger in the non-breeding and smaller in the breeding season. Moreover, during the autumn and winter months, plasma LH and testosterone concentrations were high. Outside this period, in spring and summer, the concentrations of these hormones were low. Prolactin concentrations rose around the winter solstice, after the initial increases in plasma LH and testosterone. The end of the breeding season, in early spring, was marked by a gradual decrease in plasma LH concentrations but a rapid fall in testosterone concentrations. Prolactin concentrations continued to increase and peaked near the spring equinox, several weeks after the breeding season ended, and then decreased to reach baseline values by mid-summer. These testicular and endocrine changes are consistent with observations that the emu is a short-day breeder in southwestern Australia. Reproductive activity in the male begins soon after the summer solstice, well in advance of the development of suitable breeding conditions, and is then terminated in spring before food resources become limited by the onset of the dry season.

Early events in angiogenesis: cloning an alpha-prolyl 4-hydroxylase-like gene.

To gain further insight into angiogenesis we sought to clone genes which are actively expressed during this complex process. Using the Matrigel-induced in vitro model we were able to show that although several cell-types form reticular arrays of cells on the gels (align), only endothelial cells were able to go on and form the capillary-like structures reminiscent of patent vessels. Although this alignment process did not require gene activation we show that tube formation was ultimately dependent upon gene expression occuring during the first few hours that cells are seeded onto Matrigel. We generated a cDNA library enriched for the expression of those genes and have sequenced an alpha-prolyl 4-hydroxylase-like clone (angio 0.9). This clone shares 66% overall homology to the carboxy-terminal 106 amino-acids of the published human sequence. In the region corresponding to the co-factor binding domains, His 1 and His 2, angio 0.9 has >90% homology to the published sequence. Using an RNAse protection assay we show that the level of expression of the message of this clone is five fold elevated in endothelial cells which have aligned on Matrigel. The dependence of collagen, and collagen hydroxylation in angiogenesis is well documented. Thus, our results are demonstrable proof that the principle of this approach has the potential to generate novel discoveries.

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases.

The J2E erythroid cell line, transformed by retroviral v-raf/v-myc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constitutive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.

Lumen formation during angiogenesis in vitro involves phagocytic activity, formation and secretion of vacuoles, cell death, and capillary tube remodelling by different populations of endothelial cells.

BACKGROUND: We have utilised an in vitro model of angiogenesis to investigate the morphological changes which occur during the formation of a lumen in capillary tubes. METHODS AND RESULTS: On collagen 1 gel in the presence of phorbol myristate acetate (PMA) and anti-alpha 2 beta 1 antibody, cell aggregation and alignment takes place within two hours of plating. The initial apparently homogeneous population of endothelial cells (EC) actually display at least three distinct phenotypes. One population, characterised by a phagocytic phenotype, migrated through the gel creating channels and defines the extent of the capillary network. These are later enveloped by a second population of cells characterised by intracellular vacuoles. The ultimate fate of these vacuoles is fusion with the plasma membrane. By 12 hours the original phagocytic cell population undergoes cell death, which morphologically appears apoptotic in nature. A consequence of the secretion of vacuoles and programmed cell death is the extensive remodelling of the capillary tubes, resulting in expansion of the intercellular space into a lumen. The remodelling results in 45% of the EC membrane contacting the lumenal surface at the expense of EC-EC and EC-matrix contact. A third population of cells implant between the EC involved in lumen formation and thus expand the size of the capillary tube. CONCLUSION: Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.

Effects of ischaemia on the caput epididymis and its relationship to higher epididymal obstruction: a qualitative study in the ram.

The objective of this study was to determine if localized ischaemia of the caput epididymis in the ram causes morphological changes similar to those characteristic of higher epididymal obstruction in humans. This was tested by performing unilateral occlusion of the superior epididymal artery in 10 rams. At the end of 4 (n = 5) or 28 weeks (n = 4), rams were castrated and the testes and epididymides were weighed. Analysis of histological sections at the light microscope level provided quantitative data on lumen diameter and epithelial cell height of the efferent ducts. Qualitative analysis and specific histochemical stains for identification of lipofuscin pigment provided further information on tissue changes. Electron microscopy was performed on the efferent ducts to assess ultrastructural changes. The results revealed that localized ischaemia of the proximal epididymis caused a dramatic change in tubule calibre of the efferent ducts and initial segment resulting in obstruction of the distal lumina. These changes were more severe following 28 weeks of arterial occlusion. The epithelial cells of the proximal region showed an increase in the number of lysosomes and they became active in phagocytosis of spermatozoa. Lipofuscin pigment accumulated within the epithelial cells and also in macrophage-like cells that had invaded the lumina, interstitium and intra-epithelial regions of the ducts. On the basis of these observations we conclude that the tissue changes which occur in the ram epididymis as a result of localized ischaemia show a striking similarity to those seen in men exhibiting higher epididymal obstruction. This suggests the possible implication of vascular disorders in the aetiology of obstructive azoospermia in men.

Relationship between testicular morphology and sperm production following ischaemia in the ram.

Arteriosclerosis was induced in the internal spermatic artery of rams to determine if this condition is implicated in the aetiology of testicular pathology which causes male infertility. Data were collected on sperm concentration and motility for 56 days following surgery to provide an index of testicular function. Testes were then weighed and a testicular biopsy score count was performed on histological sections to assess spermatogenic potential of seminiferous tubules. Vascular disturbance caused focal damage of the seminiferous epithelium, similar to that seen among infertile men, and a reduction in ejaculate volume, sperm concentration and sperm motility. Sperm concentration decreased following ischaemia yet was maintained to some degree by a germ-cell depleted spermatogenic epithelium. Normal testicular morphology was maintained above a testis weight of about 120 g (for an individual testis), but below this threshold spermatogenesis was severely impaired. In conclusion, these data have provided information on the relationship between testicular morphology and function following ischaemia in the ram. Furthermore, the morphological changes induced in the testis were similar to those seen among infertile men and, by their focal nature, could explain the distinction between oligozoospermia and azoospermia in men exhibiting spermatogenic arrest.

Testicular morphology and androgen profiles following testicular ischaemia in rams.

Arteriosclerotic changes were induced in the internal spermatic artery of rams to determine whether there is a link between this condition and some pathological conditions of the testes, similar to those that cause infertility in men. Eight weeks after the induction of testicular ischaemia, blood plasma was collected simultaneously from the jugular and spermatic veins after an LH injection (10 micrograms) and assayed for testosterone. The rams were then castrated and sections of the testis, ductuli efferentes and spermatic cord were examined quantitatively and qualitatively. Vascular disturbance decreased the percentage of normal spermatogenic epithelium (P < 0.01) and the diameter of the seminiferous tubules (P < 0.001). These effects were accompanied by an increase in the percentage of the interstitial region within the testis (P < 0.05). Macrophages, lymphocytes and other inflammatory cells became numerous in the interstitium as damage to the seminiferous epithelium progressed. The most striking feature of the ischaemic testis was the focal damage of the spermatogenic epithelium, that is, sections of the same testis exhibited both normal and germ cell-depleted seminiferous tubules. Concentrations of testosterone in peripheral plasma were not significantly altered by either unilateral or bilateral testicular ischaemia; however, the concentration of testosterone was higher in the experimental spermatic vein than in the contralateral spermatic vein (P < 0.05) as was the ratio of LH:testosterone (P < 0.05). Unilateral vascular disturbance of the testis did not cause damage in the contralateral testis. The ductuli efferentes of these rams also showed structural changes as a result of vascular disturbance.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythropoietin induced ultrastructural alterations to J2E cells and loss of proliferative capacity with terminal differentiation.

Erythropoietin (epo) induced differentiation of the J2E erythroid cell line is characterized by haemoglobin synthesis, together with morphological changes and an immediate increase in proliferation. In this manuscript we have shown that the size of J2E cells decreased during differentiation and the nucleus to cytoplasm ratio was reduced appreciably. Furthermore, major ultrastructural alterations occurred-mitochondria, rough endoplasmic reticulum and Golgi apparatus decreased in size and number with maturation, while nuclei condensed considerably before extrusion. The use of mitotic indices, 3H-thymidine uptakes and flow cytometry confirmed that the immature J2E cells undergo enhanced replication shortly after epo stimulation. In addition, we demonstrated that cell division ceased as the cells entered the final stages of erythroid differentiation. Thus the J2E line provides a useful model, not only for haemoglobin synthesis, but for all aspects of erythroid terminal differentiation.

A quantitative description of the epididymis and its microvasculature: an age-related study in the rat.

The morphology of the epididymal duct and, in particular, the epididymal microvasculature was examined at the light microscope level in young sexually-mature rats (3-5 months) and aged rats (18 months) to investigate the structural changes that may occur within the organ as a result of ageing, and which may predispose the organ to pathological changes. Quantitative data on the microvascular network of the epididymis (percentage of capillaries in the interstitial region, average area and surface density of the capillary lumen) were collected in 4 regions of the epididymis: the initial segment, caput, corpus and cauda. Epithelial cell height, epididymal lumen diameter, number of smooth muscle cells and percentage of smooth muscle surrounding the duct were also assessed within the same 4 regions. The data for both young and aged groups revealed a trend of decreasing capillary size from the initial segment of the epididymis to the cauda by 23%. Further, the percentage of capillaries within the interstitial region of the epididymis decreases dramatically (52%) in the same direction. The possible contribution of lymphatic capillaries to the data is discussed. The data revealed that none of the parameters assessed changed significantly up to 18 months of age. The quantitative data on the microvascular morphology of the epididymis presented in this study provide the basis for subsequent studies directed at the blood flow dynamics of the organ.

Structural and functional reserve of steroidogenic membranes in stimulated luteal cells of pregnant rats.

A comparison was made of the ultrastructural features of the remaining corpora lutea (CL) of five unilaterally ovariectomised and five control pregnant rats. Unilateral ovariectomy (ULO) was carried out on Day 8 of gestation; this treatment has been shown to double the normal rate of progesterone secretion by the remaining ovary within 8 days. On Day 16, the ovaries from both ULO and control rats were examined stereologically. The mass of luteal tissue remaining in ULO rats was only 44% of that in the controls, but their plasma progesterone concentration was 81% and not significantly different from the control value. The CL were 10% heavier in ULO rats and the amount of luteal cell cytoplasm per CL was 21% greater. The percentage of the CL occupied by luteal cells was 15% greater in ULO rats but the interstitial space was 50% less. There was no significant change in the percentage of the luteal cell cytoplasm occupied by cytosol, agranular endoplasmic reticulum (AER), mitochondria and electron-dense granules. The surface area per unit cytoplasmic volume of AER and outer and inner mitochondrial membranes (OMM and IMM, respectively) was not affected. However, expressed per CL, both the AER and IMM membrane surface areas were increased by around 30%. These morphometric changes would only account for about 30% of the reported increase in hormone secretion by the CL of ULO rats, and they suggest that the CL of Day 16 pregnant rats may have reached their optimal structural condition.

Quantitative comparison of luteal histology in the rat and rabbit: changes from mid- to late gestation.

Progesterone secretion by the corpora lutea (CL) of rats and rabbits declines from a peak, at about Day 16 of gestation, to near term (Day 22 rats and 28 rabbits). However there are major differences between the two species in CL growth and blood flow over this period. In the present work quantitative histological measurements were made of CL at these stages to examine the accompanying structural changes. Eight rats and five rabbits were examined at each stage: standard morphometric techniques were used. There was gross discrepancy between the two species in the histology of their CL at peak secretory activity. Although the proportions of the major tissue components were similar, the rabbit luteal cell (52 pl) was five times larger than that in the rat (9 pl). There was substantially less vascular and interstitial space in the rabbit, all characteristics which might affect transport processes between luteal cell cytoplasm and blood. Over the period examined, there was no change in CL volume in the rat but a 37% reduction in the rabbit due to loss of luteal cells. The vascular space in the rat, however, was reduced, whereas that in the rabbit declined only in proportion to the overall decrease in CL volume. These results show that structurally there is a substantial difference in patterns of early regression between the two species which reflect different mechanisms involved.

Angiogenesis in the developing corpus luteum of pregnant rats: a stereologic and autoradiographic study.

Within the adult mammalian ovary, angiogenesis is associated with development of the corpus luteum (CL). In this study, developing luteal tissue was examined to determine whether its vascularization involves endothelial cell replication and to what extent this proliferation contributes to forming new capillaries. Five rats each at 12, 24, 36, 48, 60, 72, 84, and 96 hr of gestation were given a subcutaneous injection of tritiated thymidine (specific activity 5 Ci/mM; 1 uCi/g body weight). One hour later they were anesthetised with sodium pentobarbitone, and the left ovary was processed for light microscopy. Sections were cut through each ovary until three newly formed CL were recognized; a 1-micron section was taken from the maximum diameter of each CL and processed for autoradiographic demonstration of thymidine labeling in endothelial cell nuclei. The same sections were also examined with stereological techniques to quantitate growth of the vascular compartment. The results show that 36.1 +/- 5.7% of endothelial cells of invading capillary sprouts divide within 12 hr of ovulation; at 24 hr, 29.0 +/- 2.8% are dividing. Within 12 hr after ovulation, blood vessels occupied 5.9 +/- 1.4% of the peripheral space of the ruptured follicle but only 1.6 +/- 0.5% in the center. However, by 36 hr these respective values were 9.3 +/- 1.6% and 8.4 +/- 1.9%. A further peak in endothelial-cell replication (31.2 +/- 5.4%), early on the 3rd day of gestation, corresponded to the very extensive anastomoses within the capillary bed established between this time (13.6% vascularity) and late on the 4th day (about 23%).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative ultrastructural characteristics relating to transport between luteal cell cytoplasm and blood in the corpus luteum of the pregnant rat.

The synthesis and secretion of progesterone by the corpus luteum (CL) may be limited or controlled by transport mechanisms operating between circulating blood and luteal cell cytoplasm. To examine this possibility, the structural features involved in transport, including membrane surface areas and diffusion distances, were quantitated in the CL of 16-day pregnant rats. One ovary from each of eight rats was fixed by perfusion via a cannula inserted into the parametrial artery, and two CL from each ovary were processed for electron microscopy and examined with standard morphometric techniques. For comparison, one CL from each of a further eight ovaries was diced into small cubes, fixed by immersion, and analyzed similarly. In perfusion-fixed CL, there was a substantial volume of vascular space (20% of the total) and interstitial space (5%) and an extensive surface area of capillaries (441 mm2 per CL). The luteal-cell membrane had numerous projections which increased its surface area by a factor of 3.08. Almost 60% of the luteal-cell surface directly faced a capillary, and a further 37% faced interstitial space which probably extended to a capillary surface. Only 3% was in direct contact with a neighboring luteal cell. Despite the extensive interstitial space the harmonic mean thickness, an estimate of likely effective diffusion distance between luteal cell cytoplasm and blood, was only 0.42 micron. This was less than half of the calculated arithmetic mean thickness owing to the presence of surface projections and an uneven capillary endothelium. Results from immersion-fixed CL were qualitatively similar; but the proportion of interstitial space was only 59% of that in perfusion-fixed CL, and the contribution of surface projections to the total area of luteal-cell membranes was significantly reduced. Collectively, these results suggest that membranes and spaces between blood and luteal-cell cytoplasm are structured so as to minimize transport distances.

Rate of blood flow and growth of the corpora lutea of pregnancy and of previous cycles throughout pregnancy in the rat.

The rate and distribution of ovarian blood flow was measured with radioactive microspheres in nulliparous rats at Days 4, 7, 10, 13, 16 and 22 of gestation; term is Day 23. Ovaries were dissected into large corpora lutea (or CL of pregnancy), small CL, assumed to be those of previous cycles, and the ovarian stroma which included follicles and additional small CL. Total ovarian blood flow was similar over Days 4-10, but then increased progressively to reach 916 +/- 69 microliter/min at Day 22. The main cause of this increase was a 786% rise in blood flow to the large CL over Days 10-22. Weight of the CL of previous cycles increased by 289% over Days 10-16 and their rate of blood flow and histological appearance were similar to those of the large CL. These results indicate that neither reduced blood flow nor vascular degeneration, as determined by light microscopic examination, initiates the reported fall in progesterone secretion near term. At least some of the CL of previous cycles are maintained and indeed grow during gestation and have a blood circulation and histological appearance characteristic of steroidogenic tissue.

Effects of handling, anaesthesia, ovariectomy and adrenalectomy on serial measurements of plasma progesterone in 16-day pregnant rats.

The basal concentrations of progesterone in plasma of 16-day pregnant rats were measured after seven different sampling procedures. Progesterone concentrations in serial samples from rats held at the time of sampling (restrained group) were compared with those obtained from rats allowed to remain free in their cage (free group). In addition, the effects on plasma progesterone concentrations of anaesthesia induced by ether or pentobarbitone sodium, and of adrenalectomy and/or ovariectomy were studied. Over the 8-h sampling period, progesterone concentrations in the plasma of restrained rats, with or without anaesthesia, were about 30% higher and more variable than those in free rats. Progesterone concentrations rose sharply over the first 30 min in restrained rats and in those treated with ether. Rats adrenalectomized the day before sampling did not show this early rise and their progesterone concentrations were similar to those of free rats. Progesterone concentrations were lowest in ovariectomized rats which had also been adrenalectomized. These findings show that adrenal secretion can increase plasma concentrations of progesterone in pregnant rats which have been handled or anaesthetized. Such a rise might well modulate the effects of experimental stimuli.

Quantitative ultrastructure of the luteal cell of the 16-day-pregnant rat and its relation to progesterone secretion.

The ultrastructure of luteal cells of five Day-16 pregnant rats were examined morphometrically to determine the relationship between the quantity of steroidogenic organelles and membranes and reported rates of progesterone secretion (2.3 micrograms/h). Each rat had 11.8 +/- 1.0 corpora lutea (mean +/- s.e.m.) with an average volume of 4.5 +/- 0.1 microliter. There were 210 000 +/- 10 000 luteal cells per CL and the luteal cell cytoplasm was composed of smooth endoplasmic reticulum (18%), mitochondria (10.6%), lipid droplets (8.9%) and granules (0.6%). The surface area of the smooth endoplasmic reticulum was 192 cm2 per CL, and that of the outer and inner mitochondrial membranes was 20 and 34 cm2, respectively. For each square micrometre of these membranes, respectively, 62, 590 and 355 molecules of progesterone would have been secreted per second. The luteal cell appears to secrete its major steroid hormone at a rate 50 times greater than that reported for the Leydig cell of the testis when secretion is expressed in terms of molecules per unit mass of steroidogenic cell or area of steroidogenic membrane.

Morphometric analysis of the corpus luteum of 16-day pregnant rats: the effect of preparative procedures on volume of luteal cells, interstitial, and vascular compartments.

The volumes of the major tissue compartments, vascular space, interstitial space, and luteal cells, in the corpus luteum (CL) of 16-day pregnant rats were determined morphometrically after different fixation procedures. Ovaries were fixed by perfusion at physiologic pressures (PF), by immersion directly in fixative (IF), by immersion after clamping blood vessels to prevent escape of blood (IFC), or by immersion after administration of heparin to prevent blood clotting (IFH). Three CL in each of eight ovaries were examined for each fixation method. Total volumes were significantly larger in CL fixed by PF and IFC, and approximated physiologic volumes as assessed from frozen ovaries. Perfusion-fixed CL had significantly larger vascular and interstitial spaces and smaller cell volumes than immersion-fixed CL. Capillary surface area varied significantly between the four groups in accordance with absolute volume of vascular space. Estimated numbers of endothelial cells and amounts of connective tissue differed significantly, presumably due to difficulties in identifying these components in immersion-fixed CL with collapsed vessels. The fixation methods used caused substantial fluid shifts between the three major compartments. A theoretical approach for considering these shifts and the likely physiologic situation was developed.

Rate of ovarian progesterone secretion and peripheral plasma progesterone levels in unilaterally ovariectomized, pregnant rats.

Peripheral plasma progesterone concentrations were serially examined in 4 sham-operated and 5 intact rats (controls) and in 5 rats that were unilaterally ovariectomized on Day 8 of gestation. The characteristic rise in progesterone levels after Day 10 appeared to be delayed by 2 days in unilaterally ovariectomized rats but by Day 16 their levels were nearly normal. In a separate experiment a venous outflow technique was used to measure the rate of progesterone secretion from one ovary on Day 16 of gestation of 9 intact rats and the remaining ovary of 5 rats, unilaterally ovariectomized 8 days earlier. The rate of progesterone secretion was almost doubled in the unilaterally ovariectomized rats due to a substantial increase in arterio-venous different in plasma progesterone levels but no change in ovarian blood flow. This doubling was sufficient to explain the maintenance of peripheral plasma progesterone levels in unilaterally ovariectomized rats without postulating increased secretion from other organs or a decrease in metabolic clearance rate of progesterone.