Female infertility in mice lacking connexin 37

The signals regulating ovarian follicle development and the mechanisms by which they are communicated are largely undefined. At birth, the ovary contains primordial follicles consisting of meiotically arrested oocytes surrounded by a single layer of supporting (granulosa) cells. Periodically, subsets of primordial follicles undergo further development during which the oocyte increases in size and the granulosa cells proliferate, stratify and develop a fluid-filled antrum. After ovulation, oocytes resume meiosis and granulosa cells retained in the follicle differentiate into steroidogenic cells, forming the corpus luteum. It has been proposed that intercellular signalling through gap junction channels may influence aspects of follicular development. Gap junctions are aggregations of intercellular channels composed of connexins, a family of at least 13 related proteins that directly connect adjacent cells allowing the diffusional movement of ions, metabolites, and other potential signalling molecules. Here we show that connexin 37 is present in gap junctions between oocyte and granulosa cells and that connexin 37-deficient mice lack mature (Graafian) follicles, fail to ovulate and develop numerous inappropriate corpora lutea. In addition, oocyte development arrests before meiotic competence is achieved. Thus, cell-cell signalling through intercellular channels critically regulates the highly coordinated set of cellular interactions required for successful oogenesis and ovulation.     

The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells

Oocyte development within avian ovarian follicles is an intricate process involving yolk deposition and the formation of extraoocytic matrices. Of these, the perivitelline membrane (pvm) not only plays a role in sperm binding but also provides mechanical support for the large oocyte's journey through the oviduct after ovulation. To date we have focused on the mechanisms for uptake of yolk precursors into oocytes of the chicken; now we extend our studies to a detailed analysis of the pvm. In the course of characterization of its major components, we obtained partial protein sequences; comparison with the GenBank database revealed that one of the pvm proteins is the homologue of mammalian zona pellucida glycoprotein 3 (ZP3), a key component in sperm binding. Following a nomenclature based on gene structure, the protein is referred to as chicken ZPC (chZPC). The chicken protein (444 residues) and murine ZP3 (424 residues) are highly conserved, with 41% of the amino acids identical. As shown by Northern blot analysis, the avian ZPC gene is expressed exclusively in the granulosa cells surrounding the oocyte, in contrast to murine ZP3, which is synthesized by the oocyte. Upon reaching a size larger than 1.5 mm in diameter, follicles accumulate chZPC in highly polarized fashion, i.e., in the space intercalated between the oocyte and the granulosa cells, as revealed by immunohistochemistry of follicle sections. ChZPC synthesis and secretion by granulosa cells was demonstrated directly by metabolic labeling and immunoprecipitation from the culture medium of granulosa cell sheets isolated ex vivo from follicles. Immunoblot analysis and glycosidase treatment of chZPC from preovulatory and freshly ovulated oocytes, as well as laid eggs, revealed that the primary product undergoes a two-step decrease in size from follicle to laid egg that is unlikely to be due to modification of the carbohydrate moiety.     

Ovarian development in mice bearing homozygous or heterozygous null mutations in zona pellucida glycoprotein gene mZP3

The plasma membrane of all mammalian eggs is surrounded by a thick extracellular coat, the zona pellucida (ZP), whose paramount function is to regulate species-specific fertilization. The mouse egg ZP is composed of only three glycoproteins, mZP1-3, that are synthesized and secreted exclusively by oocytes during their 2-3 week growth phase. Disruption of the mZP3 gene by targeted mutagenesis in embryonic stem (ES) cells yields mice heterozygous (mZP3 +/-) or homozygous (mZP3-/-) for the null mutation. As expected, male mice bearing the null mutation are indistinguishable from wild-type males with respect to viability and fertility. Female mZP3 +/- mice are as fertile as wild-type animals, but their eggs have a thin ZP (approximately 2.7 microns thick) as compared to the ZP (approximately 6.2 microns thick) of eggs from wild-type animals. On the other hand, female mZP3-/- mice are infertile and their eggs lack a ZP. The infertility apparently is due to the lack of a sufficient number of eggs in oviducts of superovulated mZP3-/- females. Light micrographs reveal that development of ovarian follicles is often retarded in mZP3-/- mice as compared to wild-type animals. This is manifested as reduced ovarian weights, reduced numbers of Graafian follicles, and reduced numbers of fully-grown oocytes in mZP3-/- females. It seems likely that the pleiotropic effects of the homozygous null mutation on ovarian development may be due, at least in part, to disruption of intercellular communication between growing oocytes and their surrounding follicle cells.     

The National Toxicology Program evaluation of genetically altered mice as predictive models for identifying carcinogens

In this study cytoskeletal antigens common to brushtail possum and tammar wallaby spermatozoa were characterised using a monoclonal antibody (PSA-10). Using indirect immunofluorescence, the PSA-10 antibody detected antigens predominantly associated with the midpiece and principal piece of mature, permeabilised marsupial spermatozoa. The principal piece determinant, shared by a variety of other species, was found to arise in the marsupial testis. Midpiece localisation of the PSA-10 epitope was detected only in marsupial spermatozoa and shown to arise in the epididymis. Immunogold labelling demonstrated that the PSA-10 antigens were predominantly associated with the fibrous sheath and midpiece fibre network of both possum and wallaby spermatozoa. Western blotting suggested that two major possum and wallaby sperm polypeptides of 158 and 182 kDa were associated with the midpiece fibre network, a cytoskeletal structure unique to marsupial spermatozoa. A 32 kDa polypeptide was associated with the principal piece fibre network and/or fibrous sheath. The finding that these marsupial sperm cytoskeletal proteins share a common linear epitope suggests that they share some sequence similarity. The midpiece fibre network of marsupial sperm, like the fibrous sheath, has been proposed to have a structural role in providing passive stiffening for the flagellum (Harding et al., 1975, 1979; Olsen, 1975). The PSA-10 monoclonal antibody may provide a tool for comparative studies of mammalian sperm cytoskeletal proteins, particularly the marsupial midpiece fibre network. It may also allow the formation of this unique marsupial cytoskeletal structure, and its fate during the fertilisation process, to be followed by immunological means.     

Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad

Oocyte loss, either directly through attrition (germ cell death) or indirectly through follicular atresia (somatic or granulosa cell death), is a fundamental event associated with defining the time of normal or premature reproductive senescence in females. Although apoptosis has been reported to function as the underlying mechanism responsible for death of both germ cells and somatic cells in the ovary, the final molecular steps which commit ovarian cells to death have not been fully elucidated. To examine if death repressor activity of the bcl-2 gene product is important for germ cell survival, we conducted studies using a Bcl-2 loss-of-function (bcl-2 -/-) transgenic mouse model. Histological analyses revealed that ovaries collected from bcl-2 -/- mice possessed numerous aberrantly formed primordial follicle-like structures containing a single layer of granulosa cells without an oocyte. Additionally, the total number of primordial follicles present which contained a healthy oocyte was markedly reduced in bcl-2 -/- mice as compared to heterozygote (bcl-2 -/+) or wild-type (bcl-2 +/+) mice, suggesting that expression of the bcl-2 death repressor gene is critical for endowment of a normal complement of germ cells and primordial follicles in the mammalian ovary.     

Further observations of the ovarian response of the tammar wallaby (Macropus eugenii) to exogenous gonadotrophins: an improved method for superovulation using FSH/LH

This study reports the development of an improved superovulation protocol in the monovulatory tammar wallaby, Macropus eugenii. Treatment with pregnant mare's serum gonadotrophin (PMSG; 10-20 IU) inhibited follicle development in the corpus luteum (CL)-bearing ovary and only 2-3 eggs per female could be recovered after ovulation induction with gonadotrophin releasing hormone (GnRH; 3 x 30 microg at 3-h intervals) or porcine luteinizing hormone (LH; 4, 5 or 8 mg) 3 days after PMSG priming. Treatment with porcine FSH (8 x 6 mg at 12-h intervals for four consecutive days) was found to override this inhibition and resulted in the recovery of 7-13 eggs per female after ovulation induction with porcine LH (4 mg on day 5). For these animals, there was no difference in numbers of developing follicles, ovulation sites and eggs recovered between the CL- and non-CL-bearing ovaries. This FSH/LH protocol was effective in both cycling and non-cycling females, and multiple ovulation occurred from about 36 h after LH treatment. After LH treatment, eggs were recovered from the oviduct at 36-50 h. At 51-57 h, 12-25% of eggs were recovered from the uterus, and by 75 h all eggs were recovered from the uterus. It is concluded that the described FSH/LH protocol used results in higher ovulation success than the PMSG/GnRH method.     

Auditory brainstem response in tammar wallaby (Macropus eugenii)

Auditory brainstem responses (ABR) elicited by click and tonal stimuli were recorded from the tammar wallaby (Macropus eugenii), a marsupial mammal. The morphology, threshold, amplitude, and latency of ABRs recorded in the tammar wallaby are similar to those of other marsupials and mammals used in auditory research, including humans. Thresholds determined by an algorithm employing cross-correlation and by conventional visual detection methods were comparable. The findings from this study indicate that tammar wallaby is a suitable model for auditory research and that algorithms employing cross-correlation are useful for detection of the ABR waveform.     

Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats

Previous studies have determined that 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocytes contained in small pre-antral (primordial and primary) ovarian follicles of Fischer 344 rats following 30 days of daily dosing with VCD. The purposes of this study were to identify the type of VCD-induced cell death occurring in small pre-antral follicles and to determine the earliest time following the onset of dosing when evidence of follicular destruction could first be detected. A significant decrease in the number of oocytes contained in small pre-antral follicles in ovaries of rats after 15 days of daily dosing (ip) with VCD (80 mg/kg) had been observed in preliminary experiments. Therefore, a study was conducted to determine the time of the onset of this follicular destruction by examination of follicular DNA integrity. Female Fischer 344 rats were dosed daily (80 mg/kg, i.p.) for 6, 8, 10, 12, or 14 days, and ovaries were removed 1, 4, or 24 hr after the final dose. Small pre-antral follicles (25-100 microns) were isolated by gentle dissociation of ovaries with collagenase, and follicles were sorted with micropipets. Genomic DNA was isolated from follicles and radiolabeled with [32P]dideoxy ATP, and the degree of fragmentation quantified by agarose gel electrophoresis and autoradiography. Degradation of DNA was evaluated by 32P content in low-molecular-weight fragments ( < 4 kilobase pairs). Degradation of DNA was not observed in follicles collected 24 hr after the final dose on any day. However, a random pattern of DNA degradation was observed, and was significantly greater (p < 0.05) compared with controls, when follicles were collected 4 hr following VCD administration on Days 10 and 12, but not on Days 6 or 8, of dosing. Although not significant, there was also evidence of DNA degradation in dosed animals on Day 14. Histological evaluation of small pre-antral follicles in ovarian sections during the early stages of VCD-induced DNA degradation (Day 10; 4 hr) demonstrated margination of chromatin along the nuclear membrane in oocytes and disruptions in focal contact between granulosa cells and oocytes, both features indicative of apoptosis. Furthermore, there was no sign of ruptured membranes in granulosa cells or oocytes or of an inflammatory response, characteristics of necrosis (pathological cell death). Whereas biochemical and morphological evidence of follicular destruction was seen 4 hr after dosing on Day 10, numbers of oocyte-containing primordial and primary follicles in VCD-treated animals were not different from controls at that time. These results demonstrate that the initial evidence of impending destruction of small pre-antral follicles is first consistently visualized following 10 days of daily dosing with VCD, although a measurable reduction in oocyte numbers has not yet occurred. Despite the fact that internucleosomal cleavage of genomic DNA was not observed, morphological evaluations support that granulosa cells and oocytes in primordial and primary follicles are destroyed via the induction of apoptosis.     

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor alpha is deleted by targeted disruption.     

Growth and cellular proliferation of antral follicles throughout the follicular phase of the estrous cycle in Meishan gilts

Meishan gilts were ovariectomized 2 h after an i.v. injection of 5'-bromo-2'-deoxyuridine (BrdU, a thymidine analogue; 5 mg/kg body weight) on Days 15-19 of the estrous cycle or 24-30 h after observed estrus (post LH, PLH). All antral follicles > or = 3 mm from one ovary were fixed in Carnoy's solution. Granulosa and thecal cell labeling indexes (LI; percentage of nuclei staining for BrdU) as well as LI of cells within the basal, middle, and antral thirds of the granulosa cell layer were estimated for each follicle. In addition, antral and granulosa cell layer volume, granulosa cell layer thickness, granulosa cell density, number of granulosa cells, and number of S-phase cells per hour were estimated for each follicle. Mean follicular diameter increased linearly (p < 0.01) from Day 15 to PLH, with a growth rate of 0.77 mm/day. Granulosa and thecal cell LI decreased (p < 0.01) from Day 15 to PLH; however, granulosa cell LI was greater (p < 0.01) than thecal cell LI on Days 15 and 16 but less (p < 0.05) than thecal cell LI on Day 19. Follicles collected from PLH gilts contained no labeled granulosa cells. Cells within the basal third of the granulosa cell layer contained fewer (p < 0.01) labeled nuclei than did cells within the middle or antral thirds. In addition, LI within the basal and middle thirds of the granulosa cell layer decreased (p < 0.01) from Days 15 to 18 and from Days 15 to 17, respectively, whereas LI within the antral third remained constant from Days 15 to 18. Granulosa cell layer thickness was greatest (p < 0.01) on Day 15, then decreased (p < 0.01) and was similar from Day 16 to PLH. Granulosa cell density was similar from Days 15 to 19, then decreased (p < 0.01) for PLH gilts. Antral and granulosa cell layer volumes increased linearly (p < 0.01) from Days 15 to 19 and Day 15 to PLH, respectively, resulting in 2.8 and 1.9 volume doublings and doubling times of 1.4 and 2.7 days, respectively. Number of granulosa cells per follicle increased linearly (p < 0.01) from Day 15 to PLH, resulting in 1.5 cell doublings and a doubling time of 3.3 days. Number of S-phase cells per follicle per hour was similar from Days 15 to 18 and then decreased (p > 0.01) from Day 18 to PLH. In summary, the percentages of proliferating granulosa and thecal cells decreased throughout the final stages of antral follicular development. Differentiation of granulosa cells occurred from the basal to the antral area as follicles matured. We proposed that, during the latter stages of follicular development, the rapid increase in follicular diameter resulted primarily from expansion of the antral cavity, whereas increases in the granulosa cell layer volume and number of granulosa cells per follicle maintained a constant granulosa cell layer thickness.     

Immunocytochemical comparison of cultured normal epithelial prostatic cells with prostatic tissue sections

The cytologic localization and cellular levels of myc oncoprotein in the human ovary during follicular growth, regression and atresia were examined by the avidin/biotin immunoperoxidase method with a specific antibody to myc oncoprotein. In primordial follicles, only the oocyte showed intense immunostaining for myc protein, whereas the granulosa cells were negative for the staining. In preantral follicles, both the oocyte and granulosa cells were moderately immunostained for myc protein. In antral and preovulatory follicles, there was no appreciable staining for myc protein in the granulosa or theca cells, while myc protein staining in the oocyte persisted with less intensity. It is of interest that myc protein expression in granulosa cells was apparent only during the preantral follicle stage. Corpora lutea during the early and mid luteal phase were negative for myc protein staining, whereas in regressing corpora lutea during the late luteal phase, peripheral theca lutein cells adjacent to the central core of scar tissue were immunostained for myc protein. Corpora albicans showed no staining for myc protein. In atretic follicles, granulosa cells and theca interna cells demonstrated positive staining for myc protein. Ovarian stromal cells were negative for the immunostaining throughout the menstrual cycle. This demonstrates that myc protein is expressed in a stage-limited manner in the human ovary during follicular growth and regression. The abundant expression of myc protein in the oocyte at the primordial and preantral follicle stages and in the granulosa cells at the preantral follicle stage suggests a role for myc expression in the initial growth of the oocyte as well as in the autonomous growth of granulosa cells during the preantral stage seemingly independent of gonadotropic stimulation. Furthermore, notable expression of myc protein in the granulosa cells and theca interna cells of atretic follicles and in the peripheral theca lutein cells of regressing corpora lutea implies the possible participation of myc expression in remodelling the ovarian local tissue following atresia and luteolysis in the human ovary.     

Plasma esterase (ES) polymorphism in the tammar wallaby, Macropus eugenii

The major plasma esterase in the tammar wallaby was identified as a carboxylesterase by inhibition studies and polymorphism with six variants was observed by isoelectric focusing (pH 4.2-4.9), followed by staining for esterase activity. Family studies demonstrated an inheritance of six codominant alleles, ESA,B,C,D,E,F, and population studies revealed marked differences in the allele frequencies in five Australian populations of tammar wallabies.     

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces genital dysmorphogenesis in the female rat

Recently, Gray and Ostby (Toxicol. Appl. Pharmacol. 133, 285-294, 1995) reported that in utero and lactational TCDD exposure causes striking abnormalities in the rat female reproductive system, including reduced fecundity and vaginal threads. The mechanism by which TCDD induces such abnormalities is unknown. Thus, we sought to determine: (1) whether TCDD reduced fecundity by destroying ovarian follicles and (2) whether the vaginal threads resulted from a TCDD-induced developmental defect during embryogenesis or abnormal vaginal opening at puberty. Pregnant Holtzman rats were treated with 1.0 microgram TCDD/kg or vehicle by a single oral dose on gestation day (GD) 11, 15, or 18. Female offspring were monitored for vaginal opening and terminated on postnatal days 2, 21, and 42. The reproductive tract was removed and evaluated for structural abnormalities. The number of primordial follicles also was determined for each ovary. TCDD exposure on GD 11, 15, or 18 did not change the day of vaginal opening, affect ovarian morphology, or reduce the number of primordial follicles. However, this exposure induced the cleft clitoris and vaginal thread originally described by Gray and Ostby (1995) in approximately 55-96% and 36-44% of the litters in our study, respectively. Histologically the thread presented as a thick cord of mesenchyme surrounded by epithelial cells. This defect was clearly visible in histological sections at birth and was noted in the closed vaginas of prepubertal animals. These data suggest that in utero and lactational exposure to TCDD does not reduce the size of the primordial follicle pool; however, it induces developmental abnormalities in the vaginal canal.     

Expression of messenger ribonucleic acid (mRNA) encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles: identification of dominant follicles by expression of 3beta-HSD mRNA within the granulosa cell layer

The objective of the present study was to examine changes in expression of mRNA encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) following estrus. Expression of 3beta-HSD mRNA was localized by in situ hybridization and quantified by image analysis. Expression of 3beta-HSD mRNA was first detected in theca interna cells of preantral follicles with a well-developed theca layer and in granulosa cells of follicles > or = 8 mm in diameter. Regardless of stage of follicular wave, expression of 3beta-HSD mRNA in granulosa cells of follicles > or = 8 mm was correlated with follicular size (r = 0.665; p < 0.01). The 36-h time period appeared to be a transition period for selection since dominant follicles were detected by size and expression of 3beta-HSD mRNA in some cows but not in others. By 48 h after wave initiation, dominant follicles could be identified by both size and expression of 3beta-HSD mRNA. Expression of mRNA for 3beta-HSD in theca cells was higher (p < 0.05) at 24 h than at 12 h and remained elevated thereafter through 96 h. In contrast to theca cells, expression of mRNA for 3beta-HSD was undetectable within granulosa cells at 12 and 24 h. At 36 h, 3beta-HSD mRNA was expressed in granulosa cells of healthy follicles > or = 8 mm, and expression was higher (p < 0.05) at 48 h compared with 36 h. Expression of 3beta-HSD mRNA levels increased further in granulosa cells (p < 0.05) at 84 and 96 h compared to 48 h. Upon detection of mRNA for 3beta-HSD in granulosa cells, high levels of expression were always found in one (dominant) follicle/cow with the exception of two cows at 36 and 84 h that expressed 3beta-HSD mRNA in two large healthy follicles. Expression of 3beta-HSD mRNA was also detectable in granulosa cells of a few large atretic follicles in which remnant granulosa cells appeared to be luteinized. Healthy follicles expressed higher (p < 0.05) levels of 3beta-HSD mRNA in both theca and granulosa cells than did atretic follicles. Expression of 3beta-HSD mRNA in theca cells was higher (p < 0.01) in dominant follicles than in other subordinate healthy follicles. These results indicate that only selected dominant follicles express 3beta-HSD mRNA within granulosa cells, and expression increased in both thecal and granulosa cells during the follicular wave. Therefore, expression of 3beta-HSD mRNA within granulosa cells may be associated with the mechanism of selection of the dominant follicle during a follicular wave and may be required for maximum steroid production during follicular dominance.     

Development of commissural neurons in the wallaby (Macropus eugenii)

We have examined the development of the laminar and areal distribution of cortical commissural neurons in a marsupial mammal, the wallaby Macropus eugenii. In this species, commissural axons approach the major cerebral commissure, the anterior commissure, via either the internal capsule or the external capsule and first cross the midline at postnatal day 14 (P14). By retrogradely labelling these axons with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) at P15, we show here that the cell bodies of these neurons are restricted to a region of cortex adjacent to the rhinal fissure. Most of these labelled neurons are located in the compact cell zone of the cortical plate, with only a few labelled cells found in the zone of loosely packed cells deep to this layer. Over the subsequent 66 days, commissural neurons are found progressively more dorsally, rostrally, and caudally, so that, by P80, they are present throughout the extent of the neocortex. At this age, they are mainly pyramidal in morphology and form a single band within the deeper part of layer 5 of the developing cortex. From P80 to adulthood, the distribution of commissural neurons has been assessed in the visual cortex by using retrograde transport of horseradish peroxidase. At P80, labelled neurons with immature pyramidal morphology are present throughout the occipital cortex; as in DiI material, somata are located in deep layer 5. At P165, previously shown to be the age when commissural axon numbers peak, widespread labelling is present in the occipital region, with labelled cells now found in two bands corresponding to layers 3 and 5. After this age, neurons become more restricted in distribution, so that, by adulthood, commissural neurons are no longer apparent throughout area 17 but are restricted to a localised region around the area 17/18 boundary. Within this region, labelling is still present in layers 3 and 5 but is more dense in layer 3. The gradual restriction of commissural fields seen here in the wallaby is similar to that reported in the neocortex in many eutherians. These findings also support studies in eutheria, suggesting that subplate neurons do not appear to play a major role in commissural development.     

Atropine and testosterone propionate induced atretic changes in granulosa cells of house rat (Rattus rattus) ovary

Degenerative changes in membrana granulosa of ovaries in R. rattus have been studied using scanning electron microscopy. Ovaries from rats treated with atropine (300 mg/kg body weight) and testosterone propionate (10 IU) were used to study sequential course of atresia in granulosa cells. Granulosa cells undergoing atresia showed degenerative changes in following order i) loosening of intercellular matrix, ii) changed morphology and texture of secretory granules, iii) destabilization of granulosa cell membranes, iv) erosion of cell membrane, v) formation of specific degenerative belts, vi) pycnosis, vii) ghost cell formation and their subsequent mixing in hazzy follicular fluid of cyst. Phenomenon of atresia, its duration, course and underlying causes have been discussed.     

Anticardiolipin antibodies and giant cell arteritis: a prospective, multicenter case-control study. Groupe de Recherche sur l''Artérite à Cellules Géantes

The common brush-tailed possum Trichosurus vulpecula is a small diprotodont marsupial common to both urban and natural environments. This is the first analysis of the neurotransmitter content of its retinal cells and, as the possum is a nocturnal forager, it was appropriate to begin with the dopaminergic amacrine cells that form an essential link in the modulation of the rod pathways subserving nocturnal vision. These results were compared with those from another diprotodont, the marsupial wallaby or quokka (Dann, 1996) to establish whether the dopaminergic systems were similar between these two diprotodont marsupials and also to compare these findings with those of other mammals. This study describes a population of amacrine cells in the possum retina that were immunolabelled with an antibody raised against tyrosine hydroxylase (TH). These TH-immunoreactive (IR) cells were located within the inner nuclear layer (INL) and their dendrites predominantly ramified within the most sclerad layers of the inner plexiform layer (IPL). The TH-IR amacrines formed a sparse cell population, of around 2400 cells, distributed over the entire retina. There was little evidence of a concentration gradient except for a slight elevation in density in the naso-temporal axis in dorsal retina. The formation of rings within the dendritic plexus, a feature common to TH-IR cells in other species, was also present in the possum and these appeared relatively frequently. This latter finding was rather unexpected since, in the marsupial quokka (Dann, 1996), the TH-IR dendrites formed fewer rings despite having the same density of TH-IR amacrines as the possum. This suggests that there may be subtle differences in the way the rod pathways are interconnected even within the same marsupial group and may also be a reflection of relative rod dominance across species.     

Programmed cell death in human ovary is a function of follicle and corpus luteum status

Although extensive investigation on follicular apoptosis (programmed cell death) has been conducted in the infraprimate ovary, there is little information regarding apoptosis and its relationship to follicular status in the human. In this study, apoptosis was investigated in 116 human ovarian follicles (primordial to dominant) and 5 corpora lutea from a total of 27 premenopausal women. Follicles and corpora lutea were evaluated for the presence of DNA fragmentation, characteristic of apoptosis, by two methods: in situ hybridization using 3' end-labeling of DNA with digoxigenin-labeled nucleotides and subsequent digoxigenin antibody and peroxidase staining, and/or biochemical analysis of low molecular weight DNA laddering. Follicle functional status was evaluated by determining follicle sizes and follicular fluid androgen/estrogen (A/E) ratios. No apoptosis was observed in 67 primordial, primary, or secondary follicles. Positive staining for DNA fragmentation was found in a few granulosa cells in 0.1- to 2-mm follicles, whereas abundant staining in granulosa was detected in 2.1- to 9.9-mm follicles. In contrast, no DNA fragmentation was detected in dominant follicles (10-16 mm). The frequency of apoptosis in follicles was calculated to be 37% in 0.1- to 2-mm follicles, 50% in 2.1- to 5-mm follicles, and 27% in 5.1- to 9.9-mm follicles. Abundant low molecular weight DNA laddering was only found in androgen-dominant follicles and not in estrogen-dominant follicles. Positive staining for DNA fragmentation and low molecular weight DNA laddering were observed in degenerating but not healthy-appearing corpora lutea. In the former, DNA fragmentation was found primarily in large luteal cells. These data suggest that follicular atresia in human ovary results from normal programmed cell death and primarily occurs in the granulosa cell layers of the early antral and < 10-mm antral follicles primarily. Furthermore, because apoptosis occurs as early as the 200-mm stage, follicle selection may begin as early as the initial formation of the antrum. The results also suggest that degeneration of the corpus luteum occurs by apoptotic mechanisms.