Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane.

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.

Bovine ovarian cortical pieces grafted to chick embryonic membranes: a model for studies on the activation of primordial follicles.

BACKGROUND: Little is known about the factors that control the initiation of growth of primordial follicles. Primordial follicles in pieces of fetal bovine ovarian cortex spontaneously activate in vitro and develop to the primary stage, but few follicles develop further. For decades, embryologists have grafted tissue to the chorioallantoic membrane (CAM) of chick embryos to study the development of various organs and structures. METHODS AND RESULTS: To test the hypothesis that grafting cortical pieces beneath the CAM membrane of 6-day-old chick embryos ('in ovo') would support the activation of primordial follicles and the growth of activated follicles to the secondary stage, ovarian cortical pieces from six bovine fetuses (6-8 months gestation) were placed either in ovo or in organ culture in serum-free medium (in vitro). Cortical pieces were retrieved after 0, 2, 4, 7, or 10 days in ovo or in vitro. Histological examination revealed a dramatic infiltration of the CAM-grafted cortical pieces with blood vessels. By day 2 in vitro, the number of primordial follicles had declined by 87% concomitant with a 3.5-fold increase in primary follicles (P < 0.01), providing evidence of the expected activation of primordial follicles. Unexpectedly, primordial follicles were not activated in CAM-grafted tissue, as shown by maintenance of their numbers and lack of increase in primary follicles during 10 days in ovo. In experiment 2, a subset of pieces was switched from culture to CAM grafts and from CAM grafts to culture on day 2. The CAM did not support the growth of primary follicles activated in vitro, apparently because the activated follicles did not survive the transfer (P < 0.05). However, primordial follicles maintained in ovo retained their ability to activate; after their removal from the CAM into culture, primordial follicles decreased in number and primary follicles increased in number within 2 days (P < 0.05). CONCLUSIONS: The CAM graft will provide a useful model for studying the factors involved in activation of primordial follicles.

The primordial to primary follicle transition.

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. A better understanding of the signals that initiate follicular growth in mammals, and of the conditions necessary for sustained growth of early preantral follicles in vitro, could have practical implications for contraception, alleviation of infertility, and regulation of the rate of follicle depletion (menopause). Our laboratory has developed two experimental systems that can be used to study factors involved in the activation of primordial follicles. In the first experimental system, small pieces of ovarian cortex, containing mostly primordial follicles, are isolated from fetal ovaries of cattle or baboons and cultured in serum-free medium. Under these conditions most primordial follicles become activated between 12 and 24 h of culture; their granulosa cells change shape, from flattened to cuboidal, and begin to express proliferating cell nuclear antigen (PCNA). During 7 days in culture, the newly-formed primary follicles and their oocytes increase significantly in diameter. This wholesale 'spontaneous' activation in serum-free medium is quite different from the much more gradual exit of primordial follicles from the resting pool that occurs in vivo and suggests that primordial follicles in vivo may be subject to a tonic inhibition of growth initiation or, alternatively, that some aspect(s) of the environment in vitro stimulates growth initiation. Recently we developed a second experimental system for studying activation of primordial follicles. Pieces of ovarian cortex from bovine or baboon fetuses were grafted beneath the developing chorioallantoic membrane (CAM) of 6-day-old chick embryos, a site known to support xenografted tissues. The cortical pieces were rapidly vascularized and histological analysis of pieces recovered after 2, 4, 7, or 10 days 'in ovo' revealed no increase in the number of primary follicles and maintenance of original numbers of primordial follicles. Therefore, grafting ovarian cortical pieces beneath the chick CAM provides an experimental system in which follicles remain at the primordial stage in a readily accessible environment and which, thus, may be used to study potential regulators of the initiation of follicle growth. The results suggest that vascularization of isolated pieces of ovarian cortex provides conditions that maintain follicular quiescence, whereas culture in vitro allows unrestrained activation of primordial follicles. Future studies with and comparisons of the in vitro and in ovo models may provide new insight into the mechanisms that regulate the primordial to primary follicle transition.

Developmental relations between sixth nerve motor neurons and their targets in the chick embryo.

The developmental relations between abducens (VI) nerves and their targets, the lateral rectus, quadratus, and pyramidalis muscles, have been examined in the chick embryo from early neural tube stages through 10 days of incubation. Sites of myoblast origins were determined by microinjection of replication-incompetent retroviruses containing the LacZ reporter into paraxial mesoderm corresponding to somitomeres 3-5. Motor neurons and axons were identified by Bodian staining, immunocytochemistry, and application of DiI and DiO to dissected peripheral nerves. Anlage of the dorsal oblique originate in somitomere 3, close to the ventrolateral margin of the mid-to-caudal mesencephalon. Precursors of the lateral rectus arise deep within somitomere 4, beside the future metencephalon (rhombomere "A"). Quadratus and pyramidalis precursors are located between and partially segregated from these other two anlage. VIth nerve axons exit rhombomeres 5 and 6 via multiple median roots, fasciculate, and by stage 17 have elongated rostrally beneath the hindbrain. Immediately caudal to a mesenchymal pre-muscle condensation located deep to rhombomere 2, the VIth nerve separates into two branches. One branch enters the rostral portion of the condensation, from which quadratus and pyramidalis muscles will segregate. This branch projects exclusively from rhombomere 5 and is the accessory abducens nerve. The other branch enters the caudal, presumptive lateral rectus, region of the condensation. This is the abducens nerve, and it projects from cells located in both rhombomeres 5 and 6. These findings indicate that specific matching of motor nerves with their presumptive targets begins prior to the differentiation and segregation of myogenic populations, and that spatial organization of developing eye muscles is initiated well before they interact with connective tissue precursors derived from the neural crest.

Periodic cone cell twists in the walleye, Stizostedion vitreum; a new type of retinomotor activity.

A typical retinomotor movements (RM) have been found in a localized area of the retina of the walleye, Stizostedion vitreum. The ellipsoid and outer segment of regions of twin cones in an oblong region of the central retina rotate, twisting about their axes at intervals throughout the 24 hr day. This twist results in a local, cyclic change of the cone mosaic, from the predominant row type to a square type. The cone cell myoids extend slightly, but the cone ellipsoids remain relatively close to the outer limiting membrane (OLM). Throughout the retina, rod cells exhibit ubiquitous extension and contraction of their myoid regions coincident with changes in illumination as is typical of fishes with duplex retinas. These retinomotor movements first appear around 6 weeks of age, and persist throughout growth, becoming more pronounced with maturity. Stizostedion vitreum is the first fish observed with this type of cone RM activity.

A short-wavelength sensitive cone mechanism in juvenile yellow perch, Perca flavescens.

The retinal cone mosaic of adult yellow perch, Perca flavescens, consists of square units each having a central single cone surrounded by four identical twin cones. No cones are present at the corners of the square units. However, the cone types and organization of the retinal mosaic in juvenile P. flavescens has not been thoroughly described. We report here the presence of small, single cones at the corner positions of the square units in juvenile P. flavescens. Not every corner, nor every unit has these cones, and their distribution over the retina is not uniform. Microspectrophotometry places the visual pigment absorbance maximum of these short single cones in the region of 400 nm. Electrophysiological measurements of spectral sensitivity demonstrate a short-wavelength mechanism in juveniles that is absent, as are the small corner cones, in adults. Possible advantages of such a mechanism to juvenile perch are discussed.

Th activation of maternal and cord blood.

Th activation of red cells is characterized by agglutination with the peanut lectin from Arachis hypogaea and is diminished by treatment with proteolytic enzymes. The first cases of Th activation were associated with bacterial infections. More recently, a high incidence of Th activation in congenital hypoplastic anemia has been reported, along with the finding that 13.5 percent of cord bloods are Th activated. The incidence of Th reactivity in newborn infants was confirmed by studying 200 paired samples of maternal and cord blood. Twenty-two (11%) of the cord samples and 13 (6.5%) of the maternal samples were Th activated. In 6 paired samples (6/22), both the mother and child had Th activation, a finding that demonstrates a high degree of concordance. Additionally, 3 (6%) of 50 pregnant women were Th positive. These findings indicate that Th activation is another of the red cell antigen alterations related to pregnancy.