The effect of detergents on the basement membrane complex of a biologic scaffold material.

The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA, glycosaminoglycans (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin ß-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC were able to infiltrate the damaged sub-basement membrane tissue, showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold.

Ultrastructural observations on sperm storage in the ovary of the platyfish, Xiphophorus maculatus (Teleostei: poeciliidae): the role of the duct epithelium.

Sperm storage is an important phenomenon occurring in viviparous and oviparous teleosts and contributes to the reproductive life history of these forms. There is a paucity of morphological studies on sperm storage in fishes. The majority of these have been confined to the light level of investigation. In this report, we describe, at the ultrastructural level, sperm storage in the viviparous platyfish, Xiphophorus maculatus. Female platyfish, as is typical of the poeciliids, are capable of storing viable sperm for up to several months within the ovary and gonoduct. We observed that sperm stored within inseminated platyfish became associated with specific epithelial cells (SACs) lining the oviduct. Two forms of association were seen: 1) sperm were found within deep surface pits and pockets, and 2) the spermatozoa were taken up and incorporated within the cytoplasm of the SACs. Junctional complexes in the form of tight junctions (zona occludens) and desmosomes at the apico-lateral surfaces of the SACs were prevalent. The junctions could have contributed to allograft survival of the haploid heterogenetic sperm cells within the female's reproductive tract. Our results shed light on the mechanism of sperm storage in the platyfish and could serve as a model for other poeciliid species and teleosts that are capable of storing sperm.

Neuropeptide Y (NPY) induces gonad reversal in the protogynous bluehead wrasse, Thalassoma bifasciatum (Teleostei: Labridae).

Female-to-male sex transformation in the protogynous marine fish Thalassoma bifasciatum is the culmination of a neuroendocrine cascade triggered by social and behavioral cues. We have previously shown that the hypothalamus and pituitary gland, key components of the neuroendocrine axis, play a significant role in gender reversal of this species. However, nothing is known about the possible involvement of other brain factors with the hypothalamo-pituitary gland axis in this fish. One such factor, neuropeptide Y (NPY) has been shown to effect a direct or indirect control of GtH-II release in gonochoristic teleosts. In view of this finding, we have investigated the role of NPY on sex reversal in T. bifasciatum. Females (n = 38) were injected intraperitoneally thrice weekly with NPY at a dose of 0.02 microgram/gbw dissolved in 0.6% saline. Controls (n = 48) received equal volumes of saline. Fish were sacrificed at 1, 4, 6, and 8 weeks. By the end of 8 weeks, 81.6% of the NPY-treated fish showed signs of gonad reversal ranging from early (n = 25) to mid- (n = 3) and late stage (n = 3). Furthermore, the effects of NPY appeared to be dependent on duration of treatment. At 6 and 8 weeks, the ovaries of four and three fish, respectively, were markedly degenerated. Our findings shed new light on the neuroendocrine control of sex reversal in T. bifasciatum and in hermaphroditic forms in general.

Human chorionic gonadotropin (hCG) induces gonad reversal in a protogynous fish, the bluehead wrasse, Thalassoma bifasciatum (Teleostei, Labridae).

Female to male successive hermaphroditism (protogyny) is common in several groups of marine fish. Thalassoma bifasciatum, the bluehead wrasse (Labridae), found in the reefs of the Caribbean normally undergoes sex reversal after receiving behavioral cues. This report deals with the successful use of human chorionic gonadotropin (hCG) in inducing gonad reversal in this species. Eighty percent (n = 40) of the treated fish showed signs of reversal in 1-6 weeks; only 11% (n = 54) of the control (nontreated) group showed signs of reversal during the same period. The number of fish undergoing reversal increased with the length of the treatment period, 55% after 1 week, 100% after 6 weeks. A bluehead color pattern, typical of the terminal male phase, also appeared more frequently in the longer treated groups. To determine the efficacy of hCG in inducing gonad reversal, morphological criteria for reversal had to be established. For the majority of treated fish, the presence of both spermatogenic cysts and degenerating oocytes was sufficient to label these gonads unambiguously as undergoing reversal. However, at the extremes of this process, i.e., onset (early) and endpoint (late) stages, ambiguity could arise in identifying a gonad as undergoing reversal, and, therefore, key criteria were established for these stages. In our hands, the most consistent and reliable criterion for the early stage reversal was appearance of male germ line cells, clusters of "spermatogonial-like" cells. For the late stage, recognizable remnants of late-state oocytes had to be present. Some details of the histological changes that occur during early, middle, and late stages of reversal are also described. It is suggested that these results with hCG shed new light on the endocrine control of gonad reversal in T. bifasciatum.

Sex reversal in wrasses. I. Uptake of testosterone by the gonads and the central nervous system and its aromatization in the CNS of Thalassoma duperrey (Teleostei: Labridae).

3H-Labelled testosterone, injected into the peritoneal cavity of T. duperrey is taken up by the central nervous system and the gonads. In the brain maximal uptake occurs within 1 to 2 h whereas maximal uptake in the gonads takes place within 1/2 to 1 h. Relatively large amount of radioactivity were eliminated into water: the elimination reached 8% of the injected dose in 4 h. The brain and the gonads are capable of aromatizing injected androgen, forming estrone, estradiol and more polar metabolites (i.e. containing more than 2 hydroxyl groups) and water soluble derivatives (possibly either the sulfates, or glucuronides). The uptake, and metabolism, may be higher in males as compared to females.

An electron microscopic study of a 'Sertoli-like' cell in the testis of a barnacle, Balanus.

The organization of a 'Sertoli-like' cell (SLC) is described in the testis of a barnacle, Balanus eburneus. Underlying a relatively thick basal lamina are 'sheet-like' cytoplasmic extensions that develop from the base of the SLC and almost completely enclose the germ cells. Junctional structures are frequently observed between the SLCs and between an SLC and a developing germ cell. Portions of the SLC extend deep into the testis and subdivide into branches. Developing sperm are seen within inpocketings of the plasmalemma of the branches. Noteworthy among the SLC's organelles are large, well-developed mitochondria, an abundant smooth endoplasmic reticulum and masses of glycogen particles. Intercellular bridges are present between germ cells which are at the same stage of development. In some respects the organization of the SLC in B. eburneus is suggestive of that in the Sertoli cell in the testis of vertebrates.

Sex differentiation of somatic tissue in the unsexualised gonad primordia of the embryos of three species of poeciliid fish.

The structure of the gonad primordia is described in the embryos of three species of poeciliid fish. Prior to the sexualisation of the germ cells, a dimorphism exists based upon the gonadal somatic features. This serves as a basis for distinguishing between male and female specimens. Presumptive males are characterised by the presence in at least one of their gonad primordia of a conspicuous stalk whereas in presumptive females, a stalk is completely lacking.