Endocrine aspects of mosquito reproduction

Blood ingestion by the female mosquito initiates a series of endocrine events that is dominated by juvenile hormone, ecdysteroids, and several peptide hormones, resulting in the maturation of a batch of eggs. The mechanisms of hormone release and their roles during the previtellogenic, vitellogenic, and postvitellogenic phases are discussed. Arch. Insect Biochem. Physiol. 35:491–512, 1997. © 1997 Wiley-Liss, Inc.     

Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro

Summary FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, >1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro. This work was supported by Grant CA-11198 and CA-20329 awarded by the National Institutes of Health, and a Biomedical Research Support Grant awarded to R. T. Mulcahy.     

Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-filled cells of the interior. We also show that matriptase potently activates hepatocyte growth factor (HGF) in vitro, and that the HGF receptor, c-Met, is co-expressed in those cells that express activated matriptase. Our observations suggest that the matriptase-HGF-c-MET pathway has the potential to be engaged, primarily in proliferative cells rather than terminally differentiated epithelial cells of the human pilosebaceous unit.     

In vitro steroid production by isolated ovarian follicles of the striped trumpeter

Ovarian follicles from striped trumpeter Latris lineata were incubated in L15 medium alone, or medium supplemented with gonadotropin (GtH) preparations (human chorionic GtH, carp maturational GtH or partially purified salmon GtH), testosterone (T) or 17-hydroxyprogesterone (17P). Levels of oestrone (E₁), 17 β -oestradiol (E₂), T, and 17,20 β -dihydroxy-4-pregnen-3-one (17,20 β P) in the medium after incubation were measured by radioimmunoassay. Basal production of E₂ was high from previtellogenic follicles, whereas little T was produced. Both T and E₂ production increased in response to treatment with GtH or steroid precursors. Vitellogenic follicles showed basal production of both T and E₂, and T but not E₂ levels generally increased in response to hormone treatment. Preparations containing follicles nearing final maturation showed low basal production of E₂ but high production of T. Treatment with steroids resulted in little change in E₂ but often very large increases in T production, whereas GtH stimulated lesser increases. 17,20 β P production was detectable from incubations of maturing follicles from two out of five fish, and in those two incubations, increased in response to treatment with 17P. E₁ was not detectable in any incubations. The results indicate that there is a shift in steroidogenesis from E₂ to T production during oocyte development, and provide further evidence that steroid biosynthesis in non-salmonids is principally regulated by substrate availability.     

Development of vitrified porcine primordial follicles in xenografts

The objective was to cryopreserve porcine primordial follicles by vitrification and to assess the development of these follicles in xenografts. Ovarian tissues containing primordial follicles were collected from neonatal (15-d-old) piglets. They were vitrified in modified tissue culture medium (TCM)-199 containing 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, 20% (v/v) fetal calf serum, and 0, 0.25, or 0.5 M sucrose. After 1 wk of storage in liquid nitrogen (LN₂), the tissues were warmed, and the morphology of follicles and oocytes was examined histologically. After vitrification in sucrose-free medium, there were 50 ± 2 (mean ± SEM; n = 10) follicles per tissue, in contrast with 108 ± 10 (n = 10) in fresh tissues. Losses were attributed to puncturing oocytes during the vitrification-warming process, as oocytes were apparently normal after treatment of the sucrose-free vitrification solution without plunging into LN₂. When tissues were vitrified in sucrose-supplemented medium, loss of oocytes decreased (P < 0.05). However, the number of abnormal oocytes having nuclear shrinkage was increased (P < 0.05) by the addition of 0.5 M sucrose; this occurred in a small number of oocytes treated with sucrose-supplemented vitrification solutions without vitrification. After 2 mo of xenografting of vitrified-warmed tissues in SCID (severe combined immune deficiency) mice, primordial follicles developed to the secondary stage (accompanied by oocyte growth), whereas there was development to the antral stage in xenografts of fresh tissues. In conclusion, primordial follicles from neonatal pigs maintained their developmental ability after vitrification and warming, although their developmental rate was slower than that of the fresh control in xenografts.     

Resumption of follicle growth in gilts after ovarian autografting

The aims of the study were to evaluate autografting of porcine ovarian tissue in terms of establishment of a blood supply, follicle survival and development, commencement of oestrous cycles and endocrine patterns in this polyovular species. Experiment 1, a preliminary study on four gilts, showed that ovarian tissue slices survived the grafting procedure and re-vascularised. In Experiment 2, a further six pre-pubertal gilts had both ovaries surgically removed and two thin cortical slices of each ovary were immediately reattached to each of the ovarian pedicles. Blood samples were taken at surgery and then weekly. Two gilts were slaughtered 2 weeks after surgery and ovarian tissue recovered. The remaining four gilts underwent daily checks for behavioural oestrus until slaughter 24 weeks after surgery. All four gilts showed standing heat at least once prior to slaughter. Plasma LH and FSH concentrations increased significantly (P<0.01) by 3 days after surgery, then fell gradually, but did not return to pre-surgery levels. Progesterone concentrations showed some evidence of cyclicity in all animals. In the grafted tissue, re-vascularisation of the tissue was apparent by 2 weeks post-grafting, although no preantral or antral follicles were observed. The tissue recovered after 24 weeks contained healthy preantral and antral follicles, luteal tissue and some large cystic follicles. It is unclear whether these cysts were the result of ovarian or hypothalamic/pituitary disturbance. In conclusion, the results of this study have shown that follicle growth and resumption of cyclicity can be achieved following ovarian autografting in pigs and indicate that this will be a useful model for investigating the mechanisms that control the early stages of follicular growth and ultimately ovulation rate in this multiovular species.     

Follicular growth and atresia in the mouse

Summary Follicles were classified on the basis of the number of layers of follicle cells, the presence and degree of development of the zona pellucida, and the presence of an antrum. Formation of an antrum in follicles with less than 7 to 8 cell layers and/or presence of necrotic cells was considered indicative of degeneration. When classified in this manner, the data suggest that follicles and their contained oocytes are committed to either normal development or atresia by the time a third layer of granulosa cells is formed. Presented in the formal symposium on Sexual Differentiation in Vitro and in vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This research was sponsored by the Division of Biomedical and Environmental Research, U.S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.