Determination of glycogen and enzymes of glycogen metabolism in human hair follicles

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles.The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.     

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.     

Self‐sterility of eggs induced by exogenous and endogenous protease in the solitary ascidian,Halocynthia roretzi

The unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, which are released naturally, are strictly self‐sterile. However, ovarian eggs isolated after spawning, which are expected to develop into UFE on the following day, are self‐fertile. Some exogenous proteases‐trypsin, chymotrypsin, papain and elastase‐induced self‐sterility in the self‐fertile ovarian eggs within an hour in vitro. The establishment of self‐sterility by the exogenous protease did not require the synthesis of new protein, or the participation of follicle cells. Some of the ovarian eggs were able to differentiate into self‐sterile eggs spontaneously in vitro. The protein synthesis inhibitors puromycin and cycloheximide had no effect on the spontaneous establishment of self‐sterility. However, several protease inhibitors such as leupeptin, soybean trypsin inhibitor (SBTI) and antipain, did inhibit the spontaneous establishment of self‐sterility. The possible participation of trypsin‐like protease in the establishment of self‐sterility in the ovary is discussed. Mol. Reprod. Dev. 52:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electron microscopy for the characterization of the vitelline coat glycoproteins of the polarized egg of Unio elongatulus

The vitelline coat (VC) glycoproteins of the Unio elongatulus egg, purified as previously described (Focarelli and Rosati, 1993: Mol Reprod Dev 35:44–51) and indicated as gp220 and gp180 by virtue of their apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The analysis confirmed the purity of our preparations and the mass of gp180, but gave a mass of 273,000 for gp220. Intact VCs and purified VC components were then visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, and rotary shadowing techniques: gp180 revealed a c-like shape and gp273 a rosette-like shape. The intact VCs were found to consist of two layers, the internal one clearly fibrous and the external one compact. Since purified preparations of gp180 spontaneously formed fibrils of similar width to those present in the inner VC layer, this layer presumably consists mainly of this component. The prevalence of gp273 in the outer layer is also suggested and discussed. Mol. Reprod. Dev. 48:511–517, 1997. © 1997 Wiley-Liss, Inc.     

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.     

Factors affecting the response to the specific treatment of several forms of clinical anestrus in high producing dairy cows

This study was designed to examine estrous response rates to the therapeutic treatment of clinical anestrus in high producing dairy cows and to identify the factors that could affect these rates. Cows with silent ovulation (Subestrus group), cystic ovarian disease (Cyst group) or ovarian hypofunction (OH group) were given specific treatment for their disorder. Data were derived from 1764 treatments in cows producing a mean of 45.4 kg of milk upon treatment including: 889 subestrous cows, 367 cystic cows and 508 cows with ovarian hypofunction. Cows showing estrus following treatment exhibited a similar pregnancy rate to cows attaining natural estrus used as reference: 33% (337/1006) and 35% (626/1796), respectively. No significant differences in pregnancy rates were observed among the Subestrus, Cyst and OH groups (34% (196/571), 34% (44/130), 32% (97/305), respectively. Based on the odds ratio, an estrous response for all groups was less likely to occur in cows that had suffered previous anestrus, compared to cows that were anestrous for the first time, whereas the likelihood of an estrous response increased in cows treated after 90 days in milk. Our results indicate that previous anestrus and a late stage of lactation can have a negative and positive effect, respectively, on the estrous response to the specific treatment of clinical anestrus shown by high producing dairy cows. Treatment targeted at each type of clinical anestrus can render similar pregnancy rates to those shown by cows in natural estrus.     

Stereological assessment of the reproductive status of female Atlantic northern bluefin tuna during migration to Mediterranean spawning grounds through the Strait of Gibraltar

The ovarian mass and gonadosomatic index (I_G) of bluefin tuna Thunnus thynnus , caught in the Strait of Gibraltar (Barbate) during migration to Mediterranean spawning grounds, were several times lower than those found in bluefin tuna from Mediterranean spawning grounds (Balearic Islands). Some of the bluefin tuna from Barbate (8.3%) were classified as immature (the most advanced oocytes present in the ovaries were early vitellogenic), and the majority (the remaining 91.6%) as non-spawning mature; the ovary contained late vitellogenic oocytes, but there was no sign of spawning activity. Stereological estimation indicated that the ovaries of spawning bluefin tuna from the Balearic Islands contained five-fold more highly yolked oocytes than bluefin tuna from Barbate. When breeding bluefin tuna cross the Strait of Gibraltar the gonad is at an incipient stage of maturation. The average batch fecundity estimated from stereological quantification of stage 4 (migratory-nucleus) oocytes in the specimens collected from Balearic was 92.8 oocytes g-1'of body mass, and the spawning frequency in this area was calculated to be 1.2 days. In specimens from Barbate a relative batch fecundity of 96.3 oocytes g -1 was estimated using stage 3 (late vitellogenic) oocyte counts.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

Preservation of caprine preantral follicle viability after cryopreservation in sucrose and ethylene glycol

Caprine preantral follicles within ovarian fragments were cryopreserved in the absence or presence of 0.5 M sucrose with or without 1 M dimethyl sulfoxide and/or 1 M ethylene glycol (EG). After being thawed, they were washed in minimum essential medium with or without 0.3 M sucrose. Histological analysis of follicle integrity immediately after cryopreservation showed consistent beneficial effects of including sucrose in the three cryoprotectant solutions analyzed when tissue was thawed without sucrose (53.9±14.8–82.4±3.2% normal vs 27.6±1.6–36.6±6.5%, P<0.05). However, in further studies, the addition of sucrose to the thaw solutions proved detrimental or of no benefit. An analysis of the cryopreserved material with calcein-AM and ethidium homodimer (markers for living and dead cells, respectively) gave comparable results to those obtained by histology. Follicles cryopreserved in EG, EG plus sucrose, or sucrose alone were cultured in vitro for 24 h following warming. During this culture period, viability fell most rapidly in material cryopreserved in sucrose alone and was no longer correlated with either the viability or integrity estimates made immediately after warming. By contrast, the viability of follicles cryopreserved in EG with sucrose and then cultured for 24 h was not significantly different from the cultured non-frozen controls. These results indicate that cryopreservation in 1 M EG plus 0.5 M sucrose combined with thawing without sucrose is effective for caprine ovarian tissue.This work was supported by CAPES/Brazil. Regiane Rodrigues dos Santos is a recipient of a grant from FUNCAP of Brazil.