Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Ultrastructural morphological injuries and maturation rates were investigated in equine oocytes exposed to vitrification solutions (VS) containing synthetic ice blockers (SIBs) during different exposure times. In experiment 1, compact cumulus-oocyte complexes (COCs; n = 30) were randomly allocated to treatments: (1) fresh fixed (control); (2) VS-1 (1.4 M dimethyl sulfoxide [DMSO] + 1.8 M ethylene glycol [EG] + 1% SIB) for 3 minutes of equilibrium time and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M sucrose + 1% SIB) for 1 minute (Eq-long); and (3) VS-1 for 1.5 minutes and VS-2 for 30 seconds (Eq-short). In experiment 2, compact (n = 248) and expanded (n = 264) COCs were evenly distributed to the following treatments: (1) immediate maturation in vitro (control); (2) vitrification using the Eq-short protocol as in experiment 1; and (3) vitrification using a stock solution containing 2.8 M formamide, 2.8 M DMSO, 2.7 M EG, 7% polyvinylpyrrolidone, and 1% SIB (Eq-short-mod). More (P < .02) oocytes with normal ultrastructural morphology were seen in fresh control and Eq-short groups than in Eq-long group. Metaphase-II (MII) rates were higher (P < .05) for oocytes with expanded cumulus than compact cumulus in the control group, and higher (P < .05) for oocytes with expanded cumulus than compact cumulus in Eq-short and Eq-short-mod groups. No difference in MII rates was detected among groups within each type of COC. In conclusion, reduction of exposure time to VS better preserved oocyte ultrastructural features, and MII rates were higher for vitrified oocytes with expanded cumulus. This study advances our knowledge on potential alternatives for vitrification of immature equine oocytes.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Steroidogenesis during induced oocyte maturation and ovulation in the African catfish,Clarias gariepinus

Changes in ovarian steroidogenesis accompanying oocyte maturation and ovulation were studied in the African catfish,Clarias gariepinus. Laboratory-reared females with postvitellogenic ovaries were treated with pimozide and LHRH-analogue. The plasma gonadotropin levels were determined by means of a homologous radioimmunoassay, the condition of the ovaries was studied by histological examination of the follicles, and the steroidogenetic capacity of the ovaries was analyzed byin vitro incubation of tissue fragments for 3 h with [³H]-pregnenolone and [³H]androstenedione as precursors. Data were collected at regular intervals between 0 and 16 h after pimozide-LHRH analogue administration.Until 4 h after the beginning of the experiments the plasma gonadotropin levels did not rise above 25 ng/ml, the ovaries remained in the stage of postvitellogenesis, and testosterone was the main end product of steroidogenesis. Four hours later the gonadotropin concentration in the blood had risen to more than 150 ng/ml, and the ovaries had entered the stage of germinal vesicle migration. At the same time steroidogenesis shifted towards the production of 17,20-dihydroxy-4-pregnen-3-one, 5-pregnane-3, 17-diol-20-one, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,17,20-triol and 5-pregnane-3,6,17,20-tetrol. During the subsequent stage of germinal vesicle breakdown the plasma gonadotropin level remained high, and the synthesis of the C₂₁-steroids showed a further increase. Simultaneously, the production of some C₁₉-steroid glucuronides was enhanced. The preovulation and especially the postovulation stages were accompanied by a gradual decrease in steroidogenic capacity of the ovaries, even though the plasma gonadotropin level remained high. It is concluded that the prematuration surge of gonadotropin influences the activity of enzymes involved in steroidogenesis, leading to a reduced C_17,20-lyase and to an augmented activity of the enzymes 20-hydroxysteroid dehydrogenase (HSD), 5-reductase, 3-HSD, 6-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3-HSD and 17-hydroxylase, gradually decreases.Not only 17,20-dihydroxy-4-pregnen-3-one, but also the 5-reduced pregnanes may be involved in inducing oocyte maturation and/or ovulation. The very polar triol and tetrol products may function, together with the steroid glucuronides as sex pheromones.A preliminary account of these results was presented at the XIII Conference of European Comparative Endocrinologists, Belgrade, September 7–12, 1986     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

Evidence that 17α,20β,21-trihydroxy-4-pregnen-3-one is a maturation-inducing steroid in spotted seatrout

Ovarian steroidogenesis during final oocyte maturation (FOM) in the spotted seatrout (Cynoscion nebulosus) was investigated by incubating ovarian fragments with tritiated pregnenolone, followed by chromatographic separation of the radioactive products. The major tritiated steroid produced during FOM comigrated with 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-dihydro-11-deoxycortisol, 20β-S) on HPLC and TLC. Only minor amounts of radioactive material coeluted with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P), 11-deoxycorticosterone (DOC), estradiol-17β and testosterone standards in the HPLC system. Additional chromatography by TLC confirmed the presence of radioactive estradiol-17β and testosterone but not 17α,20β-P and DOC. All the ovarian steroids producedin vitro during FOM were assayed for their ability to induce germinal vesicle breakdown (GVBD) of spotted seatrout oocytes. Twenty grams of ovarian tissue were incubated with human chorionic gonadotropin and exogenous pregnenolone. The steroidal products were purified by HPLC and TLC. Most of the maturation-inducing activity was confined to steroidal material which comigrated in these systems with 20β-S. This material was active at a concentration of 1 ng steroid/ml medium in the GVBD assay. Smaller amounts of material which coeluted with 11-deoxycortisol, DOC, 17α,20β-P and several minor unidentified fractions induced GVBD at concentrations of 10 ng steroid(s)/ml. The structure-activity relationships of authentic steroids in inducing GVBD of spotted seatrout oocytes was investigated. Hydroxylation at the 17α, 20β or 21 positions increased potency to induce GVBD. Steroids with multiple hydroxyl groups at the 17α and 20β positions (17α, 20β-P) and at the 17α, 20β, and 21 positions (20β-S) had maximum biological activity in the GVBD bioassay. The results suggest that 20β-S is a major maturation-inducing steroid in spotted seatrout.     

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

Ovarian follicular dynamics and hormones throughout the estrous cycle in Thai native (Bos indicus) heifers

Relatively few studies have been reported regarding the reproductive physiology of female Thai native cattle. Therefore, the objective of the present study was to evaluate the follicular dynamics and concentrations of follicle stimulating hormone (FSH), estradiol (E2) and progesterone (P4) during the estrous cycle in Thai native heifers (TNH) and to compare obtained results with those of European and Indian cattle breeds previously reported. For the detection of estrus, ovaries of all 20 heifers were examined twice daily (12 h intervals) by ultrasonography for three consecutive estrous cycles. From data of 60 estrous cycles (n = 60 estrous cycles from 20 heifers), it was found that 14 (70%) and 6 heifers (30%) had two (42 estrous cycles collected from 14 heifers) and three follicular waves (18 estrous cycles collected from 6 heifers), respectively. The days when estrus was detected, interovulatory intervals, life‐spans of corpus lutea (CL), and days for growing and regression of CLs were shorter in the two follicular waves than those in the three follicular waves (P < 0.05). In both two and thre follicular waves, larger maximum diameters and higher growth rates of the dominant follicle (DF) in an ovulatory wave were observed than those of the preceding waves without ovulation (P < 0.05). There was a progressive increase in follicular size and FSH and E2 production during follicular growth in each follicular wave. In addition, the FSH and E2 peak concentrations during the ovulatory wave were higher than those of the anovulation waves (P < 0.05). Moreover, although the ovarian follicular dynamic patterns in Thai native heifers were similar to those previously reported for European and Indian cattle breeds, the diameter of the largest preovulatory follicle (OF), subordinate follicles (SF) and CLs were smaller than those in European and Indian cattle breeds. In conclusion, when compared with European and some breeds of Indian cattle, the length of interovulatory intervals was shorter, and the sizes of dominant SF and CLs were smaller in Thai native heifers.     

Role of oocyte‐derived paracrine factors in follicular development

Mammalian oocytes secrete transforming growth factor β (TGF‐β) superfamily proteins, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 6 (BMP6) and BMP15, and fibroblast growth factors (FGFs). These oocyte‐derived paracrine factors (ODPFs) play essential roles in regulating the differentiation and function of somatic granulosa cells as well as the development of ovarian follicles. In addition to the importance of individual ODPFs, emerging evidence suggests that the interaction of ODPF signals with other intra‐follicular signals, such as estrogen, is critical for folliculogenesis. In this review, we will discuss the current understanding of the role of ODPFs in follicular development with an emphasis on their interaction with estrogen signaling in regulation of the differentiation and function of granulosa cells.