Journal of reproduction and fertility最新文献

Circulating inhibin A, inhibin B, activin A, total immunoreactive inhibin alpha-subunit (ir-alpha inhibin), LH, FSH and progesterone concentrations were measured throughout the normal ovulatory cycle and after cessation of egg laying induced by feed restriction to investigate the potential involvement of inhibins and activins in the ovulatory cycle of the domestic hen. Plasma inhibin A varied significantly (P < 0.05) during the ovulatory cycle; the concentration was highest at the preovulatory LH surge and reached a nadir 10 h later, at about the time the F(2) follicle makes the transition to become the new F(1) follicle. Plasma FSH concentrations did not change significantly throughout the cycle and showed no correlation with inhibin A. Total ir-alpha inhibin concentrations were much higher than those of inhibin A at all stages of the ovulatory cycle and showed no correlation with inhibin A or FSH. Plasma concentrations of inhibin B and of activin A were below the detection limit of the assays in all plasma samples analysed. In the feed restriction study, plasma inhibin A and total ir-alpha inhibin showed little change until the last day of oviposition (day 0) after which they fell significantly (P < 0.05) and remained low to the end of the experiment (approximately 70-78% decrease relative to day -4). Conversely, plasma FSH increased after cessation of laying and was significantly higher (P < 0.05) from day 3 to the end of the study (approximately 50% increase on day 6 relative to day -4). Plasma FSH values were negatively correlated with inhibin A (r = -0.39; P < 0.005) and total ir-alpha inhibin (r = -0.36; P < 0.005). Plasma LH and progesterone also decreased (P < 0.05) during feed restriction. The decrease in LH preceded the terminal oviposition and the associated fall in inhibin A by 2 days; there was a positive correlation between LH and inhibin A (r = 0.35; P < 0.005). Taken together these findings support (i) a role for LH in promoting inhibin A secretion by preovulatory follicles and (ii) an endocrine role for inhibin A secreted by preovulatory follicles in the maintenance of tonic FSH secretion in laying hens.

Despite extensive study of reproductive abnormalities in female hypothyroid animals, little is known of folliculogenesis and gonadotrophin secretion in spontaneously hypothyroid animals, especially in response to exogenous hormone treatment. In this study, follicular development and plasma hormone concentrations in the presence or absence of thyroxine and eCG treatment were investigated in infertile immature spontaneously hypothyroid rdw rats. Administration of thyroxine once a day from day 21 to day 29 after birth resulted in increases in body weight (P < 0.001) and ovary mass on day 30 (P < 0.01). Similar populations of both healthy and atretic antral follicles ranging from 101 to 400 micrometer in diameter were observed in control rdw and normal rats. In rdw rats, thyroxine treatment markedly increased the number of healthy antral uniovular follicles 101-400 or > 550 micrometer in diameter in the absence or presence of eCG, respectively. Combined treatment of thyroxine and eCG in rdw rats also markedly increased the number of healthy antral biovular follicles. Thyroxine treatment did not affect the population of atretic antral follicles, but resulted in decrease in the number of atretic large antral follicles (> 400 microm) in the presence of eCG. Plasma oestradiol concentrations in rdw rats given both thyroxine and eCG were significantly higher than they were in rdw rats given eCG alone (P < 0.001). There were no significant differences in plasma FSH concentrations on day 28 between rdw (10.7 +/- 1.6 ng ml(-1)) and normal rats (12.0 +/- 1.4 ng ml(-1); P > 0. 05). Although there were no significant differences in plasma LH concentrations between control rdw (1.9 +/- 0.1 ng ml(-1)) and normal rats on day 30 (1.8 +/- 0.1 ng ml(-1); P > 0.05), eCG treatment increased plasma LH to a peak concentration 52 h after injection in normal (24.9 +/- 2.4 ng ml(-1)) but not in rdw rats treated with thyroxine (4.8 +/- 0.3 ng ml(-1); P < 0.05). In conclusion, the results of the present study indicate that thyroxine treatment improves follicular development but does not rescue the defect of the preovulatory surge of LH in eCG-primed rdw rats.

Zona pellucida binding assays provide information about the fertilizing ability of spermatozoa. A zona-binding assay for canine spermatozoa using intact, denuded homologous oocytes has not been evaluated previously. In the present study, an assay using canine oocytes derived from frozen-thawed ovaries was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were performed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejaculates (0.5 x 10(6) spermatozoa in each 50 microliter droplet containing five oocytes). There was a significant difference (P < 0. 001) between all treatments. Thereafter, the same procedure was used to evaluate methods of chilling and freeze-thawing of canine semen. There was a trend (P = 0.067) for more sperm binding after 1 day of chilling compared with after 4 days of chilling. Semen samples frozen using an extender (with or without the addition of Equex STM paste) were evaluated. Equex had a significant (P = 0.034) positive effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to established in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-thawing. Furthermore, the present study showed that chilling for 4 days tended to reduce the zona-binding capacity of the spermatozoon, and that Equex STM paste had a beneficial effect on the capacity of the frozen-thawed spermatozoon to bind to the zona pellucida.

The aim of the present study was to determine whether androgens and progesterone influence the in vitro maturation of bovine oocytes as assessed by cleavage rates and competence to form blastocysts after in vitro fertilization. Bovine cumulus-oocyte complexes were cultured (n = 20 per drop) for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, eCG (2.5 iu ml(-1)) and a range of treatments that included aromatizable (testosterone; 100 nmol l(-1)) and non-aromatizable (dihydrotestosterone; 100 nmol l(-1)) androgens, an androgen antagonist (flutamide; 36 micromol l(-1)), progesterone (300 nmol l(-1)) and a progesterone antagonist (mifeprisone, RU486; 100 nmol l(-1)). Production of inhibin A, total alpha-subunit, activin A and follistatin by each group of cumulus-oocyte complexes was also measured, since inhibin-related peptides have been implicated as modulators of oocyte maturation and their production may be influenced by steroids and anti-steroids. Both testosterone and dihydrotestosterone increased oocyte cleavage rate (25%; P < 0.01) and dihydrotestosterone also increased (24%; P < 0.05) the proportion of oocytes that reached the >/= eight-cell stage. However, neither androgen affected blastocyst yield, or the proportion of blastocysts that hatched. The stimulatory effect of dihydrotestosterone on cleavage rate was reduced by flutamide but the anti-androgen had no effect when tested alone. Treatment with testosterone, but not dihydrotestosterone, decreased (P < 0.05) endogenous follistatin and increased (P < 0.05) the activin A:follistatin ratio in maturation medium. Concentrations of inhibin A, total alpha-subunit and activin A were not affected significantly by androgen or flutamide. Addition of progesterone or the anti-progestin mifepristone to cumulus-oocyte complexes had no effect on cleavage rate. However, progesterone reduced by approximately 40% (P < 0.05) the proportions of both total oocytes and cleaved oocytes that formed blastocysts. This effect was partially reversed by mifepristone. Neither progesterone nor mifepristone affected inhibin A, activin A or follistatin production. However, total alpha-subunit concentration was significantly greater in the progesterone-treated group than in the controls (50%; P < 0.05), indicating that the negative effect of progesterone on blastocyst yield may be mediated by increased inhibin alpha-subunit expression by cumulus cells.

The viability and motility of spermatozoa harvested from the epididymides of turtles were estimated to elucidate properties that might enable them to be stored over long periods of time. Spermatozoa from the painted turtle, Chrysemys picta, were analysed and compared with spermatozoa from two other turtles, Trachemys scripta and Sternotherus odoratus using the Cellsoft analysis system for videotaped images. Spermatozoa from C. picta and T. scripta, suspended in F-10 medium, showed low motility (3-6% motile) and motion velocities, whereas the motility of spermatozoa from S. odoratus was higher (40% motile). Spermatozoa from C. picta and S. odoratus, but not T. scripta, had higher motilities and motion velocities when incubated at 2 degrees C before analyses. C. picta spermatozoa were unresponsive to calcium concentrations ranging from 10(-8) to 10(-1) mol l(-1), potassium concentrations ranging from 0. 1 to 10 mmol l(-1), and to pH values in the range 5.9-8.4. Spermatozoa from C. picta were sensitive to hypo-osmotic media, and showed reduced motility at 25% of normal osmolarity and no motility at 10% of normal osmolarity. Distorted cells and missing flagellae were noted at 50% of normal osmolarity. C. picta spermatozoa were viable up to 40 days after harvest when incubated at 4 degrees C; during this time, both motility and motion velocity were increased in response to 0.5 mmol 3-isobutyl-1-methylxanthine l(-1). Spermatozoa from turtles have osmotic properties and resistance to changing chemical environments similar to spermatozoa from other vertebrates that have internal fertilization, and appear to be stable over long periods of time compared with spermatozoa from other vertebrate species.

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.