bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of acute immunoneutralization of endogenous leptin on prolactin and LH secretion during the afternoon of pro-oestrus or in steroid-treated ovariectomized female rats
Authors: Gonzalez LC , Pinilla L , Tena-Sempere M , Dieguez C , Casanueva FF , Aguilar E
Published: January 2004

Recent data indicate that leptin is involved in the control of reproductive function. Experiments were carried out to analyse the role of endogenous leptin in the regulation of LH and prolactin secretion during the afternoon of pro-oestrus and that induced by ovarian steroids in ovariectomized rats. In the first experiment, cyclic female rats were implanted with intra-auricular and intracerebroventricular (i.c.v.) cannulae and, at pro-oestrus, were injected (i.c.v.) with 10 microliters normal rabbit serum or leptin antiserum (at 13:00 and 14:00 h). Blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the second experiment, female rats in pro-oestrus were injected with normal rabbit serum or leptin antiserum at 16:00 and 18:00 h and blood samples were taken every 10 min between 18:00 and 20:00 h. In the third experiment, adult female rats that had been ovariectomized 2 weeks before were implanted with intra-auricular and i.c.v. cannulae and treated with oestradiol benzoate (30 micrograms s.c.) at 10:00 h and progesterone (2 mg s.c.) 48 h later. Normal rabbit serum (10 microliters) or leptin antiserum (10 microliters) were injected (i.c.v.) at 13:00 and 14:00 h, and blood samples were obtained at 10:00 h and at intervals of 1 h between 13:00 and 20:00 h. In the fourth experiment, hemipituitaries from ovariectomized steroid-treated female rats were incubated in the presence of leptin116-130 (an active fragment of the native molecule), GnRH or leptin + GnRH. Prolactin and LH secretion during the afternoon of pro-oestrus in females treated with leptin antiserum was similar to that observed in animals injected with normal rabbit serum. In ovariectomized female rats, the steroid-induced LH surge increased slightly after administration of leptin antiserum, whereas the prolactin surge remained unchanged. In vitro, leptin116-130 (10(-5) to 10(-8) mol l-1) inhibited LH secretion and modulated the effect of GnRH on LH release, depending on the concentration of GnRH: leptin116-130 (10(-6) mol l-1) reduced the effectiveness of 10(-7) mol GnRH l-1 and increased that of 10(-9) mol GnRH l-1. In conclusion, these experiments indicate that acute immunoneutralization of endogenous leptin does not interfere with spontaneous or steroid-induced LH and prolactin surges. In addition, the finding that leptin116-130 inhibited LH release and modulated the effectiveness of GnRH in vitro provides evidence of the direct modulatory role of leptin on LH secretion acting at the pituitary.
Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2
Authors: Garde J , Roldan ER
Published: January 2004

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.
Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos
Authors: Wrenzycki C , De Sousa P , Overstrom EW , Duby RT , Herrmann D , Watson AJ , Niemann H , O'Callaghan D , Boland MP
Published: January 2004

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos.
Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro
Authors: Fofana M , Travert C , Carreau S , Le Goff D
Published: January 2004

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.
Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist
Authors: Dufour JJ , Cognie Y , Mermillod P , Mariana JC , Romain RF
Published: January 2004

Endocrine control of follicular growth was studied in mature Romanov ewes carrying (RF+) or not carrying (R+2) the Booroola Fec gene during an oestrous cycle after gonadotrophin-dependent follicles were suppressed by treatment with an antagonist of GnRH (Antarelix, 0.5 mg per day) and superovulatory treatment was administered. The left ovary was removed after 10 days of treatment (saline or Antarelix) and the right ovary was removed at the end of the superovulatory treatment. Ewes of both genotypes treated with Antarelix had lower plasma LH concentrations than did controls from day 0 to day 10. The inhibitory effect of Antarelix on LH concentration increased with day of treatment. The variability in FSH concentrations during the initial 10 days was reduced by Antarelix treatment in both genotypes. Plasma FSH concentrations were higher in RF+ ewes than in R+2 ewes. In both genotypes, FSH concentrations varied significantly with day of treatment, with the lowest concentrations at day 8 and the highest concentrations at day 5. RF+ ewes had a greater total and atretic number of antral follicles 0.62-1.12, 1.12-2.00 and 2.00-3.00 mm in diameter (classes 2, 3 and 4) than did R+2 ewes before and after superovulatory treatment. After superovulatory treatment, the total number of atretic and non-atretic follicles > 3.00 mm in diameter (class 5) increased in both genotypes. Superovulatory treatment also increased the number of total and atretic class 4 follicles in RF+ only. Conversely, superovulatory treatment decreased the mean number of class 3 follicles in both genotypes, while the number of atretic follicles was decreased only in R+2 ewes. Antarelix treatment significantly reduced the percentage of follicles > 2.00 mm in diameter in RF+ but not in R+2 ewes. Antarelix treatment before superovulatory treatment increased the total number of class 4 follicles in both genotypes but the increase was more significant in RF+ than in R+2 ewes. These results indicate that Antarelix pretreatment favours a greater superovulatory response in Romanov ewes carrying the Fec gene because ovulatory follicles are recruited from a wider range of follicular size classes.
Hysteroscopic insemination of small numbers of spermatozoa at the uterotubal junction of preovulatory mares
Authors: Morris LH , Hunter RH , Allen WR
Published: January 2004

Mares were inseminated with motile spermatozoa suspended in 30-150 microliters Tyrode's medium directly onto the uterotubal papilla at the anterior tip of the uterine horn, ipsilateral to the ovary containing a dominant preovulatory follicle of > or = 35 mm in diameter, by means of a fine gamete intrafallopian transfer (GIFT) catheter passed through the working channel of a strobed light videoendoscope. Insemination of 10, 8, 25, 14, 11 and 10 mares with, respectively, 10.0, 5.0, 1.0, 0.5, 0.1 or 0.001 x 10(6) motile spermatozoa resulted in conception rates of, respectively, 60, 75, 64, 29, 22 and 10%. Deposition of 1.0 x 10(6) motile spermatozoa onto the uterotubal papilla began to approach the limit of successful fertilization. These doses are far lower than the 3-15 x 10(9) spermatozoa normally ejaculated by fertile stallions during mating, and the accepted minimum dose of 500 x 10(6) spermatozoa used for conventional uterine body insemination in mares. The simplicity of the technique offers a practical means of exploiting new breeding technologies that require very small numbers of spermatozoa in horse breeding.
Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture
Authors: Shores EM , Picton HM , Hunter MG
Published: January 2004

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.
Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment
Authors: Xia P , Younglai EV
Published: January 2004

The objective of this study was to investigate the relationship between oocyte morphology and follicular fluid steroid concentrations in patients being treated with intracytoplasmic sperm injection. A total of 82 IVF cycles were evaluated in patients aged 24-40 years. Oocytes at metaphase II were graded into four groups according to the status of the first polar body and the size of the perivitelline space. The proportion of oocytes at the germinal vesicle and germinal vesicle breakdown stages, and the proportion of degenerated oocytes and oocytes with a large polar body were compared with different concentrations of oestradiol, progesterone and testosterone in the follicular fluid. The association between these oocyte characteristics and the ratio of oestradiol:testosterone and oestradiol:progesterone was also analysed. The results showed that oocyte morphology, as assessed by the status of the first polar body and the size of the perivitelline space, is associated with the ratio of oestradiol:testosterone and oestradiol:progesterone but not with the absolute concentrations of oestradiol, progesterone and testosterone in the follicular fluid. A ratio of oestradiol:testosterone > 200 is the best indicator for a small proportion of grade 1 and 2 oocytes (poor quality), a large proportion of grade 3 and 4 oocytes (good quality), and a small proportion of oocytes with cytoplasmic inclusions. These results will be of clinical use in evaluating oocyte quality.
Difference in localization of eosinophils and mast cells in the bovine ovary
Authors: Reibiger I , Spanel-Borowski K
Published: January 2004

The bovine ovary contains a considerable number of leucocytes which can be located with an antibody against the CD18 molecule. In the present study, subtyping and cell counting were carried out on histological sections stained with Sirius red for eosinophils and with toluidine blue for mast cells. The CD18(+) cells were identified immunohistologically. Eosinophils and mast cells contributed considerably to the CD18(+) pool. The number of eosinophils in the corpus luteum increased rapidly in early development to approximately 90% of the CD18(+) cells, and decreased to 30% during secretion and to 10% during regression. Mast cells were not detectable in the follicles, the corpus luteum and the periphery of the cortex, but were observed in the interstitial cortical stroma and the medulla. The number of mast cells in these regions, which corresponded to 60-76% of the CD18(+) cells, did not change significantly throughout the oestrous cycle. It is concluded that eosinophils are selectively recruited at the periovulatory period and that mast cells are unevenly distributed.
Influence of season and low-level oestradiol immunoneutralization on episodic LH and testosterone secretion and testicular steroidogenic enzymes and steroidogenic acute regulatory protein in the adult ram
Authors: Price CA , Cooke GM , Sanford LM
Published: January 2004

The regulation of LH-dependent and -independent increases in testosterone secretion by key proteins in the testes of adult rams was investigated. Serial blood samples were collected from groups of four control and passively immunized (oestradiol antiserum for 3 weeks) rams and the animals were gonadectomized in either the non-breeding season (April) or the breeding season (September). LH pulse frequency and basal (interpulse) concentrations were several times greater (P < 0.01) in the breeding season than in the non-breeding season. Neither of these parameters nor LH pulse amplitude were affected by oestradiol immunization. Parameters of testosterone episodic secretion and response to an injection (i.v.) of 15 micrograms NIH-LH-S25 were also greater (P < 0.05) in the breeding season and, with the exception of pulse frequency, in immunized rams versus controls. Substrate utilization established that testosterone biosynthesis was predominantly via the 5-ene pathway. Increases in blood testosterone concentration in the breeding season were associated with a fivefold higher (P < 0.01) activity of cytochrome P450 17alpha-hydroxylase/C-17,20 lyase (P450(17alpha)) and a 65% higher (P < 0.05) relative amount of mRNA for cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) in the testis. Of the steroidogenic enzyme activities examined, only that for 17beta-hydroxysteroid dehydrogenase (17beta-HSD) tended to be increased by oestradiol immunization. Blood concentrations of cholesterol lipoproteins and expression of the testicular low density lipoprotein receptor were not affected by season or immunization. The amount of steroidogenic acute regulatory protein (StAR) mRNA was 65% higher (P < 0.01) in the breeding season and 20% higher (P < 0.01) in immunized rams versus controls. These results indicate that greater LH stimulation may increase testosterone biosynthesis in the breeding season by increasing StAR mRNA (and presumably delivery of cholesterol to P450scc) and the activity of P450(17alpha), and possibly that of P450scc (activity not measured). More moderate increases in StAR mRNA and 17beta-HSD activity may explain, in part, the increases in testosterone secretion with oestradiol immunization.
Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa
Authors: Johnston SD , McGowan MR , Phillips NJ , O'Callaghan P
Published: January 2004

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.
Effect of nutrition and superovulation on oocyte morphology, follicular fluid composition and systemic hormone concentrations in ewes
Authors: O'Callaghan D , Yaakub H , Hyttel P , Spicer LJ , Boland MP
Published: January 2004

The objective was to determine the effect of dietary intake on follicle and oocyte morphology in unstimulated and superovulated ewes. Fifty-four ewes were fed grass meal at 0.5, 1.0 or 2.0 times maintenance energy requirements (M) for 32 days. Oestrous cycles were synchronized using progestagen pessaries and either unstimulated or superovulated with 200 mg pig FSH. The ewes were killed and ovaries were collected either 36 or 12 h before the anticipated LH surge. Serum progesterone concentrations in ewes on day 10 after withdrawal of the pessary were lower in ewes fed 2.0M than in ewes fed 0.5M or 1.0M (P < 0.05). LH pulse frequency tended to be higher in ewes fed 2M than 1M (1.0 +/- 0.3 versus 0.3 +/- 0.2 pulses per 8 h) on day 6 after removal of the pessary but the effect was not significant. In unstimulated ewes, more follicles (>/= 3 mm) were observed when the animals were killed in ewes fed 2.0M (3.5 +/- 0.3) than in ewes fed 0.5M (2.4 +/- 0.3) or 1.0M (2.4 +/- 0.5; P < 0. 05). Fewer follicles were observed in superovulated ewes on 0.5M (7. 5 +/- 1.2) than in ewes on 1.0M (12.0 +/- 0.5) or 2.0M (12.3 +/- 1. 4; P < 0.05). Follicular fluid progesterone concentrations were higher in ewes fed 0.5M compared with those fed 1M or 2M (P < 0.05). Insulin-like growth factor (IGF)-I concentrations were higher in follicular fluid from ewes on 1M compared with either those on 0.5M or 2M (P < 0.05), whereas IGF-II concentrations were lower in follicular fluid from ewes on 2M compared with those on 1M or 0.5M (P < 0.05). Superovulation increased follicular fluid progesterone, oestradiol, IGF-I and IGF-II concentrations (P < 0.01). Concentrations of the 34, 22 and 20 kDa IGF binding proteins were lower in follicles from superovulated ewes compared with unstimulated ewes (P < 0.05). Oocytes from superovulated ewes showed abnormalities such as premature activation of cumulus expansion and vacuolation of the nucleolus and increased frequency of detachment of interchromatin-like granules from the nucleolar remnant. Collectively, these results indicate that both high and low dietary intakes can alter systemic and follicular fluid hormone concentrations. Relative to dietary effects, the effects of superovulation were greater and involved substantial increases in follicular fluid hormone concentrations and abnormal oocyte morphology.
Ultrastructural evidence of transplacental transport of immunoglobulin G in bitches
Authors: Stoffel MH , Friess AE , Hartmann SH
Published: January 2004

In dogs, passive immunity is conferred to fetuses and neonates by the transfer of maternal immunoglobulin G through the placenta during the last trimester of pregnancy and via the mammary gland after parturition, respectively. However, morphological evidence of transplacental transport is still lacking. The aim of the present study was to localize maternal immunoglobulin G in the labyrinthine zone and in the haemophagous zone of the canine placenta by means of immunohistochemistry and immunocytochemistry. In the labyrinthine zone, immunoglobulin G was detected in all the layers of the materno-fetal barrier including the fetal capillaries. Immunoreactivity was particularly prominent in maternal basement membrane material as well as in the syncytiotrophoblast. However, this evidence of transplacental transport of immunoglobulin G originated from a limited number of unevenly distributed maternal vessels only. In the cytotrophoblast of the haemophagous zone, immunoglobulin G was localized to phagolysosomes at various stages but was never detected within fetal vessels. The results indicate that maternal immunoglobulin G is degraded in cytotrophoblast cells of the hemophagous zone and, therefore, that transplacental transport is restricted to a subpopulation of maternal vessels in the labyrinthine zone.
Postnatal growth rate and gonadal development in circadian tau mutant hamsters reared in constant dim red light
Authors: Lucas RJ , Stirland JA , Mohammad YN , Loudon AS
Published: January 2004

The role of the circadian clock in the reproductive development of Syrian hamsters (Mesocricetus auratus was examined in wild type and circadian tau mutant hamsters reared from birth to 26 weeks of age under constant dim red light. Testis diameter and body weights were determined at weekly intervals in male hamsters from 4 weeks of age. In both genotypes, testicular development, subsequent regression and recrudescence exhibited a similar time course. The age at which animals displayed reproductive photosensitivity, as exhibited by testicular regression, was unrelated to circadian genotype (mean +/- SEM: 54 +/- 3 days for wild type and 59 +/- 5 days for tau mutants). In contrast, our studies revealed a significant impact of the mutation on somatic growth, such that tau mutants weighed 18% less than wild types at the end of the experiment. Our study reveals that the juvenile onset of reproductive photoperiodism in Syrian hamsters is not timed by the circadian system.
Localization of calcium and zinc in the sperm storage tubules of chicken, quail and turkey using X-ray microanalysis
Authors: Holm L , Ekwall H , Wishart GJ , Ridderstrale Y
Published: January 2004

Sperm storage tubules from the utero-vaginal junction of chickens, quails and turkeys were analysed for calcium and zinc using X-ray microanalysis of ultra-rapidly frozen tissue in a scanning electron microscope. This technique enabled the tubular fluid surrounding the stored spermatozoa and the intracellular content of the cells of the sperm storage tubules to be analysed separately and, by using standards with known concentrations, their elemental concentrations were estimated. The mean (+/- SEM) concentration of calcium in the tubular fluid from chickens, quails and turkeys was 17 +/- 3, 19 +/- 3 and 17 +/- 4 mmol kg(-1) wet weight, respectively. The intracellular calcium concentration of the cells of the tubules did not differ significantly from these values and was also similar in the mucosal epithelial cells of the utero-vaginal junction. Zinc was localized in the cells of turkey sperm storage tubules and tubular fluid, but at low concentrations. No zinc could be detected in corresponding structures from chickens and quails. The concentration of calcium in the tubular fluid is within the range known to inhibit the motility of spermatozoa, supporting this function for calcium during storage. Zinc is known to depress turkey sperm metabolism and it may also be involved in inducing quiescence of spermatozoa during storage in this species.
Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation
Authors: Carroll J
Published: January 2004

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.
Number of chromocentres in the nuclei of mouse Sertoli cells in relation to the strain and age of males from puberty to senescence
Authors: Krzanowska H , Bilinska B
Published: January 2004

Nuclei of mouse Sertoli cells were examined on air-dried toluidine blue-stained preparations to analyse factors influencing the aggregation of heterochromatin into chromocentres. For the CBA strain males tested at 1.3, 2, 3, 6, 9 and 12 months of age, mean numbers of heterochromatin bodies were 4.8, 2.4, 2.1, 1.8, 1.6 and 1. 4, respectively; two chromocentres predominated from 2 to 9 months of age. In the KE strain, heterochromatin aggregation was significantly accelerated; nuclei containing only one chromocentre were predominant from 6 months. The number of chromocentres did not change with the stages of the seminiferous cycle, and after 1 month of cryptorchid condition. Cryptorchidism resulted in disruption of spermatogenesis and Sertoli cell dysfunction, as demonstrated by the lack of immunohistochemically detectable androgen receptors. The difference in the number of chromocentres between KE and CBA Sertoli cells persisted after 3 days of in vitro culture, but unidentified cells with numerous chromatin bodies were also observed. Testing recombinant inbred strains indicates that at least two genes are involved in the difference in the number of chromocentres between progenitor KE and CBA strains; however, no correlations were found with 15 marker loci or with parameters linked to reproduction. Of the eight strains tested, AKR and C3H showed a 'CBA-like' chromocentre pattern; C57BL, B10.BR, B10.BR-Y(del) and KP were 'KE-like'; and BALB/c and DBA/2 were intermediate. The results showed that centromere aggregation in the Sertoli cell progresses throughout the life of a male in a strain specific manner; however, its functional significance remains unknown.
Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo
Authors: Bruck I , Bezard J , Baltsen M , Synnestvedt B , Couty I , Greve T , Duchamp G
Published: January 2004

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles </= 29 mm was significantly higher at A1 than at A2. The number of oocytes at the germinal vesicle stage was significantly higher at A2 (41.5%) than at A1 (17.8%). Meiosis-activating sterols (FF-MAS and T-MAS) were identified in follicular fluid recovered at A2. Follicular concentrations of FF-MAS and T-MAS were unaffected by treatment with crude equine gonadotrophins. The present study demonstrates that follicular aspiration during oestrus allowed a new follicular population to develop and resulted in a higher degree of synchronization of oocyte development with respect to cumulus expansion and nuclear maturation. The availability of a more homogeneous population of oocytes might facilitate a better optimization of in vitro maturation and in vitro fertilization techniques in mares. Administration of crude equine gonadotrophins during early dioestrus did not affect the growth of small follicles, the oocyte yield after aspiration or oocyte maturation in vivo.
In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture
Authors: Lin M , Zhang X , Murdoch R , Aitken RJ
Published: January 2004

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.
Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation
Authors: Zhang C , Duan E , Cao Y , Jiang G , Zeng G
Published: January 2004

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.
Effect of administration of eCG to postpartum cows on folliculogenesis in the ovary ipsilateral to the previously gravid uterine horn and uterine involution
Authors: Sheldon IM , Dobson H
Published: January 2004

This study tested the hypothesis that increased oestradiol secretion by large follicles in the ovary ipsilateral to the previously gravid uterine horn has a local effect to increase the rate of uterine involution. Cows were administered an i.m. water placebo (n = 19), 250 iu equine chorionic gonadotrophin (eCG) (n = 18) or 750 iu eCG (n = 20) 14 days post partum (day 0). Transrectal ultrasonography at the time of treatment and 2, 4 and 6 days later monitored uterine horn diameter and ovarian structures. Blood samples collected contemporaneously were assayed for 15-keto-13, 14-dihydro-prostaglandin F(2alpha) and oestradiol concentration. For control cows, accumulated diameter of the largest follicle in the ovary ipsilateral to the previously gravid uterine horn, compared with the contralateral ovary, was smaller on day 0 (P < 0.05) and days 2, 4 and 6 (P < 0.001). There were no significant differences in the eCG-treated animals. There were 12, 8 and 11 cows with a plasma oestradiol concentration < 1 pg ml(-1) on day 0 in the control, 250 and 750 iu eCG treatment groups, respectively. For control cows, the peripheral oestradiol concentrations were higher on day 6 compared with days 0, 2 and 4 (P < 0.05); for cows treated with 250 iu eCG, concentrations were higher on days 4 and 6 compared with day 0 (P < 0.05); and for cows treated with 750 iu eCG, concentrations were higher on days 2 and 4 compared with day 0 (P < 0.01). Treatment with eCG, or the presence of a follicle > 8 mm in diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the rate of uterine involution or plasma 15-keto-13,14-dihydro-prostaglandin F(2alpha) concentration. In conclusion, administration of eCG to increase follicular growth and oestradiol production overcame the inhibition of follicular growth in the ovary ipsilateral to the previously gravid uterine horn, but did not affect uterine involution.
Anovulation in non-reproductive female Damaraland mole-rats (Cryptomys damarensis)
Authors: Molteno AJ , Bennett NC
Published: January 2004

Within colonies of Damaraland mole-rats (Cryptomys damarensis), anovulation in non-reproductive females is thought to play an important role in maintaining reproductive skew. Pituitary sensitivity and ovarian structure were examined in three groups of females that differed with respect to their social environment and breeding status to determine whether anovulation is due to inhibitory social cues or is merely the result of a lack of copulatory stimulation. The contribution of gonadal steroid negative feedback to neuroendocrine differences in the reproductive systems of the respective groups was also investigated. LH secretion after a 0.5 micrograms GnRH challenge in females that had been removed from the presence of the breeding individuals for at least 6 months (removed non-reproductive females) was significantly higher than in non-reproductive females in the colony, but significantly lower than in reproductive females. In both removed non-reproductive females and reproductive females, corpora lutea were observed in ovaries of seven of eight females, indicating that ovulation occurs spontaneously in subordinate females on removal from the breeding pair. Circulating progesterone concentrations in removed non-reproductive females were significantly higher than in non-reproductive females, indicating that circulating progesterone is not responsible for infertility in non-reproductive females. Indeed, after hystero-ovariectomy, reproductive females continued to show significantly greater GnRH-stimulated LH secretion than non-reproductive females. Thus, differential inhibition of gonadotrophin secretion in breeding and non-breeding females occurs independently of gonadal steroids. It is concluded that female Damaraland mole-rats are spontaneous ovulators and that anovulation results from inhibitory social cues within the colony, not a lack of copulatory stimulation. Since non-reproductive females are infertile, inhibition of the hypothalamo-pituitary-gonadal axis has the potential to play a causal role in maintaining reproductive skew in colonies of C. damarensis.
Classification of small type B/C follicles as primordial follicles in mature rats
Authors: Meredith S , Dudenhoeffer G , Jackson K
Published: January 2004

In the present study, follicles were classified according to the morphology of their granulosa cells. Type B follicles contained only flattened granulosa cells; type B/C follicles had a mixture of flattened and cuboidal granulosa cells in a single layer, and type C follicles had a single layer of cuboidal granulosa cells. The primary objectives of the study were to determine whether 5-bromo-2-deoxyuridine incorporation into type B/C follicles was a marker for initiation of growth and how long type B/C follicles could remain at the same stage before transformation to type C follicles. Female Holtzman rats received bromo-deoxyuridine for 7 days. After the infusion (day minipumps were removed = day 0), rats were ovariectomized on days 0 (n = 9), 30 (n = 8), 90 (n = 8) and 150 (n = 9). The numbers of type B, B/C and C follicles within one ovary were determined using modified fractionator counting. Analysis over all times demonstrated that there were more (P < 0.0001) type B/C (941 +/- 61 per ovary) than type C (140 +/- 18 per ovary) or type B (159 +/- 19 per ovary) follicles. The numbers of type B and type C follicles did not differ from each other at any time. Only one of 34 rats evaluated had bromo-deoxyuridine-labelled type B follicles. On day 150, 57% of the bromo-deoxyuridine-labelled type B/C follicles remained from day 0. It is concluded that (1) DNA synthesis in granulosa cells of type B/C follicles is not a reliable indicator of impending growth; and (2) type B and type B/C follicles are both components of the pool of primordial follicles.
Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes
Authors: Strom Holst B , Larsson B , Linde-Forsberg C , Rodriguez-Martinez H
Published: January 2004

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.