Reproduction

Stirling, D.; Waterman, M. R.; Simpson, E. R.

doi: 10.1530/jrf.0.0910001pmid: 1847419

Summary. A radiolabelled cRNA was synthesized using a 1·4 kb cDNA complementary to mRNA encoding bovine basic fibroblast growth factor (bFGF) as a template, and used as a probe to investigate the expression of mRNA encoding bFGF in bovine ovarian tissue, and luteal cells in primary culture. Northern analysis of poly(A +) RNA prepared from follicles and corpora lutea of various stages revealed a major mRNA species of 7 kb in corpora lutea of all stages, the amount of which was higher late in the luteal phase. No hybridizable message was detectable in follicles of any size. When luteal cells were established in primary culture, expression of the 7 kb mRNA species was maintained. This expression was increased markedly when cells were treated with LH/hCG or Bt2cAMP. Prostaglandin F-2α treatment caused a marked decrease in the basal content of this 7 kb mRNA, and also severely impaired the ability of LH to stimulate this expression.Keywords: ovary; corpus luteum; RNA; bFGF; cow

Shaw, J. M.; Kola, I.; MacFarlane, D. R.; Trounson, A. O.

doi: 10.1530/jrf.0.0910009pmid: 1995866

Summary. This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1·5, 3·0 or 4·5 m-DMSO. Rapidly cooled straws containing 4·5 m-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4·5 m-DMSO was not influenced by straw handling as 91–92% formed blastocysts in vitro, 77–78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing <4.5 m-DMSO ice formation occurred throughout (1·5 m-DMSO), or in parts (3·0 m-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3·0 or 1·5 m-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3·0 m-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4·5 m-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.Keywords: dimethyl sulphoxide; rapid freezing; ice formation; handling; chromosome rearrangements; mouse; embryos

Cook, D. L.; Parfet, J. R.; Smith, C. A.; Moss, G. E.; Youngquist, R. S.; Garverick, H. A.

doi: 10.1530/jrf.0.0910019pmid: 1899886

Summary. Two experiments were conducted to (1) investigate developmental endocrinology of ovarian follicular cysts (cysts) in cattle and (2) evaluate effects of cysts on hypothalamic and hypophysial characteristics. Cysts were induced with oestradiol-17β (15 mg) and progesterone (37·5 mg) dissolved in alcohol and injected s.c. twice daily for 7 days. Cysts were defined as the presence of follicular structures (which may or may not have been the same structure) of 2·0 cm in diameter or greater that were present for 10 days without ovulation and corpus luteum development.In Exp. 1, 22 non-lactating, non-pregnant Holstein cows were allocated to 3 groups. Beginning on Day 5 (oestrus = Day 0) of the oestrous cycle, 7 cows (Controls) were treated with twice daily s.c. injections of ethanol (2 ml/injection) for 7 days. Luteolysis was then induced with PGF-2α and blood samples were collected daily every 15 min for 6 h from the morning after the PGF-2α injection (Day 13) until oestrus. Steroids to induce cysts were injected as previously described into the remaining cows (N = 15). Three blood samples were collected at 15-min intervals every 12 h throughout the experimental period. Additional blood samples were collected every 15 min for 6 h on a twice weekly basis. After steroid injections, follicular and luteal structures on ovaries were not detected via rectal palpation for a period of 36 ± 4 days (static phase). Then follicles developed which ovulated within 3–7 days (non-cystic; N = 7) or increased in size with follicular structures present for 10 days (cystic; N = 8). Mean (± s.e.m.) concentrations of LH, FSH, oestradiol-17β and progesterone in serum remained low and were not different during the static phase between cows that subsequently developed cysts or ovulated. During the follicular phase, mean serum concentration of LH (ng/ml) was higher (P < 0·1) in cows with cysts (2·9 ± 0·2) than in cows without cysts (1·1 ± 0·1) or control cows (1·4 ± 0·2). In addition, LH pulse frequency (pulses/6 h) and amplitude (ng/ml) were higher (P < 0·1) in cows with cysts (3·6 ± 0·3 and 2·2 ± 0·3, respectively) than in non-cystic (2·3 ± 0·2 and 1·0 ± 0·2, respectively) and control (1·8 ± 0·1 and 1·1 ± 0·2, respectively) groups during the follicular phase.In Exp. 2, 20 non-lactating, non-pregnant dairy cows were used: 15 cows received exogenous steroids as previously described. Hypothalamic and hypophysial tissues were collected after diagnosis of cystic structures in 11 cows (cystic group). The remaining 4 cows in the steroid-treated group ovulated and were assigned to the control group in addition to 5 non-steroid treated cows. Hypothalamic and hypophysial tissues were collected during the late-luteal phase (Days 16–18) from these control cows (N = 9). Anterior pituitary concentrations (μg/g) of LH (60·5 ± 11·0, 44·6 ± 11·7), FSH (30·2 ± 4·0, 22·1 ± 4·6) and receptors for GnRH (17·2 ± 2·2, 23·4 ± 2·6 m × 10−10/mg protein) did not differ between cows with cysts and control cows, respectively. Content of GnRH (ng) in the combined preoptic area and hypothalamus proper was higher (P < 0·05) in control cows (37·7 ± 6·6) than cows with cysts (18·6 ± 6·1). In the pituitary stalk median eminence, GnRH content (ng) tended to be higher (P ≥ 0·1) in cows with cysts (38·5 ± 9·6) compared with control (21·1 ± 15·2) cows.Secretory patterns (mean concentration, pulse frequency and amplitude) of LH were therefore increased during the follicular phase in cows which developed cysts compared to cows which subsequently ovulated. In addition, hypothalamic GnRH content, but not pituitary characteristics, appeared to be altered in cows with cysts.Keywords: ovary; follicular cysts; dairy cattle; gonadotrophins; hypothalamus; pituitary

Brown, J. L.; Bush, M.; Packer, C.; Pusey, A. E.; Monfort, S. L.; O'Brien, S. J.; Janssen, D. L.; Wildt, D. E.

doi: 10.1530/jrf.0.0910029pmid: 1899889

Summary. Pituitary–gonadal function was examined in male lions free-ranging in the Serengeti Plains or geographically isolated in the Ngorongoro Crater of Tanzania. Lions were classified by age as adult (6·1–9·8 years), young adult (3·3–4·5 years) or prepubertal (1·4–1·6 years, Serengeti Plains only). Each animal was anaesthetized and then bled at 5-min intervals for 100 min before and 140 min after i.v. administration of saline or GnRH (1 μg/kg body weight). Basal serum LH and FSH concentrations were similar (P > 0·05) among age classes and between locations. In Serengeti Plains lions, net LH peak concentrations after GnRH were ∼ 25% greater (P < 0·05) in prepubertal than in either adult or young adult animals. GnRH-stimulated LH release was similar (P > 0·05) between adult and young adult lions, and these responses were similar (P > 0·05) to those measured in Ngorongoro Crater lions. Basal and GnRH-stimulated testosterone secretion was higher (P < 0·05) in adult than in young adult lions and lowest (P < 0·05) in prepubertal lions. Age-class differences in testosterone production were related directly to the concentrations of LH receptors in the testis (P < 0·05). Basal and GnRH-stimulated testosterone secretion and gonadotrophin receptor concentrations within age classes were similar (P > 0·05) between lions of the Serengeti Plains and Ngorongoro Crater. Lower motility and higher percentages of structurally abnormal spermatozoa were observed in electroejaculates of young adult compared to adult Serengeti Plains males (P < 0·05) and were associated with decreased steroidogenic activity. In contrast, there were no age-related differences in ejaculate characteristics of Ngorongoro Crater lions. Seminal quality in the Crater population was poor in adult and young adult animals and was unrelated to alterations in pituitary or testicular function. In summary, only seminal quality in adult male lions was affected by location, whereas age significantly affected both basal and GnRH-stimulated testosterone secretion and seminal quality (Serengeti Plains only) in sexually mature males. The striking seminal/endocrine differences among pride (breeding) males of different ages raises questions about the impact of age on individual reproductive performance in this species.Keywords: lion; GnRH; LH; testosterone; receptors; testis; spermatozoa

Mate, K. E.; Rodger, J. C.

doi: 10.1530/jrf.0.0910041pmid: 1847424

Summary. Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33°C, or room temperature when long-term sperm survival (> 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.Keywords: marsupial; spermatozoa; acrosome reaction; capacitation; ionophore

Jenner, L. J.; Parkinson, T. J.; Lamming, G. E.

doi: 10.1530/jrf.0.0910049pmid: 1847425

Summary. Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P < 0·05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.Keywords: uterus; oxytocin; receptors; cattle; oestrous cycle; pregnancy

Halhali, A.; Acker, G. M.; Garabédian, M.

doi: 10.1530/jrf.0.0910059pmid: 1995862

Summary. Intraluminal injection of female rats at Day 5 of pseudopregnancy with 10–500 ng 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) significantly increased the uterine weight and induced decidual reaction. This effect was observed as early as the 3rd day after 1,25-(OH)2D3 injection. It was detectable only in the injected left horn and not in the non-injected right horn. A 500 ng dose of 25-(OH)D3 had no such effect. The present in-vivo results suggest that 1,25-(OH)2D3 may play a physiological role in endometrial cell differentiation into decidual cells, a crucial step in the process of blastocyst implantation.Keywords: 1,25-Dihydroxyvitamin D3; uterus; endometrial cells; decidualization; fertility; rat

Pellestor, F.; Sèle, B.

doi: 10.1530/jrf.0.0910065pmid: 1995863

Summary. Epidemiological and mouse cytogenetic studies have suggested the existence of a positive relationship between the frequency of chromosomal abnormalities and the duration of male sexual abstinence. A cytogenetic analysis of human spermatozoa was performed with 4 men after periods of sexual abstinence ranging from 2 to 15 days. Using hamster eggs for penetration, 934 sperm chromosome complements were obtained. The overall frequency of sperm chromosomal abnormalities was 9·4%. There was no correlation between the length of sexual abstinence and each type of chromosomal abnormality. Our results do not support the hypothesis of an increased risk of aberration with duration of abstinence.Keywords: sexual abstinence; man; sperm chromosomes; hamster egg penetration assay; ageing

Van Vliet, Renata A. C.; Walton, J. S.; Wildeman, A. G.; Betteridge, K. J.; Gibbins, Ann M. Verrinder

doi: 10.1530/jrf.0.0910073pmid: 1995864

Summary. Total cellular RNA was isolated from conceptus tissue obtained from 22 superovulated cows 18 days after artificial insemination. Total RNA was also isolated from luteal tissue from 3 cyclic cows 7 and 8 days after oestrus. Luteal and conceptus RNA were simultaneously subjected to formaldehyde–agarose gel electrophoresis and transferred to nitrocellulose by bidirectional diffusion blotting. Northern blots were probed using cDNAs specific for bovine oxytocin and bovine β-actin gene sequences. Hybridization of the oxytocin cDNA to RNA was consistently observed on autoradiographs as a 0·6 kilobase (kb) band in lanes containing corpus luteum RNA, but was not detected in lanes containing conceptus RNA. The presence of conceptus RNA on the blots was confirmed by hybridization of the actin cDNA to conceptus RNA, which resulted in a 2·0 kb band on autoradiographs. These results suggest that oxytocin is not synthesized by the bovine conceptus on Day 18 of gestation.Keywords: oxytocin; conceptus; messenger RNA; β-actin; cattle

Guilbault, L. A.; Grasso, F.; Lussier, J. G.; Rouillier, P.; Matton, P.

doi: 10.1530/jrf.0.0910081pmid: 1995865

Summary. Dairy heifers were superovulated in the presence (dominant group, N = 8) or absence (non-dominant group, N = 6) of a dominant follicle at the start of a superovulatory treatment on Days 7–12 of the oestrous cycle (Day 0 = oestrus). Daily ultrasonographic observations of ovaries (recorded on videotape) starting on Day 3 were used to assess the presence or absence of a dominant follicle (diameter > 9 mm, in a growing phase or at a stable diameter for < 4 days) and to monitor follicular development before and during treatment. The number of CL estimated by ultrasonography (7·1 ± 1·8 vs 13·5 ± 1·4) or by rectal palpation (6·9 ± 2·0 vs 16·3 ± 1·6) and mean progesterone concentrations (32·5 ± 19 vs 80·7 ± 16 ng/ml) after treatment were lower (P < 0·01) in the dominant than in the non-dominant group. Based on number of CL, two populations of heifers were identified in the dominant group, i.e. those that had a high (dominant–high, N = 4; > 7 CL) or a low (dominant–low, N = 4; < 7 CL) response to treatment. During treatment, the increases in number of follicles 7–10 mm and > 10 mm in diameter occurred sooner and were of higher magnitude in the non-dominant than in the dominant–high or dominant–low groups (P < 0·01). At the expected time of ovulation 6–7 days after the start of treatment, there was a rapid decrease in number of follicles 7–10 mm and > 10 mm in diameter in the dominant–high and non-dominant groups but not in the dominant–low group. Compared with the dominant–high group, differences in profiles of changes in diameter of largest (F1) and second largest (F2) follicles indicated that emergence of the dominant F1 follicle before treatment was delayed by 1–2 days in the dominant–low group. These results suggest that the presence of a dominant follicle before superovulation treatment may decrease the superovulatory response and/or alter the maturation process of growing follicles during treatment, especially when emergence of the dominant F1 follicle occurred within 3 days of the start of treatment.Keywords: follicle; dominance; superovulation; ultrasonography; cattle