Reproduction

Tucci, J.; Beck, F.

doi: 10.1530/jrf.0.1120001pmid: 9538324

The interaction between parathyroid hormone-related protein (PTHrP) and the parathyroid hormone (PTH)/PTHrP receptor is thought to play a role in the growth and differentiation of various tissues throughout fetal development in the rodent. The aim of the present study was to define the patterns of expression of PTHrP and of the PTH/PTHrP receptor in the rat uterus during the early stages of normal pregnancy, and following artificial induction of a decidual reaction. Using hybridization histochemistry, we have shown that the receptor gene is switched on early in pregnancy (by 1.5 days post coitum) in the endometrial stromal cells that surround the lumen. These cells include the anti-mesometrial subepithelial stromal cells that are destined to become decidualized. This pattern continues until 5.0 days post coitum, when PTHrP is switched on in antimesometrial luminal epithelial cells that line the implantation chamber. Stromal cells underlying the implantation chamber then downregulate transcription of the receptor gene, and within 12 h differentiate into decidual cells. A similar pattern was seen in uteri in which a decidual reaction had been induced artificially. Therefore, it may be postulated that in early pregnancy the endometrial stroma initiates transcription of the gene for the PTH/PTHrP receptor and is thus 'primed' for the PTHrP signal from the luminal epithelial cells. Some time after receiving the signal, the endometrial stromal cells downregulate the receptor gene, and this appears to be a trigger for the terminal differentiation of the stromal cells into decidual cells. These results suggest that PTHrP, acting through the PTH/PTHrP receptor, plays a role in the initiation of a decidual reaction during early pregnancy by regulating the differentiation of endometrial stromal cells into decidual cells.

Wakayama, T.; Whittingham, D. G.; Yanagimachi, R.

doi: 10.1530/jrf.0.1120011pmid: 9538325

Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to −20°, −50° or −196°C and storage for 1–28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95–100%) and developed to the two-cell stage (89–100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80–94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at −196°C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to −196°C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.

Singh, J.; Pierson, R. A.; Adams, G. P.

doi: 10.1530/jrf.0.1120019pmid: 9538326

Heifers were studied to determine whether computer-assisted quantitative echotexture analysis of ultrasound images reflect functional and endocrine characteristics of dominant and subordinate follicles at specific stages of development. Heifers were examined using transrectal ultrasonography each day until ovariectomy on day 3 (n = 8) and day 6 (n = 9) of wave 1, day 1 of wave 2 (n = 7), or after onset of pro-oestrus ≥ 17 days after ovulation (n = 8) to obtain growing, early-static, late-static and regressing dominant follicles of wave 1, subordinate follicles, preselection follicles and preovulatory dominant follicles. Ultrasound images of the follicles were obtained in vitro and analysed using custom-developed computer algorithms. Mean pixel (picture element) values (grey-scale: black = 0, white = 255) for the follicle wall and stroma increased (P < 0.05) progressively from the growing to the regressing phases of the dominant follicle of wave 1. The antrum and wall of subordinate follicles had higher (P < 0.05) mean pixel values than that of the corresponding dominant follicles. Pixel heterogeneity (a measure of variation of grey-scale values of pixels) of images of the follicle antrum and wall increased (P < 0.05) progressively during the early-static to regressing phases. A progressive increase (P < 0.05) in the slope of the regression line of pixel values for the follicle wall was detected from the growing to the regressing phases of the dominant follicle of wave 1. The regression line of the wall of the preovulatory dominant follicle had the lowest (P < 0.05) slope. Oestradiol concentration in the follicular fluid decreased (P < 0.05) from the growing to the late-static phase, while a marked decrease (P < 0.05) in the androstenedione concentration was recorded between the growing and the early-static phases of the dominant follicle. Progesterone content did not increase until follicles were in the final stages of regression. Pixel heterogeneity of the antrum and wall, and the slope of the follicle wall regression line were negatively correlated (P < 0.001) with oestradiol and the oestradiol:progesterone ratio in follicular fluid. The results of this study support the hypothesis that echotexture characteristics of ultrasound images of the follicle antrum and wall are correlated with the functional and endocrine status of a follicle.

Adam, C. L.; Findlay, P. A.; Kyle, C. E.; Young, P.

doi: 10.1530/jrf.0.1120031pmid: 9538327

Ovariectomized, oestradiol-implanted Soay ewe lambs from 21 September (aged 21 weeks) had restricted (liveweight maintenance) (n = 4) or unrestricted food (n = 4); ovary-intact lambs had unrestricted food (n = 8). LH activation in ovariectomized lambs on restricted and unrestricted food and onset of ovulatory cycles in ovary-intact lambs all occurred on 7 December (sed 8.8 days) (32 weeks), but at different liveweights (24.2, 17.9 and 18.3 kg, respectively, sed 1.22). LH pulse frequency was similar in ovariectomized lambs on restricted and unrestricted food. From 29 August (aged 18 weeks), Soay ewe lambs in seasonally advanced decreased artificial daylength were given restricted food, unrestricted food, or food was restricted for 8 weeks and then unrestricted (n = 8 per group). Ovarian cycles started 3 weeks earlier than in lambs in natural photoperiod on similar dates for all three groups (14, 18 and 19 November, respectively, sed 5.5 days) (29 weeks), but at different liveweights (16.2, 20.7, and 18.4 kg, respectively, sed 0.87). From 1 August, Suffolk × Greyface ewe lambs (aged 16 weeks) had restricted food, unrestricted food, or food restricted for 8 weeks and then unrestricted (n = 8 per group). By 1 November (29 weeks), 0/8 lambs on restricted food (29.3 ± 0.92 kg) but 8/8 lambs on unrestricted food and 5/8 lambs on 8 weeks of restricted food had ovulated (mean dates: 16 October ± 2.5 days (27 weeks, 40.1 ± 1.02 kg), and 1 November ±3.0 days (29 weeks, 35.5 ± 1.23 kg), respectively. Thus, nutritional growth restriction during the 11 weeks preceding normal puberty delayed pubertal date in the improved breed but did not influence the timing of puberty in the unimproved Soay breed within the weight range studied.

Jewgenow, K.; Penfold, L. M.; Meyer, H. H. D.; Wildt, D. E.

doi: 10.1530/jrf.0.1120039pmid: 9538328

About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40–90 μm) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0°C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at 0 h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P >0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P <0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2′-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% ± 2.9%, 18.8% ± 1.9% and 16.2% ± 1.6% were intact after 0 h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2′-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.

Lennard, S. N.; Gerstenberg, C.; Allen, W. R.; Stewart, F.

doi: 10.1530/jrf.0.1120049pmid: 9538329

Northern blot and in situ hybridization techniques have demonstrated a marked increase in mRNA encoding epidermal growth factor (EGF) in the endometrium of mares, coincident with the start of interdigitation between the allantochorion and endometrium during placentation. In the present study, the unusually high EGF expression in the epithelium of the endometrial glands was shown to be maintained until at least day 250 of gestation (term = 320–340 days) in mares carrying normal horse conceptuses. However, in mares carrying failing donkey-in-horse pregnancies created by embryo transfer, EGF expression was severely retarded in those areas of the endometrium that were heavily infiltrated with lymphocytes and which showed a failure of placental development. Specific receptors for EGF were also detected in tissue homogenates from pregnant mares using125I-labelled human EGF. Binding was high in the fetal membranes (allantochorion), both before implantation (days 30–34) and in the fully developed placenta (days 150–250), and was equivalent to the level of binding to homogenates of adult liver and kidney. Binding was much reduced in endometrial homogenates before implantation and from non-pregnant mares but increased after implantation to reach values equivalent to those exhibited by the fetal membranes. Scatchard analysis of displacement curves indicated a single class of high-affinity binding sites in the fetal membranes and pregnant endometrium sampled at day 150 of pregnancy and chemical cross-linking of the receptor – 125I-labelled EGF complexes in fetal membranes revealed two radiolabelled bands of 170 kDa and 150 kDa. A large excess of insulin-like growth factor I (IGF-I) failed to displace any labelled EGF from the tissue homogenates. The marked and sustained upregulation of endometrial EGF expression during pregnancy in mares, and the presence of EGF receptors in the fetal allantochorion and maternal endometrium, suggest a possible role for EGF in the marked growth of these two tissues during placentation in equids.

Williams, E. D.; Major, B. J.; Martin, T. J.; Moseley, J. M.; Leaver, D. D.

doi: 10.1530/jrf.0.1120059pmid: 9538330

Parathyroid hormone-related protein (PTHrP) was detected at 32.8 ± 3.9 pmol l−1 in uterine luminal fluid from immature rats treated with oestradiol. As mRNA encoding PTHrP has previously been localized to implantation sites in pregnant rats, the role of luminal PTHrP during pregnancy was explored. Infusion of a parathyroid hormone (PTH)/PTHrP receptor antagonist, [Asn10,Leu11]PTHrP(7–34) amide, into the uterine lumen during pregnancy in rats resulted in excessive decidualization. This effect was also observed after intrauterine infusion of a monoclonal antibody raised against PTHrP. The effect of infusion of PTH/PTHrP receptor antagonist was dependent upon successful implantation, was dose-dependent and confined to the treated horn. A decrease in the number of apoptotic decidual cells in antagonist-infused uterine horns compared with vehicle or non-infused horns was detected immunohistochemically at day 13 of pregnancy, and this decrease is likely to contribute to the 'over-decidualization' observed. In pseudopregnant rats, infusion of PTH/PTHrP receptor antagonist into the uterine lumen resulted in an increase in uterine wet weight of the infused horn compared with the non-infused horn, indicating a direct effect on deciduoma formation. Thus, activation of the PTH/PTHrP receptor by locally produced PTHrP appears to be crucial for normal decidualization during pregnancy in rats.

Campbell, B. K.; Baird, D. T.; Webb, R.

doi: 10.1530/jrf.0.1120069pmid: 9538331

The study reports the development of a serum-free culture system for sheep thecal cells that overcomes the problem of spontaneous luteinization and the use of this system to study the control of proliferation and differentiation. Theca cells were isolated by enzymatic dispersion from small follicles (< 3.5 mm) and the effect of plating densities (25–100 × 103 cells per well), LH (0.001–100 μg l−1), insulin (1–5000 μg l−1), insulin-like growth factor I (IGF-I) analogue (1–100 μg LR3-IGF-Il−1) and epidermal growth factor (EGF) (0.005–50 μg l−1) on the number of cells and androstenedione and progesterone production were determined. Plating density had a marked effect on the pattern of hormone secretion with densities between 50 and 75 × 103 cells per well resulting in a high androstenedione: progesterone ratio at optimum doses of LH (0.1 μg l−1: P < 0.001). In the first 48 h, the production of both androstenedione and progesterone was stimulated in a dose-dependent manner by LH (P < 0.001). However, the production of androstenedione was ten times higher than that of progesterone and was more sensitive to LH (ED50 value 0.08 μg l−1 for androstenedione and 1 μg l−1 for progesterone). From 48–144 h of culture higher doses of LH (> 1 ng ml−1) inhibited androstenedione (P < 0.001) and stimulated progesterone (P < 0.001) and resulted in a marked change in cell morphology, thus reflecting both functional and morphological luteinization. At optimum doses of LH, both insulin and IGF stimulated cell proliferation (P < 0.001) and androstenedione production (P < 0.001) in a dose responsive manner and there was a significant (P < 0.001) interaction between them. In contrast, both insulin and IGF-I inhibited (P < 0.001) progesterone production in a dose responsive manner. EGF stimulated cell proliferation (P < 0.001) and progesterone production (P < 0.001), but inhibited androstenedione production (P < 0.001), in a dose responsive manner. In conclusion, this culture system exhibits physiologically relevant responses to known in vivo modulators of follicle development. The biphasic nature of the theca cell response to LH emphasises the exquisite sensitivity of theca cells to LH stimulation and highlights the importance of dose–response relationships in the gonadotrophic control of ovarian function.

De Rensis, F.; Cosgrove, J. R.; Foxcroft, G. R.

doi: 10.1530/jrf.0.1120079pmid: 9538332

The principal aim of this study was to investigate the ontogeny of an opioidergic mechanism mediating the suckling-induced inhibition of LH secretion during lactation in sows. In contrast to an increase in LH secretion in response to naloxone treatment on days 10 and 11 of lactation (P < 0.05), a single injection of 2 mg naloxone kg−1 at 39, 51, 63 or 75 h post partum had no effect. However, the last of four injections of 2 mg naloxone kg−1 given at 12 h intervals to group IV sows did elicit a positive LH response (P < 0.05). Multiple injections of 1 mg naloxone kg−1 at 3 h intervals over 30 h on day 10–11 consistently increased (P < 0.05) mean plasma LH with no evidence of induced refractoriness to repeated use of the antagonist. Similarly, naloxone did not affect mean plasma prolactin in the immediate postpartum period, but either repeated naloxone treatments on day 10–11, or single naloxone injections on day 10 or 11 of lactation decreased plasma prolactin (P < 0.05). Therefore, the regulation of LH and prolactin secretion in lactating sows changes with time post partum. An opioid-dependent mechanism is an important component of the suckling-dependent regulation of LH and prolactin secretion in established lactation, but not during the first 72 h postpartum period.

Setchell, B. P.; Plöen, L.; Ritzen, E. M.

doi: 10.1530/jrf.0.1120087pmid: 9538333

The effect of two class III antiarrhythmic drugs (Almokalant, Astra-Hässle and Dofetilide, Pfizer) on fluid secretion by rat testes has been examined. Both drugs reduced fluid secretion, whether this was measured by the amount of rete testis fluid that could be collected 22 h after unilateral efferent duct ligation, or by the difference in mass between the ligated and unligated testes, or by the difference in amount of supernatant fluid after the parenchyma of the ligated and unligated testes had been dispersed and centrifuged. The secretion of potassium, calculated from the amount of potassium in the supernatant fluids from the ligated and unligated testes was also reduced by the drugs, whereas the secretion of androgen-binding protein and inositol was unaffected. The concentration of potassium in the secreted fluid, calculated from the amount and composition of the supernatant fluids, was not affected by treatment of the rats with Almokalant, but was increased in rats treated with Dofetilide and, in these, the concentration of sodium was reduced and that of magnesium and inositol was increased and the concentration of total protein was unaffected. The concentration of androgen-binding protein in secreted fluid was increased in rats treated with Almokalant, while the concentration of testosterone was unaffected. Histological examination of testes from treated rats revealed phagocytosis of stage 19 spermatids in tubules at stages VIII–IX after 2 days, at stages IX–XI after 4 days and at stages VIII–XIV after 7 days, apparently owing to an effect on spermiation. It appears that these drugs interfere with potassium-mediated fluid secretion by the testis, leading to the other changes seen.