Summary.

The presence of a thread in a limited region of the rat uterus causes a gain in weight throughout the length of the uterine horn which bears it. In addition, such a thread lessens, but does not inhibit completely, the decidual cell response which can be induced by uterine trauma. This inhibition increases progressively with the length of time that the device has been present. A mechanism is suggested to explain this effect.

Summary. Ovine placental lactogen and the SBU-3 antigen (derived from a trophoblast membrane preparation), two proteins of widely different structure, function and destination, were shown by ultrastructural immunogold techniques to localize in identical structures in the sheep placentome throughout most of pregnancy. Both were restricted to the ultrastructurally similar membrane-bounded granules in the chorionic fetal binucleate cell and the syncytium at the fetomaternal interface. The Golgi body from which the granules form was also doubly labelled but only in the binucleate cell, never the syncytium. This provides further evidence that the binucleate cells migrate and fuse to form the syncytium. The two proteins were homogeneously distributed in the granules and would be released together by exocytosis. Only the lactogen reaches the fetal and maternal circulations so the SBU-3 may have some more local function.

In early pregnancy the SBU-3 antigen is found by itself in the granules, indicating that the association with the lactogenic hormone is not obligatory. Neither antigen was found consistently in the otherwise ultrastructurally similar interplacentomal binucleate cell granules, corroborating the presence of two functional populations of binucleate cells.

The recent identification of toll-like receptor (TLR) signaling within ovarian granulosa cells has broad implications for ovarian physiology. Functions of TLRs within granulosa cells of the laying hen are of particular interest due to the method of transovarian transmission of Salmonella enteritidis, which results in egg contamination. This study utilized hen granulosa cells to evaluate the expression and function of Gallus TLR-signaling at distinct stages of follicular maturity. Data presented herein demonstrate the presence of TLR2, TLR4, and TLR15 mRNAs in undifferentiated granulosa cells from prehierarchal follicles and differentiated granulosa cells from preovulatory follicles, together with mRNAs encoding adaptor proteins and signaling components required for TLR signaling gene. Treatment with lipopolysaccharide (LPS) or LH, in vitro, led to the differential regulation of TLRs based on the stage of follicle maturation, with the largest (F1) follicle granulosa cells having the most rapid response. Furthermore, treatment with LPS resulted in attenuation of agonist-induced progesterone synthesis in undifferentiated, but not differentiated, granulosa cells. Additionally, undifferentiated granulosa cells were significantly more sensitive to LPS-induced apoptosis than differentiated granulosa cells from the F1 follicle. Together, these data provide evidence for a complete and functional TLR signaling pathway in hen granulosa cells, with effects on steroidogenesis and cell viability dependent upon stage of maturation. These differences may reflect the susceptibility of granulosa cells at early stages of maturation to undergo apoptosis in response to select pathogenic stimuli, thus attenuating transovarian transmission, whereas granulosa cells from preovulatory follicles are comparably resistant to LPS-mediated apoptosis.

We reported previously that active immunization of heifers using a synthetic peptide-based inhibin vaccine (bIα(1–29)Tyr³⁰) can enhance ovarian follicular development and ovulation rate during spontaneous oestrous cycles. To extend this study, we investigated the effect of inhibin immunization more closely by monitoring plasma hormone profiles and ovarian activity in bIα(1–29)Tyr³⁰-immunized and control (ovalbumin-immunized) heifers (n = 6 per group) over three consecutive oestrous cycles, which were synchronized and shortened by administering a PGF_2α analogue at intervals of 14 days. Blood samples were collected at 2–8 h intervals for 40 days and the ovaries were examined daily using ultrasonography. Repeated-measures anova showed that inhibin immunization significantly increased plasma FSH concentration (by 52% overall; P < 0.01) and ovulation rate (by 58%; P < 0.01). Both immunized and control heifers showed the same cyclic pattern of plasma FSH (treatment × time interaction; not significant), indicating that the increase in plasma FSH was sustained throughout the cycle. Immunization did not affect the concentration or pattern of secretion of LH, oestradiol or progesterone and had no influence on the timing of the LH surge or ovulation after PG injection. While inhibin immunization increased the number of 'large' (i.e. growing to ≥ 10 mm diameter) follicles that developed during both the preovulatory (by 90%, P< 0.02) and postovulatory (by 190%, P< 0.01) period, there was no difference between groups in the temporal pattern of growth or regression of large follicles or of corpora lutea. These observations confirm a physiological role for ovarian inhibin as a component of the ovarian feedback mechanism controlling FSH secretion in heifers, and support the hypothesis that active immunization of heifers against inhibin enhances ovarian follicular development and ovulation rate by promoting a sustained increase in pituitary FSH secretion.

Summary. The distribution of α₂-PEG, a human analogue of β-lactoglobulin, in endometrium at different phases of the cycle was determined using immunohistochemistry with monoclonal and polyclonal antibodies. In the epithelial cells of glands in the functional zone of the endometrium, α₂-PEG was first detectable from Days 19 to 21 during the mid-luteal phase and maximal immunostaining was observed during the end of the late luteal phase. Intense staining in the glandular secretions and weaker staining in surface luminal epithelial cells during this period were observed. A minor population of basal glands contained α₂-PEG during the follicular phase. These results suggest that α₂-PEG synthesis by the glandular epithelium of the regenerated endometrium is hormonally regulated. Maximal staining occurring during the late luteal phase suggests that regulation may be related to the hormonal requirement for pre-decidualization rather than that required for histologically defined glandular epithelial secretion.

Keywords: endometrium; human; menstrual cycle; pregnancy-associated endometrial α₂-globulin; pregnancy proteins

In the placenta, cortisol is inactivated by NADP⁺- and NAD⁺-dependent isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD). Decreased placental 11βHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11βHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD⁺-dependent 11βHSD activities by 52 ± 4% and 45 ± 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26–45), mid- (days 46–90) and late gestation (days 91–135), or the 30 days prior to mating, had no significant effect on NAD⁺-dependent, placental 11βHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 ± 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD⁺-dependent placental 11βHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11βHSD activities.

Blood samples, morphometric measurements and behavioural data were collected weekly for 52 weeks from six adult Eld's deer stags exposed to natural fluctuations in photoperiod (38°N latitude). Mean (± sem) serum LH concentrations reached peak values in the autumn (October), three months before FSH and testosterone concentrations reached peak values in early winter (January). Prolactin concentrations were inversely related (r = −0.733, P < 0.001) to LH and directly related to daylength, and maximal concentrations were observed during mid-summer (July) and minimal concentrations during early winter (January). The temporal pattern of circulating inhibin was positively correlated with FSH (r = 0.88, P < 0.001), but lagged behind the seasonal FSH increase by 1–3 weeks. Antler length, body weight and chest girth were maximal during pre-rut (December–January). Maximal scrotal circumference and combined testes volume were observed in mid-winter (February), whereas peak neck girth and behavioural aggression occurred 1–3 months later (March–May). On the basis of quarterly electroejaculation results, motile spermatozoa were produced in all seasons. However, the greatest number of motile spermatozoa per ejaculate was observed during the winter and spring, whereas the highest incidence of sperm pleiomorphisms (> 80%) was detected in the autumn. Histological assessments of the regressed testis (July) revealed fewer germ cells undergoing spermatogenesis and an increased incidence of degenerating and abnormal cell types. In summary, Eld's deer exhibit a circannual hypothalamic–pituitary–gonadal cycle with onset of pituitary activation occurring during the autumn and winter, whereas gonadal activity peaks during the winter and spring as daylengths are increasing. Marked circannual variations in circulating prolactin suggest that Eld's deer may use photoperiodic cues to modulate seasonal fertility; however, the existence of an endogenous seasonal rhythm operating independently of photoperiod cannot be excluded.

Plasma LH, FSH and testosterone were measured in blood samples collected via remote catheterization from six adult Eld's deer stags every 10 min for 8 h before and 2 h after GnRH (1 μg kg⁻¹, i.v.) administration. Blood samples were collected within 2 weeks of the summer solstice (21 June), autumn equinox (22 September), winter solstice (21 December) and spring equinox (20 March). Marked seasonal variations in basal LH, FSH and testosterone concentrations were observed. From autumn, well-defined LH pulses were temporally associated with small, but detectable pulses in testosterone. During the winter transition into the breeding season, episodic LH pulses were also temporally associated with corresponding testosterone surges that lasted 2–3 h. High amplitude, low frequency testosterone surges were also observed during the spring, but often in the absence of detectable LH pulses. Basal LH and testosterone concentrations decreased during the summer and, although LH pulses were detected, associated testosterone pulses were absent. Only 37% of LH pulses occurred coincidentally with FSH pulses, and FSH pulses were generally less prominent. The increases in LH and FSH above basal concentrations after GnRH treatment were significant (P < 0.05) for all seasons. Increases in testosterone after GnRH treatment were greatest during the winter and spring, but testosterone also increased to a lesser extent during the autumn (P < 0.05). In contrast, testosterone concentrations were not different before and after GnRH treatment during summer. The net LH increase after GnRH treatment was similar (P > 0.05) for all seasons, whereas the proportional increase in LH was greatest (P < 0.05) during the summer and autumn when basal concentrations were lowest. Although the net FSH increase after GnRH treatment ranged only from 20 to 40 ng ml⁻¹ during all seasons, the proportional rise in FSH after GnRH treatment tended to be highest during the summer and autumn when basal concentrations were lowest. Basal testosterone secretion and the net increase in testosterone after GnRH treatments were greatest during the spring rut (P < 0.05). Thus, in Eld's deer, it appears that pituitary gonadotrophs seasonally adjust their basal secretory 'set point', but the capacity to respond above basal concentrations is constrained to a maximum net increase in both LH and FSH, regardless of season. Because both basal LH and the net releasable pool of LH remain relatively constant, increased testosterone secretion after GnRH treatment probably represents increased testicular sensitivity to LH.

This study tested the efficacy of assisted reproduction (synchronization of oestrus and intrauterine artificial insemination (AI)) in contributing to the captive propagation of an endangered species, the Eld's deer (Cervus eldi thamin). Semen was collected from males preselected on the basis of under-represented genotype. Motility of spermatozoa after thawing from ejaculates diluted with BF5F extender (8% glycerol), frozen on dry ice in 0.5 ml straws and stored in liquid nitrogen was 60–70%. Intravaginal progesterone-releasing devices (controlled internal drug release, CIDR-type G) were inserted into 20 adult Eld's deer hinds for 14 days. In all hinds, semen (7.5–10 × 10⁶ motile spermatozoa per uterine horn) was deposited by laparoscopy performed 70 h after removal of the CIDR device. Ovarian activity, before and after AI, was monitored by analysing pregnanediol-3α-glucuronide (PdG) concentrations in voided urine collected three to seven times per week. During the period of CIDR device insertion, urinary PdG profiles were equal to, or above, normal luteal phase concentrations in all hinds. Within 48 h of device withdrawal, PdG concentrations returned to baseline values in 17 of the 20 females, and the onset of behavioural oestrus occurred at this time in 12 hinds. On the basis of sustained increases in urinary PdG, 9 of the 20 hinds were diagnosed as pregnant by 90 days after AI, all of which delivered offspring after a mean gestation of 241.1 days (range, 235–245). Seven singletons (two females, five males) were born alive and survived, and one singleton and one set of twins were stillborn (three females). This is the greatest number of pregnancies and offspring produced in a single AI trial for any endangered mammal. These results demonstrate that genotype preselection can be combined with assisted reproductive technologies, including use of frozen semen, to produce genetically valuable offspring useful for conserving a rare species.