Cycle In Sheep Research Articles

Cervical expression of hyaluronan synthases varies with the stage of the estrous cycle in the ewe

The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.

Changes in Ovarian Follistatin Levels During the Oestrous Cycle in Sheep may Serve as an Intraovarian Regulator

The expression and concentration of follistatin and activin change during oestrous cycle suggesting their involvement in the regulation of follicular development. The aim of this study was to determine the level, source and potential role of follistatin in the sheep ovary. Follistatin in ovarian venous blood, measured by radioimmunoassay, remained at its low level from follicular phase (day -1 and 0) to mid-luteal phase (days 11-13) phase but were significantly elevated during the late luteal phase (days 14 and 15) when corpora lutea underwent regression. Western blot analyses of follicular fluid at day 15 of the cycle showed two strong bands at 42 and 45 kDa and weakly stained bands at 39 and 31 kDa. At day 0, these bands became weaker and the 39 kDa band became undetectable. However, there were no differences in follistatin concentrations between ovaries with and without functional corpus luteum (CL) during the whole luteal phase. In addition, although the ovaries of Booroola ewes normally contain more corpora lutea than those of normal merino ewes, follistatin concentrations in both jugular and ovarian venous blood were similar in Booroola and normal merino ewes. It is concluded that the secretion of follistatin from the ovary is not related to the formation of CL or high ovulation rate of Booroola ewes. The elevation in follistatin concentration in follicular fluid and ovarian blood during late luteal phase may indicate a dual role of follistatin in the luteolysis of existing CL and development of new follicle cohort.

1 PROGESTERONE AS THE DRIVING REGULATORY FORCE BEHIND SERUM FSH CONCENTRATIONS AND ANTRAL FOLLICULAR GROWTH IN CYCLIC EWES

Ovarian antral follicles in sheep grow in an orderly succession, producing typically 3 to 4 follicular waves per 17-day estrous cycle. Each wave is preceded by a transient increase in circulating FSH concentrations. The mechanism controlling the number of recurrent FSH peaks and emerging follicular waves remains unknown. During the ewe's estrous cycle, the time between the first 2 FSH peaks and days of wave emergence is longer than the intervals separating the ensuing FSH peaks and follicular waves. The prolonged inter-peak/inter-wave interval occurs early in the luteal phase when low levels of progesterone are secreted by developing, or non-fully functional, corpora lutea. The purpose of the present study was to determine the effect of varying progesterone (P4) levels on circulating concentrations of FSH and antral follicular development in sheep. Exogenous P4 (15 mg per ewe i.m.) was administered twice daily to 6 cycling Rideau Arcott × Dorset ewes from Day 0 (ovulation) to Day 4 (the mean duration of the inter-wave interval); 6 animals served as controls. Follicular growth was monitored in all animals by daily transrectal ultrasonography (Days 0 to 9). Jugular blood samples were drawn twice a day from Day 0 to 4 and then daily until Day 9 to measure systemic concentrations of P4 and FSH. The first FSH peak post-ovulation was detected on Day 1.4 ± 0.2 and 4.0 ± 0.2 in treated and control ewes, respectively (P < 0.05). The next FSH peak(s) occurred on Days 3.4 ± 0.3 and 5.2 ± 0.2 in the treatment group and on Day 5.5 ± 0.3 in controls. Consequently, the treatment group had, on average, 3 follicular waves emerging on Days 0, 3, and 6, whereas the controls produced 2 waves emerging on Days 0 and 5 (P < 0.05).We then retrospectively analyzed and compared daily serum concentrations of P4 and FSH obtained in cyclic Western White Face ewes (Columbia × Rambouillet) that had 3 (n = 10) or 4 (n = 19) follicular waves per cycle. Mean P4 concentrations were greater (P < 0.05) in sheep with 4 waves per cycle compared with their counterparts, which had 3 waves of follicular growth. Interestingly, the ewes with 3 waves exceeded (P < 0.05) all animals with 4 follicular waves in mean serum FSH concentrations on Days 0 to 2, 6 to 7, and 9 to 15 post-ovulation. In summary, creation of mid-luteal phase levels of P4 in metestrus shortened the time to the first post-ovulatory FSH peak in ewes, resulting in emergence of one more follicular wave compared with control animals during the same time frame. The ewes exhibiting 4 waves of follicular emergence had greater serum levels of P4 but lower FSH concentrations compared with sheep with 3 waves per cycle. Therefore, progesterone appears to be a key endocrine signal governing the control of periodic increases in serum FSH concentrations and the number of follicular waves in cyclic sheep. This study was funded by OMAFRA and NSERC grants. Appreciation is extended to Norman C. Rawlings, Susan Cook, and Sekallu Srinivas (University of Saskatchewan) and the staff at Ponsonby Sheep Research Station.

Cell turnover and gene activities in sheep mammary glands prior to lambing to involution

Mammary glands are special tissue characterized by proliferation of the epithelium, during puberty and pregnancy and by programmed cell death, during involution. In this study, apoptosis was identified by TUNEL staining and then related to cell proliferation, as determined by Ki-67 staining. The apoptotic index was at its highest at 8 days of involution, whereas the proliferation index was at its highest during lactation. Caspase-3 was immunolocalised only in mast cells and along the basal membrane in the mammary tissue at −10 days from lambing, 150 days of lactation and at 8 days of involution. This finding could indicate that caspase-3 is not involved in sheep mammary gland apoptosis, but that other proteins – such as apoptosis inducing factor (AIF) – can trigger apoptosis, through the mitochondrial pathway, in a caspase-independent manner. The expression of genes involved in the regulation of lactation and apoptosis was also investigated and determined relatively to −10 days from lambing. The relative expression level of LALBA, reached its maximum during lactation, whereas the expressions of BCL2, BCL2L1, BAX, STAT5A, STAT3, IGFBP5 and FOXO3A, increased significantly during involution in correlation with apoptotic index. This work shows for the first time the turnover of mammary cells and the interaction of their signals during the complete lactation cycle in sheep. The data on gene expression can contribute to elucidate the mechanisms controlling milk production and cell turnover in this species.

Prepubertal changes in the synthesis, storage and release of the somatostatin in the hypothalamus of female lambs: A morphofunctional study

It is assumed that hypothalamic somatostatin plays a role in the preovulatory phase of the oestrous cycle in sheep. The aim of the study was to investigate the processes of synthesis, storage and release of somatostatin in hypothalamic neurons, in immature female lambs, in the period approaching to puberty. Experiments were carried out on 10 prepubertal (17 weeks old) and 10 peripubertal (32 weeks old) ovary-intact lambs. Morphofunctional changes in the somatostatin neurons were assayed with immunohistochemistry and hybridisation in situ. Computer image analysis was used to determine the density of both reactions and the percentage of the area exhibiting immunohistochemical staining. These parameters express the content of immunoreactive (ir) somatostatin and expression of mRNA for pre-pro-somatostatin (PPS). Two populations of ir somatostatin perikarya were localized in the hypothalamus: a very large number of perikarya in the periventricular (PEV) nucleus, and single cell bodies in the arcuate (ARC) nucleus. Only ir somatostatin fibres, but no perikarya were seen in the ventromedial (VM) nucleus and preoptic area. The analysis of mRNA PPS showed perikarya filled with silver grains localized in the PEV, ARC and VM. There were differences in the content of ir somatostatin and the intensity of the PPS mRNA signal between the two periods investigated. In the median eminence, the content of ir somatostatin in the terminals decreased in the peripubertal compared to the prepubertal group ( P < 0.001). In the PEV, the content of ir somatostatin in the perikarya and the expression of PPS mRNA decreased in the peripubertal compared to the prepubertal group ( P < 0.001). In the ARC, the content of ir somatostatin in the perikarya increased ( P < 0.001), but expression of PPS mRNA decreased ( P < 0.001) in the peripubertal compared to the prepubertal group. There were no differences in the expression of PPS mRNA in the VM. We concluded, that the different secretory activity of the two hypothalamic populations of somatostatin neurons can be related to their different physiological functions in the prepubertal period of female lambs.

Expression of endothelial nitric oxide synthase in the ovine ovary throughout the estrous cycle

This study was conducted to evaluate the expression of endothelial nitric oxide synthase (eNOS) in ovarian follicles and corpora lutea (CL) throughout the estrous cycle in sheep. Three experiments were conducted to (1) immunolocalize eNOS protein, (2) determine expression of mRNA for eNOS and its receptor guanylate cyclase 1 soluble beta3 (GUCY1B3), and (3) co-localize eNOS and vascular endothelial growth factor (VEGF) proteins in the follicles and/or CL throughout the estrous cycle. In experiment 1, ovaries were collected from ewes treated with FSH, to induce follicular growth or atresia. In experiment 2, ovaries were collected from ewes treated with FSH and hCG to induce follicular growth and ovulation. In experiment 3, ovaries were collected from superovulated ewes to generate multiple CL on days 2, 4, 10, and 15 of the estrous cycle. In experiments 1 and 2, the expression of eNOS protein was detected in the blood vessels of the theca externa and interna of healthy ovarian follicles. However, in early and advanced atretic follicles, eNOS protein expression was absent or reduced. During the immediate postovulatory period, eNOS protein expression was detected in thecal-derived cells that appeared to be invading the granulosa layer. Expression of eNOS mRNA tended to increase in granulosa cells at 12 and 24 h, and in theca cells 48 h after hCG injection. In experiment 3, eNOS protein was located in the blood vessels of the CL during the estrous cycle. Dual localization of eNOS and VEGF proteins in the CL demonstrated that both were found in the blood vessels.

Male-induced short oestrous and ovarian cycles in sheep and goats: a working hypothesis

The existence of short ovulatory cycles (5-day duration) after the first male-induced ovulations in anovulatory ewes and goats, associated or not with the appearance of oestrous behaviour, is the origin of the two-peak abnormal distribution of parturitions after the "male effect". We propose here a working hypothesis to explain the presence of these short cycles. The male-effect is efficient during anoestrus, when follicles contain granulosa cells of lower quality than during the breeding season. They generate corpora lutea (CL) with a lower proportion of large luteal cells compared to small cells, which secrete less progesterone, compared to what is observed in the breeding season cycle. This is probably not sufficient to block prostaglandin synthesis in the endometrial cells of the uterus at the time when the responsiveness to prostaglandins of the new-formed CL is initiated and, in parallel, to centrally reduce LH pulsatility. This LH pulsatility stimulates a new wave of follicles secreting oestradiol which, in turn, stimulates prostaglandin synthesis and provokes luteolysis and new ovulation(s). The occurrence of a new follicular wave on days 3-4 of the first male-induced cycle and the initiation of the responsiveness to prostaglandins of the CL from day 3 of the oestrous cycle are probably the key elements which ensure such regularity in the duration of the short cycles. Exogenous progesterone injection suppresses short cycles, probably not by delaying ovulation time, but rather by blocking prostaglandin synthesis, thus impairing luteolysis. The existence, or not, of oestrous behaviour associated to these ovulatory events mainly varies with species: ewes, compared to does, require a more intense endogenous progesterone priming; only ovulations preceded by normal cycles are associated with oestrous behaviour. Thus, the precise and delicate mechanism underlying the existence of short ovulatory and oestrous cycles induced by the male effect appears to be dependent on the various levels of the hypothalamo-pituitary-ovario-uterine axis.

Gap Junctional Connexin 37 Is Expressed in Sheep Ovaries

The objective of current study was to evaluate the expression of Cx37 in ovarian follicles and in corpora lutea (CL) during the estrous cycle in sheep. Ovine Cx37 was cloned and characterized to design speciesspecific probe and primers. In Exp. 1, ovaries were collected on d 13, 14, 15, and 16 of the estrous cycle, or from FSH-induced ewes at 0, 2, 4, 8, 12, 24, and 48 h after hCG treatment on d 15 of the estrous cycle. In Exps. 2 and 3, CL were collected on d 5, 10, and 15 of the estrous cycle, or at 0, 4, 8, 12, and 24 h after prostaglandin F2alpha (PGF2alpha)-induced luteal regression on d 10 of the estrous cycle, respectively. Ovarian tissues (e.g., granulosa cells, theca cells, ovarian follicles, and /or CL) were used for Cx37 immunostaining followed by image analysis or for determination of Cx37 mRNA expression by real-time RT-PCR. We demonstrated that (1) Cx37 protein was expressed in granulosa and cumulus oocyte complex compartments, ovarian blood vessels, and on the luteal cell borders, (2) expression of Cx37 mRNA was greater in granulosa than in theca cells of preovulatory follicles, (3) Cx37 mRNA expression in granulosa but not theca cells was affected by hCG treatment, (4) Cx37 protein and mRNA expression were dependent on the stage of luteal development, and (5) Cx37 expression changed during PGF2alpha- induced luteal regression. Thus, Cx37 may play a role in follicular development and ovulation as well as in luteal tissue growth, differentiation, and regression.

The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep

The aim of the present study was to investigate the temporal relationship between the secretory pattern of serum LH and FSH concentrations and waves of ovarian antral follicles during the luteal phase of the estrous cycle in sheep. The growth pattern of ovarian antral follicles and CL were monitored by transrectal ultrasonography and gonadotropin concentrations were measured in blood samples collected every 12 min for 6 h/d from 7 to 14 d after ovulation. There were two follicular waves (penultimate and final waves of the cycle) emerging and growing during the period of intensive blood sampling. Mean and basal LH concentrations and LH pulse frequency increased ( P < 0.001) with decreasing progesterone concentration at the end of the cycle. Mean and basal FSH concentrations reached a peak ( P < 0.01) on the day of follicular wave emergence before declining to a nadir by 2 d after emergence. None of the parameters of pulsatile LH secretion varied significantly with either the emergence of the final follicular wave or with the end of the growth phase of the largest follicle of the penultimate wave of the cycle. However, mean and basal LH concentrations did increase ( P < 0.05) after the end of the growth phase of the largest follicle of the final follicular wave of the cycle. Furthermore, the end of the growth phase of the largest follicle of the final wave coincided with functional luteolysis. In summary, there was no abrupt or short-term change in pulsatile LH secretion in association with the emergence or growth of the largest follicle of a wave. We concluded that the emergence and growth of ovarian antral follicles in follicular waves do not require changes in LH secretion, but may involve changes in sensitivity of ovarian follicles to serum LH concentrations.

Conceptus signals for establishment and maintenance of pregnancy

Establishment and maintenance of pregnancy results from signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and requires progesterone produced by the corpus luteum. In most mammals, hormones produced by the trophoblast maintain progesterone production by acting directly or indirectly to maintain the corpus luteum. In domestic animals (ruminants and pigs), hormones from the trophoblast are antiluteolytic in that they act on the endometrium to prevent uterine release of luteolytic prostaglandin F2α. In cyclic and pregnant sheep, progesterone negatively autoregulates progesterone receptor gene expression in the endometrial luminal and superficial glandular epithelium. In cyclic sheep, loss of the progesterone receptor is closely followed by increases in epithelial estrogen receptors and then oxytocin receptors, allowing oxytocin to induce uterine release of luteolytic prostaglandin F2α pulses. In pregnant sheep, the conceptus trophoblast produces interferon tau that acts on the endometrium to inhibit transcription of the estrogen receptor alpha gene directly and the oxytocin receptor gene indirectly to abrogate development of the endometrial luteolytic mechanism. Subsequently, sequential, overlapping actions of progesterone, interferon tau, placental lactogen, and growth hormone comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function to maintain pregnancy in sheep. In pigs, the conceptus trophoblast produces estrogen that alters the direction of prostaglandin F2α secretion from an endocrine to exocrine direction, thereby sequestering luteolytic prostaglandin F2α within the uterine lumen. Conceptus estrogen also increases expression of fibroblast growth factor 7 in the endometrial lumenal epithelium that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the fibroblast growth factor 7 receptor. Strategic manipulation of these physiological mechanisms may improve uterine capacity, conceptus survival, and reproductive health.

Conceptus signals for establishment and maintenance of pregnancy

Establishment and maintenance of pregnancy results from signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and requires progesterone produced by the corpus luteum. In most mammals, hormones produced by the trophoblast maintain progesterone production by acting directly or indirectly to maintain the corpus luteum. In domestic animals (ruminants and pigs), hormones from the trophoblast are antiluteolytic in that they act on the endometrium to prevent uterine release of luteolytic prostaglandin F2α. In cyclic and pregnant sheep, progesterone negatively autoregulates progesterone receptor gene expression in the endometrial luminal and superficial glandular epithelium. In cyclic sheep, loss of the progesterone receptor is closely followed by increases in epithelial estrogen receptors and then oxytocin receptors, allowing oxytocin to induce uterine release of luteolytic prostaglandin F2α pulses. In pregnant sheep, the conceptus trophoblast produces interferon tau that acts on the endometrium to inhibit transcription of the estrogen receptor alpha gene directly and the oxytocin receptor gene indirectly to abrogate development of the endometrial luteolytic mechanism. Subsequently, sequential, overlapping actions of progesterone, interferon tau, placental lactogen, and growth hormone comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function to maintain pregnancy in sheep. In pigs, the conceptus trophoblast produces estrogen that alters the direction of prostaglandin F2α secretion from an endocrine to exocrine direction, thereby sequestering luteolytic prostaglandin F2α within the uterine lumen. Conceptus estrogen also increases expression of fibroblast growth factor 7 in the endometrial lumenal epithelium that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the fibroblast growth factor 7 receptor. Strategic manipulation of these physiological mechanisms may improve uterine capacity, conceptus survival, and reproductive health.

Progesterone and placental hormone actions on the uterus: insights from domestic animals.

Progesterone is unequivocally required for maternal support of conceptus (embryo/fetus and associated extraembryonic membranes) survival and development. In cyclic sheep, progesterone is paradoxically involved in suppressing and then initiating development of the endometrial luteolytic mechanism. In cyclic and pregnant sheep, progesterone negatively autoregulates progesterone receptor (PR) gene expression in the endometrial luminal (LE) and superficial glandular epithelium (GE). In cyclic sheep, PR loss is closely followed by increases in epithelial estrogen receptor (ERalpha) and then oxytocin receptor (OTR), allowing oxytocin to induce uterine release of luteolytic prostaglandin F2alpha pulses. In pregnant sheep, the conceptus produces interferon tau (IFNtau) that acts on the endometrium to inhibit transcription of the ERalpha gene and thus development of the endometrial luteolytic mechanism. After Day 13 of pregnancy, the endometrial epithelia do not express the PR, whereas the stroma and myometrium remain PR positive. The absence of PR in the endometrial GE is required for onset of differentiated function of the glands during pregnancy. The sequential, overlapping actions of progesterone, IFNtau, placental lactogen (PL), and growth hormone (GH) comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function during gestation. In pigs, estrogen, the pregnancy-recognition signal, increases fibroblast growth factor 7 (FGF-7) expression in the endometrial LE that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the receptor for FGF-7. Strategic manipulation of these physiological mechanisms may offer therapeutic schemes to improve uterine capacity, conceptus survival, and reproductive health of domestic animals and humans.

Conceptus signals for establishment and maintenance of pregnancy

Establishment and maintenance of pregnancy results from signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and requires progesterone produced by the corpus luteum (CL). In most mammals, hormones produced by the trophoblast maintain progesterone production by acting directly or indirectly to maintain the CL. In domestic animals (ruminants and pigs), hormones from the trophoblast are antiluteolytic in that they act on the endometrium to prevent uterine release of luteolytic prostaglandin F2 alpha (PGF). In cyclic and pregnant sheep, progesterone negatively autoregulates expression of the progesterone receptor (PR) gene in the endometrial luminal (LE) and superficial glandular epithelium (GE). Available evidence in cyclic sheep indicates that loss of the PR is closely followed by increases in epithelial estrogen receptors (ER) and then oxytocin receptors (OTR), allowing oxytocin to induce uterine release of luteolytic PGF pulses. In pregnant sheep, the conceptus trophoblast produces interferon tau (IFN tau) that acts on the endometrium to inhibit transcription of the ER alpha gene directly and the OTR gene indirectly to abrogate development of the endometrial luteolytic mechanism. Subsequently, sequential, overlapping actions of progesterone, IFN tau, placental lactogen (PL) and growth hormone (GH) comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function to maintain pregnancy in sheep. In pigs, the conceptus trophoblast produces estrogen that alters the direction of PGF secretion from an endocrine to exocrine direction, thereby sequestering luteolytic PGF within the uterine lumen. Conceptus estrogen also increases expression of fibroblast growth factor 7 (FGF-7) in the endometrial LE that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the FGF-7 receptor. Strategic manipulation of these physiological mechanisms can offer therapeutic schemes to improve uterine capacity, conceptus survival and reproductive health.

Prolactin cycles in sheep under constant photoperiod: evidence that photorefractoriness develops within the pituitary gland independently of the prolactin output signal.

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01-0.04 mg kg-1 day-1; 12-24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean +/- SEM, 35.3 +/- 0.6 and 37.0 +/- 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22-30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 +/- 0.4 and 37.6 +/- 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.

Prenatal programming of reproductive neuroendocrine function: fetal androgen exposure produces progressive disruption of reproductive cycles in sheep.

In the agonadal, androgenized ewe testosterone before birth produces a precocious pubertal rise in circulating LH and abolishes the LH surge mechanism. The present study tested two predictions from this model in the ovary-intact female: 1) prenatal androgen exposure produces early ovarian stimulation; and 2) despite early ovarian stimulation, progestogenic cycles would not occur because of the abolition or disruption of the LH surge. Pregnant ewes were injected with testosterone propionate twice per week from either d 30-90 (T60 group; 100 mg/injection) or d 60-90 (T30 group; 80 mg/injection) of gestation (term, 147 d). Control ewes received no injections. At birth, the androgenized and control lambs were divided into two groups: ovary-intact to determine the effects of prenatal androgen on the timing of puberty and subsequent ovarian function, and ovariectomized to assess the timing of the pubertal decrease in sensitivity to estrogen negative feedback and the subsequent increase in LH. Neonatally orchidectomized, estrogen-treated males were included for comparison of the timing of this pubertal rise in LH secretion. Neuroendocrine puberty (determined on the basis of LH increase) was advanced in the androgenized females to a similar age as in males. Repeated progesterone cycles of the same duration and number occurred in the ovary-intact ewes, and they began at the same time as for control females, thus negating both predictions. Differences appeared during the second breeding season, when reproductive cycles were either absent (T60) or disrupted (T30 group). Our findings reveal that exposure to androgens in utero causes a progressive loss of cyclic function in adulthood.

Use of high-resolution transrectal ultrasonography to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes

Transrectal ovarian ultrasonographic studies have shown that, in cattle, follicular wave emergence is associated with a large increase in the number of small antral follicles (4–6 mm in diameter); an analogous association has not been found for small follicles (2–3 mm in diameter) in the ewe. In previous studies in ewes, accurate assessment of the number of follicles has been limited to follicles ≥2 or 3 mm in size. Newer, high-resolution equipment allowed us to identify follicles ≥0.4 mm and to quantify all antral follicles ≥1 mm in diameter in seven cyclic Western White Face ewes. This allowed us to expand the small follicle pool examined, from 1 to 4 follicles/day (2–3.5 mm in diameter) in earlier studies, to 8–18 follicles/day (1–3 mm in diameter). Total number of small follicles (≥1 and ≤3 mm in diameter) increased between Days −1 and 0 (Day 0=day of ovulation), and declined between Days 1 and 3 ( P<0.05). There were no significant changes in the number of small or medium (4 mm in diameter) follicles around days of follicle wave emergence (±2 days). The 1–3 follicles in the 2–3 mm size range, which constituted a follicle wave (i.e. grew to ≥5 mm in size before regression or ovulation), were the only small follicles to emerge in an orderly succession during the estrous cycle, approximately every 3–5 days. Thus, unlike in cattle, there is no apparent increase in numbers of small follicles at follicle wave emergence in cyclic sheep, and little evidence for selection of recruited follicles and follicular dominance.

Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep.

Associations between granins (secretogranin II (SgII) and chromogranin A and B (CgA and CgB)) and gonadotrophins (LH and FSH) have been reported in rodents and they may interact to facilitate differential storage and secretion of LH and FSH. This study investigated the relationship between granins and gonadotrophins in sheep at different stages of the oestrous cycle. Thirty-four cycling ewes had their oestrous cycles synchronised, and were divided into late luteal (LL; n=5) and early (EF; n=4), mid (n=3) and late (LF; n=11) follicular stages, and 24-53 h (n=5), 80-100 h (n=3) and 120-144 h (n=3) after the preovulatory LH surge (PS). LHbeta mRNA levels were low in LF ewes (when plasma levels and pulse frequency of LH were high) but had increased by 80-100 h PS. In contrast, FSHbeta mRNA levels decreased during the follicular phase and plasma FSH concentrations followed a similar pattern, to peak at 24-53 h PS due to low plasma oestradiol levels. While alpha-gonadotrophin subunit (alpha-GSU), SgII and CgA mRNA levels did not change, CgB mRNA levels were elevated in EF ewes and had declined in ewes around the surge. Four distinctly sized mRNA transcripts ( approximately 1.3, 2.0, 2.8 and 3.2 kb) were observed for CgA mRNA, while a double band was observed for LHbeta mRNA that was subsequently reduced to a single band after 3'-poly(A) tail truncation. The long and short LHbeta transcripts were prevalent in follicular and luteal ewes respectively. Numbers of LH(+ve)/FSH(-ve) granules stored within gonadotrophs were not different in LL and LF ewes (even though proportions of LH(+ve) granules were higher in LF ewes), but were reduced at 24-53 h PS. The majority of LH(+ve) granules also contained SgII, although few CgA(+ve) granules were found. Granule partitioning was evident whereby FSH and CgA were located near the periphery, and LH and SgII throughout the matrix. In conclusion, increases in both storage of LH(+ve) granules and secretion of LH in LF ewes despite constant LHbeta mRNA levels was facilitated, at least in part, by improved LHbeta mRNA transcript stability. Fewer LH(+ve)/FSH(-ve) granules were in storage after the PS, which was mirrored by a reduction in LH pulsatile release. Surprisingly, in view of results in rodents indicating significant changes, SgII and CgA mRNA levels did not change over the oestrous cycle in sheep. Conversely, CgB mRNA levels decreased around the time of PS. These novel results illustrate major differences in granin-gonadotrophin interactions between sheep and rodents.

Mechanisms for Ovarian Cycle Disruption by Immune/inflammatory Stress

This review summarizes highlights of our experiments investigating mechanisms, mediators and sites by which endotoxin disrupts reproductive neuroendocrine activity and interferes with the estrous cycle of sheep. Endotoxin, or lipopolysaccharide (LPS), is a commonly used model for immune and inflammatory stress. When administered to ovary-intact ewes, endotoxin interrupts the follicular phase of the cycle by interfering with several steps in the preovulatory chain of endocrine events. One such step is the development of high frequency LH pulses, which provide an essential stimulus for the preovulatory increase in estradiol secretion from the ovarian follicle. Follow-up experiments in ovariectomized ewes demonstrate that endotoxin inhibits pulsatile LH secretion at both the hypothalamic and pituitary levels, suppressing pulsatile GnRH secretion and reducing pituitary responsiveness to GnRH. This disruption of GnRH and LH pulsatility is mediated by pathways that include the synthesis of prostaglandins and cortisol, both of which are increased by endotoxin. It is postulated that a prostaglandin-mediated pathway disrupts the cycle during immune and inflammatory stress, whereas a separate cortisol-mediated pathway reinforces this disruption and also participates more generally in suppressing cyclicity during other stressful situations that activate the hypothalamo-pituitary-adrenal axis.

Three-dimensional geometric comparison of partial and complete flexible mitral annuloplasty rings

Background: It has previously been shown in sheep that mitral annular physiologic dynamics during the cardiac cycle are abolished by complete ring annuloplasty, but recent clinical studies suggest that flexible partial ring annuloplasty preserves normal mitral annular dynamics. Methods: Eight radiopaque markers were sutured equidistantly around the mitral anulus in 3 groups of sheep: no-ring control animals (n = 16); animals with a flexible Tailor partial ring annuloplasty (n = 6; St Jude Medical, Inc, St Paul, Minn); and animals with a flexible Duran ring annuloplasty (n = 7; Medtronic, Inc, Minneapolis, Minn). After 7 to 10 days&' recovery, 3-dimensional marker coordinates were measured by biplane cinefluoroscopy. Mitral annular area and folding (defined as displacement of the mitral anulus from a least-squares plane) and mitral annular septal-lateral and commissure-commissure dimensions were calculated from the 3-dimensional marker coordinates throughout the cardiac cycle every 17 ms. Results: In the no-ring control group mitral annular area varied from 8.0 ± 0.2 to 7.2 ± 0.2 cm2 (10% ± 2%), and the septal-lateral and commissure-commissure dimensions varied from 27.7 ± 0.4 to 25.9 ± 0.4 mm (7% ± 1%) and from 38.2 ± 0.8 to 36.4 ± 0.8 mm (5% ± 1%), respectively (mean ± standard error of the mean, P <.001 for all comparisons). In the Duran ring annuloplasty and Tailor partial ring annuloplasty groups, the anulus was fixed in size throughout the cardiac cycle (area = 4.8 ± 0.1 and 5.3 ± 0.3 cm2, septal-lateral = 21.8 ± 0.7 and 22.0 ± 0.8 mm, and commissure-commissure = 27.7 ± 0.7 and 31.2 ± 1.7 mm). Mitral annular folding did not differ significantly between the control and Tailor partial ring annuloplasty groups but was dampened in the Duran ring annuloplasty group. Conclusions: Partial Tailor flexible ring annuloplasty fixed mitral annular area and dimensions throughout the cardiac cycle in sheep; however, it preserved physiologic mitral annular folding dynamics, which might be important in terms of long-term valve function and prevention of left ventricular outflow tract obstruction.

Regulation of oxytocin receptor gene expression in sheep: tissue specificity, multiple transcripts and mRNA editing

The increase in uterine oxytocin receptor concentrations over the late luteal phase of the oestrous cycle in sheep is thought to play an important role in the regulation of the duration of the cycle by facilitating the effect of oxytocin on uterine prostaglandin release. Experiments indicated that oxytocin receptor mRNA expression in the endometrium was high at oestrus compared with at days 2, 7 and 12 of the oestrous cycle. The amount of oxytocin receptor mRNA expression in the pituitary gland did not show any significant differences during the oestrous cycle. Oxytocin receptor cDNA was obtained and characterized from ovine uterine endometrium on day 15 of the oestrous cycle, using RT-PCR techniques, to study the mechanisms underlying the resolution of oxytocin receptor expression. The cDNA sequence for the oxytocin receptor gene in sheep was found to be similar to that described previously, except for a difference of seven nucleotides. These nucleotide differences resulted in changes in four of the deduced amino acids in the oxytocin receptor sequence. The heterogeneity of the different sized oxytocin receptor transcripts in sheep is due, at least in part, to the alternative use of polyadenylation sites. Northern hybridization confirmed that the oxytocin receptor gene is expressed in ovine corpus luteum. The investigations on oxytocin receptor gene expression indicate that the patten of oxytocin receptor gene expression in sheep is not only tissue-specific, but also highly function-related. Evidence was obtained of mRNA editing in both the coding and the 3'-untranslated (3'UTR) regions of oxytocin receptor gene transcripts in ovine endometrium; this was the first demonstration of this phenomenon for oxytocin receptor mRNA. The present results indicate that the observed differences in oxytocin receptor mRNA sequences for the different oxytocin receptor populations in endometrium are due to mRNA editing. mRNA editing of oxytocin receptor transcripts may be reflected in changes in the amino acid composition of the carboxyl terminus of the receptor, which would explain the differences in the observed responses to an oxytocin challenge.