Oestrous Cycle In Sheep Research Articles

Association of faecal volatiles and steroids with behavioural expression during oestrous cycle of sheep (Ovis aries).

This study was investigated to determine the faecal volatiles, steroid hormones and their correlation with behavioural signs during oestrous cycle in sheep (Ovis aries). This experiment was monitored from pro-oestrous to met-oestrous phases for correlation of endocrine dependence biochemical constituents in faeces and blood to detection of oestrous biomarkers. Medroxyprogesterone acetate sponges were used for 8 days to bring uniformity of oestrus in sheep. Faeces were collected during different phases of cycle and subjected to determination of fatty acids, minerals, oestrogens and progesterone analysis. Likewise, blood samples were also obtained for enzymatic and no-enzymatic antioxidants. The results revealed that faecal progesterone and oestrogen levels significantly increased during pro-oestrous and oestrous phase, respectively (p < .05). The blood plasma enzymatic levels showed significant demarcation during the oestrous phase than other periods (p < .05). There were also reported marked variations in volatile fatty acids across different phases of the oestrous cycle. Interestingly, methyl octanoate, methyl cis-10 pentadecenoate and methyl heptadecanoate volatiles were found to be specific to oestrus period. In addition, methyl hexanoate, methyl palmitoleate and methyl cis-9 oleate were identified in met-oestrus, indicating their possible role to be an oestrous biomarker. It is concluded that the pattern of volatile compounds and faecal steroids, in association with behavioural patterns, could be measured as a non-invasive method of heat detection in sheep.

Expression and functional analysis of the Follistatin-like 3 (FSTL3) gene in the sheep ovary during the oestrous cycle.

Follistatin‐like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA‐Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.

Seventy years of progestagen treatments for management of the sheep oestrous cycle: where we are and where we should go

Management of the ovine oestrous cycle is mainly based on the use of exogenous hormones to mimic or enhance (progesterone and its analogues) or manipulate (prostaglandin F2α and its analogues) the activity of the corpus luteum, combined with the application of other hormones mimicking the pituitary secretion of gonadotrophins (e.g. equine chorionic gonadotrophin). These protocols have been applied without major change for decades but, now, there are two reasons to reconsider them: (1) our greatly improved knowledge of the dynamics of ovarian physiology, following the application of transrectal ultrasonography, indicates that modification of the protocols may improve fertility yields and (2) increasing concerns about animal health and welfare, food safety and the environmental impact of the treatments, as evidenced by public opinion and therefore market forces. Here, we offer an overview of these issues, introduce an updated protocol and suggest ways for future improvements to the protocols.

Expression of oestrogen receptor, androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle.

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real-time polymerase chain reaction (RT-qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real-time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro-oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro-oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro-oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time- and tissue-specific profiles along the oestrous cycle.

Nutritional status affects the microRNA profile of the hypothalamus of female sheep.

Recent studies on the seasonal regulation of the oestrous cycle in sheep have focussed mainly on the responses to photoperiod. However, the brain systems that control reproductive activity also respond to nutritional inputs, although the molecular mechanisms involved are not completely understood. One possibility is that small, non-coding RNAs, such as micro-RNAs (miRNAs), have significant influence. In the present study, the amounts and characteristics of miRNAs in hypothalamus from oestrous and anestrous ewes, fed low- or high-nutrient diets, were compared using Illumina HiSeq sequencing technology. In total, 398 miRNAs, including 261 novel miRNAs, were identified in ewes with an enhanced nutritional status (HEN), whereas 384 miRNAs, including 247 novel miRNAs, were identified in the ewes with a lesser nutritional status (HAN). There were eight conserved and 140 novel miRNAs expressed differentially between the two libraries. Based on quantitative real-time polymerase chain reaction, six miRNAs were assessed to verify the accuracy of the library database. Moreover, the correlation between the miRNA target and several upstream and downstream genes in the oestrus-related pathways were also verified in hypothalamus nerve cells. According to the results, nutritional status plays an important role in oestrous regulation in sheep, and the hypothalamic processes and pathways induced by nutritional signals (folic acid and tyrosine) are different from those induced by photoperiodic regulation of oestrus. We have expanded the repertoire of sheep miRNAs that could contribute to the molecular mechanisms that regulate the initiation of oestrous cycles in anestrous ewes in response to the influence of nutritional status.

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.

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.

Effects of Breed on Follicular Dynamics and Oestradiol Secretion during the Follicular Phase in Sheep

Current study evaluates effects from breed and management background on follicular dynamics and endocrine output during the follicular phase of sheep oestrous cycle. Follicular phases were synchronized with three cloprostenol doses, 10 days apart, in three groups of 10 females of different non-prolific Spanish breeds (Manchega, Rubia del Molar and Negra de Colmenar). Development of all follicles reaching antral diameters >or=2 mm was assessed by daily transrectal ultrasonographies, whereas follicular function was evaluated by measurement of plasma oestradiol concentrations. All the ovulatory follicles were present at induced luteolysis in Manchega sheep, while a 93.7% were detected in Rubia del Molar and Negra de Colmenar ewes. The mean size of these ovulatory follicles was similar between breeds at 0 h, but their growth rates were higher in Manchega ewes, reaching a larger size at oestrous detection than in Negra de Colmenar and Rubia del Molar sheep (p < 0.05). Conversely, the oestradiol levels increased with time in Rubia del Molar and Negra de Colmenar (p < 0.05); whilst remained stable in Manchega females. However, the patterns of follicular turnover were similar between breeds. These results indicate that, though differences in follicular size and size distribution, patterns of follicular turnover in sheep are affected neither by the breed nor by the background of management and selection.

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.

Replenishment of LH stores of gonadotrophs in relation to gene expression, synthesis and secretion of LH after the preovulatory phase of the sheep oestrous cycle.

The pattern of replenishment of LH secretory granule stores in sheep pituitary gonadotrophs, after an induced LH surge, was determined by immunogold localisation at the ultrastructural level by electron microscopy. Twenty-four Welsh Mountain ewes were initially synchronised with progestagen devices for 14 days before luteolysis was induced by a prostaglandin F(2 alpha) analogue, cloprostenol. A further 24 h later, a preovulatory LH surge was induced by intravenous injection of a GnRH agonist, buserelin. Animals were divided into four groups (n=6) and blood sampled at 2 h intervals from 4 h prior, to 18 h after, buserelin administration and then at infrequent intervals (1 to 8 h) thereafter until death. Pulse profiles of LH were also obtained by an additional collection of blood samples within a 6 h window directly preceding death. Groups of animals were killed at 24, 48, 72 or 96 h after buserelin treatment. Pituitaries were dissected and processed for transmission electron microscopy and frozen for later molecular biological analysis. A characteristic preovulatory surge of LH was observed in all animals. The cytoplasm of gonadotrophs, in animals killed 24 h after buserelin treatment, was completely empty of secretory granules. This was associated with diminutive pituitary LH content, low pituitary GnRH binding levels and an almost complete absence (one pulse in one animal) of LH pulsatile secretion. Despite the lack of apparent secretory activity, clusters of exposed LH beta label present within the cytoplasm at this time and constant LHbeta mRNA expression levels irrespective of tissue collection time, suggest that the cell is actively synthesising LHbeta. The formation of sparse numbers of small LH beta immuno-labelled electron-dense secretory granules was apparent at 48 h after buserelin treatment, and replenishment of LH beta immuno-labelled granule stores continued until total granule numbers had increased two-fold (P<0.01) by 96 h post-treatment. Affiliated with granule replenishment was a significant increase in pituitary LH content (P<0.01), pituitary GnRH binding levels (P<0.01) and the restoration of LH pulsatile secretion. Despite the replenishment of granule stores with time, cytoplasmic area did not vary. These results suggest that restoration of pulsatile LH secretion after a preovulatory LH surge is related to replenishment of LH beta secretory granule stores and an increase in GnRH binding levels.

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.

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.

Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P < 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles > 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.

Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P &lt; 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles &gt; 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.

Steroidogenic factor-1 (SF-1) and the regulation of expression of luteinising hormone and follicle stimulating hormone b-subunits in the sheep anterior pituitary in vivo

Luteinising hormone (LH) and follicle stimulating hormone (FSH) comprise a common a-subunit and hormone-specific β-subunit, are expressed in gonadotroph cells of the anterior pituitary and during the sheep oestrous cycle, are regulated by gonadotrophin releasing hormone (GnRH), ovarian peptides and steroids. Transcription factor steroidogenic factor-1 (SF-1) transactivates the common a-subunit and LHβ subunit in other species. We investigated whether SF-1 regulates β-subunit expression and the regulation of SF-I expression in vivo in sheep. Immunocytochemistry co-localised SF-1, LH and FSH to sheep pituitary gonadotrophs and electrophoretic mobility shift assays (EMSA) demonstrated that SF-1 bound to the LHβ promoter in vitro. No SF-1 DNA binding site was found in the FSHβ promoter. No difference in mean levels of SF-1, FSHβ, LHβ and GnRHr mRNA was measured between the luteal and follicular phases of the oestrous cycle. However SF-1 mRNA levels were correlated to those of LHβ in individual luteal phase animals ( r = 0.88, p < 0.05), when the transcription rate of LHβ was significantly higher ( p < 0.01). GnRH antagonist treatment of luteal animals did not produce a significant reduction in mRNA levels of LHβ, SF-1 and FSHβ. Administering oestradiol benzoate with GnRH antagonist reduced levels of SF-I and FSHβ mRNA from luteal values ( p < 0.005). We conclude that: (1) expression of SF-1 is not solely dependent on GnRH and is downregulated by oestradiol; (2) the LHβ transcription rate is increased during the luteal phase but is not related to levels of LHβ or SF-1 mRNA; and (3) SF-1 does not appear to directly regulate expression of FSHβ, but may regulate expression indirectly.

Regulation of gonadotrophin-releasing hormone receptor expression in the ewe

Abstract The interaction of gonadotrophin-releasing hormone (GnRH) with its receptor is of crucial importance to gonadotroph function, although until recently, little was known of the structure of the GnRH receptor (GnRH-R). This report has addressed the steps leading to the cloning of the sheep GnRH-R cDNA and the endocrine mechanisms associated with the regulation of GnRH-R mRNA expression. Differences between species and between in vivo and in vitro studies are apparent. The expression of GnRH-R mRNA was shown to increase prior to the gonadotrophin surge and decrease thereafter; these changes were correlated with GnRH binding in the pituitary and follicle oestradiol production. In the early follicular phase of the sheep oestrous cycle, oestradiol appears to be the primary regulator of GnRH-R mRNA, whereas in the periovulatory period, the action of GnRH itself is more important.

Mobilization of LH secretory granules in gonadotrophs in relation to gene expression, synthesis and secretion of LH during the preovulatory phase of the sheep oestrous cycle.

Changes in LH secretory granules in pituitary gonadotrophs throughout the sheep oestrous cycle were determined by immunogold localisation of LH at ultrastructural level by electron microscopy. Oestrous cycles in Welsh Mountain ewes were initially synchronised with progestagen sponges and studies carried out in the subsequent cycle. Animals were allocated at random to six groups each of five animals, one killed on day 12 of the luteal phase and the other groups after prostaglandin (PG)-induced luteal regression at PG plus 18 h (early follicular phase), oestrus (PG plus 33.6 +/- 1.0 h), oestrus plus 9 h just before the preovulatory LH surge, 1 h after GnRH agonist-induced LH surge at PG plus 48 h (mid-LH surge) and oestrus plus 24 h, after the preovulatory LH surge. Blood samples collected throughout confirmed the pulsatile secretion of LH before and the timing in relation to the preovulatory LH surge. Pituitaries were dissected and processed for transmission electron microscopy and frozen for later extraction of mRNA. Only a single type of LH cell was present in the sheep pituitary. In the luteal phase, LH-immunopositive secretory granules were distributed throughout the cytoplasm in 80% of cells while in 20% of cells granules were polarised to the region of the cell next to a vascular sinusoid. The percentage of polarised cells increased during the follicular phase to 45% at oestrus, 75% at oestrus plus 9 h just before the LH surge and 90% in mid-LH surge. Cell size increased in parallel with polarisation. Gonadotrophs after the LH surge were almost totally devoid of LH granules but prominent LH beta immunoreactivity was observed in the rough endoplasmic reticulum. Analysis of granule diameters revealed a single class of granules with a maximum diameter of 300 nm. Polarised cells had significantly fewer 130-150 nm granules than non-polarised cells, suggesting preferential exocytosis of LH-containing granules of this size from polarised cells. Northern analysis showed that LH beta mRNA levels decreased from luteal through the follicular phase. These results suggest that the preovulatory LH surge in sheep is not related to a change in synthesis of LH but to a progressive recruitment of gonadotrophs into a releasing state, priming, as indicated by polarisation of secretory granules to the region of the cell next to the vascular system.

Ovulation rate and the concentrations of gonadotrophins and metabolic hormones in ewes infused with glucose during the late luteal phase of the oestrous cycle.

The positive relationship between nutrition and ovulation rate was investigated in sheep infused intravenously with glucose. Ovulation rate increased (2.0 +/- 0.0 vs 2.4 +/- 0.3) when ewes were given an infusion of glucose (60-65 mM/h) for five days in the late luteal phase of the oestrous cycle. The effect of glucose was obtained without any significant change in LH secretion. The concentration of FSH in glucose-infused ewes was lower during the infusion (luteal phase) but higher during the early follicular phase. These data suggest that the change in ovulation rate occurred without increased gonadotrophin support to the follicle during the late luteal phase, which is the period of the sheep oestrous cycle during which improved nutrition increases ovulation rate. There were no changes in GH or prolactin, but changes in circulating glucose and insulin levels were detected. We conclude that insulin, because of its role in cell growth and metabolism, is involved in mediating ovulation responses to nutritional stimuli, either directly or more likely by the stimulation of insulin-mediated glucose uptake.

Role of Endogenous Opioids for Regulation of the Oestrous Cycle in Sheep and Cattle

Role of Endogenous Opioids for Regulation of the Oestrous Cycle in Sheep and Cattle

Changes in progesterone secretion following treatment with transforming growth factor (TGF- ) during the follicular phase of the sheep oestrous cycle

The effects of transforming growth factor alpha (TGF-alpha) on ovarian steroid secretion were investigated. Three crossbred ewes synchronized for oestrus with ovarian autotransplants were infused with TGF-alpha (30 micrograms in 12 h) via the ovarian artery for 12 h before withdrawal of progestagen pessary. Three ewes were used as controls. Jugular and ovarian venous blood samples were taken at intervals of 10 min at two stages during the follicular phase (21-27 h and 38-42 h after pessary withdrawal) and every 2 h from 44 to 86 h. Plasma LH and FSH concentrations, and ovarian secretion rates of inhibin, androstenedione, oestradiol and progesterone were determined using radioimmunoassays. LH pulse amplitude increased in ewes treated with TGF-alpha in the early follicular phase (0.92 +/- 0.25 micrograms l-1 in controls versus 3.10 +/- 0.35 micrograms l-1 in TGF-alpha treated ewes; P < 0.05) and remained high in the late follicular phase. Plasma FSH concentrations were high during the follicular phase in ewes treated with TGF-alpha (P < 0.05). The infusion of TGF-alpha had no significant effect on the ovarian rate of secretion of androstenedione and, although the secretion rates of oestradiol and inhibin were consistently lower in TGF-alpha-infused ewes, the differences were not significant. The ratio of secretion of androstenedione to oestradiol was greater during the follicular phase in TGF-alpha-treated ewes (P < 0.05), suggesting that the efficiency of aromatization had been impaired.(ABSTRACT TRUNCATED AT 250 WORDS)