Sheep Ovaries Research Articles

257 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitro matured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus–oocyte complexes from slaughterhouse sheep ovaries were matured in vitro for 27 h. After in vitro maturation, oocytes (n = 77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r = 0.87; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitro maturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r = 1; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitro maturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitro matured sheep and goat oocytes. This work was supported by the following grant: INIA: RZ2007-00013-00-00. M. Muñoz and D. Martín are sponsored by RYC08-03454 and PTA2007-0268-I, respectively.

Expression of PDGFs and Their Receptors in the Fetal and Adult Sheep Ovary.

Platelet derived growth factors (PDGFs) and their receptors are key regulators of cell growth and angiogenesis, especially during fetal development. In rodents and humans, expression of PDGF mRNA was observed in specific ovarian compartments and the addition of PDGF to follicles in culture increased their size and enhanced the transition from primordial to primary follicles. Differences in the pattern of PDGF expression were noted depending on the species or age studied. However the effect of follicular health on expression has not been examined in depth. The objectives of this study were to determine the cell specific gene expression of PDGF ligands (PDGFA, B and C) and their receptors (PDGFRA and B) during ovarian and follicular development in sheep. Ovaries were collected from fetuses on days 55, 75 and 95 of gestation and from ewes at 4 weeks, 10 months, 2 years and 8 years of age and examined using in situ hybridization. During fetal life, PDGFA expression was sometimes associated with the vasculature and extra ovarian rete. Consistent expression was noted in the surface epithelium and in oocytes in both ovigerous cords and formed follicles. No expression was observed in granulosa cells of formed follicles or connecting rete. Fetal expression of PDGFB was similar to PDGFA at days 55 and 75; however no expression was apparent in the rete and by day 95 expression was largely confined to the surface epithelium. Expression of PDGFC was concentrated in the surface epithelium and cells surrounding the vasculature, with little or no expression observed in oocytes, follicles or in cells of the rete system. Postnatally, PDGFA and B were expressed in oocytes at all stages of follicular development from primordial to large antral follicles and in granulosa cells from the primary stage. Granulosa cell expression of PDGFC was evident in large preantral and small antral follicles. Thecal expression was not apparent until the large preantral stage and was initially restricted to PDGFB. At antral stages PDGFB was expressed predominantly in the theca interna while PDGFA was expressed in both theca interna and externa. PDGF receptors were not expressed in follicles until after antrum formation with PDGFRA expression in both theca interna and externa while PDGFRB expression was limited to theca interna. In general, atresia resulted in decreased expression of PDGFA and B in granulosa cells, increased expression of PDGFA and B in the thecal layers, increased expression of PDGFC in the granulosa and the appearance of PDGFC expression in the thecal layers. Only PDGFA expression was consistently noted in components of the ovarian rete system. Expression of PDGFA, B and C and PDGFRA and B was apparent throughout the CL, with receptor expression often associated with the vasculature. Cells associated with the vasculature showed expression of PDGFA, B and C, as well as PDGFA and B. Changes in expression patterns at different stages of follicular development along with changes associated with follicular atresia are consistent with a role for these factors in regulation of follicular development. (poster)

EFEITO DO DIÂMETRO FOLICULAR SOBRE A QUALIDADE DOS OÓCITOS OBTIDOS DE OVÁRIOS DE OVELHAS (Ovis aries) E CABRAS (Capra hircus)

This study aimed to evaluate the influence of follicular diameter on the quality of oocytes from ovaries of female sheep and goats. The ovaries of sheep (156 units) and goats (105 units) from slaughterhouse were used. The follicles were measured, aspirated and divided into three classes of follicular diameter: class 1 (CI) – 2 to 3 mm, class 2 (CII) – 4 to 5 mm and class 3 (CIII) – 6 mm. The recovered oocytes were evaluated and classified according to morphological aspect in 5 quality groups: grade 1 (GI), grade 2 (GII), grade 3 (GIII), naked (N) and atresia (A). Of the 468 follicles aspirated from sheep, 327 CI, 84 CII, CIII 57 and 83 GI, 78 GII, GIII 95, 119 N, 93 A were found, and from the 422 follicles aspirated from goats 197 CI, 132 CII, 92 CIII  and 64 GI, 70 GII, 91 GIII, 123 N, 74 A were found. The data did not show significant correlation between quality of oocytes and diameter of follicles (P > 0.05). Under the conditions observed in this study it was concluded that the follicular diameter has no influence on the quality of cumulus-oocyte complex (COC) recovered from the ovaries of female sheep and goats.KEYWORDS: Aspirated follicle, cumulus cells, oocyte quality.

Whole sheep ovary cryopreservation: evaluation of a slow freezing protocol with dimethylsulphoxide

To evaluate a slow freezing method for whole ovary cryopreservation by evaluating effects of added cryoprotectant. Sheep ovaries were isolated during surgery, flushed with either Ringer-Acetate or dimethylsulphoxide and cryopreserved by slow freezing. After rapid thawing, viability was assessed by ovarian in vitro perfusion, cell culture, histology and fluorescent live-dead assay. Production of cyclic AMP and progesterone was slightly higher in the dimethylsulphoxide group. Cultured ovarian cells from dimethylsulphoxide-preserved ovaries secreted larger amounts of progesterone than cells from Ringer-Acetate preserved. Light microscopy of ovarian biopsies obtained after perfusion, revealed well-preserved tissue in the dimethysulphoxide group but not in the Ringer-Acetate group. The density of small follicles and ovarian cell viability were higher in dimethysulphoxide ovaries compared to Ringer-Acetate ovaries. Equilibrium with its protective effect can be achieved by slow freezing protocol, with an additional protective effect by the presence of dimethylsulphoxide.

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.

First Evidence of Bone Morphogenetic Protein 1 Expression and Activity in Sheep Ovarian Follicles1

Bone morphogenetic protein (BMP) 1 is a vertebrate metalloproteinase of the astacin family. BMP1 plays a key role in regulating the formation of the extracellular matrix (ECM), particularly by processing the C-propeptide of fibrillar procollagens. BMP1 also promotes BMP signaling by releasing BMP signaling molecules from complexes with the BMP-antagonist chordin. As a result of BMP1's dual role in both ECM formation and BMP signaling, we hypothesized that BMP1 could play a role in ovarian physiology. Using the sheep ovary as a model system, we showed that BMP1 was expressed in the ovary throughout early fetal stages to adulthood. Furthermore, in adult ovaries, BMP1 was expressed along with chordin, BMP4, and twisted gastrulation, which together form an extracellular regulatory complex for BMP signaling. Within ovine ovaries, immunohistochemical localization demonstrated that BMP1 was present in granulosa cells at all stages of follicular development, from primordial to large antral follicles, and that the levels of BMP1 were not affected by the final follicle selection mechanism. In cultured granulosa cells, BMP1 expression was not affected by gonadotropins, but BMP4 and activin A had opposing effects on the levels of BMP1 mRNA. BMP1 appeared to be secreted into the follicular fluid of antral follicles, where it is able to exert procollagen C-proteinase and chordinase activities. Interestingly, BMP1 activity in follicular fluid decreased with follicular growth.

Serum progesterone concentrations, follicular development and time of ovulation using a new progesterone releasing device (DICO ®) in sheep

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO ®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO ® ( n = 9) and CIDR-G ® ( n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO ® ( n = 8) and CIDR-G ® ( n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO ® and CIDR-G ® devices, respectively. In Experiment 3, the re-utilization of DICO ® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO ® ( n = 11) for a period of 6 days. The re-used DICO ® devices induced a lower serum progesterone concentration than the new devices ( P < 0.05). However, the re-used DICO ® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO ® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO ® devices for 6 days was applied with ( n = 8) or without ( n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO ® or CIDR-G ® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.

The effects of melatonin on in vitro oocyte competence and embryo development in sheep

The aim of this study was to assess the effects of melatonin on the in vitro maturation and fertilization of sheep oocytes, and on the in vitro culture of the embryos. Oocytes from sheep ovaries collected at the slaughterhouse were divided into four groups, two of which were treated with either 10E–5 M (M5) or 10E–6 M (M6) melatonin, while the other two groups served as untreated controls (C5 and C6). After in vitro fertilization with fresh ram semen, the embryos produced in each group were divided into two sets, one cultured with melatonin (M5M, C5M, M6M and C6M), and the other without melatonin (M5C, C5C, M6C, and C6C). A melatonin concentration of 10E–6 M increased maturation rate (82.5% vs. 73.7% for M6 and C6, respectively; P &lt; 0.05) and tended to improve cleavage rate 36 hours after in vitro fertilization (79.4% vs. 72.6% for M6 and C6, respectively, P = 0.08). A higher melatonin concentration (10E–5 M) did not have significant effects on those parameters. Blastocyst rates on day 8 did not differ significantly among groups.

335. X-LINKED INHIBITOR OF APOPTOSIS PROTEIN (XIAP) EXPRESSION PATTERNS IN THE SHEEP OVARY

At reproductive age, the ovary undergoes continual cyclicity of follicles due to multiple positive and negative signals that promote follicle growth and development, selection for ovulation, or atresia. The majority of follicles undergo atresia, a degenerative process involving granulosa cell apoptosis. This process is executed by caspases, which are cysteine proteases. Caspases are potently inhibited by XIAP providing a potential mechanism to control follicular atresia. The study objective was to test the hypothesis that XIAP will show elevated expression in healthy antral follicles compared to atretic antral follicles and will show an inverse relationship with active caspase-3 immunoreactivity in the same antral follicle during the sheep estrous cycle. Reproductively mature Romney ewes (n = 9) had estrous cycles synchronized with a prostaglandin F2α analogue, then tissue was collected on days 14, 15 and 16 of the subsequent natural cycle. Analysis involved determining the presence and localization of XIAP using in situ hybridization histochemistry and immunohistochemistry then comparing XIAP expression patterns with distribution of active caspase-3 protein. XIAP mRNA was not detected in primordial, primary and secondary follicles. In contrast, XIAP protein was present from the primary stage onwards. Antral follicles showed positive XIAP mRNA and protein expression in both granulosa and thecal cell layers and antral follicles on the same tissue section showed variable expression. All day 14 antral follicles were positive for XIAP mRNA expression irrespective of the level of active caspase-3 immunoreactivity, whereas an inverse relationship between active caspase-3 and XIAP was apparent in the majority of day 15 and 16 antral follicles. XIAP protein was widely expressed in active caspase-3 negative antral follicles and indicated a negative correlation with the onset of active caspase-3 expression in the majority of follicles. These results indicate that XIAP may regulate follicular atresia and act as an indicator of follicular health in the sheep ovary.

Ovarian function 6years after cryopreservation and transplantation of whole sheep ovaries

Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.

Involvement of the SLIT/ROBO pathway in follicle development in the fetal ovary

In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 expression and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells, while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary, SLIT-ROBO signalling may require an autocrine and paracrine interaction. Furthermore, at the time of increased SLIT-ROBO expression, there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall, these results suggest, for the first time, that the SLIT-ROBO pathway is expressed at the time of follicle formation during fetal ovary development.

Inhibitor of Differentiation (Id) Genes Are Expressed in the Steroidogenic Cells of the Ovine Ovary and Are Differentially Regulated by Members of the Transforming Growth Factor-β Family

Inhibitor of differentiation (Id) proteins act during embryogenesis and development to repress gene transcription required for lineage commitment, while promoting cell growth. Growth factors belonging to the TGFbeta superfamily of signaling molecules, notably the bone morphogenetic proteins (BMPs) and activin, can regulate Id expression in these tissues. Id expression and function in adult physiology is less well determined, and we hypothesized a role for Id proteins in the adult mammalian ovary. Immunohistochemistry for Id1, Id2, Id3, and Id4 in the sheep ovary revealed consistent expression in granulosa and thecal cells of ovarian follicles throughout development. In atretic follicles, Id proteins were selectively down-regulated in thecal cells (P < 0.0001). Additionally, Id1 was universally up-regulated in the cumulus cells adjacent to the oocyte. Immunohistochemistry for phospho (p)-smad 1/5/8 signaling components (stimulated by BMPs) showed a punctate pattern of expression whereas p-smad 2/3 (stimulated by activin) was ubiquitously expressed in follicles. Neither pathway, however, displayed differential staining in line with Id1 cumulus-specific expression, suggesting a more complex relationship between Id1 expression and TGFbeta signaling in these cells. Nevertheless, in vitro, stimulation of ovine granulosa cells with BMP6 or activin A led to a respective increase and decrease in Id1 (P < 0.0001), Id2 (P < 0.0001), Id3 (P < 0.0001), and Id4 (P < 0.05) transcripts, and Id1 gene expression was further manipulated by the oocyte-secreted factors BMP15 and growth differentiation factor 9 (P < 0.001). These data confirm that TGFbeta signaling can regulate Id gene expression in the sheep ovarian follicle and suggest a functional role for the Id family in the mammalian ovary.

Evidence for a potential role of neuropeptide Y in ovine corpus luteum function

Neuropeptide Y (NPY) is a neurohormone that is typically associated with food intake, but it has also been reported to affect the production of progesterone from luteal tissue in vitro. However, NPY has not been previously immunolocalized in the ovine ovary or in the corpus luteum (CL) of any species, and the effects of this neurohormone on luteal function in vivo are not known. Thus, we performed fluorescent immunohistochemistry (IHC) to localize NPY in the ovine ovary and used avidin-biotin immunocytochemistry (ICC) to further define the intracellular localization within follicles and the CL. We then infused NPY directly into the arterial supply of the autotransplanted ovaries of sheep to determine the in vivo effect of exogenous NPY on ovarian blood flow and on the luteal secretion rate of progesterone and oxytocin. Immunohistochemistry revealed that the NPY antigen was localized to cells within the follicles and CL, in the nerve fibers of the ovarian stroma, and in the vessels of the ovarian hilus. In the follicle, the NPY antigen was localized to nerves and vessels within the theca interna layer, and strong staining was observed in the granulosal cells of antral follicles. In the CL, NPY was localized in large luteal cells and in the vascular pericytes and/or endothelial cells of blood vessels, found dispersed throughout the gland and within the luteal capsule. In vivo incremental infusions of NPY at 1, 10, 100, and 1,000 ng/min, each for a 30-min period, into the arterial supply of the transplanted ovary of sheep bearing a CL 11 d of age increased ( P ≤ 0.05) ovarian blood flow. The intra-arterial infusions of NPY also increased ( P ≤ 0.05) in a dose-dependent manner the secretion rate of oxytocin, which was positively correlated ( P ≤ 0.05) with the observed increase in ovarian blood flow. The infusions of NPY had a minimal effect on the secretion rate of progesterone, and similar intra-arterial infusions of NPY into sheep with ovarian transplants bearing a CL over 30 d of age had no significant effect on ovarian blood flow or on the secretion rate of progesterone. These results suggest that NPY acts on the luteal vascular system and the large luteal cells to rapidly stimulate blood flow and the secretion of oxytocin, respectively, which collectively implies a putative role for NPY during the process of luteolysis when increasing amounts of oxytocin are secreted from the ovine CL in response to uterine pulses of prostaglandin F2α.

Ovarian follicle development in Booroola sheep exhibiting impaired bone morphogenetic protein signalling pathway

The role of bone morphogenetic proteins (BMPs) in the regulation of ovarian function has been extensively investigated but the mechanism of regulation is not well understood. The aim of this study was to investigate the effect of mutation in the BMP receptor in Booroola sheep on the number of primordial follicles and rate of follicle recruitment in comparison with that in normal merino sheep in vivo. Whole sheep ovaries at the time of birth, 1.5 and 5 years old were collected and processed for the follicle quantification, using computerised stereological methods and statistical analyses. At birth, the total number of primordial follicles in Booroola sheep was significantly lower than in merino sheep. At 1.5 and 5 years, a reversed pattern in favour of Booroola ewes was seen with significantly more primordial follicles than merino. In parallel, the rate of primordial follicle recruitment to developing cohort was substantially lower in Booroola ewes with only 51 and 66% of primordial follicle consumption at 1.5 and 5 years respectively compared to 92 and 97% in merino ewes. On other hand, the mean numbers of developing primary follicles were smaller in Booroola sheep at the time of birth, yet, Booroola ewes possess more primary follicles than merino at 1.5 years. These findings suggest that attenuation of the intraovarian signalling pathway of BMPs may in fact be a successful means of rationalising follicle consumption, preventing unnecessary loss of follicles from the initial primordial follicle pool, hence increasing reproductive longevity and fertility.

Localization of X-Linked Inhibitor of Apoptosis Protein mRNA in Sheep Ovary.

Localization of X-Linked Inhibitor of Apoptosis Protein mRNA in Sheep Ovary.

Development of the Rete Ovarii in the Sheep Ovary.

Development of the Rete Ovarii in the Sheep Ovary.

Expression of Follicle-Stimulating Hormone Receptor Variants During the Estrous Cycle of the Ewe.

Several splice-variants of the follicle stimulating hormone receptor (FSHR) have been identified in sheep. These include a G protein-coupled (FSHR-1), a putative dominant negative (FSHR-2), and a growth factor type-I (FSHR-3) form. These variants likely couple to different signaling pathways and may be involved in FSH responsiveness at different stages of follicular development. However, with the exception of FSHR-1, the roles of variant forms of the FSHR during follicular development in the ewe are unknown. Thus, our objective was to examine expression patterns of variant forms of the FSHR at different times during the estrous cycle and in different sized follicles in the ewe. In Exp. 1, 8 cycling Suffolk-cross ewes were euthanized 24 (n=3), 36 (n=3) and 48 (n=2) hr after the onset of estrus. In Exp. 2, 7 anestrous Suffolk-cross ewes received CIDRs for 14d, followed by treatment (n=3) with PMSG (500IU), or no PMSG (n=4; CIDR-only). Ewes were euthanized 24 (n=4; 2 CIDR-only, 2 PMSG-treated) or 36 (n=3; 2 CIDR-only, 1 PMSG-treated) hr after CIDR removal. Ovaries were harvested from each ewe and all visible follicles were aspirated. Granulosa cells (GC) were isolated from follicular fluid; each isolate was pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm) or preovulatory (≥ 6.1 mm). Total RNA was extracted from GC and reverse-transcribed. The resulting cDNA was subjected to qPCR using primers designed to amplify each FSHR variant specifically. For Exp. 1, FSHR-3 was the most highly expressed (P≤0.05) variant in small and medium follicles. Expression of FSHR-2 and FSHR-3 was higher (P≤0.05) in medium follicles compared to small and preovulatory follicles. Expression of FSHR-1 was not significantly different between follicle size classes, but was higher (P≤0.05) than FSHR-2 in small follicles. No differences in FSHR variant expression were found at different times after onset of estrus, nor were FSHR variants expressed at significantly different levels in preovulatory follicles. For Exp. 2, application of CIDRs during anestrous appeared to cause slight differences in expression patterns of FSHR variants. The most highly expressed variant (P≤0.05) in small and medium follicles was still FSHR-3; however, FSHR-1 expression was higher (P≤0.05) in large versus small and medium follicles, while no differences were found in experiment 1 for FSHR-1 in different sized follicles. Thus, progesterone treatment during anestrous may enhance expression of the G protein-coupled form of the FSHR as follicles develop. FSHR-2 was expressed at the lowest (P≤0.05) levels of any variant, but was higher (P≤0.05) in large compared to medium and small follicles. No significant differences in FSHR variant expression were found within large follicles. This is the first study to examine expression patterns of FSHR-1, FSHR-2, and FSHR-3 during development of follicles in the mature sheep ovary. Our results indicate that FSHR-3 is the predominant variant of the FSHR expressed by GC during the ewe estrous cycle, and thus FSH may have important effects on follicular development through other forms of its receptor in addition to the G protein-coupled form. (poster)

Morphological analysis of primordial follicles from sheep ovaries cryopreserved in EG, DMSO and their association

Morphological analysis of primordial follicles from sheep ovaries cryopreserved in EG, DMSO and their association

Comparative metabolism of benzo(a)pyrene by ovarian microsomes of various species

Knowledge of the ability of the female reproductive system to metabolize polycyclic aromatic hydrocarbons (PAHs) is critical to the diagnosis and management of female infertility and for risk assessment purposes. The PAHs are a family of widespread pollutants that are released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, PAHs become activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. The extent of susceptibility to PAH exposure may depend on the ability of animals to metabolize these chemicals. The present study has been undertaken to assess whether any differences exist among mammals in the metabolism of benzo(a)pyrene (BaP), a prototypical PAH compound. Microsomes isolated from the liver and ovaries of rats, mice, goats, sheep, pigs, and cows were incubated with 5 microM BaP. Postincubation, samples were extracted with ethyl acetate and analyzed for BaP/metabolites by reverse-phase HPLC with fluorescence detection. The rate of metabolism (pmol of metabolite/min/mg protein) was found to be more in liver than in ovary in all the species studied (P < 0.05). The differences in metabolite concentrations were statistically significant (P < 0.0001) among the various species in both organs studied. Multiple species comparison also revealed that the differences were statistically significant (P < 0.001) between rodents (rat and mouse) and higher mammals (ewe, sow, and cow). Even among the higher mammals, in a majority of the cases, the differences in metabolite concentrations were significantly different (P < 0.001) both in ovary and liver. The BaP metabolites identified were 4,5-diol; 7,8-diol; 9,10-diol; 3-hydroxy BaP; and 9-hydroxy BaP. The rodent microsomes produced considerably higher proportion of BaP 4,5-diol and 9,10-diol than did cow, sow, goat, and sheep. However, microsomes from higher mammals converted a greater proportion of BaP to 3-hydroxy and 9-hydroxy BaP, the detoxification products of BaP. Overall, our results revealed a great variation among species to metabolize BaP.

Expression of the orexigenic peptide ghrelin in the sheep ovary

Ghrelin has been implicated in the control of cell proliferation in reproductive tissue. Here, we provide evidence that both ghrelin mRNA and protein are present in ovarian follicles. Persistent expression of ghrelin was also demonstrated in sheep ovary throughout the estrous cycle and pregnancy. In fact, the relative mRNA levels of ghrelin varied depending on the stage of the cycle, with the highest expression during the development of the corpora lutea (CL) and minimal expression in the regressing CL. A similar pattern was seen during pregnancy. Dynamic changes in the profile of ghrelin expression during the estrous cycle and throughout pregnancy suggest a precise regulation of ovarian expression of ghrelin, which could represent a potential role for ghrelin in the regulation of luteal development. In conclusion, the presence of the ghrelin signaling system within the sheep ovary especially in the oocytes opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.