Preovulatory Follicle Development Research Articles

Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

66 RELATIONSHIP BETWEEN PLASMA CHLORIDE LEVELS AND FOLLICLE DIAMETER IN CYCLING SPANISH PUREBRED MARES

Several studies in cows have shown that plasma levels of chloride are positively correlated with the diameter of the preovulatory follicle (FD), establishing at the same time a significant positive association between plasma levels and intraoviducal concentration of this electrolyte (Hugentobler et al. 2007). These relationships may reflect the influence of changes in systemic metabolic plasma on follicular fluid, physiological events that might be closely related to the quality of the oocyte and granulosa cells of the preovulatory follicle (LeRoy et al. 2004). However, these mechanisms remain unknown in the mare. The objective of this research was to investigate the relationship between plasma chloride concentrations and growth of FD during the ovulatory period in the Spanish purebred mares. This research was approved by the Animal Ethics Committee of the CEU-Cardenal Herrera University. A total of 30 reproductive cycling Spanish purebred mares, aged 4 to 17 years old, were studied. Plasma concentrations of chlorine and follicular growth (FD) were determined daily by extracting blood samples and monitoring of ovarian ultrasound all mares, from Day –5 to the day of ovulation. Plasma chloride concentrations were evaluated by an analyzer with selective electrodes Vetlyte® (IDEXX, Hoofddorp, the Netherlands). To analyse the effect of cycle day on FD and concentrations of chloride, ANOVA was used. The interrelations between both parameters were expressed by Pearson correlation coefficient. The FD increased significantly, from 27.02 ± 2.113 to 40.23 ± 1.377 cm, during the 5 days prior to ovulation. Plasma chloride decreased from 105.6 ± 0.928 mmol L–1 on Day –5 to 104.5 ± 0.128 mmol L–1 on Day –1, and increased significantly, to 107.4 ± 1.442 mmol L–1, on the day of ovulation. No significant correlations between FD and chloride concentrations (r = 0.21) were found. Unlike what happens in the cow, plasma chloride levels affect preovulatory follicle development in Spanish purebred mares to a lesser extent. Nevertheless, plasma chloride or any other electrolytes have some kind of involvement in the dynamics of growth and maturation of oocytes.

Progesterone exposure of seasonally anoestrous ewes alters the expression of angiogenic growth factors in preovulatory follicles

Small-dose, multiple injections of GnRH given to seasonally anoestrous ewes induce final stages of the preovulatory follicle development, but result in an high incidence of defective CL unless animals are primed with progesterone, which completely eliminates luteal dysfunction. Progesterone priming upregulates luteal vascularization; however, its effect on follicular angiogenesis is poorly understood. This study tested the hypothesis that progesterone priming of seasonally anoestrous ewes treated with dose multiple injections of GnRH eliminates defective luteal function by altering the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2, angiopoietin (ANG)-1, ANG-2, and TIE-2 during early and late preovulatory follicle development. Ten seasonally anoestrous ewes were given 20 mg of progesterone im 3 days before the start of GnRH treatment; 10 other animals served as controls. Intravenous injections of 500 ng GnRH were given to all animals every 2 hours for 28 hours, followed at 30 hours with a 300-μg GnRH bolus injection to synchronize the preovulatory LH surge. Ovaries were collected at 24 and 46 hours after the start of GnRH treatment. Small (2–2.5 mm) and large (>2.5 mm) follicles were analyzed for protein and mRNA expression of the angiogenic factors using immunohistochemistry and in situ hybridization assays. Progesterone priming did not have an influence on angiogenic factor levels in small follicles. However, progesterone-primed animals showed significantly (P ≤ 0.05) higher levels of VEGF, VEGFR-2, ANG-1, and ANG-2 in large follicles compared with nonprimed ones. These data suggest that progesterone priming alters the expression of angiogenic factors in large preovulatory follicles, ensuring adequate luteal development and function.

The Ephrin Signaling Pathway Regulates Morphology and Adhesion of Mouse Granulosa Cells In Vitro1

Follicle-stimulating hormone (FSH)-mediated changes in granulosa cell adhesion and morphology are essential for preovulatory follicle development, given the dramatic changes in follicle size and granulosa cell number that occur during this transition. Members of the Eph-ephrin family of cell-positioning and adhesion molecules, a family that consists of ephrin ligands and their Ephrin (Eph) receptors, regulate cell location, adhesion, and migration during embryonic development and tumor growth. However, very little is known about ephrin signaling during folliculogenesis. We have found that FSH increases the expression of several members of the Eph-ephrin family and that this signaling regulates granulosa cell morphology and adhesion. FSH induced increased mRNA levels of the ephrin ligand, ephrin-A5 (Efna5), and its receptors, Eph receptors A3, A5, and A8 (Epha3, Epha5, and Epha8, respectively), in granulosa cells. Immunofluorescence studies indicated that EFNA5 and EPHA5 are located in the membrane of granulosa cells of developing mouse follicles. Eph-ephrin signaling directly affected granulosa cell morphology and adhesion. Recombinant EFNA5 reduced cell spreading and increased cell rounding in mouse primary granulosa cells and in a rat granulosa cell line, whereas EPHA5 reduced granulosa cell adhesion in both model systems. Both FSH and forskolin also increased Efna5 and Epha5 mRNA levels in rat and human granulosa cell lines, indicating that FSH regulates these genes via the cAMP-dependent protein kinase A pathway and that this regulation is conserved across different species. The present study identifies Eph-ephrin signaling as a novel FSH-mediated pathway regulating granulosa cell morphology and adhesion.

Physiological status alters immunological regulation of bovine follicle differentiation in dairy cattle

Lactation in dairy cattle is associated with a multitude of endocrine, metabolic and immunological changes that not only influence animal health, but also affect fertility, and in particular ovarian function. We have previously generated a global transcriptomic profile of bovine follicular tissue using RNA sequencing. This study aimed to: identify key immune-related transcriptional changes that occur during follicle differentiation and luteinisation using ingenuity pathway analysis (IPA); and determine if a compromised model of stress (i.e. lactation) influences the temporal expression of these genes. Ovarian follicular tissue from Holstein–Friesian non-lactating heifers (n=17) and lactating cows (n=16) was compared at three stages of preovulatory follicle development: (A) the newly selected dominant follicle in the luteal phase (Selection); (B) the follicular phase before the LH surge (Differentiation), and (C) the preovulatory phase after the LH surge (Luteinisation). IPA revealed an over-representation of immune-related pathways in theca compared with granulosa cells during differentiation; these were related to leucocyte extravasation and chemotaxis. Conversely, luteinisation was characterised by over-representation of immune-related pathways in granulosa compared with theca cells; these were related to inflammation and innate immune response. Notably, comparison of follicles from heifers and lactating cows revealed a large number of differentially expressed genes associated with immune cell subpopulations and chemotaxis. In conclusion, identification of immune-related canonical pathways during follicle development supports the hypothesis that ovulation is an inflammatory event. This process is influenced by the physiological status of lactation and likely contributes to compromised peri-ovulatory follicle function by impairing the inflammatory process of ovulation.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of β-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

Ovarian Response to Oestrous Synchronization Protocol Based on Use of Reduced Doses of Cloprostenol in Cyclic Goats

The objective of this study was to assess the efficacy of two reduced doses vs a high/luteolytic dose of cloprostenol on luteolytic activity and synchronization of oestrus in cyclic goats. Experiment 1, included 24 goats randomly allocated to three groups: control group (group H) received a single high dose of cloprostenol (87.5 μg; 1.0 ml; i.m.) and M and L groups, which received half (43.75 μg; 0.5 ml) and a third (26.25 μg; 0.3 ml) of the highest dose, respectively. Experiment 2, included 24 goats randomly assigned to the same experimental groups. Each group was treated using two injections of cloprostenol administered 10 days apart to synchronize oestrus. Transrectal ultrasonographic scanning (US) was performed to detect the presence, size and development of corpora lutea and ovarian follicles. Furthermore, detection of oestrus was performed every 12 h between 24 and 72 h after the second injection of cloprostenol, and the luteolytic effect was verified by US. In Experiment 1, all goats that had corpora lutea at timing of treatment regressed their corpora lutea. In Experiment 2, the occurrence of oestrus and the interval between treatment to onset of oestrus were: 100%, 49.5 ± 3.0 h; 100%, 51.0 ± 3.0 h; and 75%, 56.0 ± 3.5 h for H, M and L groups, respectively. The development of preovulatory follicles and occurrence of subsequent corpora lutea were similar among groups. In summary, the use of 26.25 μg of cloprostenol is effective for the synchronization of oestrus in cyclic goats.

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Nutritionally Induced Relationships Between Insulin Levels During the Weaning‐to‐Ovulation Interval and Reproductive Characteristics in Multiparous Sows: I. Luteinizing Hormone, Follicle Development, Oestrus and Ovulation

To get more insight in how insulin secretion patterns and corresponding insulin-like growth factor-1 (IGF-1) levels are related to luteinizing hormone (LH) secretion, follicle development and ovulation, 32 multiparous sows were fed either a dextrose plus lactose-containing diet at 4 h intervals (DL; each 150 g/day) or an isocaloric control diet at 12 h intervals (CTRL; containing soybean oil) during the weaning-to-ovulation interval (WOI). Insulin parameters (basal, peak levels and mean insulin) and IGF-1 levels during the WOI were similar for both treatments, but the insulin secretion pattern differed (related with feeding frequency and meal sizes). Oestrus and ovulation characteristics were not influenced by treatment. The LH surge was higher in CTRL compared with DL sows (3.73 vs 3.00 ng/ml; p = 0.03). Average diameter (6.5 vs 6.1 mm; p = 0.08) and uniformity (CV: 11 vs 15%, p = 0.02) of follicles ≥3 mm at day 4 after weaning was higher in CTRL compared with DL sows. Basal insulin levels were positively related with follicle diameter at ovulation (β = 0.05 mm/(μU/ml); p = 0.04) and negatively related with LH surge level (β = -0.07 (ng/ml)/(μU/ml); p = 0.01). Insulin area under the curve (AUC) (β = 0.037 (ng/ml)/1000 μU; p = 0.02) and IGF-1 levels (β = 0.002 (ng/ml)/(ng/ml); p < 0.01) were positively related to basal LH level around the LH surge. From these data, we conclude that insulin and IGF-1 levels during the WOI are related to LH secretion and follicle development. Not only the absolute level of insulin seems important, but also the pattern within a day in which insulin is secreted seems to affect LH secretion and development of pre-ovulatory follicles.

Atrial natriuretic peptide is an antiapoptotic factor for human granulosa luteinized cells with impact on the results of COH/IVF in women undergoing IVF program

To investigate human granulosa luteinized cells as a local source of atrial natriuretic peptide (ANP) and its relationship to cell viability, development of preovulatory follicles and fertilization rate of oocytes. Indirect immunofluorescent technique, proANP kit, 4'6-Diamidino-2-phenylindole (DAPI)-DNA staining and Caspase-3 activity assay were used to examine localization patterns, concentrations in follicular fluids (FFl) and antiapoptotic role of ANP, respectively. ANP is expressed on granulosa cells with membrane, submembrane and specific granular cytoplasmic localization. Significant differences in the mean concentrations of ANP in FFl and in the mean levels of apoptotic human granulosa luteinized cells (GLC) were found between women with high and low immunoreactive ANP expression (P < 0.05). Concentrations of ANP correlated inversely and significantly with the percentage of apoptotic GLC isolated from the same women (r = -0.2645; P < 0.05). Enhanced in vitro production of ANP by granulosa cells (after sodium nitroproside and 2Bu-cGMP treatment), as well as after treatment with human recombinant ANP was associated with significant suppression of Caspase-3 activity compared to control cells (P < 0.05). Concentrations of ANP in FFl correlated positively and significantly with the number of punctured follicles and oocytes retrieved (r = 0.44716, r = 0.57854; P < 0.05) and with the fertilization rate of oocytes in vitro (r = 0.59773; P < 0.05). Human granulosa luteinized cells are a local source of ANP in the preovulatory human follicle, which acts as a survival factor through cGMP signaling, and its levels in follicular fluids are positively associated with the number of punctured follicles, isolated oocytes and their fertilizability in vitro.

Targeted Disruption of Mapk14 (p38MAPKα) in Granulosa Cells and Cumulus Cells Causes Cell-Specific Changes in Gene Expression Profiles that Rescue Cell-Oocyte Complex Expansion and Maintain Fertility

Abstract MAPK14 (p38MAPKα) is critical for FSH and prostaglandin E (PGE)2 signaling cascades in granulosa cells (GCs) and cumulus cell-oocyte complexes (COCs) in culture, indicating that this kinase might impact follicular development and COC expansion in vivo. Because Mapk14 knockout mice are embryonic lethal, we generated GC specific Mapk14 knockout mice (Mapk14gc−/−) by mating Mapk14fl/fl and Cyp19a1-Cre mice. Unexpectedly, the Mapk14gc−/− female mice were fertile. Analyses of gene expression patterns showed that amphiregulin (Areg) and epiregulin (Ereg), two key regulators of ovulation and COC expansion, were up-regulated in the GCs but down-regulated in cumulus cells of the mutant mice in vivo. COCs from the mutant mice expanded and expressed matrix-related genes, if cultured with AREG, but not when cultured with forskolin or PGE2, the latter being a key factor regulating MAPK14 activity in cumulus cells. Conversely, when GCs from the Mapk14gc−/− mice were cultured with forskolin, they produced more Areg and Ereg mRNA than did wild-type GCs. These results indicate that disruption of Mapk14 selectively alters the expression of Areg and other genes in each cell type. Greater AREG and EREG produced by the GCs appears to by-pass and compensate for the critical need for MAPK14 signaling and induction of Areg/Ereg (and hence matrix genes) by PGE2 in cumulus cells of the mutant mice. In conclusion, although MAPK14 is not overtly essential for preovulatory follicle development or events associated with ovulation and luteinization in vivo, it does impact gene expression profiles.

Targeted Disruption of Mapk14 (p38MAPKα) in Granulosa Cells and Cumulus Cells Causes Cell-Specific Changes in Gene Expression Profiles that Rescue COC Expansion and Maintain Fertility

MAPK14 (p38MAPKalpha) is critical for FSH and prostaglandin E (PGE)2 signaling cascades in granulosa cells (GCs) and cumulus cell-oocyte complexes (COCs) in culture, indicating that this kinase might impact follicular development and COC expansion in vivo. Because Mapk14 knockout mice are embryonic lethal, we generated GC specific Mapk14 knockout mice (Mapk14gc(-/-)) by mating Mapk14(fl/fl) and Cyp19-Cre mice. Unexpectedly, the Mapk14gc(-/-) female mice were fertile. Analyses of gene expression patterns showed that amphiregulin (Areg) and epiregulin (Ereg), two key regulators of ovulation and COC expansion, were up-regulated in the GCs but down-regulated in cumulus cells of the mutant mice in vivo. COCs from the mutant mice expanded and expressed matrix-related genes, if cultured with AREG, but not when cultured with forskolin or PGE2, the latter being a key factor regulating MAPK14 activity in cumulus cells. Conversely, when GCs from the Mapk14gc(-/-) mice were cultured with forskolin, they produced more Areg and Ereg mRNA than did wild-type GCs. These results indicate that disruption of Mapk14 selectively alters the expression of Areg and other genes in each cell type. Greater AREG and EREG produced by the GCs appears to by-pass and compensate for the critical need for MAPK14 signaling and induction of Areg/Ereg (and hence matrix genes) by PGE2 in cumulus cells of the mutant mice. In conclusion, although MAPK14 is not overtly essential for preovulatory follicle development or events associated with ovulation and luteinization in vivo, it does impact gene expression profiles.

Global Transcriptome Analysis of Porcine Oocyte, Granulosa, and Theca Cells to Identify Potential Secreted Factors and Receptors Involved in Cell-to-cell Interactions During Pre-Ovulatory Follicle Development.

Global Transcriptome Analysis of Porcine Oocyte, Granulosa, and Theca Cells to Identify Potential Secreted Factors and Receptors Involved in Cell-to-cell Interactions During Pre-Ovulatory Follicle Development.

MAPK14 (p38MAPKalpha) Is Essential for PGE2-Induced COC Expansion in Culture, but Is Not Essential for Pre-Ovulatory Follicle Development In Vivo.

MAPK14 (p38MAPKalpha) Is Essential for PGE2-Induced COC Expansion in Culture, but Is Not Essential for Pre-Ovulatory Follicle Development In Vivo.

Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle

To determine intrafollicular hormone levels and characterize the mRNA expression of the insulin-like growth factor (IGF) receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus. Experimental animal study. Academic medical center. Adult rhesus macaques. Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hours) or 3, 6, 12, or 24 hours after an ovulatory hCG bolus. IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time reverse transcriptase-polymerase chain reaction. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and enzyme-linked immunosorbent assay. IGF1, IGF2, and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1, and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, -5, and -6 and PAPP-A mRNA levels increased after hCG administration, while insulin receptor and IGFBP4 mRNA levels decreased after hCG administration. IGFBP3 and -6 and PAPP-A protein increased after hCG administration. Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.

Oocyte-specific deletion of complex and hybrid N-glycans leads to defects in preovulatory follicle and cumulus mass development

Complex and hybrid N-glycans generated by N-acetylglucosaminyltransferase I (GlcNAcT-I), encoded by Mgat1, affect the functions of glycoproteins. We have previously shown that females with oocyte-specific deletion of a floxed Mgat1 gene using a zona pellucida protein 3 (ZP3)Cre transgene produce fewer pups primarily due to a reduction in ovulation rate. Here, we show that the ovulation rate of mutant females is decreased due to aberrant development of preovulatory follicles. After a superovulatory regime of 48 h pregnant mare's serum (PMSG) and 9 h human chorionic gonadotropin (hCG), mutant ovaries weighed less and contained approximately 60% fewer preovulatory follicles and more atretic and abnormal follicles than controls. Unlike controls, a proportion of mutant follicles underwent premature luteinization. In addition, mutant preovulatory oocytes exhibited gross abnormalities with approximately 36% being blebbed or zona-free. While 97% of wild-type oocytes had a perivitelline space at the preovulatory stage, approximately 54% of mutant oocytes did not. The cumulus mass surrounding mutant oocytes was also smaller with a decreased number of proliferating cells compared with controls, although hyaluronan around mutant oocytes was similar to controls. In addition, cumulus cells surrounding mutant eggs were resistant to removal by either hyaluronidase or incubation with capacitated sperm. Therefore, the absence of complex and hybrid N-glycans on oocyte glycoproteins leads to abnormal folliculogenesis resulting in a decreased ovulation rate.

Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

BackgroundThe objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.MethodsMature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).ResultsOut of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.ConclusionThese data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.

Glucogenic supply increases ovulation rate by modifying follicle recruitment and subsequent development of preovulatory follicles without effects on ghrelin secretion

This study determined the effects of short-term energy inputs on ghrelin secretion and possible links with changes in the follicle population or the ovulation rate. Oestrous cycle was synchronized in 16 Manchega sheep using progestagen sponges and cloprostenol. Half of the animals were treated from days 0 to 4 by the oral administration, twice daily, of 200 ml of a glucogenic mixture containing 70% of glycerol, 20% of 1,2-propanediol and 10% of water; the control group received 200 ml water. The mean (+/-S.E.M.) plasma glucose increased immediately after the first administration (3.9+/-0.3 vs 3.0+/-0.1 mmol/l in control group, P<0.05), remaining statistically different during the treatment. However, plasma ghrelin levels were similar in both groups. On the other hand, the results indicated that short-term energy inputs modify ovulation rate (1.9+/-0.1 vs 1.3+/-0.2 in control group, P<0.05) by increasing the number of follicles able to be selected to ovulate during the period of treatment (>or=4 mm in size; 5.9+/-0.6 vs 4.3+/-0.4 at day 2, P<0.05). After sponge withdrawal, the number of these follicles decreased throughout follicular phase (5.8+/-0.8 to 1.5+/-0.4, P<0.0005) while the number of large follicles increased (>or=6 mm in size; 0.8+/-0.4 to 2.0+/-0.3, P<0.05); this would indicate an active growth of preovulatory follicles that were not found in the control group. Thus, the increases of ovulation rate by high-energy inputs would be caused by an enhancement in the developmental competence of preovulatory follicles.

Effects of Daidzein on mRNA Expression of Gonadotropin Receptors and P450 Aromatase in Ovarian Follicles of White Silky Fowls

Effects of daidzein on expression of mRNAs of gonadotropin receptors (FSHR, LHR) and P450 aromatase (P450arom) were evaluated in ovarian follicles of white silky fowls. The hens were 13 months old in the post-peak period of egg laying and were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at 10 mg/kg for 7 consecutive weeks. The mRNA expression of related genes was measured by semi-quantitative RT-PCR in the granulosa layers of the preovulatory follicle (PRF: Fl, F2 ...) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF) and atretic follicle (ATF). Results showed that daidzein supplementation significantly increased the number of SYF and LWF (p<0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F3 to Fl, but LHR mRNA displayed opposite developmental changes. P450arom mRNA was highest in the SYF, but was very low in the granulosa layers after follicles finished selection. Treatment with daidzein resulted in increased mRNA expression of FSHR in F3 granulosa layer, LHR in granulosa layers of F3 to Fl and P450arom in LWF (p<0.05). These results indicated that dietary supplementation of daidzein up-regulated mRNA expression of gonadotropin receptors and P450arom to improve the development of preovulatory follicles in white silky fowls after the peak-laying period.

Plasma concentrations of prolactin in brushtail possums (Trichosurus vulpecula) in different physiological states

Prolactin (Prl) has been implicated in reproduction in many mammalian species and is illustrated by the distinctive patterns of secretion during the breeding season, the oestrous cycle and lactation. The recent development of a homologous RIA for measuring the circulating Prl concentrations in brushtail possums has facilitated the reliable measurement of Prl in plasma during different physiological states in this species for the first time. Determination of Prl concentrations during lactation involved the collection of weekly blood samples from eight female possums from the time of parturition through either one or two consecutive lactational cycles. Prl was at baseline levels during early lactation (weeks 0-14 post-partum), and then increased markedly to maximum concentrations at weeks 19-21 before returning to nadir levels at a time coincident with the weaning of pouch young (weeks 23-27). The profile of Prl secretion over the oestrous cycle and in particular at the time of the preovulatory LH surge was obtained from 14 possums during the reproductive cycle, in which preovulatory follicle development and ovulation were monitored by laparoscopy. There was no distinct daily pattern of Prl secretion during the oestrous cycle; however, in 3/4 possums in which a typical preovulatory LH surge was measured, a biphasic preovulatory Prl surge was also observed. The preovulatory Prl surge commenced 2-6 h prior to, and had returned to baseline close to the onset of, the preovulatory LH surge, and a second surge of Prl occurred concomitantly with the delayed preovulatory FSH surge. Seasonality of Prl levels was established from weekly blood samples collected from six barren female possums, and concentrations of Prl were lower during the breeding season compared to the non-breeding season. Additionally, a circadian pattern of Prl secretion was evident in both female and male possums, with Prl levels higher in the morning compared to the afternoon. In conclusion, interpretation of endogenous secretory patterns suggests that Prl may be important during late lactation and at impending ovulation, but the involvement of the circannual rhythm of Prl in the regulation of seasonality in the brushtail possum remains to be determined.