Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression

A growing body of evidence indicates that intrafollicular progesterone receptor signaling pathways are obligatory for follicle rupture. However, the intrafollicular localization and regulation of progesterone receptor expression during the periovulatory period in cattle are not known. In this study, we determined the effect of the preovulatory gonadotropin surge on localization and expression of progesterone receptor mRNA in bovine periovulatory follicular and luteal tissue. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 6, 12, 18, 24 (preovulatory follicles) and 48 h (CL) after a GnRH-induced LH surge (n=5-8 per timepoint). Expression of progesterone receptor mRNA was detected in periovulatory follicular and luteal tissue at all timepoints examined. Relative levels of progesterone receptor mRNA were dramatically upregulated within 6h after the LH surge compared to all other time points (P<0.0001). In situ hybridization analysis revealed that the significant increase in progesterone receptor mRNA expression was localized to the granulosal layer of preovulatory follicles. Our results indicate that progesterone receptor mRNA expression is upregulated specifically in the granulosal layer of bovine preovulatory follicles following the LH surge. Progesterone receptor signaling pathways may help mediate the effects of the preovulatory LH surge on follicle rupture in cattle.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.     

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.     

Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals

Involvement of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied during the last decade. In all mammalian species, IGF-I stimulates granulosa cell proliferation and steroidogenesis. The concentrations of IGF-I and -II do not vary during terminal follicular growth and atresia. In contrast, the levels of IGFBP-2 and -4, as well as IGFBP-5 in ruminants, dramatically decrease and increase during terminal follicular growth and atresia, respectively. These changes are responsible for an increase and a decrease in IGF bioavailability during follicular growth and atresia, respectively. They are partly explained by changes in ovarian expression. In particular, expression of IGFBP-2 mRNA decreases during follicular growth in ovine, bovine and porcine ovaries, and expression of IGFBP-5 mRNA dramatically increases in granulosa cells of bovine and ovine atretic follicles. Changes in IGFBP-2 and -4 levels are also due to changes in intrafollicular levels of specific proteases. Recently, we have shown that the pregnancy-associated plasma protein-A (PAPP-A) is responsible for the degradation of IGFBP-4 in preovulatory follicles of domestic animals. Expression of PAPP-A mRNA is restricted to the granulosa cell compartment, and is positively correlated to expression of aromatase and LH receptor. From recent evidence, the bone morphogenetic protein (BMP) family would also play a key role in ovarian physiology of domestic animals. In particular, we and others have recently shown that a non-conservative substitution (Q249R) in the bone morphogenetic protein-receptor type IB (BMPR-IB) coding sequence is fully associated with the hyperprolific phenotype of FecB(B)/FecB(B) Booroola ewes. BMP-4 and GDF-5, natural ligands of BMPR-IB, strongly inhibit secretion of progesterone by ovine granulosa cells in vitro, but granulosa cells from FecB(B)/FecB(B) ewes are less responsive than those from FecB(+)/FecB(+) to the action of these peptides. It is suggested that in FecB(B)/FecB(B) ewes, Q249R substitution would impair the function of BMPR-IB, leading to a precocious differentiation of granulosa cells and of follicular maturation. Interestingly, recent findings have described mutations in BMP-15 gene associated with hyperprolific phenotypes in Inverdale and Hanna ewes, suggesting that the BMP pathway plays a crucial role in the control of ovulation rate.     

Steroids as local regulators of ovarian activity in domestic animals

The presented overview gives clear evidence for steroids as local regulators of follicular and luteal activity. In the follicle, estrogen receptor-alpha (ERalpha) and ERbeta expression are demonstrated in cow, ewe and pig. Besides species specific effects in general, there is evidence that estradiol-17beta (E(2)) exerts a dose-dependent inhibition on the secretion of progesterone (P(4)) by both theca interna cells (TI) and granulosa cells (GC). GC enhance the ability of the TI to produce androstendione by supplying them with progestin precursor. Androgen produced by TI enhances the ability of the GC to make E(2), and high concentrations of E(2) in the preovulatory follicle inhibit 3beta-HSD in both TI and GC and thus, may promote the use of the pathway Delta(5) for TI androgen production. The authors suggest that E(2) acts within the follicle to exert positive feedback on androgen and E(2) production, and exerts mitotic and anti-atretic or anti-apoptotic effects on follicular cells. Parts of the E(2)-mediated local action are regulated by stimulating effects on hormone receptors (LH, FSH, oxytocin). Gap junctions permit transfer of nutrients and cytokines to and from the avascular GC and oocyte, and formation is stimulated by estrogens. In bovine corpus luteum (CL) there is evidence that P(4) may directly regulate the production of P(4), oxytocin and prostaglandins (PGs) in a cycle dependent fashion. In most of domestic animal species, there is clear evidence for CL production of E(2) with clear stimulatory and luteotropic effects on P(4), and an intraluteal circuit that involves paracrine effects of E(2), oxytocin and PGF(2alpha) (especially in pigs). In contrast, there are species (ruminants, mares) in which the evidence for important local effects of E(2) is less clear, although expression of ERalpha, ERbeta and progesterone receptor (PR) is documented. Progesterone is very important for the regulation of CL lifetime by effects on the endometrium and release of the luteolytic signal PGF(2alpha). In conclusion, steroids as local regulators of ovarian activity are now documented and may stimulate further research in this field.     

Induction of the Expressions of Antioxidant Enzymes by Luteinizing Hormone in the Bovine Corpus Luteum

Luteoprotective mechanisms of luteinizing hormone (LH) involved in the maintenance of bovine corpus luteum (CL) function have not been completely clarified. Since antioxidant enzymes are well documented as antiapoptotic factors in the CL of many mammals, we hypothesized that the luteoprotective action of LH is mediated by stimulating the local production and action of antioxidant enzymes. To test the above hypothesis, in the present study, we examined the mechanisms involved in the luteoprotective actions of LH. Cultured bovine luteal cells obtained from the CL at the mid-luteal stage (days 8–12 of the estrous cycle) were treated with LH (10 ng/ml), onapristone (OP; a specific progesterone receptor antagonist, 100 μM) and diethyldithiocarbamate [DETC; an inhibitor of superoxide dismutase (SOD), 100 μM] for 24 h. LH in combination with or without OP significantly increased the mRNA and protein expressions of manganese SOD (Mn-SOD) and catalase (CATA) and SOD activity. While LH alone significantly increased the mRNA and protein expressions of SOD containing copper and zinc (Cu,Zn-SOD), OP in combination with or without LH significantly decreased the mRNA and protein expressions of Cu,Zn-SOD. In addition, Cu,Zn-SOD, Mn-SOD and CATA mRNA expressions were higher at the mid luteal phase than the other luteal phases. LH in combination with DETC significantly decreased LH-increased cell viability. The overall results suggest that LH increases cell viability by LH-increased antioxidant enzymes, resulting in maintenance of CL function during the luteal phase in cattle.     

Monitoring follicular development in cattle by real-time ultrasonography: a review.

The application of real-time ultrasonography to monitoring ovarian function in mammals has advanced the understanding of follicular dynamics and its regulation. Follicular development is a wave-like sequence of organised events. The waves consist of the synchronous growth of small (4 to 5 mm) antral follicles, followed by the selection and growth of one dominant follicle which achieves the largest diameter and suppresses the growth of the subordinate follicles. In the absence of luteal regression, the dominant follicle eventually regresses (becomes atretic) and a new follicular wave begins. The dominant follicle regulates the growth of the subordinate follicles, because the appearance of the next wave is accelerated if the dominant follicle is ablated, and delayed if the lifespan of the dominant follicle is prolonged. During bovine oestrous cycles, two or three successive waves emerge, on average, on the day of ovulation (day 0) and day 10 for two-wave cycles, and on days 0, 9 and 16 for three-wave cycles. During the oestrous cycle there are thus two or three successive dominant follicles, and the last of these ovulates. Ovarian folliculogenesis is a complex process involving interactions between pituitary gonadotrophins, ovarian steroids and non-steroidal factors. Subtle changes in the hormonal milieu regulate folliculogenesis and the emergence of a follicular wave is preceded by a small increase in the concentration of plasma follicle-stimulating hormone. The mechanisms that promote the selection of a dominant follicle have not been elucidated, but considerable progress has been made in understanding follicular development and its regulation. Most treatments designed to control the development of follicular waves have been based on the physical or hormonal removal of the suppressive effect of the dominant follicle, and the consequent controlled induction of the emergence of a new follicular wave. The studies reviewed here describe current methods for regulating the bovine ovarian cycle, interesting models for future studies, and information that may be used for improving reproductive efficiency.     

Risk factors associated with Mycoplasma capricolum subspecies capripneumoniae and morbillivirus infection in small ruminants in Tanzania

Tropical Animal Health and Production - Mortality of domestic small ruminants caused by contagious caprine pleuropneumonia (CCPP) and Peste des petits ruminants (PPR) is frequently reported in...     

Characteristics of Ovarian Follicle Development in Domestic Animals

In most domestic animals the later stages of follicle development occurs in a wave‐like pattern during oestrous cycles (cattle, sheep, goats, horses and buffalo) or periods of reproductive activity (llamas and camels). A follicle wave is the organized development of a cohort of gonadotrophin‐dependent follicles all of which initially increase in size, but most of which subsequently regress and die by atresia (subordinate follicles). The number of remaining (dominant) follicles is specific to the species and is indicative of litter size. Follicle waves develop during both luteal and follicular phases and it is the dominant follicle(s) of the last follicular wave that ovulates. However, there are cases where dominant follicles from the last two follicle waves can ovulate (sheep and goats). There are exceptions to the organized wave‐like pattern of follicle growth where follicle development is apparently continuous (pigs and chickens). In these animals many follicles develop to intermediate diameters and at specific times follicles that are destined to ovulate are selected from this pool and continue growing to ovulation. Understanding the pattern of follicle development in different species is increasingly important for designing improved methods to manipulate reproduction in domestic animals.     

Regional differences in concentrations of LHRH receptors,LH and FSH in the bovine adenohypophysis

Pituitaries from intact luteal phase (INT) and ovariectomized (OVX) cows were collected at slaughter to determine whether differences exist among regions of the bovine adenohypophysis in the concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and receptors for luteinizing hormone releasing hormone (LHRH). Each adenohypophysis was divided into three paired regions (anterior, AT; medial, M; posterior, PT) by first making a midsagittal cut followed by two transverse cuts of approximately equal size. Values for all variables were similar between paired regions. Mean LHRH receptor, LH and FSH concentrations were greater in OVX than INT adenohypophyseal regions. Receptor and gonadotropin concentrations differed among all three regions and were greatest in the AT, intermediate in the M and lowest in the PT regions of the adenohypophysis. There were significant correlations between LHRH receptor concentrations and concentrations of LH and FSH among the three adenohypophyseal regions for both INT and OVX cows. Therefore, to accurately characterize LHRH receptors from the bovine adenohypophysis, a midsagittal-half of the gland should be used for analysis.     

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor-α, interleukin-1α and interferon-τ

When animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F_2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF_2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF_2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF_2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF_2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF_2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF_2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts

Recently GnRH, GnRH-R systems has been demonstrated in various extrahypothalamic and extrapituitary reproductive tissues in different mammalian species, where GnRH acts in an autocrine and or paracrine manner and modulates different biological processes. GnRH-R mRNA has also been demonstrated in bovine ovaries (follicle and corpus luteum) and normal and carcinogenic human endometrium/endometrial cells. This is the first study elucidating presence of GnRH-R mRNA and GnRH-R protein in bovine uterus and oviducts in follicular and luteal phases of the estrous cycle and further localizing the receptors to endometrial and oviductal epithelial cells. To our knowledge this is the first report demonstrating GnRH-R mRNA and protein in mammalian oviducts. We used gene-specific primers and monoclonal GnRH-R antibody to test GnRH-R mRNA and GnRH-R protein through RT-PCR and immunobloting. Immunohistochemistry was employed to localize these receptors to endometrial and oviductal epithelial cells. GnRH-R mRNA and receptor protein were expressed at expected molecular weights of 920bp and 60kD, respectively. Densitometry analysis revealed that expression levels for GnRH-R protein in uterus and oviducts were similar to bovine pituitary. The presence of GnRH receptors in bovine uterus and oviducts is intriguing and it would be imperative to examine the functional role of this system in the regulation of reproductive processes.     

Lysophosphatidic Acid Synthesis and its Receptors’ Expression in the Bovine Oviduct During the Oestrous Cycle

Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early‐luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early‐luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus–oocyte complex.     

Expression of mRNA for the angiopoietin-tie system in granulosa cells during follicular development in cows

Recent findings indicate that the changing profile of angiopoietins (ANPT) and their receptor Tie2 are closely associated with development and regression of the vascular network in the cyclic ovary. We previously reported that mRNA expression for the ANPT-Tie system in theca interna changes during bovine follicular development and atresia, and both ANPTs affect steroidogenesis in the preovulatory follicle. The aim of this study was to investigate mRNA expression for ANPT1, ANPT-2 and Tie2 in granulosa cells (GC) during follicular development in the cow. Bovine follicles were classified according to the estradiol-17beta (E(2)) concentration in follicular fluid (FF) as follows: (1) E(2)<0.5, (2) 0.5180 ng/ml FF. Semi-quantitative RT-PCR analysis revealed that the expression of ANPT-1 mRNA was not detected in most of the follicle with E(2)<5 ng/ml (diameter of 5-10 mm), but clearly detected in all follicles with E(2)>5 ng/ml (diameter of >10 mm). The mRNA expression for ANPT-2 was drastically decreased in the follicles with E(2)>5 ng/ml. Tie2 mRNA expression remained unchanged at the different stages of follicular development. The present data show that ANPT-1 becomes predominant in the follicle producing high levels of E(2), indicating the possible switch-over from ANPT-2 (antagonist) to ANPT-1 (agonist). Thus, the result suggests that the ANPT-Tie system in bovine GC may stimulate E(2) secretion rather than angiogenesis in the late stages of follicular development.     

Expression of Angiopoietin (ANPT)-1, ANPT-2 and their Receptors in Dominant Follicles during Periovulatory Period in GnRH-Treated Cow

Ovarian follicular vasculature is involved in follicular development and ovulation. Angiopoietin (ANPT)-Tie system is important for vascularization of the tissue surrounding the developing follicles and corpus luteum (CL). To determine how the expression of ANPT-1, ANPT-2 and their receptors in the follicles would be associated with the ovulatory process, the present study was conducted to examine mRNA expressions of ANPT-1, ANPT-2 and their receptors during the periovulatory phase in gonadotrophin-releasing hormone (GnRH)-treated cows. The ovaries were collected by transvaginal ovariectomy (n = 5, cows/group) and the follicles (n = 5, one follicle/cow) were classified into following groups: before GnRH administration [before luteinizing hormone (LH) surge]; 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (peri-ovulation); and early CL (days 2-3). The mRNA expression was analysed by quantitative real-time PCR (rotor-gene 3000). Angiopoietin-1 expression rapidly decreased at 3-5 h and kept low level at 10 h after GnRH treatment compared with that before GnRH, and returned to the level before LH surge in the follicles >20 h after GnRH treatment. The levels of ANPT-2 mRNA decreased at 10 and 25 h after treatment compared with other periods. The ratio of ANPT-2/ANPT-1 (an index for destabilization of blood vessels) increased in the follicles at 3-5 h after GnRH treatment only. Both of Tie-1 and Tie-2 receptor expressions decreased in the follicles at 25 h after GnRH treatment. The results of the present study indicated that mRNA expressions of ANPT-1, ANPT-2 and their receptors changed in the bovine follicles during periovulatory period. These results suggest that angiopoietin-Tie system is associated with the initiation of vasculature of follicle that grows towards ovulation.     

Histomorphological and immunohistochemical study of angiogenesis and angiogenic factors in the ovary of the mare

Cyclical ovaries of 18 mares were examined histologically and immunohistochemically for vascular endothelial growth factor A and B (VEGF A; VEGF B), angiopoietin1 and 2 (Ang1; Ang2), vascular endothelial growth factor receptor 1 and 2 (VEGF-R1; VEGF-R2), angiopoietin receptor (Tie2) and von Willebrand factor. The most intensive coexpression of the examined factors and receptors was detected in the periovulatory period, when a distinctive ovarian angiogenesis takes place, being essential for tertiary follicle maturation and for the endocrine function of the Corpus luteum. Based on the immunohistochemical results, VEGF A, Ang2, VEGF-R2 and Tie2 in particular seem to play a significant role on angiogenesis during follicular and luteal development in the mare, while Ang1 supports vessel stabilisation. The findings of luteal regression and follicular atresia showed that, in the absence of VEGF A, Ang2 and its receptor Tie2 contribute substantially to vessel regression and therefore to luteolysis and follicular atresia.     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.