In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n=37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6+/-0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5+/- 0.6 versus day 10 = 10.4 +/-0.6 primary follicles/section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.     

CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR‐mediated signaling in preovulatory follicles

Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C‐type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression. Mol. Reprod. Dev. 79: 795–802, 2012. © 2012 Wiley Periodicals, Inc.     

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.     

Presence of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles and improvement of in vitro preantral follicle survival and development with GH

This study aimed to demonstrate the expression of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles in order to investigate the effects of GH on the survival and development of preantral follicles. The ovaries were processed for the isolation of follicles to study GH-R mRNA expression or to localization of GH-R by immunohistochemical analysis. Pieces of ovarian cortex were cultured for 7 days in minimum essential medium⁺ (MEM⁺) in the presence or absence of GH at different concentrations (1, 10, 50, 100, and 200 ng/mL). High expression levels of GH-R mRNA were observed in granulosa/theca cells from large antral follicles. However, preantral follicles do not express mRNA for GH-R. Immunohistochemistry demonstrated that the GH-R protein was expressed in the oocytes/granulosa cells of antral follicles, but any protein expression was observed in preantral follicles. The highest (P < 0.05) rate of normal follicles and intermediate follicles was observed after 7 days in MEM⁺ plus 10 ng/mL GH (70%). In conclusion, GH-R mRNA and protein are expressed in caprine antral follicles, but not in preantral follicles. Moreover, GH maintains the survival of goat preantral follicles and promotes the development of primordial follicles.     

Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.     

Estrogen receptor alpha and beta expression in the porcine ovary

In order to investigate the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the porcine ovary, in situ hybridization was applied. Specific ovine ERalpha and bovine ERbeta cDNA probes were labeled with [-32S]dCTP. In the porcine ovary, positive signals for ERbeta were found in both granulosa and theca cells of all types of antral follicles as well as in the corpora lutea at all stages of regression. ERalpha mRNA was limited exclusively to the granulosa cells of preovulatory follicles and was present in a few cells of early corpora lutea. Thus, we showed differential expression of ERalpha and ERbeta at the mRNA level. Large antral follicles and early corpora lutea are the site, where both forms of estrogen receptor are expressed.     

Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles

Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.     

Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.     

Bovine oocytes in secondary follicles grow and acquire meiotic competence in severe combined immunodeficient mice

Cortical tissues containing only primordial and primary follicles, or secondary follicles 140-190 microm in diameter, were collected from bovine ovaries and xenografted under the kidney capsules of female severe combined immunodeficient (SCID) mice. Histological examination revealed that all grafts were well vascularised and contained surviving follicles at 4 or 6 weeks after grafting. Primordial and primary follicles survived but did not develop beyond the one-layer stage. Secondary follicles, on the other hand, had formed antra at 4 weeks after grafting. The mean diameter of secondary follicles, which was 165.2 +/- 17.0 microm (n = 42) before grafting, had developed to 442.9 +/- 77.9 microm (n = 37) and 592.9 +/- 116.0 microm (n = 45) in diameter at 4 and 6 weeks after grafting, respectively. The mean diameter of oocytes, which was 55.1 +/- 4.9 microm (n = 42) before grafting, also increased significantly (4 weeks: 105.6 +/- 6.3 microm; 6 weeks: 122.2 +/- 2.6 microm; p < 0.05). Oocytes were recovered from follicles that had developed to more than 400 microm in diameter after 6 weeks, and were subjected to subsequent mature culture. Of these oocytes, 34% (11/32) resumed meiosis and 6% (2/32) matured to the second metaphase. Follicular fluid in bovine antral follicles developed in SCID mice had the 69 kDa protein, which was detected by anti-mouse albumin antibody but not by anti-bovine albumin antibody in immunoblotting analysis. These results demonstrated that bovine secondary follicles develop to the antral stage in SCID mice, and that the oocytes in the follicles acquire the meiotic competence.     

Mouse oocytes suppress cAMP-induced expression of LH receptor mRNA by granulosa cells in vitro

Mouse oocytes suppress follicle-stimulating hormone (FSH)–induced luteinizing hormone receptor (LHR) messenger ribonucleic acid (mRNA) expression in cultured granulosa cells. The objective of this study was to assess the mechanism by which oocytes suppress FSH-induced LHR expression. The effect of cumulus cell–denuded, germinal-vesicle-stage oocytes, isolated from antral follicles, on FSH-induced cyclic adenosine monophosphate (cAMP) production by cultured granulosa cells was determined by radioimmunoassays. In addition, the effect of oocytes on 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells was assessed by RNase protection assays. Oocytes had no detectable effect on FSH-induced cAMP production. However, oocytes dramatically suppressed 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells. It was concluded that the mechanism by which oocytes suppress FSH-induced steady-state expression of LHR mRNA is not by inactivating FSH, preventing functional interactions of FSH with its granulosa cell receptors, or by interfering with the signal-transduction mechanisms required for FSH-dependent cAMP production. In addition, since oocytes suppressed the 8Br-cAMP–induced increase in steady-state expression of mRNA for LHR, oocyte-derived factors probably suppress expression by acting downstream of FSH-induced elevation of granulosa cell cAMP. Mol. Reprod. Dev. 49:327–332, 1998. © 1998 Wiley-Liss, Inc.     

Differential expression of ZPC in the bovine ovary,oocyte, and embryo

Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished. Mol. Reprod. Dev. 49:435–443, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural localisation of calcium deposits in pig ovarian follicles

Calcium intracellular signaling regulates many intracellular events including oocyte maturation. This signaling is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. In this study, intracellular calcium deposits were detected in follicle-enclosed pig oocytes using the combined oxalate-pyroantimonate method. These deposits were observed in the nucleus, the mitochondria, the cytoplasm, and on the surface of lipid droplets. The amount of calcium deposits was expressed as a percentage of the area of the respective cellular compartment, which is covered with calcium deposits on ultrathin sections. The distribution of calcium deposits in oocytes changed during folliculogenesis. The amount of calcium deposits in nuclei (1.11% of the area of oocyte nuclei) and cytoplasm (1.02%) in oocytes from secondary and early antral follicles (0.90% nuclei; 0.99% cytoplasm) is significantly lower (P < 0.05) than the amount of calcium deposits in these compartments in oocytes from primary follicles (2.51% nuclei; 2.34% cytoplasm) or antral follicles with growing oocyte (2.91% nuclei; 2.21% cytoplasm). The amount of calcium deposits in mitochondria of oocytes from primary follicles (1.27%) or antral follicles with growing oocyte (1.14%) is significantly lower (P < 0.05) than in the nucleus (2.51% in oocytes from primary follicles; 2.91% in growing oocytes from antral follicles) or cytoplasm (2.34% in oocytes from primary follicles; 2.21% in growing oocytes from antral follicles). The amount of calcium deposits in the cytoplasm of fully-grown oocytes (1.46%) dropped to levels significantly lower (P < 0.05) than those observed in the oocyte nucleus (2.29%). On the basis of these data, we can conclude that the population of follicles on pig ovaries differs in the distribution and concentration of calcium deposits in oocytes, and these changes may be involved in the regulation of the meiotic competence of oocytes.