Oocyte-somatic cell interaction plays a crucial role in preantral folliculogenesis. Although topical research has focused on oocyte-granulosa cell interaction over the past decade, an oocyte-thecal cell regulatory loop may also play an important role during the preantral stage. Formation of the thecal cell layer is a key event that occurs during preantral folliculogenesis. Granulosal factors (e.g. IGF-I and KL) appear to stimulate the recruitment of thecal cells from stromal cells. Oocyte-derived GDF9 appears to indirectly modulate thecal cell differentiation, perhaps through regulating the granulosal IGF-I and KL expression. Theca-produced androgens stimulate granulosa cell proliferation and preantral follicle growth. GDF9 enhances preantral follicle growth by up-regulating thecal androgen production, suggesting that a threshold level of androgens derived from thecal cells is necessary for preantral follicle growth, and that the androgen production may be controlled by the oocyte-derived GDF9. The challenge ahead is not to only understand the precise nature of these interactions, and to elucidate how dysregulation in these interactions may lead to ovarian pathologies such as polycystic ovary syndrome and gonadotropin poor-responsiveness.
Coordination of extra- and intrafollicular signals is required for the development of ovarian follicles and for the production of functional oocytes. Oocytes play an active role in this coordination. Oocytes produce two families of growth factors: members of the transforming growth factor beta (TGFβ) superfamily, including bone morphogenetic protein (BMP) 6, BMP15, and growth differentiation factor 9 (GDF9); and members of fibroblast growth factors (FGFs), including FGF8. These oocyte-derived paracrine factors, in coordination with the other intrafollicular signals, regulate the development and function of oocyte-associated cumulus cells. In this review, we first summarize the role of oocytes in follicular development and ovulation, focusing on the effects of oocyte-derived paracrine factors on the development and function of cumulus cells. In addition, we summarize recent findings on the coordination of oocyte-derived signals with other intrafollicular signals, such as estrogen and epidermal growth factor (EGF) receptor signals, in cumulus cell development and function, and discuss the potential mechanisms driving this coordination.
Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following preovulatory gonadotropin stimulation. In response to the preovulatory luteinizing hormone (LH) increase, oocytes undergo GVBD, followed by first polar body extrusion. Although the preovulatory surge of LH is the primary event responsible for the induction of maturation of the oocyte, LH does not act directly on the oocyte due to the absence of functional LH receptors in germ cells. Instead, actions of LH are mediated either by paracrine factors secreted by LH-responsive somatic cells or by the transport of cellular messengers from granulosa/cumulus cells to oocytes through intercellular gap junctions. In addition to the nuclear maturation exemplified by GVBD and extrusion of the first polar body to complete the first meiotic division, oocytes also undergo cytoplasmic maturation characterized by cytoplasmic changes essential for monospermic fertilization, processing of the sperm, and preparation for development to preimplantation embryos. In this review, we summarize our recent works on the identification and characterization of novel LH-inducible ovarian factors for nuclear and cytoplasmic maturation of oocytes.
Follicular development and ovulation process are complex events in which a sequential process is started by two pituitary hormones, FSH and LH. Recent studies using microarray analysis have shown that numerous endocrine factors are expressed and specifically activate the signal transduction cascades to upregulate or downregulate the expression of target genes in granulosa cells and cumulus cells. Especially, the PI 3-kinase-AKT pathway during the follicular development stage and the ERK1/2 pathway after LH surge are essential for initiating the dramatic changes in follicular function. The proliferation of granulosa cells and cell survival are dependent on the FSH-induced PI 3-kinase-AKT pathway in a c-Src-EGFR dependent manner. On the other hand, the transient activation of ERK1/2 by LH surge is dependent on de-novo transcription and translation. EGF-like factors, especially amphiregulin, that are expressed in a cAMP-PKA-CREB dependent manner, act on EGFR expressed on granulosa cells and cumulus cells. The phosphorylated EGFR induces the RAS-cRAF-MEK-ERK1/2 pathway in both cell types. One of the ERK1/2 target molecules is C/EBP that is a member of transcription factors increasing the expression of genes involved in granulosa cell luteinization, cumulus expansion and oocyte maturation. These signaling cascades upregultaed by FSH or LH in granulosa cells play important roles in follicular development and ovulation process.
Although injected freeze-dried hamster spermatozoa can develop into male pronuclei even after 12 months of storage at 4ºC, the developmental competence of hamster pronuclear oocytes is not well understood. Furthermore, production of live offspring from freeze-dried spermatozoa is limited in some animals, such as mice, rabbits and rats. Here, we report the birth of hamster offspring following intracytoplasmic injection with freeze-dried spermatozoa. The integrity of the sperm DNA after freeze-drying and rehydration is very important for the developmental competence of hamster embryos produced by injecting freeze-dried spermatozoa into oocytes. This study used the TUNEL method to examine DNA fragmentation and the chromosomal integrity of spermatozoa after freeze-drying using two freezing media: M2 medium, and a Tris-HCl buffered solution containing 50 mM EGTA (EGTA solution). The rate of DNA fragmentation when hamster spermatozoa freeze-dried in EGTA solution was used was significantly (P < 0.05) lower than that in M2 medium (4.3% vs. 41.4%), and the chromosomal integrity in EGTA solution was higher in EGTA solution than in M2 medium (81.1% vs. 41.0%). The percentage of morulae/blastocysts derived from hamster spermatozoa freeze-dried in EGTA solution was significantly (P < 0.05) higher than that derived from spermatozoa freeze-dried in M2 medium (62.2% vs. 12.5%). After transfer to foster mothers, 3 of 23 morulae/blastocysts developed into live offspring.
After screening 269 crude drugs for their ability to inhibit the development of mouse zygotes, we found Coptis rhizome and Phellodendron bark to have inhibitory effects. We examined the effects of both extracts and of berberine, a major component of these plants, on in vitro development of zygotes and on full-term fetal development in the mouse. Mouse zygotes were cultured in medium containing water-soluble extracts of Coptis rhizome or Phellodendron bark, or berberine at various concentrations for 5 days and the potential of zygotes to develop to blastocysts was examined. In addition, superovulated mice were intramuscularly injected with berberine and mated, and examined for the in vivo development of fertilized eggs to blastocysts and full-term fetuses. In vitro development of zygotes to blastocysts was almost completely inhibited when they were cultured in medium containing more than 0.1 μg/ml Coptis rhizome, 10 μg/ml Phellodendron bark, or 0.01 μg/ml berberine chloride or berberine sulfate. When superovulated and mated females received 100 μg berberine chloride once a day for 2 to 14 days, the proportions of recovered blastocysts and full-term fetuses were significantly decreased. The present study indicates the potential use of berberine as a contraceptive for animals.
Conventional freezing procedures are not appropriate for surgically retrieved spermatozoa from the epididymis or testis because of their low numbers. Techniques for the cryopreservation of small numbers of spermatozoa have not been fully established. We tried to develop a cryopreservation method for a single spermatozoon using Cryotop, which has a simple structure and is easy to handle. Different parameters influencing the freezing procedure, types of container, sources of spermatozoa, and cryoprotectants were evaluated. The sperm recovery rate after thawing was similar between the sperm frozen using Cryotop or zona pellucida as containers (98.0% vs. 88.0%). Freezing of motile single spermatozoa obtained from ejaculates and testes were evaluated for recovery rate (90.0% vs. 95.0%) and motility rate (44.4% vs. 42.1%), which were not significantly different. The survival rate was significantly higher when sperm were treated with sucrose rather than with SpermFreeze (65.3% vs. 37.3%, P < 0.01). Cryotop was a highly effective tool for the cryopreservation of a single spermatozoon, and sucrose was determined to be an efficient cryoprotectant.
The number of cumulus–oocyte complexes (COCs) in a droplet is known to affect oocyte maturation rates. In this study, we investigated whether droplet size or the number of COCs examined in in vitro maturation (IVM) affect mouse oocyte maturation, fertilization, or early embryonic development. Moreover, we aimed to determine the optimal, practical culture condition of IVM that could be achieved without changing the culture medium composition. The droplet sizes used were 20, 50, 100, and 200 μl, and the numbers of COCs examined were 1, 5, 10, 20, and 50. The groups with oocyte maturation rates exceeding 75% were treated with in vitro fertilization and in vitro culture. Compared with in vivo-matured oocytes, the 20 COCs/100-μl group did not exhibit significant differences in developmental competence and quality. We also observed progression to blastocysts in the 5-COC group, but the zygotes were likely to form multiple pronuclei. The results indicate: (1) droplet size and numbers of COCs examined in IVM affect oocyte quality; (2) the 20 COCs/100-μl condition effectively stimulated maturation in all experiments in this study; and (3) culture of 5 COCs in IVM can also produce blastocysts.
In mammals, the mechanisms regulating the initiation of follicle development remain poorly understood. We previously reported that testosterone induced the activation of porcine primordial follicles after 7 days of culture. Herein, the effect of KIT ligand (KL) on the viability, activation and development of porcine primordial follicles was examined. Ovarian strips containing primordial follicles were cultured for 7 days in a medium supplemented with KL (10, 100, 250 or 500 ng/ml). The percentage of degenerated follicles was significantly decreased (39 ± 2%) in the 100 ng/ml KL-treated group compared to the control (56 ± 1%), whereas almost all of the primordial follicles degenerated at higher concentrations of KL. No primordial follicles developed in culture at any concentration of KL. When ovarian strips were cultured in medium supplemented with testosterone (10–6 M) + KL (100 ng/ml), primordial follicles developed to early secondary follicles (5 ± 3%), however the total percentages of developing follicles in the testosterone group (21 ± 5%) and the testosterone + KL group (28 ± 5%) were not significantly different. These results suggest that KL does not promote primordial follicle activation, although it does promote follicle viability in culture.
The purpose of this study was to examine the effects of different fractions of bovine follicular fluid (bFF) and fetal bovine serum (FBS) as maturation medium supplements on development to the blastocyst stage of bovine oocytes matured and fertilized in vitro. Three bFF fractions obtained by ultracentrifugation were designated as the 1st, 2nd and 3rd fractions, and four FBS fractions, also obtained by ultracentrifugation, were designated as the 1st, 2nd, 3rd and 4th fractions. Cumulus-oocyte complexes (COCs) were divided into two groups: those with homogeneous ooplasm and those with heterogeneous ooplasm. COCs were cultured in basic maturation medium with a bFF fraction or FBS fraction. In oocytes with homogeneous ooplasm, the rates of development to the blastocyst stage with bFF-1st and bFF-2nd were significantly higher than that with bFF. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with bFF-2nd was significantly higher than that with bFF. In oocytes with homogeneous ooplasm, the rate of development to the blastocyst stage with FBS-1st was significantly lower than that with FBS. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with FBS-3rd was significantly higher than that with FBS. The results indicate that bFF-2nd and FBS-3rd, obtained by ultracentrifugation, are effective maturation medium supplements, as they promoted the development of high-quality matured bovine oocytes.
The present study analyzed tumor protein p53 gene (TP53) polymorphism as a factor in uterine implantation and establishment of pregnancy in four old-aged Japanese Black cows with long-term infertility. They had G allele or A allele in exon 6 of the gene. Oocytes were collected from these females using ovum pick up (OPU) and fertilized in vitro. Only some of the oocytes from individuals having G allele had developmental competence to the blastocyst stage, while no oocytes having the A allele had the competence to develop to the blastocyst stage. In conclusion, we suggest that a cause of long-term infertility in old-aged Japanese Black cows may be a polymorphism of TP53 affecting the competence of embryonic development.