Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro.

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2mM glutamine and 1% antibiotic-antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (5 ng/mL): Control, FSH (0.5 microg/mL), LH (5 microg/mL), estradiol-17beta (E2, 1 microg/mL), FSH+LH, and FSH+LH+E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected (P=0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased (P<0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH+LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH+LH+E2+EGF group (83.5+/-3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended (P=0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased (P=0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.

In vitro maturation of bitch oocytes from advanced preantral follicles in synthetic oviduct fluid medium: serum is not essential.

The ability of oocytes from preantral follicles to mature in vitro was assessed using a synthetic oviduct fluid (SOF) medium. Advanced preantral follicles (approximately 210 microm diameter) were isolated from the ovaries of domestic bitches and assigned to one of four treatment groups: (1) SOF (n = 230); (2) SOF + 3 mg/ml bovine serum albumin (+BSA, n = 220); (3) SOF + 20% fetal bovine serum (+FBS, n = 227); or (4) SOF + 3 mg/ml BSA + 20% FBS (+BSA+FBS, n = 232), then cultured for up to 72 h. A group of control follicles was not cultured (n = 103). The percentages of oocytes reaching metaphase I to metaphase II stages (MI to MII) did not differ between treatments at each culture period. Within treatments, the percentages of oocytes at MI to MII stages did not differ with duration of culture. However, when compared to the control group (0.97%) the percentages of oocytes at MI to MII increased (P < 0.05) in the SOF group after 48 h (10.0%) and 72 h (12.2%) of culture. In the +BSA (10.1%) and +FBS (9.7%) groups, the percentages of oocytes at MI to MII increased (P < 0.05) above control values only after 72 h of culture. The percentage of oocytes at MI to MII did not significantly increase in the +BSA+FBS group (3.9,6.6 and 7.6% at 24,48 and 72 h of culture, respectively) compared to the control group. These results indicate that under the described conditions supplementation of culture medium with BSA or FBS is not essential, and the simple medium SOF can support nuclear maturation of a small proportion of bitch oocytes in vitro.

Effects of external pH on hormonally regulated ovarian follicle maturation and ovulation in Atlantic croaker.

In vitro studies of ovarian follicle maturation and ovulation in teleost fishes typically are conducted within a narrow range (7.5-7.8) of constant external (medium) pH, although there is evidence that pH can influence ovulation. Therefore, this study with Atlantic croaker investigated the effects of external pH on hormonally regulated in vitro maturation and ovulation as well as changes in the pH of ovarian fluid during in vivo maturation and ovulation. For the in vitro experiments, follicles were first incubated with human chorionic gonadotropin (hCG) to induce maturational and ovulatory competencies, and then with maturation-inducing hormone (MIH) to induce completion of maturation and ovulation. At a constant external pH within the range of 7.0-8.2, the lower pH levels (7.0-7.3) generally inhibited or slowed down hormonally induced maturation and ovulation whereas higher pH (7.6-8.2) facilitated these processes. When ovarian follicles were incubated at a constant pH of 7.6 during the priming incubation with hCG, changing the external pH during the incubation with MIH had relatively little effect on oocyte maturation or ovulation. Thus, the inhibitory effect of constant low levels of external pH (7.0-7.3) on maturation and ovulation may be primarily due to disruptions in the gonadotropin-dependent acquisition of maturational and ovulatory competencies. The pH of ovarian fluid remained constant at 8.5 during in vivo ovarian follicle maturation and ovulation. Subsequent in vitro tests showed that external pH of 8.5 enhances hormonally induced maturation and ovulation relative to pH of 7.6. These observations suggest that attention should be paid to the pH of incubation media used in basic research and in biotechnological applications relying on in vitro maturation and ovulation in teleosts. Further, an understanding of the physiological significance of the enhancing effect of alkaline pH on maturation and ovulation will require determination of the intrafollicular pH around the oocyte during the periovulatory period.

Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker.

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F(2alpha) and PGE(2), whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 microM) and PGF(2alpha) (5 microM) did not induce maturation, and NDGA (10 microM) did not affect MIH-dependent maturation. However, IM (100 microM) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 microg/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 microM; H7, 50 microM) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 microM) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF(2alpha) restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1). LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2). PKC is downstream of genomic activation.

Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker.

Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-microm segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P<0.05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study.

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles.

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.