Women with reduced ovarian reserve or advanced maternal age have an altered follicular environment.

OBJECTIVE: To determine whether altered follicular environment is associated with ovarian reserve or maternal age. DESIGN: Prospective study examining follicular fluid (FF) composition and follicular cell metabolism. SETTING: University research department and private IVF clinic. PATIENT(S): Women (n = 54) undergoing routine IVF treatment were allocated to one of three groups based on ovarian reserve and maternal age. INTERVENTION(S): Surplus FF, granulosa cells (GC), and cumulus cells (CC) were collected. MAIN OUTCOME MEASURE(S): Follicular fluid concentrations of carbohydrates, hormones, and selected ions. Metabolic analysis and gene expression of GCs and CCs. RESULT(S): Compared to women <35 years with normal ovarian reserve, FF glucose levels were significantly decreased and lactate and progesterone (P4) concentrations significantly increased in women with reduced ovarian reserve or advanced maternal age, whereas GC and CC glucose uptake, lactate production, and phosphofructokinase platelet gene expression were significantly increased. Granulosa cell P4 production from women with reduced ovarian reserve or advanced maternal age was decreased; however, in CCs the reverse was observed with increased gene expression in P4 receptor, prostaglandin E receptor-2, cytosolic phospholipase A2, and tumor protein 53. CONCLUSION(S): Women with either reduced ovarian reserve or advanced maternal age have altered follicular cell metabolism, FF metabolites, and P4 production. This perturbed environment may be responsible for impaired oocyte developmental competence and subsequent embryo development.

Transforming growth factor-ß (TGFß) in porcine seminal plasma.

Bioactive factors in seminal plasma induce cellular and molecular changes in the female reproductive tract after coitus. An active constituent of seminal plasma in mice and humans is the potent immune-modulating cytokine transforming growth factor-ß (TGFß). To investigate whether TGFß is present in boar seminal plasma, TGFß(1) and TGFß(2) were measured by immunoassay. High levels of TGFß(1) and TGFß(2) were detected in 100% of seminal fluid samples from 73 boars. Both were predominantly in the active, not latent form. Interferon-γ (IFNγ) and lipopolysaccharide (LPS), agents that interact with TGFß signalling, were detectable in 5% and 100% of samples, respectively. TGFß(1) and TGFß(2) concentrations varied widely between boars, but correlated with each other and with sperm density, and remained relatively constant within individual boars over a 6-month period. Frequent semen collection substantially diminished the concentration of both TGFß isoforms. Using retrospective breeding data for 44 boars, no correlation between TGFß content and boar reproductive performance by artificial insemination (AI) with diluted semen was found. It is concluded that TGFß is abundant in boar seminal plasma, leading to the speculation that, in pigs, TGFß may be a male-female signalling agent involved in immune changes in the female reproductive tract elicited by seminal fluid.

Relationship between cumulus cell apoptosis, progesterone production and porcine oocyte developmental competence: temporal effects of follicular fluid during IVM.

The objective of the present study was to determine the temporal effects of sow follicular fluid (FF) in vitro on cumulus cell viability and function, as well as oocyte nuclear and cytoplasmic maturation. Cumulus-oocyte complexes (COCs) recovered from the ovaries of prepubertal pigs were matured in medium with (+FF) or without (-FF) follicular fluid for the first 22 h of IVM. At 22 h of IVM, each group of COCs was then transferred to medium with or without FF and matured for another 22 h, forming four treatment groups (-FF/-FF, -FF/+FF, +FF/-FF and +FF/+FF). The concentration of progesterone in spent IVM medium and the incidence of cumulus cell apoptosis in individual COCs were determined at 22 and 44 h of IVM. Cumulus expansion was also recorded at 44 h of IVM. Finally, the ability of oocytes to complete meiosis to the MII stage and form blastocysts after IVF and embryo culture was assessed. Maturation with FF for part or the whole of IVM increased cumulus expansion and progesterone production and decreased the incidence of cumulus cell apoptosis compared with the -FF/-FF group (P < 0.05). The changes were greatest for the +FF/+FF group and intermediate for the -FF/+FF and +FF/-FF groups. Regression analysis revealed a negative association between cumulus cell progesterone production and the incidence of cumulus cell apoptosis (P < 0.001). Meiotic maturation was enhanced when FF was present during the first half of IVM. Oocytes matured in the presence of FF during the first and/or second half of IVM displayed an increased ability to form blastocysts compared with the -FF/-FF group (P < 0.05). The extent of the increase was similar for all FF-supplemented groups. The results show that FF exerts several beneficial effects at different times during IVM and suggest that a major role of FF is to provide protection from oxidative stress. We propose that the incidence of cumulus cell apoptosis in COCs must be kept below a certain threshold to ensure adequate functionality, including steroidogenic activity, is maintained for the acquisition of oocyte developmental competence.

Serum testosterone bioassay evaluation in a large male cohort.

OBJECTIVE: To assess if a cell-based readout of androgen action in serum demonstrates a closer association with recognized classical parameters of androgen action in men than current measures of serum testosterone (T). DESIGN: To develop, validate and utilize a mammalian cell-based assay to measure specifically bioactive T and determine if this measure is a physiologically relevant fraction of serum T. MEASUREMENTS AND PARTICIPANTS: We have developed a specific serum T bioassay using human prostate cancer cells. A rapid 5-min exposure to 100% serum followed by serum withdrawal confers specificity of the assay to serum T and provides sufficient sensitivity to measure T in male serum samples. Matrix effects were experimentally discounted as a confounding issue. A total of 960 male serum samples from the Florey Adelaide Male Ageing Study (FAMAS) with previous comprehensive cohort data and serum measurements were utilized. RESULTS: Bioassay T measurement in the 960 FAMAS serum samples returned a median of 10.7 nmol/l (1.7-45.4), and was most closely related to immunoassayed total T, but not immunoassayed bioavailable T or calculated free T. Immunoassayed total T demonstrated a positive association with isometric grip-strength (R(2) = 0.127, P < 0.001), self-reported sexual desire (R(2) = 0.113, P < 0.001) and erectile function (R(2) = 0.085, P < 0.05) while bioassay T did not. CONCLUSIONS: While cellular bioassays offer a rapid and sensitive means of identifying the androgenic potential of complex environmental compounds, the utility of such assays in defining a clinically relevant fraction of serum T distinct from total T needs further investigation.

Effect of follicle size and dibutyryl cAMP on the cAMP content and gap junctional communication of porcine prepubertal cumulus-oocyte complexes during IVM.

The lower ability of oocytes from prepubertal pigs to yield viable embryos than those from adult pigs appears, in part, a consequence of their reduced ability to accumulate cAMP during IVM. The present study examined the cAMP content of oocytes and cumulus-oocyte complexes (COCs), cumulus expansion and gap junctional communication (GJC) in COCs from 3- and 5-8-mm follicles during IVM. The effect of 1 mm dibutyryl cAMP (db-cAMP) treatment for the first 22 h of IVM was also examined for both follicle size classes. The cAMP concentration of oocytes from 5-8-mm follicles was threefold greater than that in oocytes from 3-mm follicles following 11 h of IVM (11.9 +/- 5.9 v. 3.6 +/- 1.8 fmol, respectively; P < 0.05). In the presence of db-cAMP, the cAMP content of oocytes from 3- and 5-8-mm follicles was no longer significantly different at 11 h IVM. The cAMP concentration of intact COCs from 5-8-mm follicles was significantly higher than that in COCs from 3-mm follicles at 11 h (1110.6 +/- 318.0 v. 116.9 +/- 55.7 fmol, respectively; P < 0.05). Despite maturation with db-cAMP, the cAMP content in COCs from 3- and 5-8-mm follicles at 11 h of IVM remained significantly different (15.1 +/- 4.9 v. 196.2 +/- 33.3 fmol, respectively; P < 0.05). The COCs from 3-mm follicles displayed lower cumulus expansion than did COCs from 5-8-mm follicles at both 11 h (cumulus expansion index (CEI) 1.0 +/- 0.1 v. 1.8 +/- 0.1, respectively; P < 0.01) and 22 h (CEI 1.9 +/- 0.3 v. 2.9 +/- 0.2, respectively; P < 0.05) of IVM. The level of cumulus cell-oocyte GJC decreased during IVM, with the number of GJC significantly greater in COCs from 3-mm compared with 5-8-mm follicles at both 6 h (613 +/- 55 v. 304 +/- 44 fluorescence intensity (FI), respectively; P < 0.05) and 11 h (644 +/- 99 v. 337 +/- 38 FI, respectively; P < 0.05) of IVM. By 22 h of IVM, the GJC of COCs from 3-mm follicles had decreased (227 +/- 18 FI) and was no longer significantly different to that of COCs from 5-8-mm follicles (139 +/- 15 FI; P > 0.05). Dibutyryl cAMP had no effect on the cAMP content, cumulus expansion or GJC of the whole COC. In conclusion, the results of the present study demonstrate that COCs from 3-mm follicles accumulate less intraoocyte and inter-COC cAMP, display lower cumulus expansion and maintain their cumulus cell-oocyte GJC for longer during IVM than do COCs from 5-8-mm follicles.

Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs.

The present study compared the distribution and steroid composition of 3-, 4- and 5-8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5-8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5-8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5-8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5-8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion.

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes.

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.

Effects of milrinone and butyrolactone-I on porcine oocyte meiotic progression and developmental competence.

Inappropriate coordination of oocyte nuclear and cytoplasmic maturation is thought to contribute to the poor efficiency of embryo production in vitro. With the aim of improving this coordination, the effects of milrinone, an inhibitor of type 3 phosphodiesterases, and butyrolactone-I, a selective inhibitor of cdc2 kinases, on porcine oocyte maturation were investigated. Oocytes recovered from slaughterhouse-derived ovaries of prepubertal animals were treated with the inhibitors for 24 h. At concentrations of 50 and 250 microm, milrinone reversibly inhibited meiotic progression in 57% and 71% of oocytes, respectively. The presence or absence of milrinone in the medium used to wash oocytes for 30 min did not alter the inhibitory effect of the 24 h treatment. At concentrations of 25 and 50 microm, butyrolactone-I inhibited meiotic progression in 61% and 66% of oocytes, respectively, but the effect was not fully reversible in the absence of follicle-stimulating hormone (FSH). The presence of FSH during the butyrolactone-I treatment period increased the ability of oocytes to subsequently complete meiosis at 44 h without changing the inhibitory effect at 24 h. Following in vitro fertilisation at 44 and 50 h, treatment with butyrolactone-I and milrinone, alone or in combination, did not alter embryo cleavage rate, blastocyst formation rate or blastocyst cell number. Despite the different actions of milrinone and butyrolactone-I, the present study demonstrates that these reagents inhibit meiotic progression to a similar extent in the presence of FSH while maintaining developmental competence in porcine oocytes.

Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion.

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex, which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II and is currently the primary candidate molecule for the cumulus-expansion enabling factor. This study was conducted to determine whether GDF9 is the mouse cumulus-expansion enabling factor. Cumulus-oocyte complexes were collected from mice, and the oocyte was microsurgically removed to generate an oocytectomized (OOX) complex. OOX complexes treated with FSH alone or recombinant mouse GDF9 alone failed to expand, whereas expansion was induced in the presence of FSH by GDF9, TGFbeta1, or coculture with oocytes. A specific GDF9-neutralizing antibody, mAb-GDF9-53, neutralized the expansion of OOX complexes in response to GDF9 but not the expansion of OOX complexes cocultured with oocytes. Using real-time RT-PCR, hyaluronan synthase 2 (HAS2) mRNA expression by OOXs was up-regulated 4- to 6-fold by oocytes and GDF9. Monoclonal neutralizing antibody-GDF9-53 attenuated GDF9-induced OOX HAS2 expression but not oocyte-induced HAS2 expression. A TGFbeta antagonist neutralized TGFbeta-induced, but not oocyte-induced, expansion of OOX complexes, and when combined with monoclonal neutralizing antibody-GDF9-53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the bone morphogenetic protein receptor II extracellular domain, which is a known GDF9 antagonist, completely antagonized GDF9-induced expansion but only partially neutralized oocyte-induced expansion. This study provides further evidence that like TGFbeta, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFbeta alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion.

Seminal plasma regulates corpora lutea macrophage populations during early pregnancy in mice.

In mice, exposure of the uterus to seminal plasma at mating initiates an inflammatory response within the endometrium, which is characterized by production of cytokines that recruit and activate leukocytes. We hypothesized that this seminal plasma-induced inflammatory response would extend to the ovary, increasing leukocyte abundance within corpora lutea and potentially enhancing progesterone synthesis. Female mice mated to males with their seminal vesicles surgically removed exhibited fewer macrophages within corpora lutea on the day after mating, compared with females mated to vasectomized or normal, intact males. The mean number of F4/80-positive macrophages and major histocompatibility complex (MHC) class II-positive activated macrophages was approximately 2-fold fewer in the absence of seminal vesicle fluid. The effects of seminal plasma on macrophage abundance subsided by Day 4 and were not accompanied by a change in serum progesterone levels during luteinization (Days 1, 2, or 4 after mating) or luteolysis (Days 6 or 9). In vitro secretion of progesterone from corpora lutea cultured with or without LH also did not differ between treatment groups. There was no effect of seminal plasma deficiency in males on the number of ovulated ova or corpora lutea in females. These results imply that seminal plasma exposure of the female reproductive tract at mating augments the macrophage population of newly formed corpora lutea, although these additional macrophages seem not to play a role in steroidogenesis and may instead be involved in tissue remodeling within corpora lutea.

Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels.

In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to approximately 40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.

Relationship between donor animal age, follicular fluid steroid content and oocyte developmental competence in the pig.

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73% P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL(-1); P < 0.005) and androstenedione (70 v. 16 ng mL(-1); P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17beta-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed 'oocyte capacitation'. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.

The influence of seminal plasma on ovarian function in pigs--a novel inflammatory mechanism?

Seminal plasma is increasingly recognised as contributing to the reproductive process in roles apart from that of providing nutritive support and transport for spermatozoa. Seminal components elicit inflammatory responses in the female reproductive tract, including altered patterns of cytokine secretion, which have consequences for early embryo development and implantation. This review examines evidence, generated principally in the porcine model, for a more recently recognized role for seminal plasma in regulating the temporal kinetics of ovulation, corpus luteum development and steroid production in the ovary. Molecular mechanisms that operate to facilitate communication via a novel semen-uterine-ovarian axis are postulated. A better understanding of these events may facilitate development of strategies to ensure maximal fertility and reduce embryo mortality in the pig and other polyovular species.

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation.

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.

Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation.

The differential regulation of cAMP levels within the oocyte and somatic (cumulus) cell compartments of the bovine follicle, and the subsequent regulation of oocyte meiotic maturation was examined through specific cell-type localisation of phosphodiesterases (PDEs). Selective PDE inhibitors were used to modulate cAMP levels in each of the two follicular compartments and to examine their effects on oocyte meiotic maturation. Ovaries were obtained from an abattoir and cumulus-oocyte complexes (COC) were aspirated from antral follicles into culture medium supplemented with 4 mg/ml BSA and 2mM 3-isobutyl-1-methylxanthine (IBMX). COC, denuded oocytes (DO), or mural granulosa cells (MGC) were cultured either with or without forskolin or FSH, in the presence of specific PDE inhibitors; either milrinone (PDE3 inhibitor), cilostamide (PDE3 inhibitor), or rolipram (PDE4 inhibitor). COC/DO cultures were assessed for meiotic progression and cAMP content, and MGC for cAMP production. The type 3 PDE inhibitor, but not the type 4, prevented spontaneous meiotic maturation and elevated intraoocyte cAMP in cultured denuded oocytes. In contrast, the type 4 PDE inhibitor had no effect on the oocyte, but elevated mural granulosa and cumulus cell cAMP production. The results of this study indicate that specific PDE subtypes are differentially localised within the two compartments of the bovine follicle-the type 3 PDE in the oocyte and the type 4 PDE in the granulosa cells. In addition, oocyte cAMP levels are primarily regulated in bovine oocytes by its degradation by PDE, whereas granulosa cell cAMP levels are controlled mainly by active adenylate cyclase, with both sources able to participate in oocyte meiotic regulation.