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Insemination transmits to the female reproductive tract constituents of seminal plasma that target uterine epithelial cells to activate a cascade of inflammatory and immunological changes. Experiments in rodents show seminal factor signalling acts to 'condition' the female immune response to tolerate the conceptus, and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. The active factors in seminal plasma are identified as members of the transforming growth factor-beta family, with the relative balance of active moieties influencing the precise character of the female tract response. Experiments in rodents show that disruption of seminal plasma priming causes foetal growth retardation and changes in placental structure, with long-term consequences for the growth of the neonate. Recent studies indicate a similar physiological function and molecular basis for seminal plasma actions in the pig. In gilts, seminal plasma elicits an endometrial response characterised by recruitment of inflammatory leukocytes and induction of several pro-inflammatory cytokines and cyclo-oxygenase-2. The consequences are evident throughout the pre-implantation period of early pregnancy with altered leukocyte populations and cytokine parameters seen for at least 9 days. Exposure to semen also alters the dynamics in pre-implantation embryo development with an increase in the number of embryos and in their viability. Furthermore seminal plasma influences the temporal kinetics of ovulation, corpus luteum development and steroid production in the ovary. Dissecting the actions of seminal plasma may facilitate development of strategies to ensure maximal fertility and reduce embryo mortality in the pig.

Summary 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 10AE7 nmol/l (1AE7–45AE4), 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 AE127, P

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.

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus–oocyte complexes (COCs) were isolated from large (>1.5 mm) and small (0.7–1.5 mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 ± 9.3 versus 6.4 ± 2.7, mean ± S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.

Seminal plasma (SP) acts to influence the uterine endometrium after mating, activating synthesis of embryotrophic cytokines and inflammatory changes that condition the tract for embryo implantation and establishing pregnancy. The objective of this study was to investigate in pigs whether the ovary might also be responsive to SP exposure. Prepubertal gilts were synchronised with exogenous gonadotrophins and received transcervical treatment with pooled boar SP or PBS; then the ovarian tissue was recovered at 34 h (preovulation) and on days 5 and 9 after treatment. The ovarian response was assessed by measuring ovulation rate, number and size of corpora lutea, ovarian leukocyte populations, progesterone productionin vivo, as well as responses of retrieved granulosa cells culturedin vitro. In SP-treated gilts, leukocyte recruitment into the ovarian tissues was increased fourfold at 34 h, with macrophages comprising the most abundant cell lineage. There was no effect of SP on the number of oocytes ovulated; however, the weight of corpora lutea was increased in SP-treated gilts. SP also induced an increase in plasma progesterone content seen from day 5 to at least day 9 after treatment. In addition, granulosa cells and thecal tissue retrieved from preovulatory follicles of SP-treated gilts were more responsivein vitroto growth factor- and gonadotrophin-stimulated cell proliferation and progesterone synthesis. These results suggest that uterine exposure to SP influences immune cell trafficking in the ovary and enhances steroidogenesis in early pregnancy. The effects of SP on ovarian function potentially contribute to reproductive success in the pig.

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 × CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 ± 5.1%) and 20 (77.3 ± 5.6%) IU rhFSH groups compared with control embryos (96.7 ± 1.0%). The number of inner cell mass cells was reduced ( P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 mM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P , 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P , 0.05) increased embryo development to the blastocyst stage by 33%‐39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P , 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential. cumulus cells, cyclic adenosine monophosphate, embryo, meiosis, phosphodiestrases

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.

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.

Our current perspectives on the relationship between the oocyte and its surrounding somatic cells are changing as we gain a greater understanding of factors regulating folliculogenesis. It is now widely accepted that the oocyte plays a very active role in promoting follicle growth and directing granulosa cell differentiation. The oocyte achieves this, in part, by secreting soluble paracrine growth factors that act on its neighboring granulosa cells, which in turn regulate oocyte development. In preantral follicles, the oocyte directs granulosa cells to regulate oocyte growth, and oocytes may also directly drive follicle growth. In antral follicles, the oocyte governs the behaviour of cells in its immediate vicinity, thereby actively regulating its own microenvironment. As such, the oocyte establishes and maintains the distinct cumulus lineage of granulosa cells. This oocyte-cumulus cell interaction, in general, prevents luteinization of cumulus cells by promoting growth, regulating steroidogenesis and inhibin synthesis, and suppressing luteinizing hormone receptor expression. Conversely, mural granulosa cells in antral follicles, which have no direct physical contact with the oocyte and, presumably, experience a more diffuse concentration of oocyte-secreted factors, proceed to a different phenotype. In the ovulating follicle, oocyte-secreted factors also play vital roles in enabling cumulus cell expansion and regulating extracellular matrix stability, thus facilitating ovulation. The identities of these oocyte-secreted growth factors regulating such key ovarian functions remain unknown, although growth differentiation factor-9 (GDF-9), GDF-9B and/or bone morphogenetic protein-6 (BMP-6) are likely candidate molecules, probably forming complex local interactions with other related members of the transforming growth factor-beta (TGF-beta) superfamily. Elucidating the nature of oocyte-somatic cell interactions at the various stages of follicle development will have important implications for our understanding of factors regulating folliculogenesis, ovulation rate and fecundity.

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 meioticmodulating 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, calceinAM. 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 initia l0hl eveluntil 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 ;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. cumulus cells, cyclic adenosine monophosphate, meiosis, oocyte development, phosphodiesterases

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

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.

Purpose: Determine the short-term effects of creatine supplementation on performance of military tasks, thermoregulation, and health risks. Methods: Male military personnel were randomly assigned to a creatine (CR; N = 8) or a placebo (CON; N = 8) supplementation group. Testing was conducted at baseline, after a 6-day load phase (20 g/d), and after 4 weeks of taking 6 g/d. Measurements included body composition, liver/ kidney function tests, core body temperatures during a 10-mile march and 5-mile run, and performance on physical tasks. Results: Serum and urine creatine increased significantly in the CR group. Body mass and number of pull-ups performed increased significantly in the CR group but not the CON group by week 4. No significant differences between the CR and CON groups were found for other performance measures, body composition, core body temperature, or other biochemical measures. Conclusion: Creatine supplementation increased body mass and pull-up performance but did not cause acute health problems. Creatine did not increase core temperature compared with placebo under the environmental conditions of the study, and it is unlikely that creatine will enhance the overall readiness or performance of soldiers.

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.

Ovarian follicular development in the bovine estrous cycle is characterized by a wave-like pattern of successive periods of follicle growth and regression, with the majority of estrous cycles in cows comprising either two or three waves per cycle. Each wave involves recruitment of a cohort of antral follicles, followed by selection of a single dominant follicle that progresses to the pre-ovulatory stage, while the remaining follicles of the wave, the subordinate follicles, undergo atresia. The pituitary gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone, are the primary drivers of follicle recruitment and growth, with molecules produced locally within the follicle having secondary but essential roles in dominant follicle selection, while promoting atresia of subordinate follicles [1]. Bidirectional communication between the oocyte and adjacent cumulus granulosa cells results in expansion and mucification of the cumulus-oocyte complex necessary for ovum release at ovulation, mediated by oocyte-secreted peptides (growth differentiation factor 9, bone morphogenic protein [BMP] 15, fibroblast growth factor [FGF] 8) [2, 3]. In turn, molecules from cumulus cells (epidermal growth factor–like peptides, cAMP, and pyruvate) trigger meiotic and cytoplasmic maturation of the oocyte essential for fertilization and embryo development [4]. Similarly, paracrine signals and substrates passing between mural granulosa cells and theca cells of the follicle wall contribute to both granulosa and theca cell proliferation and differentiation essential for continued follicle growth and maturation leading to ovulation. Theca cells supply androgens as precursors for conversion to estrogens by aromatase located exclusively in granulosa cells, and growth factors (insulin-like growth factors [IGFs] and FGFs) that act synergistically with the estrogens to further enhance follicle growth. In turn, estrogens and other growth factors produced by granulosa cells exert regulatory actions back on the theca cells. These closely coordinated activities of the three follicle cell types—oocyte, granulosa cells, theca cells—comprise a regulatory network that results in an intrafollicular milieu necessary for follicle growth and ovulation of a developmentally competent ovum as culmination of the final follicular wave of the cycle [5

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA 1 sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [ 3 H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased (3)H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [(3)H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.

Leptin, a hormonal product of the Lep gene, is expressed by adipocytes and is thought to play a role in regulating food intake and reproduction. The leptin protein has been localized in many reproductive tissues, including the ovary. Several publications indicate that the ovary is directly affected by leptin and that leptin may be a factor linking obesity and reproductive dysfunction. In this study, the effect of systemic leptin administration on ovulation in the rat ovary, both in vivo and in vitro, was investigated. Ip administration of leptin (30 μg at 3 hourly intervals for 15 h) to immature gonadotropin-primed rats caused a decline in ovulation in vivo, from 15.9 ± 2.0 oocytes in the control animals to 5.3 ± 1.6 oocytes in the leptin-treated animals (P < 0.001). Plasma progesterone and estradiol levels were analyzed immediately before ovulation, and neither was altered significantly in animals receiving the leptin treatment. Food consumption and body weight decreased following leptin treatment; however, a loss in body weight alone (pair-fed controls) was insufficient to explain the decrease in ovulation observed in the leptin-treated animals. In vitro perfusion of FSH-primed whole ovaries showed that treatment with leptin in combination with LH significantly decreased ovulations from 5.7 ± 1.6 per ovary perfused with LH alone to 1.3 ± 0.6 in those with LH and 1 μg/ml leptin (P < 0.05). Progesterone and estradiol levels in the samples taken during the perfusion period were unaffected by leptin treatment. In summary, leptin administration resulted in fewer ovulations, both in vivo and in vitro, but did not influence steroid levels. Systemic leptin administration at these doses can therefore inhibit ovulation, a process that occurs through a direct effect on the ovary.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.

Follicle growth is regulated by the combined actions and interactions of pituitary gonadotropic hormones and local intraovarian paracrine and/or autocrine agents, including the peptide growth factors, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF). The present study was undertaken to determine a) whether the previously demonstrated inhibitory effect of FSH on DNA synthesis was related to its ability to cause cumulus mucification as a differentiated response incompatible with continued proliferation, and b) whether increased IGF-binding proteins (IGFBP) secreted in response to FSH competed with IGF receptors, thereby inhibiting response to exogenous IGF-I. To determine the effects of cumulus mucification in modulating the mitogenic response to IGF-I, two other agents that induce cumulus mucification by different mechanisms, EGF and dibutyryl cAMP (dbcAMP), were compared with FSH. To determine the possible role of IGFBP in modulating the mitogenic response to IGF-I, an IGF-I analogue that does not bind to IGFBP, long arg3-IGF-I (LR3-IGF-I), was compared with native IGF-I for efficacy in stimulating DNA synthesis in the absence and presence of each of the above agonists. Both IGF-I and LR3-IGF-I stimulated [3H]thymidine incorporation in cumulus cells to a much greater extent than in mural granulosa cells. Incorporation in mural cells was increased by each of FSH, EGF, and dbcAMP acting by itself, and in most instances was considerably enhanced by the combined action of these agents with each of the IGF-I forms. In contrast, the considerably greater stimulatory effect of both IGF-I and LR3-IGF-I on cumulus cells was markedly decreased by each of FSH, EGF, and dbcAMP. These findings suggest that the inhibition of IGF-I-stimulated DNA synthesis in cumulus cells is a consequence of induction of cumulus cell differentiation (mucification) by FSH and EGF rather than through competition between IGF-I receptor and IGFBP secretion induced by these agents.

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.

The present study was aimed at determining whether insulin-like growth factor-1 (IGF-1) is a component of fetal bovine serum (FBS) that enables porcine cumulus cells to expand in response to FSH in vitro. Cumulus-oocyte complexes (COC) obtained from 4- to 6-mm follicles of prepubertal gilts were cultured at 39 degrees C for 24 h in media that contained human recombinant IGF-1 (50 ng/ml), FBS (15% v:v), or their combination, with or without FSH (1.5 microg/ml), and cumulus expansion was scored microscopically. Expansion was FSH dependent and was observed only when IGF-1, FBS, or both were present. The proportion of FSH-stimulated COC exhibiting full expansion in response to IGF-1 alone did not differ significantly (p > 0.05) from the proportion in those cultured with FBS or IGF-1+FBS (79 +/- 2.8% vs. 84 +/- 2.2% or 76 +/- 6.2%, respectively). In a concentration-response study, FSH-stimulated expansion was observed in a significant proportion of COC (32 +/- 2.8% vs. 0% control) at 1 ng/ml IGF-1, with the proportions increasing dose-dependently to maximal values between 10 and 75 ng/ml IGF-1, and decreasing at higher IGF-1 concentrations. Exposure of COC to an IGF-1 receptor (IGF-1R)-neutralizing antibody (Ab) for 90 min before addition of FSH and FBS dose-dependently inhibited cumulus expansion, with maximal inhibition at 10 microg/ml (1 +/- 1.0% vs. 68 +/- 1.1% control). In the absence of FBS or IGF-1, some COC had a tendency toward slight expansion when cultured with FSH, and the Ab completely inhibited that effect, suggesting that this may be due to endogenous IGF-1 production by the COC. The Ab effect was reversible and was eliminated by washing twice with fresh medium followed by culture for an additional 24 h in the presence of FSH and FBS. Expression of IGF-1R mRNA in the isolated oocyte and cumulus cells was determined by reverse tramscriptase polymerase chain reaction using sequence-specific primers. The IGF-1R message was detected in both the oocyte and cumulus cells. Collectively, these observations suggest that IGF-1 is a component of serum that enables cumulus cells to expand in response to FSH in vitro, and that the effect is receptor mediated. Since IGF-1 is present in the follicle in vivo, it may have a physiological role during gonadotropin-induced cumulus expansion.

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 microg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6-8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[35S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes.

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulus–oocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 μM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 μg/ml cytochalasin D or 1.9 mM 6-dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol- and DMAP-treated oocytes dividing. Blastocyst development did not vary with 18.4 ± 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two-cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome. Dev. Genet. 21:160–166, 1997. © 1997 Wiley-Liss, Inc.

During embryo implantation in the human trophoblast cells invade the endometrium and plasminogen activators (PA) are implicated in this tissue invasion process. Our studies on surplus embryos from patients undergoing in-vitro fertilization (IVF) treatments showed that PA activity was present in secretions of blastocysts but not embryos at the 2-4-cell stage. The secretion of this PA activity by blastocysts was increased by the addition of epidermal growth factor (EGF) to the embryo cultures. This PA activity had a molecular weight similar to that of urokinase-type PA (uPA). The mRNA for uPA was present in blastocysts, as determined by the reverse-transcription and polymerase chain reaction (RT-PCR) technique. These data indicate that uPA is produced by human preimplantation embryos and may play a role in early human development and embryo implantation.

Effects of gonadotropins and peptide growth factors on 3H-thymidine incorporation, as an indicator of proliferation of granulosa cells and cumulus cells from bovine ovarian follicles during in vitro culture, were determined. Incorporation of 3 H-thymidine in cumulus cells during 24-h culture in protein-free medium was about 100-fold greater than in granulosa cells from small follicles. Small but significant (p < 0.05) increases in incorporation of 3 H-thymidine in cumulus cells (2.5-fold), but not in granulosa cells, resulted from addition of FSH, LH, or epidermal growth factor to culture media. Much larger, highly significant (p < 0.01) increases in 3 H-thymidine incorporation resulted from exposure of both cumulus cells (18-fold) and granulosa cells (6-fold) to insulin-like growth factor-I (IGF-I). Addition of IGF-I together with FSH resulted in additive increases in 3 H-thymidine incorporation in granulosa cells over a wide range of each agonist. In contrast, addition of FSH markedly decreased the stimulatory effect of a maximally effective concentration of IGF-I on incorporation in cumulus cells. Addition of estradiol, either alone or with IGF-I, had no consistent effect on 3 H-thymidine incorporation in either cumulus cells or granulosa cells. Differentiated functions of cumulus and granulosa cells, including cumulus expansion (an indicator of hyaluronic acid secretion) and progesterone secretion, were investigated under the same culture conditions. FSH was highly effective in stimulation of cumulus cell expansion, with maximal expansion occurring at the lowest concentration tested (0.04 izg/ml). LH was effective only at considerably higher concentrations consistent with degree of contamination with FSH. IGF-I was ineffective in inducing cumulus expansion, either alone or together with FSH. FSH and IGF-I by themselves each stimulated progesterone secretion by both cumulus and granulosa cells, with further additive stimulatory effects by the two agents in combination. On a per cell basis, cumulus cells produced considerably more progesterone than granulosa cells under both basal and hormone-stimulated conditions. The results suggest that IGF-I has a dual role in follicle regulation-that it acts additively with FSH as an autocrine regulator of granulosa cell growth and, in the absence of FSH, as a paracrine regulator of cumulus cell proliferation in small antral follicles. The effect of FSH, by inducing cumulus expansion, may terminate further cumulus cell proliferation before ovulation.

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide get electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0-6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen ; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between Cl and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components.

The present study was undertaken to determine transforming growth factor alpha (TGF alpha) gene expression in various components of medium-sized porcine ovarian follicles by reverse transcription-polymerase chain reaction, and to localize the peptide during folliculogenesis by immunocytochemistry. A strong signal for TGF alpha transcript was detected in cumulus and granulosa cells, whereas the signal in theca cells was very weak but detectable. No TGF alpha mRNA was detected in the oocyte. Immunolocalization studies revealed intense TGF alpha staining in cumulus and granulosa cells of all stages of follicular development. TGF alpha immunostaining was also observed in the oocytes of primordial and primary follicles. In antral follicles, immunostaining was observed only in cumulus and mural granulosa cells, whereas theca cells and oocytes exhibited very little or no detectable staining. TGF alpha immunostaining was absent in follicles undergoing atresia. No TGF alpha immunostaining was observed when the antibody was omitted from the procedure, and the staining was substantially reduced when the primary antibody was preabsorbed with the same mass of TGF alpha peptide, suggesting the specificity of the staining procedure. These results suggest a local follicular production of TGF alpha in the porcine ovary and thus a role for TGF alpha of follicular origin in the regulation of follicular development in an autocrine/paracrine fashion.

The present study was undertaken to determine the expression of the genes for epidermal growth factor receptor (EGF-R), and two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) in isolated large (LLC) and small (SLC) porcine luteal cells by reverse transcription-polymerase chain reaction (RT-PCR), and to localize their peptides in whole corpora lutea (CL) as well in isolated LLC and SLC by immunocytochemistry. RT-PCR revealed strong cDNA signals for EGF-R and EGF in both LLC and SLC, and for TGF-alpha in LLC. The signal for TGF-alpha message in SLC was relatively weaker but detectable. Immunocytochemistry revealed intense EGF-R staining in LLC and SLC in both isolated and intact CL preparations. On the other hand, immunoreactive EGF and TGF-alpha appeared to be present only in LLC in intact CL sections, and isolated luteal cell preparations confirmed their presence in LLC and absence in SLC. These results suggest an autocrine/paracrine role for EGF and TGF-alpha in luteal functions.

The present study was undertaken to determine the expression of genes for epidermal growth factor (EGF) and its receptor (EGF-R) in various components of medium-sized porcine ovarian follicles by reverse transcription-polymerase chain reaction (RT-PCR), and to localize their peptides during folliculogenesis by immunocytochemistry. A strong band for EGF mRNA transcript was detected in the oocyte, whereas the signal in cumulus, granulosa, and theca cells was very weak but detectable. In contrast, a very strong EGF-R mRNA signal was observed in cumulus, granulosa, and theca cells, whereas the signal in the oocyte was very weak. EGF peptide was localized in the oocyte, cumulus, and granulosa cells of all stages of follicle. In the oocyte, the intensity of immunostaining was more pronounced in primordial and primary follicles, compared to antral follicles. In large antral follicles, immuno-staining was pronounced in granulosa cells, whereas theca cells showed little or no detectable staining for EGF. EGF staining was also observed in the cumulus and granulosa cells of follicles undergoing atresia. EGF-R immunostaining was observed in the oocytes of primordial and primary follicles, and in cumulus, granulosa, and theca cells of all stages of follicle, including atretic follicles. In large antral follicles, the intensity of immunostaining was more pronounced in theca cells than in granulosa cells, and the oocyte showed little or no detectable staining. No immunostaining was observed when the primary antibody was replaced with preimmune serum (EGF), or preabsorbed with the control peptide (EGF-R), confirming the specificity of the staining procedures. These results suggest a local follicular production of EGF and its receptor in the porcine ovary, and thus a role for EGF of follicular origin in the regulation of follicular development in autocrine/paracrine fashion. © 1995 Wiley-Liss, Inc.

Glycine is the amino acid with the highest concentration in sow oviduct fluid, uterine fluid and follicular fluid. In this study, the effect of glycine on in vitro maturation, fertilization and early embryonic development in pigs was evaluated. In addition, the expression of T-protein, a component of the glycine cleavage enzyme system, was studied in the oocytes, cumulus cells, granulosa cells and oviductal epithelial cells using the methods of reverse transcription-polymerase chain reaction (RT-PCR). The results showed that supplementation of porcine oocytes with glycine during in vitro maturation improved on the development of embryos in vitro. After fertilization, glycine also enhanced embryo cleavage rate in the presence, but not in the absence, of oviductal cells. PCR product corresponding to T-protein was found in uncultured oocytes, cumulus cells, granulosa cells and oviductal epithelial cells. These results indicate that glycine is an important amino acid for the in vitro oocyte maturation, fertilization and early embryonic development in pigs.

To determine whether altered follicular environment is associated with ovarian reserve or maternal age.Prospective study examining follicular fluid (FF) composition and follicular cell metabolism.University research department and private IVF clinic.Women (n = 54) undergoing routine IVF treatment were allocated to one of three groups based on ovarian reserve and maternal age.Surplus FF, granulosa cells (GC), and cumulus cells (CC) were collected.Follicular fluid concentrations of carbohydrates, hormones, and selected ions. Metabolic analysis and gene expression of GCs and CCs.Compared to women35 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.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.

Seminal plasma derived factors are implicated in mediating inflammation in the female reproductive tract following insemination at mating. During inflammation, leukocytes are activated to express adhesion receptors resulting in adherence to each other and for the ECM as well as for various cell types. The present study describes the purification of a leukocyte cell-cell adhesion regulator derived from seminal vesicle fluid. Seminal vesicle fluid proteins were chromatographed by cation exchange, hydrophobic interaction and reversed phase. Chromatography on Phenyl Superose resolved two distinct forms of cell-cell adhesion regulation, type I and II. Reversed phase chromatography of fractions inducing type I adhesion resulted in the isolation of a 15kDa adhesion inducing protein (pAIF-1). The N-terminal sequence contained a hydrophobic consensus sequence which exists in: two bovine seminal vesicle proteins (BSPA3, PDC 109); IGF-II receptor; fibronectin; and the cation independent mannose-6-phosphate receptor.

The study examined whether co-culture with oviductal epithelial cells was of benefit to ovine in vitro fertilization (IVF) and embryo culture procedures utilizing a well characterized culture system based on a synthetic oviductal fluid medium (SOFM) supplemented with serum in a 90% N2, 5% O2, 5% CO2, atmosphere at 38.6°C. Two experiments were carried out. In Experiment 1, comparison was made between the frequency of fertilization and development of in vitro matured (IVM) oocytes cultured in the absence (Group 1) or presence of oviductal cells for a 24 h (Group 2), 48 h (Group 3) or 96 h (Group 4) period post insemination. In Experiment 2, comparison was made between the development of IVM oocytes fertilized and cultured in vitro for 7.5 days in the absence or presence of oviductal cells with IVM oocytes which had been fertilized in vitro for 20 h in the presence of oviductal cells and then transferred to the oviducts of a recipient ewe, 30 h post oestrus, for a 6.5 day period of in vivo culture. Similar rates of fertilization (54–58%) and blastocyst development from cleaving zygotes (48–69%) were achieved in both experiments in vitro with no evident benefit of including oviductal cells. In fact, fewer (P = 0.02) blastocysts developed from cleaved embryos when co-cultured for the 96 h period (Group 4). Whilst the blastocyst development rates obtained in vitro (45–58%) were similar to, or higher than (P = 0.03), those obtained in vivo (43%), more than 50% of in vitro cultured embryos in both experiments showed evidence of fragmentation and/or irregular cleavage as well as a lack of firm compaction at the morula stage. Also the blastocysts that had not hatched, or hatched by 7.5 days of in vitro culture had significantly fewer cells than those cultured in vivo (P < 0.003). The results confirm that SOFM supplemented with serum can support the fertilization and development of ovine oocytes to the blastocyst stage in vitro, but that development is compromised compared with that obtained in vivo and that these defects were not offset by including oviductal cells in the culture media. However, it should be noted that the composition of the medium and gaseous atmosphere developed for the IVF embryo culture procedures are recognized as being suboptimal to the maintenance of the viability of somatic cells, highlighting the difficulties of selecting culture conditions suited for both embryonic and somatic cells development, a primary requisite for realizing the potential beneficial effects of co-culture on embryo viability.

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on 3H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. 3H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in 3H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.

This study investigated the mechanisms underlying 2-hydroxyestradiol (2-OHE2) effect on luteal steroidogenesis using serum-free cultures of mixed luteal cells from day 8 pseudopregnant rats. Initially, interactions between 2-OHE2 and LH or dibutyryl (db)cAMP on progesterone production were investigated. LH (250 ng/ml) and 2-OHE2 (2.5 microg/ml) had comparable effects on progesterone accumulation, while dbcAMP (5 mM) was more stimulatory. When applied together, 2-OHE2 did not synergize with LH or dbcAMP to further enhance progesterone accumulation. Furthermore, in time course experiments, the dose-dependent effect of 2-OHE2 was to reduce and eventually abolish the time-dependent increase in cAMP accumulation. In contrast LH stimulated cAMP accumulation at all times. Experiments in which cells were co-treated with 2-OHE2, 22-OH-cholesterol and cyanoketone, or with 2-OHE2 and 22-OH-cholesterol or pregnenolone indicated that 2-OHE2 not only had a stimulatory effect on the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase enzymes, but it also appeared to inhibit the 20alpha-hydroxysteroid dehydrogenase leading to a relative increase in progesterone accumulation. Experiments with hormone antagonists suggested that the actions of 2-OHE2 were not mediated by the estrogen, alpha- or beta-adrenergic receptors. The results of this study support the concept of a physiological role for catecholestrogens in rat luteal steroidogenesis.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A4), and estradiol-17 beta (E2) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the above-mentioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 micrograms/ml) together and LH (2 micrograms/ml) and FSH (1.5 micrograms/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E2 (1 microgram/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A4 (1 microgram/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-beta 1 had no effect on GVBD or cumulus expansion.(ABSTRACT TRUNCATED AT 250 WORDS)

To compare the IVF rates of oocytes retrieved from small follicles (2 mL in volume) with those of oocytes retrieved from large follicles and to test the effect of adding gonadotropins to the IVF medium on the fertilization rates of oocytes from small follicles.Oocytes were retrieved with endovaginal ultrasound (US) guidance from patients undergoing infertility treatment in our IVF program. Oocytes were grouped according to the volume of the originating follicle and subjected to our routine procedure for IVF. HMG was added to the IVF medium for some of the oocytes from small follicles.Toronto Fertility and Sterility Institute is affiliated with the University of Western Ontario and University of Toronto and is equipped for RIA, endovaginal US monitoring and oocyte retrieval, and for processing and culturing gametes and embryos.Infertile patients admitted to our IVF program.Patients underwent ovarian stimulation with hMG before oocyte retrieval. No other interventions were introduced to the processing and culturing the gametes and embryos except the addition of hMG to the medium of some of the small follicle-originated oocytes with the informed consent from the patients.Rates of fertilization, cleavage of the fertilized embryos before replacement, and meiotic status of some of the oocytes from small follicles.Most of the oocytes from small follicles did not complete the first meiotic division; they had low rates of fertilization and cleavage compared with oocytes from large follicles, and these rates were improved by the addition of hMG to the IVF medium.Oocytes from small follicles are probably less mature and require a more physiological environment to achieve normal rates of fertilization and cleavage.

The role of tumor necrosis factor (TNFoa) in ovarian function was investigated using in vitro culture of theca and granulosa cells isolated from gilt follicles (4-6 mm) and small (SLC) and large (LLC) luteal cells from mid-cycle corpora lutea. TNFa did not affect basal accumulation of progesterone (P) by theca cells after 72 h of culture. However, TNFa (0.1-100 ng/ml) caused a marked dose-dependent noncytotoxic inhibition (p < 0.05) of LH or LH + insulin (I)-stimulated P accumulation by theca cells after 72 h. Maximal inhibitions averaged 87 + 6% at 5 ng/ml TNFa for LH-stimulated P and 69 ± 4% at 50 ng/ml TNFa for LH + I-stimulated P. The inhibitory effect of TNFa, evident by 24 h after culture, progressively increased on Days 2 and 3 of culture. The effect of TNFao on theca cells was mediated by cAMP generation as evidenced by TNFa inhibition of LH-induced cAMP accumulation and P accumulation in response to LH and forskolin but not dibutyryl cAMP. Consistent with this, TNFca had no effect on increased P accumulation by theca cells in the presence of 22-hydroxycholesterol or pregnenolone alone, but inhibited further increases in P accumulation stimulated by LH plus sterol substrates. Unlike that in theca cells, FSH-induced P accumulation in granulosa cell cultures was slightly enhanced (p < 0.05) by low doses of TNFa (0.1, 0.5, and 1.0 ng/ml) after 72 h, while higher doses (5-50 ng/ml) did not alter P accumulation. Furthermore, in experiments with luteal cells, TNFot had no effect on P accumulation in cultures of SLC or LLC after 36 h. The effect of TNFa on porcine theca cells may indicate a role in preventing premature follicular luteinization; together with previous evidence, this suggests a complex and possibly evolving physiological role for TNF during follicular development.

Superovulation is a major limiting factor in the contributions that embryo transfer can make to the genetic improvement of cattle. This review analyzes the limitations of superovulation of cattle, including factors which influence ovulatory response of donors, those which influence fertilization and embryo viability, and certain aspects of animal treatment and management that determine success in terms of efficiency of viable embryo production. Recent research findings on which new approaches to superovulation may be based, and areas in which future research may be expected to yield further improvements are discussed.

Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.

The resumption of meiosis results in synthesis of tissue-type plasminogen activator (tPA) in the rat and mouse oocytes (Haurte et al., Cell 43:551-558, 1985). The present study demonstrates that freshly ovulated rat oocytes released their tPA into the surrounding medium upon in vitro activation by sperm penetration or treatment with a calcium ionophore. The presence of a neutralizing monoclonal anti-tPA antibody during in vitro activation by the calcium ionophore inhibited the activation-induced zona hardening and also preserved the ability of the oocyte to be penetrated by sperm subsequent to activation. Rat oocytes undergo zona hardening during in vitro maturation in the absence of serum, presumably as a result of spontaneous cortical granule release, based on findings in mice and hamsters. In the present study, the anti-tPA antibody prevented the zona hardening and enhanced partition by spermatozoa of rat oocytes that were matured in vitro without serum. Collectively, the observations reported have suggest a possible role of tPA released during the cortical granule reaction in the zona reaction, which contributes to the block to polyspermy.

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).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.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, P0.001), self-reported sexual desire (R(2) = 0.113, P0.001) and erectile function (R(2) = 0.085, P0.05) while bioassay T did not.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.

The corpora lutea of several species contain estrogen receptors, but the role of estrogens in luteal function is unclear in most species. In this study, we investigated the direct effect of estradiol-17�3 (E,) and catecholestrogens (2-OHE2 or 4-OHE,) on rat and pig luteal steroidogenesis using in vitro cultures of small (SLC) and large (LLC) luteal cells prepared by elutriation. SLC and LLC were cultured at 37#{176}C for 36 ii in serum.free media and treated with E,, 2-OHE,, or 4-OHE,; LH; forskolin (FORS); dibutyryl cAMP (dbcAMP); or combinations thereof. In the rat, E2 (2.5-10 �ag/ml) inhibited progesterone (P4) production by both cell types dose.dependently. P4 production by rat SLC increased with increasing dose of 4-OHE2 up to the 2.5jLg dose, then decreased to near control level at the l0-�.ig dose. In LLC, P4 production in the presence of 4-OHE2 decreased initially (up to 2.5 pg/mI 4-OHE2), then increased at the 10-pg dose. LH, FORS, and dbcAMP stimulated P4 production by SLC and LLC. For SLC, the stimulatory effects of LH and 4-OHE, (2.5 pg) were comparable but lower than those of FORS and dbcAMP. For LLC, the effects of 4.OHE, (10 �tg), LH, and FORS were comparable but lower than those of dbcAMP. In time-course experiments, E2 inhibition of P4 production was observed at 36 and 72 h but not 6 h of culture for SLC and at all time points for LLC. On the other hand, 4.OHE, (at appropriate concentrations) stimulated P4 production by both cell types at 6 and 36 h of culture, but the effect at all concentrations became inhibitory at 72 h. In the pig, E2 tended to inhibit P4 production by SLC and LLC, particularly at 72 h, but stimulated dose-dependent increases in pregnenolone (P,) production by both cell types. For SLC, 2-OHE, or 4-OHE, equally inhibited P4 production dose-dependently. However, 2-OHE, was less effective than 4.OHE2 in inhibiting P4 production by LLC. In the same experiments, catecholestrogen treatments were generally associated with concomitant decreases in P5 production by SLC and LLC. As well, all three steroids caused dose.dependent increases in androstenedione (A) production by LLC; no A was detectable in SLC cultures. Timecourse experiments showed that E2 (10 pg/mI) inhibited P4 production at 36 and 72 h but not 6 h in SLC cultures, and at 72 h only for LLC, while 4.OHE2 (10 pg/ml) inhibited both cell types at all time points. Experiments to determine the mechanism of action of the estrogens revealed that E, and catecholestrogen act at a point distal to cAMP generation since LH, FORS, or dbcAMP were unable to reverse the inhibitory activity of 4-OHE2 on SLC and LLC while E, inhibited basal and secretagogue. stimulated P4 production by SLC. Our data suggest that in vivo luteotrophic effects of E2 in the rat may be mediated by catecholestrogens. In the pig, there was no evidence for a direct luteotrophic effect of E2. Catecholestrogens inhibited pig basal and secretagogue-stimulated P4 production more effectively than did E,, apparently due to dual sites of action in the steroid biosynthetic pathway.