A retrospective analysis of adverse effects of an in vivo fluoroquinolone antibiotic enrofloxacin treatment on oocyte quality in the common marmoset.

Here we report a retrospective analysis of negative effects of routine enrofloxacin treatment of recurrent diarrhea on the ovary and the developing oocytes of the common marmoset, a small New World primate. The most deleterious effect on oocytes was observed about two months post treatment suggesting that the enrofloxacin effect is on early growing follicles. Manifestations of toxicity included decreased numbers of growing follicles and recovered culturable oocytes, as well as signs of early atresia of granulosa cells. In addition, increased amounts of holed stroma after treatment strongly suggested increased death of the early growing follicles. Of the oocytes judged to be of adequate quality for culture, maturation rates were not affected but fertilization of in vitro matured MII oocytes and subsequent cleavage rates were severely reduced in the enrofloxacin treated animals. Further, the arrested oocytes, which failed to mature or fertilize, showed obvious meiotic spindle abnormalities.

Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes.

Chimerism associated with placental sharing in marmosets has been traditionally analysed using conventional chromosome staining on metaphase spreads or polymerase chain reaction. However, the former technique requires the presence of proliferating cells, whereas the latter may be associated with possible blood cell contamination. Therefore, we aimed to develop a single-cell analysis technique for sexing marmoset cells. We applied fluorescent in situ hybridisation (FISH) to cell nuclei using differentially labelled X and Y chromosome-specific probes. Herein we present the validation of this method in metaphase cells from a marmoset lymphoblastoid cell line, as well as application of the method for evaluation of cross-sex chimerism in interphase blood lymphocytes and haematopoietic bone marrow cells from marmosets of same- and mixed-sex litters. The results show conclusively that haematopoietic cells of bone marrow and leucocytes from blood are cross-sex chimeric when the litter is mixed sex. In addition, single samples of liver and spleen cell suspensions from one individual were tested. Cross-sex chimerism was observed in the spleen but not in liver cells. We conclude that FISH is the method of choice to identify cross-sex chimerism, especially when combined with morphological identification of nuclei of different cell types, which will allow a targeted tissue-specific analysis.

Body weight-associated differences in ovarian morphology in captive common marmoset (Callithrix jacchus).

In captivity, Callithrix jacchus (common marmoset) is on average heavier than their wild-living counterparts, and has a tendency to produce triplet litters rather than the normal twins. To provide initial basic information about possible weight-related differences among the ovaries, a morphometric study of follicular phase ovaries from 48 young adult marmosets has been carried out. Nearly 90% of these ovaries were found to contain some degree of luteal tissue composed of large and/or small cells. The luteal structures, follicles of all stages, and stroma were subjected to morphometric analysis, and these results were compared with body weight, circulating triglyceride, androstenedione, and total estrogens. Where only large luteal cells were present, the median body weight was the highest (only this group included animals over 500 g) compared with mixed, or only small luteal cells, or absence of luteal cells. Furthermore, in this group plasma triglycerides were significantly higher compared to other groups, suggesting possible role of triglycerides in promoting luteinisation. Plasma androstenedione was also a critical discriminating factor, and was elevated where large luteal cells were present even as a mixture with small cells suggesting the large luteal cells to be the likely major ovarian source of this hormone and its metabolites. Additionally, the ovaries with large luteal cells compared to those containing only small or no luteal cells, had lower primordial follicle reserve associated with high levels of atresia and luteinisation among growing non-ovulatory follicles, indicating an accelerated activation, but at the same time a suboptimal environment for follicular growth.

Critical estradiol dose optimization for oocyte in vitro maturation in the common marmoset.

The aim of the present study was to critically evaluate the effect of different concentrations of estradiol (E2) during IVM of common marmoset (Callithrix jacchus) oocytes from antral follicles. The doses tested were 0, 0.1, 1, or 10 µg/mL E2 (referred to as 0 E2, 0.1 E2, 1 E2, and 10 E2 groups). After a preincubation, the concentration of E2 in IVM drops under oil was approximately 20% of the amount added (0.02; 0.2 and 1.9 µg/mL, respectively) because of absorption into the oil. Oocyte progression to metaphase II was significantly higher in the 0.1 E2 group than that in the absence of E2. With progressively higher doses, the maturation rate tended to decrease suggesting an overdose effect. Furthermore, the total first cleavage rate was significantly higher in the 0.1 E2 group than that in the 0 E2 group and decreased progressively with further increases in E2 concentration, with the 10 E2 group showing the same low rate as without E2. The oocytes which failed to cleave, after maturation in 10 E2, showed obvious signs of overdose with the highest rates of degeneration and abnormal spindle form, and an absence of embryo progression. In contrast to these obvious negative effects on the oocyte, 10 E2 was the only group in which a significant increase in radial cumulus expansion was observed. The concentration 0.1 E2, which is 10 times lower than the most commonly used E2 dose, produced the best results in all oocyte factors evaluated. These results represent the first study for a primate species showing a strong positive effect of E2 on oocyte maturation and embryo development, but only at the optimal concentration, and emphasize the critical limits of the optimal concentration range.

Gross morphometry of the heart of the Common marmoset.

The Callithrix jacchus is a Brazilian endemic species that has been widely used as an experimental model in biomedical research. Anatomical data are necessary to support experimental studies with this species. Eleven hearts of C. jacchus from the German Primate Centre (DPZ) have been studied in order to characterize their gross morphometry and compare them with other animal models and human. Biometric data were also obtained. The mean values for morphometry of the hearts did not show any significant difference between male and female. The relative heart weight was similar to human, bovine and equine species. Considering those aspects, the C. jacchus could be used as non-human primate experimental model for biomedical studies on heart.

Numerical chromosome disorders in the common marmoset (Callithrix jacchus)--comparison between two captive colonies.

BACKGROUND: Chromosomal analyses were performed for marmosets from two colonies - Deutsches Primatenzentrum (DPZ) and Biomedical Primate Research Centre (BPRC). Chlorine-based disinfectants are used in DPZ; no chemical disinfection is applied in BPRC. METHODS: The rates of chromosomal non-disjunction, polyploidy and endoreduplication were investigated after G-banding. RESULTS: For DPZ monkeys, the mean rates of non-disjunction were 7.6% for bone marrow and 11.3% for lymphocytes. The polyploidy level was 2.5% in bone marrow and 0.8% in blood. Frequency of endoreduplication in bone marrow and in leucocytes was 0.5% and 0.8%, respectively. For BPRC, the rate of non-disjunction in leucocytes (1.3%) was significantly lower than that for DPZ; the polyploidy rate (0.2%) in blood was lower than that in DPZ; endoreduplication was not observed. CONCLUSION: The levels of chromosomal disorders are elevated for DPZ colony. We suggest that the increased rate of chromosomal disorders in DPZ marmosets can be related to the chemical disinfection of their environment.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): a model for studying sperm subpopulations and among-donor variations.

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.

Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization--what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects?

A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.

Quantification of ZP1, ZP2 and ZP3 mRNA of marmoset monkey (Callithrix jacchus) oocytes from periantral and antral follicles.

In mammals, the oocyte and preimplantation embryo are protected by the zona pellucida (ZP) consisting mainly of ZP glycoproteins, which are responsible for sperm binding, induction of the acrosome reaction and zona pellucida hardening to prevent polyspermia. The ZP proteins become increasingly important as possible predictors for in vitro cultured oocytes competence. As little is known about the stage-dependent expression of ZP1, ZP2 and ZP3 in marmoset monkey (Callithrix jacchus) oocytes, mRNA expression was investigated with real-time RT-PCR. Total-RNA was isolated from three different classes of marmoset oocytes; Class 1 oocytes from periantral follicles (<600 µm, n = 10), Class 2 oocytes from small antral follicles (600-1000 µm, n = 10) and Class 3 oocytes from large antral follicles (>1000 µm, n = 9). Compared with Class 1 oocytes mRNA expression of ZP1, ZP2 and ZP3 in Class 2 oocytes was significantly decreased. In Class 3 oocytes, the transcription of ZP1, ZP2 and ZP3 genes showed also a significant decrease compared with Class 1 oocytes. In this study a differently regulated expression of the ZP genes during late folliculogenesis with an obvious downregulation of ZP1, ZP2 and ZP3 could be demonstrated for the first time in the marmoset monkey.

A case report of spontaneous opening of congenitally fused labia in a female common marmoset (Callithrix jacchus) followed by pregnancy and birth of twins.

A first case of spontaneous opening of congenitally fused labia (CFL phenotype) in a captive common marmoset followed by pregnancy and birth is presented here. The occurrence of this phenotype has been previously published in captive marmosets, but so far the etiology is unknown.

Epidermal growth factor effects on marmoset monkey (Callithrix jacchus) oocyte in vitro maturation, IVF and embryo development are altered by gonadotrophin concentration during oocyte maturation.

BACKGROUND: This is the first study of the effect of epidermal growth factor (EGF) on marmoset monkey oocytes matured in vitro. METHODS: We have evaluated the effects of 10 ng/ml EGF in combination with 1 or 10 IU/ml of gonadotrophins (FSH/hCG 1:1 ratio) during in vitro maturation (IVM) of marmoset oocytes. Immature cumulus-oocyte complexes (COCs) were retrieved from ovarian antral follicles of unprimed monkeys. COCs from six animals (n= 268) used in this study were randomly distributed among four experimental groups: (A) 1 FSH +1 hCG; (B) 10 FSH +10 hCG; (C) 1 FSH +1 hCG + EGF; and (D) 10 FSH +10 hCG + EGF (where 1 and 10 are concentrations, IU/ml). After IVM, oocytes were fertilized in vitro and embryos were allowed to progress up to 87-88 h. RESULTS: the highest rate of total and radial cumulus expansion was observed in Group A, with the lowest in Group B (P < 0.05). Neither maturation nor fertilization rate were affected by gonadotrophin concentration or presence of EGF. Addition of EGF increased degeneration and decreased first cleavage rate, which was significantly lower in Group C than Group A (P < 0.005). Interestingly, in the EGF groups some embryos cleaved faster than without EGF. CONCLUSIONS: The effects of EGF are highly dependent on concentration of gonadotrophins present in IVM medium. EGF has a negative effect on oocytes in the presence of low gonadotrophins, but contrastingly partially protects oocytes from the negative effects of high gonadotrophins. We propose that these observed negative effects of EGF may suggest use of an inappropriate dose of growth factor.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm.

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C. jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the "Simple" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the "Simple" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the "Simple" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the "Simple" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species.

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus).

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 (>1.5mm) and small (0.7-1.5mm) 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.

Effects of human pituitary FSH isoforms on mouse follicles in vitro.

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors.

BACKGROUND: Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. METHODS: Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-alpha-tubulin antibody combined with a DNA fluorochrome. RESULTS: 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. CONCLUSIONS: This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.

Comparison of the effects of Percoll and PureSperm on the common marmoset (Callithrix jacchus) semen.

Density gradient centrifugation is a widely used technique for the separation of motile from non-motile sperm, for the removal of contaminating agents such as bacteria and viruses, and for the removal of seminal plasma or cryoprotectant. In the choice of a density gradient medium for a new species, it is important to perform toxicity tests. The present study was carried out to evaluate the potential toxic effects of two silica-based density gradient products (Percoll and PureSperm), on the sperm of the common marmoset. We assessed two different batches of Percoll (polyvinylpyrrolidone (PVP)-coated colloidal silica particles) and one of PureSperm (saline-coated colloidal silica suspension) by means of a computer-aided sperm analysis on semen collected by vibrostimulation. The results showed that although some of the sperm patterns of movement and viability changed significantly over time, and provide a first description of marmoset sperm motility changes under capacitating conditions, there was no significant difference in the sperm treated with Percoll or PureSperm in comparison with the control. We conclude that simple exposure to either of these products does not have a negative effect on viability or motility of marmoset sperm.

Oocyte-sperm interaction in the course of IVF: a scanning electron microscopy analysis.

Interaction between the spermatozoon and the zona pellucida during the first steps of fertilization was analysed on approximately 500 polyploid and unfertilized IVF oocytes by scanning electron microscopy (SEM). Oocytes demonstrate high inter- and intra-individual variations in size and morphology which do not correlate either with the maturity of the oocytes or with the age of the women. During gamete interaction, corona radiata cells are widely dispersed around the zona but are still in contact with it through cytoplasmic filaments. Channels between granulosa cells guide spermatozoa towards the zona. In the course of fertilization, different types of attachment of the spermatozoon to the oocyte occur. Most commonly, a flat, tangential attachment of the sperm head to the surface of the zona appears, which is then followed by intrusion into the zona in precisely this horizontal position. However, vertical binding with penetration by the tip of the head first also occurs. In oocytes where large, cluster-like numbers of bound spermatozoa are visible, vertical binding and penetration is the most usual position. In the process of gamete interaction, both spermatozoa and zona pellucida are actively involved. Spermatozoa, including their tails, which are attached to the zona, are overgrown by filaments of zona material. These filaments of the zona are made of granules, which are the basic components of zona material. After the removal of the zona pellucida by laser, the oolemma becomes visible. It is covered by microvilli of highly variable numbers. Between these microvilli, cortical granules are evident, and appear even before sperm penetration.

In vitro development of marmoset monkey oocytes by pre-antral follicle culture.

A technique for in vitro maturation of oocytes from small ovarian follicles of marmoset monkeys (Callithrix jacchus) has been developed. We employed a two-step culture system for primary follicles (45-85 microm) and a one-step culture technique for secondary follicles (>85 microm). The two-step technique started with the culture of stromal tissue fragments for 2 days. Thereafter, mechanically isolated follicles were transferred to a culture system where they attached to the culture surface and grew for up to a further 12 days. Significant growth of the small follicles and their oocytes was only achieved with gonadotrophins in the medium. Oocytes with a mean diameter of 39 microm from follicles <85 microm reached a mean diameter of 90 microm by the end of the two-step culture. After in vitro maturation, 19% of oocytes from these follicles had progressed to the germinal vesicle breakdown (GVBD). Follicles between 85 and 170 microm in diameter were isolated from the stroma and placed directly in the culture. Oocytes from these follicles had a mean diameter of 64 microm. The maximum size the oocytes reached in culture was related to the age of the females (pre-pubertal females: 102 +/- 1.3 microm; adults: 96 +/- 1.4 microm). Twenty-seven per cent of oocytes from pre-pubertal ovaries achieved GVBD and nearly two-thirds of these progressed to polar body stage. From adult ovaries, only 12% progressed to GVBD and one-third of these to polar body stage. It is possible to develop mature oocytes in vitro from marmoset secondary pre-antral follicles (>85 microm). From primary follicles, although near full size oocytes were developed, maturation capacity was incomplete.

Congenitally caused fused labia in the common marmoset (Callithrix jacchus).

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.

Progress toward understanding follicle development in vitro: appearances are not deceiving.

The interactive factors that influence the developmental progress of a follicle and determine whether it will progress to ovulation or toward atresia, are highly complex. In vitro models are being developed that are intended to provide a simplified environment to facilitate understanding of the dynamics of the processes involved. The purpose of this overview is to evaluate progress to date and to focus attention on issues that need more careful consideration to improve the usefulness of the models. Basically, two approaches exist. One, attached follicle culture, employs either enzyme-digested or mechanically harvested follicles depending on the method but allows attachment of the follicles to the culture surface. This produces a rounded or flattened structure (depending on culture conditions) that is no longer an intact follicle. During this culture, the cells reorganize themselves, some remaining in contact with the oocyte and others attaching to the culture surface and proliferating. The other approach, intact 3-dimensional follicle culture, employs mechanically dissected preantral follicles that are cultured as free-floating intact structures. Intact follicle culture emulates the in vivo developmental pattern of the follicle more closely than a non-intact structure can, and thereby provides a favorable model to investigate the interaction between hormonal and paracrine factors in the development of the follicle in isolation from systemic effects. For example, intact follicle culture has begun to be used to investigate the local effects of several different steroids. In addition, the local effects of inhibin, activin, and follistatin and their interactions with locally produced growth factors and steroids as well as synergy with gonadotrophins are beginning to be investigated. In our laboratory, the focus is on the roles of gonadotrophins at different stages of follicle development, particularly the effect of FSH isoforms in modulating follicle development in vitro. Finally, an important issue that urgently needs to be addressed, for future studies of in vitro follicle development, is the rationalization and standardization of follicle culture conditions.