Summary. The development of a culture system for individual mouse ovarian follicles using a low concentration of homologous serum, human follicle-stimulating hormone (hFSH) Follicles cultured in groups without being in direct contact with each other showed much poorer growth than those grown individually, but the inhibition was not uniform and some follicles grew larger than others in the group. Follicles that contacted each other directly in culture tended to fuse into one mass and their growth was substantially inhibited. Even under these conditions, one follicle often continued to grow slowly while the others degenerated. Such alteration of growth patterns suggests interfollicular paracrine control and may be a means of three-dimensional spacing of follicle growth within the ovary, as well as part of the mechanism of follicle selection. The dose\p=n-\ response curve based on the mean growth trajectory of follicles cultured individually, produced increasing rates of growth with 12\m=.\5\p=n-\100miu hFSH ml\m=-\1. Higher concentrations of hFSH did not increase growth rate further, but oestradiol secretion continued to increase with increasing hFSH up to the maximum used (2000 miu ml\m=-\ 1).

This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte meiotic competence and the time course of nuclear maturation are profoundly influenced by their follicular origin, and marginally by FSH treatment.

Carbohydrates attached to the protein core of glycoprotein hormones influence a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, FSH is produced and released as an array of isoforms that differ from each other in the structure of their oligosaccharide attachments. In this review, we discuss how carbohydrate heterogeneity can impact on FSH action in different in vitro and in vivo systems. We present evidence for diverse effects of distinct charge isoforms at the target cell level, including differential and unique effects on various end responses, and discuss how the use of multiple cell-type assays has allowed identification of some specific effects of FSH isoforms on different cell populations and follicle compartments as well as oocyte maturation. Finally, we discuss recent information on the ability of naturally occurring and laboratory manufactured FSH isoforms to evoke particular effects on granulosa cell function and ovarian follicular maturation in vivo. Such studies have provided evidence that the type(s) of FSH signal delivered may in fact regulate distinct biological outcomes irrespective or in addition to outcomes dictated solely by clearance rate differences.

This study was conducted to investigate the relationships between oocyte meiotic competence, follicle size, and occyte-somatic cell associations in the marmoset monkey (Callithrix jacchus). Follicles were excised from ovaries of nonstimulated adult cyclic females (n = 6) collected on Day 7 of the follicular phase. Follicles were separated into size groups: large preantral (260-400 microns), periantral (420-640 microns), small antral (660-1000 microns), large antral (1020-2000 microns), and preovulatory (> 2000 microns). Partially naked and cumulus/granulosa-enclosed oocytes (n = 473) were released from follicles and cultured in Waymouth's medium with 10% fetal calf serum, 1 microgram/ml human (h) FSH, and 10 micrograms/ml hLH. Somatic cells remaining after 46 h were removed, and oocytes were fixed after 48 h and mounted for viewing. Chromatin staining and microtubulin fluorescence labeling were used to assess progression of meiotic maturation and spindle normality. The follicle size distribution and oocytesomatic cell associations are reported. Competencies of oocytes to achieve germinal vesicle breakdown (GVBD) and metaphase II (MII) increased significantly (p < 0.001) with follicular size but not with the association of somatic cells. Marmoset oocytes from antral follicles resumed (GVBD) and completed (MII) meiotic maturation with high frequencies (98% and 72%, respectively), with no significant differences among size groups of antral follicles. GVBD competence was virtually absent in oocytes from preantral follicles (2%) and was acquired coincidentally with antrum formation (60%), although MII competence was attained after the completion of antrum formation. Partially naked oocytes from small antral follicles matured with a high incidence of spindle and meiotic abnormalities (44%). Marmoset oocyte meiotic competencies are notably higher than in any other nonhuman primate species studied, and a possible explanation for this phenomenon in relation to the stage of antrum formation is offered.

Penile vibrostimulation (PVS), a noninvasive repeatable method, has been shown in the squirrel monkey to yield semen of higher quality than rectal probe electro‐ejaculation (RPE). The present study aimed at establishing the conditions for PVS to collect ejaculates from marmoset monkeys. Ten adult males were trained on the appropriate handling before each was subject to six to 12 PVS tests. Ejaculation was stimulated using a FertiCare® personal vibrator fitted with a 2 cm × 0.5 cm i.d. glass tube. The stimulus was repeatedly applied over a frequency of 75–95 Hz and amplitude of 1–2 mm for up to 20 min. Ejaculates were analyzed for volume, total sperm number, sperm concentration, and proportion of living and motile sperm. Ejaculates were obtained in 31 of 88 PVS tests; 87.1% of the ejaculations occurred at 80–85 Hz frequency and 1–1.5 mm amplitude. In 18 tests ejaculates were produced within 49.7 seconds. Ejaculates were characterized by (mean values): volume 31.9 μl, total sperm number 34.2 × 106/ejaculate, concentration 1,154.2 × 106 sperm/ml, live sperm 74.6%, motile sperm 59.6%. Total number and concentration of spermatozoa were significantly enhanced in singly living males. PVS yielded three to four times more spermatozoa than comparable previously published values for RPE. Enhancing the success rate by preselecting males for responsiveness may render PVS the sperm collection method of choice in marmoset monkeys. Am. J. Primatol. 52:149–154, 2000. © 2000 Wiley‐Liss, Inc.