Age-related decline of fertility in women is the result of the decline in both quantity and quality of the resting ovarian follicle pool. The aim of the present study was to determine whether the decline of follicle quality with age is reflected by ultrastructural changes in the resting follicle pool. Ovarian biopsy specimens were obtained by laparoscopy from seven healthy women aged 25-32 yr (young group) and from 11 healthy women aged 38-45 yr (advanced-age group). A total of 182 resting follicles from the young group were compared with 81 resting follicles from the advanced-age group for signs of age-related changes by transmission-electron microscopy. The ooplasmic fraction of vacuoles was increased (P = 0.02), and the fraction of mitochondria decreased (P = 0.005), in the advanced-age group. Also, the density of the mitochondrial matrix (P < 0.001) and the frequency of dilated smooth endoplasmic reticulum (SER; P = 0.001) and Golgi complex (P = 0.02) were increased with age. The frequencies of ruptured mitochondrial membranes (P = 0.001) and dilated SER (P = 0.003) were increased with age in the granulosa cells. Overall follicle-quality scores, which should reflect atretic changes, were not different for the young and advanced-age groups. In conclusion, in resting follicles, the morphological changes with age are different from the changes seen in quality decline by atresia. The morphological changes with age specifically involved the mitochondria, the SER, and the Golgi complex, and they may be the cause of atresia on initiation of follicular growth because of the substantial increase in metabolic requirements.

In humans, follicle quantity and quality decline with age by atresia. In the present study we aimed to describe the quality of the follicle pool through an ultrastructural investigation of resting follicles in young healthy women. From ovarian biopsies of 7 women aged 25-32 yr, 182 small follicles were morphometrically assessed for various signs of atresia. Morphometric variables were analyzed by principal components analysis (PCA) to demonstrate correlations between variables and to construct an objective follicle score. One third of small follicles consisted of primordial follicles. Nucleus:cell ratios remained constant for oocytes and granulosa cells from primordial to primary follicles, suggesting that follicles up to primary stages belong to the resting pool. The distribution of follicle quality scores as derived from PCA showed that most follicles were of good quality and with little signs of atresia. Atresia in resting follicles appears to be a necrotic process, starting in the ooplasma. Early atresia was characterized by increasing numbers of multivesicular bodies and lipid droplets, dilation of smooth endoplasmic reticulum and Golgi, and irregular mitochondria with changed matrix density. In progressive atresia mitochondrial membranes ruptured, oocyte nuclear membranes were indented or ruptured, and the ooplasma showed extensive vacuolarization. The early involvement of mitochondria in this process suggests that damage is induced by oxygen radicals. PCA follicle quality scores can be reliably approximated using a reduced number of seven morphometric variables, which were selected by stepwise forward analysis. The algorithm to calculate these follicle scores is presented.

Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes.

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cycle and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.

The Sertoli cells of pig fetuses from 35 days postcoitum until 1 mo after birth have been investigated by light and electron microscopy in decapitated animals and their control littermates, as well as in untreated animals. Until 52 days postcoitum, Sertoli cells change in form during the formation of sex cords but from then onwards they are rather uniform. They primarily display an elongated nonindented nucleus with one or more prominent nucleoli, a well-developed Golgi apparatus, and in the basal compartment below or beside the nucleus, a large lipid droplet. There are large quantities of rough endoplasmic reticulum (RER) from 52 days postcoitum onwards, often with complex whirl forms and a parallel arrangement of profiles with relatively few ribosomes. After birth their numbers seem to be somewhat less, and by 1 mo after birth the RER profiles are often shorter and almost free of ribosomes. Clustered ribosomes are found in large quantities throughout the period under investigation. Especially in the early fetal period, the endoplasmic reticulum (ER) profiles show prominently filled cisternae. Mitochondria are mostly long and slender, or small and ovoid. Most have lamellar cristae, but mixed lamellar-tubular cristae can also be seen. Between decapitated, control and untreated animals no obvious ultrastructural differences could be observed. The peritubular cell sheath surrounding the sex cords did not show signs of differentiation into a layer of myoid cells.