Basal membrane remodeling during follicle histogenesis in the rat ovary: contribution of proteinases of the MMP and PA families.

In mammalian females, follicular units arise from the fragmentation of ovigerous cords, which spread over the first three postnatal days in the rat. The mechanisms underlying such a process of epithelial remodeling involve a specific balance between basal membrane (BM) deposition and degradation that has as yet not been precisely described. We have investigated the contribution of proteases in BM remodeling by localization of transcripts, protein, or enzymatic activity. In addition, we have analyzed BM deposition at the ultrastructural level and by immunofluorescence detection of BM components. At birth, when fragmentation occurred, epithelial cells displayed an upregulation of membrane type 1-matrix metalloproteinase (MT1-MMP) and urokinase-type plasminogen activator (uPA), as well as laminin alpha1 mRNAs. Although MMP2 expression was restricted to mesenchymal cells throughout development, in situ zymography showed that gelatinase-MMP2 activity colocalized with BM deposition inside deepening clefts in the areas of ovigerous cord fragmentation. In the days following birth, gelatin and plasminogen-casein zymography showed an increased enzymatic activity of MMP2 and uPA, respectively. In organotypic cultures of 21-day postconception ovaries, serine protease inhibitors like aprotinin could efficiently block follicle histogenesis. In addition, our results show that the well described and great wave of oocyte attrition characteristic of the days following birth closely correlates with BM remodeling. Altogether, our data show that during follicle histogenesis, ovigerous cord fragmentation results from an acute BM component deposition in deepening clefts and that BM homeostasy involves proteinases of the MMP2/MT1-MMP/TIMP3 and plasminogen/uPA families.

Unaltered development of the initial follicular waves and normal pubertal onset in female rats after neonatal deletion of the follicular reserve.

In rats, the pool of primordial follicles is established within the first 3 d postnatally (dpn). Immediately after their differentiation, a subset of follicles begins to grow and constitutes the initial follicular waves. In this study we investigated the development of these early growing follicles after deletion of the primordial follicle pool induced by 1.5 Gy gamma-irradiation at 5 dpn. Within only 24 h, i.e. at 6 dpn, 99% of the primordial follicles disappeared, whereas most of the growing follicles remained unaffected. The study of these surviving follicles throughout the immature period has shown that their subsequent growth proceeded normally, as assessed by proliferating cell nuclear antigen immunostaining and follicular counts. No modification in the process of follicular atresia, studied by terminal deoxynucleotidyltransferase-mediated deoxy-UTP-fluorescein nick end labeling and Southern blot of DNA fragmentation analysis, was observed. Complementary analysis, by either in situ hybridization for inhibin subunits, P450 aromatase, and LH receptor mRNAs or plasma dosages of 17beta-estradiol and inhibin B, further showed that follicular maturation was unaltered. In line with these observations, pubertal onset was normal, regarding both age and ovulation rate. Nevertheless, as a consequence of the nonrenewal of the growing pool, the follicular complement was practically exhausted at puberty, and 90% of the females evidenced sterility by 4 months. Altogether, our results demonstrate that the deletion of the primordial follicle pool has induced no modification in the growth pattern of the early growing follicles that develop as their counterparts in control ovaries. Within the immature period, the initial follicular waves ensure the ovarian functionality and thus play a key role in the initiation of reproductive life.

Establishment of the reproductive function and transient fertility of female rats lacking primordial follicle stock after fetal gamma-irradiation.

In mammals, the primordial follicle stock is not renewable, and its size, therefore, limits the reproductive life span of the female. In this study we have investigated the morphological and functional differentiation of dysgenesic ovaries in female rats exposed in utero to 1.5 Gy gamma-irradiation. As a consequence of the severe depletion in oocytes, females evidenced premature ovarian failure from 6 months on. Nevertheless, puberty onset and fertility at the beginning of reproductive life were similar to those of controls. The differentiation and evolution of the entire follicular population were followed during the immature period, using follicle counts, in situ hybridization of follicular maturation markers, and analysis of atresia. Primordial follicles were much more affected by irradiation (1.4-1.9% of controls) than growing follicles (30-45% of controls). As the very low number of primordial follicles remained constant throughout this period, it may be considered that the growing follicle pool plays the role of follicular reserve, permitting the transient normal fertility of irradiated females. Within the neonatal period, primary and secondary follicles, as revealed by proliferating cell nuclear antigen immunostaining, remain quiescent longer in irradiated than in control ovaries. Consequently, the majority of the most mature follicles (i.e. the first follicular wave) characterized by a high expression of aromatase transcripts during the infantile period, are missing in irradiated ovaries. Concomitantly, the 17beta-estradiol plasma peak is absent, and plasma FSH levels are higher than those in control females. In conclusion, these observations emphasize that the female reproductive life span depends not merely on the size of the primordial follicle stock, but also on the entire follicle complement as well as follicular dynamics during the immature period.

Different patterns of anti-Müllerian hormone expression, as related to DMRT1, SF-1, WT1, GATA-4, Wnt-4, and Lhx9 expression, in the chick differentiating gonads.

In mammals, anti-Müllerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells after birth. In birds, AMH starts to be expressed in indifferent gonads of both sexes at a similar level and is later up-regulated in males. We previously demonstrated that, unlike in mammals, the onset of AMH expression occurs in chick embryo in the absence of SOX9. We looked for potential factors that might be involved in regulating AMH expression at different stages of chick gonad differentiation by comparing its expression pattern in embryos and young chicken with that of DMRT1, SF-1, WT1, GATA-4, Wnt-4, and Lhx9, by in situ hybridization. The results allowed us to distinguish different phases. (1) In indifferent gonads of both sexes, AMH is expressed in dispersed medullar cells. SF-1, WT1, GATA-4, Wnt-4, and DMRT1 are transcribed in the same region of the gonads, but none of these factors has an expression strictly coincident with that of AMH. Lhx9 is present only in the cortical area. (2) After this period, AMH is up-regulated in male gonads. The up-regulation is concomitant with the beginning of SOX9 expression and a sex dimorphic level of DMRT1 transcripts. It is followed by the aggregation of the AMH-positive cells (Sertoli cells) into testicular cords in which AMH is coexpressed with DMRT1, SF-1, WT1, GATA-4, and SOX9. (3) In the females, the low level of dispersed medullar AMH expression is conserved. With development of the cortex in the left ovary, cells expressing AMH accumulate in the juxtacortical part of the medulla, whereas they remain dispersed in the right ovary. At this stage, AMH expression is not strictly correlated with any of the studied factors. (4) After hatching, the organization of left ovarian cortex is characterized by the formation of follicles. Follicular cells express AMH in conjunction with SF-1, WT1, and GATA-4 and in the absence of SOX9, as in mammals. In addition, they express Lhx9 and Wnt-4, the latter being also found in the oocytes. (5) Moreover, unlike in mammals, the chicken ovary retains a dispersed AMH expression in cortical interstitial cells between the follicles, with no obvious correlation with any of the factors studied. Thus, the dispersed type of AMH expression in indifferent and female gonads appears to be bird-specific and not controlled by the same factors as testicular or follicular AMH transcription.