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Endocrinology Vol. 141, No. 11 4284-4294
Copyright © 2000 by The Endocrine Society

ARTICLES

Loss of Oocytes in Dazl Knockout Mice Results in Maintained Ovarian Steroidogenic Function but Altered Gonadotropin Secretion in Adult Animals

J. R. McNeilly, P. T. K. Saunders, M. Taggart, M. Cranfield, H. J. Cooke and A. S. McNeilly

Medical Research Council Human Reproductive Sciences Unit, University of Edinburgh Center for Reproductive Biology (J.R.M., P.T.K.S., A.S.M.), Edinburgh, Scotland EH3 9ET; Medical Research Council Human Genetics Unit, Western General Hospital (M.T., H.J.C.), Edinburgh, Scotland EH4 2XU; and Center for Proteins and Peptides, School of Biological and Molecular Sciences, Oxford Brookes University (M.C.), Headington, Oxford, United Kingdom OX3 0BP

Address all correspondence and requests for reprints to: Dr. J. R. McNeilly, Medical Research Council, Human Reproductive Sciences Unit, University of Edinburgh Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, Scotland EH3 9ET. E-mail: j.mcneilly{at}hrsu.mrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (-/-) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from -/- females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3ß-hydroxysteroid dehydrogenase, 17{alpha}-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor {alpha} (ER{alpha}) and ERß together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ERß, inhibin {alpha}, and ßB-subunits, and -/- mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/- females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/- females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/- mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE MAMMALIAN female, fertility requires the production of mature oocytes, and it is by the process of folliculogenesis that mature fertilizable oocytes are produced. Within the ovary, each follicle consists of an oocyte surrounded by somatic cells. A complex cascade of events involving both intra- and extraovarian factors leads to ovulation. During this period, there is coordination between the growth and development of the oocyte, differentiation of the somatic cells to become granulosa cells or thecal cells, followed by proliferation and development to produce the steroidogenically active follicle (1, 2).

In the mouse during fetal life, the ovary consists of somatic cells arranged in cords, with each cord surrounded by mesenchymal cells and filled with masses of rete ovary-derived cells and oocytes (3). Within the first 3 days of birth, primordial follicles are formed that consist of oocytes with attendant somatic squamous pregranulosa cells (4, 5). Almost immediately, folliculogenesis is initiated, and a subset of these primordial follicles is recruited and begins to grow. The mechanisms involved in this initial recruitment are as yet unknown; however, studies in the mouse, rat, cow, and human have demonstrated that although follicle growth can be initiated in the absence of gonadotropins, few follicles develop beyond two layers of granulosa cells (6, 7, 8, 9). However, the availability of transgenic mouse models of ovarian failure may help to identify the intraovarian factors involved (10). The rate of recruitment of primordial follicles into the growth phase is greatest between 7–21 days and thereafter decreases and remains stable for the rest of the reproductive life of the animal (11). Until the formation of an antrum, regulation of development appears to be intrafollicular with various crucial factors, e.g. growth differentiation factor-9 (GDF-9) and Kit ligand, involved in signaling from the oocyte to the granulosa cells and vice versa (12, 13, 14). In fact, an oocyte is necessary for preantral growth up to the induction of a thecal layer, which occurs once the follicle has achieved two layers of granulosa cells. This thecal layer provides a source of aromatizable androgen to the adjacent granulosa cells essential for estrogen production (15, 16, 17).

Available evidence suggests that the presence of oocytes is critical for the induction of effective thecal-granulosa cell interaction and hence for the initiation of steroidogenesis (18). The requirement of oocytes for the maintenance of steroidogenesis is not clear. The Dazl knockout (-/-) mouse offers us a unique opportunity to address this question. Although on embryonic day 15 the ovaries appear normal in -/- females, there is a marked reduction in oocyte numbers on E19 and a complete absence of follicles and ova in the adult ovary (19). The aim of this study was to determine the effect of early oocyte loss on the capacity of the ovary to produce steroids and inhibins and the consequences on plasma and pituitary gonadotropin secretion using intact and ovariectomized adult -/-, heterozygous (+/-), and wild-type (+/+) Dazl females.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
In all studies the female mice used were from lines generated by a conventional knock out approach in which the Dazl gene was disrupted by a neomycin-resistant marker after homologous recombination in embryonic stem (ES) cells, causing the loss of exons 6 and 7 and all but five amino acids of exon 5. Chimeric males were generated by selection of ES cells containing the targeted mutation using a PCR-based assay. Mating these males to MF1 females generated heterozygotes that when intercrossed gave homozygous (-/-) animals (7). Animals were weaned at 3 weeks of age and genotyped at 6 weeks by extraction of tail DNA and subsequent PCR. They were maintained on a 14-h light, 10-h dark photoperiod under normal animal facility conditions. All studies were approved by the Home Office (United Kingdom) and undertaken under license. Adult females (8–12 weeks of age) expressing all three genotypes (+/+, +/-, and -/- for the Dazl gene) were caged in sibling groups and age matched before this study. Vaginal smears were collected daily for at least 7 days, equivalent to at least one estrous cycle from intact females of all three genotypes (n = 6–10/genotype), to determine whether they were exhibiting normal estrous cycles. During the next cycle, +/- and +/+ animals only were killed by CO2 asphyxiation when an estrogenic type smear, similar to that observed in all -/- animals, was observed. The final vaginal smear was air-dried, fixed for 1–2 min in 70% ethanol, then stained with hematoxylin, dehydrated, and mounted in Pertex (Cellpath, Hemel Hempstead, UK) as a record of vaginal cytology at the time of tissue collection. Blood samples were collected by cardiac puncture after killing the animals with CO2, and plasma was separated by centrifugation at 3000 rpm for 15 min and frozen at -20 C until assayed for LH, FSH, inhibin A and B, estradiol, and progesterone. Pituitaries were collected and stored at -20 C until homogenized in 0.5 ml PBS (Sigma, St. Louis, MO) before assay for LH and FSH. Ovaries and uteri were removed, weighed, and fixed in Bouin’s fixative for 2–5 h, then transferred to 70% ethanol. Concurrently, females (n = 6–10) from the three genotypes had the stage of the estrous cycle determined by vaginal smear before being ovariectomized under IsoFlo anesthesia (Mallinckrodt, Inc., Harefield, UK). After removal, the ovaries were weighed and fixed in Bouin’s as previously described. Seven days after surgery the animals were killed by CO2, and plasma, pituitaries, and uteri were collected as previously described. Ovaries were also removed from day 16 females after animals were killed by CO2 and were fixed in Bouin’s fixative; the animals were genotyped retrospectively from tail DNA and PCR as described previously.

Immunocytochemistry
After processing, ovaries and uteri were embedded in paraffin wax and sectioned at 5 µm thickness before detection of the antigens listed in Table 1Go (20, 21, 22, 23, 24). Antigen retrieval (25) was required for the successful detection of some antigens using 0.01 M citrate pH 6, or 0.05 M glycine-EDTA, pH 3.5. Before incubation of the sections with primary antibody, all slides were blocked with either 1:5 normal rabbit serum/Tris-buffered saline if the primary antibody was monoclonal or 1:5 normal swine serum/Tris-buffered saline if it was polyclonal. Similarly, primary antibodies, diluted in the appropriate blocking buffer, were added to the sections, which were coverslipped with Gelbond (FMC Bioproducts, Rockland, ME) before incubation overnight at 4 C. Detection using a biotinylated second antibody (swine antirabbit or rabbit antisheep for polyclonal primary antibodies; rabbit antimouse for monoclonal primary antibodies; DAKO Corp., Copenhagen, Denmark) was followed by the avidin-biotin-horseradish peroxidase system (DAKO Corp.) and visualized by 3',3'-diaminobenzidine. For sections incubated with anti-Mullerian hormone/Mullerian inhibiting substance (AMH/MIS) antiserum, the avidin-biotin-alkaline phosphatase system (DAKO Corp.) was used with nitro blue tetrazolium (Sigma) and x-phosphatase (5-bromo-4-chloro-3-indolyl-phosphate; Sigma) visualization. Control sections were incubated with normal rabbit serum or normal mouse serum in place of the primary antibody. The specificity of the antibodies was confirmed in previous studies using antigen preadsorption. All sections were counterstained with hematoxylin, then dehydrated and mounted in Pertex (Cell Path, Hemel Hempstead, UK). Sections were photographed using an Olympus Corp. Provis microscope (New Hyde Park, NY), and Kodak 420 digital camera (Eastman Kodak, Inc., Rochester, NY), and montages were assembled using Photoshop 5 (Adobe Systems, Inc., San Jose, CA).


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  		Table 1. Antisera used for immunocytochemical studies on ovarian and uterine tissues

 
Immunoassays
The concentrations of plasma and pituitary LH and FSH were measured by RIA using reagents supplied by the NIDDK with all samples for each hormone assayed in duplicate in one assay. The reference preparations used were rat LH RP-1 and rat FSH RP-3, and the minimum detectable concentrations were 0.2 and 1.2 ng/ml for LH and FSH, respectively. The intraassay coefficients of variation were less than 6% (26). As the volume of plasma available for assay was limited, not all samples were assayed for inhibin A and B, estradiol, and progesterone; however, there were sufficient samples to allow meaningful values (n = 4–6) to be obtained in each treatment group. In some cases the samples were assayed as single samples. Inhibins A and B were measured using two-site enzyme-linked immunosorbent assays (27, 28) and were validated for mouse plasma (29). All samples were assayed in a single assay, and the minimum detectable concentrations were 1 pg/ml (inhibin A) and 8 pg/ml (inhibin B), respectively, with coefficients of variation of less than 10%.

Estradiol and progesterone were determined after solvent extraction using sensitive RIA methods modified for use with mouse and sheep plasma, as previously described (30, 31). All samples were measured in the same assay with a coefficients of variation of less than 8%, and the minimum detectable concentrations for estradiol and progesterone were 4.6 pg/ml and 0.5 ng/ml, respectively.

Statistical analysis
All data were analyzed by ANOVA and Student’s paired t tests using the GB Stat program (Dynamic Microsystem, Inc., Silver Spring, MD). P <= 0.05 compared with the appropriate control was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of genotype on uterine morphology
The degree of hypertrophy of the uterine endometrium gives an indication of the circulating estrogenic steroid level. Despite a lack of oocytes, uteri from intact -/- females were grossly similar to those from intact estrous +/- females, with no differences in endometrial thickness between the genotypes noted. However, uteri from intact estrous +/+ females were significantly heavier (P < 0.01 and P < 0.05, respectively) compared with those from -/- and +/- animals (Fig. 1AGo, wet weight). There was a similar pattern of expression of estrogen receptor {alpha} (ER{alpha}; Fig. 1BGo, a and b) and ERß (Fig. 1BGo, c and d) in all three genotypes, with intense staining of both glandular and stromal endometrium.



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  		Figure 1. A, Changes in uterine wet weights in wild-type (+/+), heterozygous (-/+), and homozygous (-/-) Dazl-1 knockout mice before ({blacksquare}) and after ({square}) ovariectomy. a–c, Significant differences between uteri from ovary-intact mice; d–f, significant differences after ovariectomy. B, Immunostaining for ER{alpha} (B, a and b) and ERß (B, c and d) in the uteri removed from these intact wild-type (WT) +/+ and knockout (KO) -/- adult mice. There was no difference among +/+, +/- (results not shown), and -/- in the distribution of ER{alpha} or ERß immunostaining.

 
After ovariectomy, there was a highly significant (P < 0.01) decrease in uterine wet weights in all three genotypes (Fig. 1AGo). Interestingly, uteri from -/- females weighed significantly (P < 0.05) less than those from both +/+ and +/- after ovariectomy. There was no significant difference in uterine weight between ovariectomized +/- and +/+ females, although the +/- females tended to be lighter. There was no difference in body weight among the three genotypes.

Functional competence of ovarian cells and expression of steroidogenic enzymes
Evidence that the -/- ovary, despite a lack of oocytes, secretes sufficient estrogenic steroid to maintain a proliferative uterine endometrium led us to investigate the functional activity of the cells in the -/- ovaries compared with those in the other two genotypes. Cellular proliferation, as determined by immunocytochemistry using proliferating cell nuclear antigen (PCNA) antiserum (Fig. 2Go, a and b) showed few dividing cells in the -/- ovary, whereas in the +/+ and +/- ovaries (+/- data not shown), extensive cellular proliferation was observed in all follicular tissues, consistent with the presence of actively proliferating granulosa cells with a much lower rate of cell division in the ovarian stroma. This absence of functionally competent follicular tissue was confirmed by the presence of few MIS-positive cells in the -/- ovaries (Fig. 2gGo), with MIS being expressed only in granulosa cells of small follicles in normal +/+ and +/- ovaries (Fig. 2Go, c and e). Similarly, stromal cells and corpora lutea were immunopositive for sulfated glycoprotein-1 (SGP-1) in +/+ and +/- ovaries (Fig. 2Go, d and f), whereas ovaries from -/- females did not contain any organized structures that expressed SGP-1 (Fig. 2hGo).



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  		Figure 2. Morphological appearance of ovaries in adult +/+, +/-, and -/- Dazl mice. Sections were immunostained with antibodies directed against PCNA (a and b), AMH/MIS (c, e, and g), and SGP-1 (d, f, and h). Granulosa cells surrounding preantral as well as antral (*) follicles were present in ovaries from both +/+ (a, c, and d) and +/- (e and f) females, whereas no follicular structures were present in ovaries lacking Dazl (-/-; b, g, and h). Granulosa cells in developing follicles were immunopositive for PCNA (a, arrows) consistent with a high rate of cell division in the somatic cells of the follicle, whereas very few PCNA-positive cells (b, arrows) were present in ovaries from -/- mice. The histological appearance of ovaries from +/+ and +/- mice were indistinguishable; both contained small follicles (arrowheads) in which granulosa cells were immunopositive for AMH/MIS and contained antral follicles (*), the granulosa cells of which did not express the protein. Stromal cells (S) and corpora lutea were immunopositive for SGP-1. In contrast, ovaries from -/- females did not contain organized structures expressing either AMH/MIS (g) or SGP-1 (h). Magnifications: a and b, x20; c, d, e, f, g, h, x10.

 
Ovarian sections from +/+ and +/- females, stained for 3ß-hydroxysteroid dehydrogenase (3ßHSD), showed intense cytoplasmic staining in both stromal and thecal cells. The first appearance of 3ßHSD-immunopositive granulosa cells was in antral follicles (Fig. 3aGo; data for +/- ovaries not shown). In ovarian tissue from -/- females (Fig. 3bGo) groups of intensely stained cells were dispersed throughout the tissue, consistent with a lack of structural organization. The intensity of staining and the variation in size of these steroidogenically active cells varied among -/- animals (data not shown). The pattern of expression of 17{alpha}-hydroxylase was identical to that of 3ßHSD in +/+ (Fig. 3cGo), +/-, and -/- (Fig. 3dGo) mouse ovaries.



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  		Figure 3. Expression of steroidogenic enzymes and ERß within the cytoplasm of cells in ovaries from both +/+ and -/- mice. Immunoexpression of 3ßHSD in the +/+ ovary (a) was intense both within the stroma and in thecal cells surrounding the developing follicles, and expression in GC was initiated in antral follicles (*). Expression of 3ßHSD was maintained in the -/- ovaries (b) and was localized to groups of cells, the size and abundance of which varied within the ovarian structures between animals. Similarly, 17{alpha}-hydroxylase expression was in only thecal and interstitial stroma (s) in +/+ ovaries (c), with no immunopositive cells observed in the granulosa cells (*); however, in the -/- animals, groups of positive cells were observed throughout the ovarian tissue. In ovaries from +/+ females, expression of aromatase was observed within GC of large antral follicles, where it was more abundant in those cells close to the thecal layer (e, arrowheads). In corpora lutea (g), variable immunopositive staining was observed, ranging from immunonegative (§) to strongly immunopositive (arrows). Expression of aromatase was observed in groups of cells in all ovaries from -/- females, but the intensity of immunopositive staining varied between ovaries from weak (f, arrowheads) to strong (h, arrows). Granulosa cells (*) from +/+ ovaries 1) expressed abundant ERß from small preantral to large preovulatory follicles. In -/- ovaries (j), expression of ERß was localized to clusters of cells (arrowheads), which varied throughout the tissue. ERß-immunopositive cells were also observed in the oviduct (od). Magnification, all x40.

 
In ovaries from +/+ and +/- females, aromatase cytochrome P450-positive cells were present in the peripheral granulosa cells of large preovulatory follicles, with the most intense staining observed at the granulosa/thecal junction (Fig. 3eGo, arrowhead). In corpora lutea, there was variable expression of aromatase ranging from immunonegative to strongly immunopositive cells (Fig. 3gGo, arrows). The potential of cells within the -/- ovaries to synthesize estrogen was endorsed by the detection of aromatase-positive cells in -/- ovarian tissue (Fig. 3Go, f and h). The intensity of staining and its distribution in the ovarian mass varied between ovaries, with some positive cells forming clusters as shown in Fig. 3gGo. ERß was expressed in the nuclei of granulosa cells throughout follicular development in both +/+ (Fig. 3iGo) and +/- ovaries and in occasional clusters of cells within -/- ovaries (Fig. 3jGo).

In ovaries collected from +/+ and +/- females on day 16 postpartum, there were many preantral follicles with intensely positive stained granulosa cells for both activin/inhibin ßB and inhibin {alpha} (Fig. 4Go, a and c; data for +/- not shown). Both subunits were also detected in -/- ovaries, with groups of immunopositive staining cells distributed throughout the ovarian stroma (Fig. 4Go, b and d). In adult -/- ovaries, although a few inhibin {alpha}-positive cells were present (Fig. 4hGo), they were weakly stained and tended to lack the cluster formation observed in the day 16 postpartum tissue. In both +/+ and +/- ovaries, inhibin {alpha} was strongly expressed in granulosa cells (Fig. 4Go, e–g).



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  		Figure 4. Inhibin {alpha}- and ß-subunits were expressed in +/+, +/-, and -/- ovaries. Sections were obtained from ovaries recovered on day 16 (a–d) or during adulthood (e–h). On day 16 immunopositive staining for inhibin/activin ßB (a) and inhibin {alpha} (c) subunits was detected in granulosa cells (gc) of preantral follicles of ovaries from immature +/+ mice. Both subunits (b, ßB; d, {alpha}) were also detected in the ovaries of day 16 -/- females, where they were present in groups of cells (arrows) distributed throughout the ovarian stroma. In ovaries from adult +/+ (e) and +/- (f) mice, there was no obvious difference in the pattern of expression of inhibin {alpha}-subunit, which was present in granulosa cells of small follicles (arrowheads), but was most abundant in large antral follicles (blue arrows). At higher power, the contrast among the structure of the adult +/+ ovary (g), which contained organized follicles; the granulosa cells (gc), which were immunopositive for inhibin {alpha}; and the -/- ovaries (h), in which very few cells randomly distributed within the ovarian tissue (arrows) expressed inhibin {alpha}-subunit, is clearly seen.

 
Plasma gonadotropin concentrations
LH. Plasma and pituitary LH concentrations in intact and ovariectomized females are shown in Fig. 5Go, a and c. In intact -/- females, plasma LH concentrations were significantly (P < 0.01) elevated compared with those in both +/+ intact and +/- intact females (Fig. 4aGo). After ovariectomy, plasma LH concentrations in +/+ and +/- females were significantly (intact +/+ vs. ovariectomized +/+, P < 0.01; intact +/- vs. ovariectomized +/-, P < 0.01) higher than their respective intact levels. The increase in plasma LH in ovariectomized +/- females tended to be lower than those in the other two groups. Furthermore, ovariectomy in the -/- females did not lead to an increase in plasma LH concentrations.



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  		Figure 5. Changes in mean (±SEM) plasma and pituitary concentrations of LH (a and c) and FSH (b and d) in ovary-intact ({blacksquare}) and ovariectomized ({square}) adult +/+, +/-, and -/- Dazl mice. Within the intact and ovariectomized mice, different letters indicate significant differences between groups.

 
Pituitary LH in intact females from all three genotypes mirrored the secretion pattern of plasma LH with pituitaries from intact -/- females containing highly significantly (P < 0.01) greater amounts of LH than those from +/+ and +/- females. After ovariectomy, pituitary LH levels in +/+ and +/- females rose significantly (P < 0.01) compared with levels in their respective intact groups. As observed in the pattern of plasma LH secretion, only in ovariectomized -/- females was there no significant increase in pituitary LH. No differences in pituitary LH were observed between +/+ and +/- females, either intact or ovariectomized; however, there was significantly (P < 0.05) less LH in +/+ pituitaries after ovariectomy than in pituitaries from -/- females.

FSH. Plasma and pituitary FSH concentrations are shown in Fig. 4Go, b and d, respectively. A pattern of secretion similar to that of LH was noted, with no difference in plasma FSH in +/+ and +/- intact females, but intact -/- females had significantly (P < 0.01) elevated circulating FSH. Despite this increased FSH secretion, there was a significant (P < 0.01) rise in plasma FSH in -/- females after ovariectomy, which was similar to that observed in +/- females. Although there was a significant (P < 0.01) increase in plasma FSH in ovariectomized +/+ females, it was significantly (P < 0.05) lower than that observed in both +/- and -/- females. It is interesting to note that the greatest postovariectomy rise in plasma FSH concentrations was observed in the +/- females.

Pituitary FSH was significantly (P < 0.01) higher in intact -/- females than in intact +/+ and +/- females. Although both plasma and pituitary FSH in +/- females appeared lower than the +/+ value, they failed to achieve significance. Ovariectomy resulted in a rise in pituitary FSH in all genotypes; however, only in the +/+ and +/- females was this rise significant (P < 0.01) compared with those in their respective intact groups. Although there appeared to be an increase in pituitary FSH after ovariectomy in the -/- females compared with their intact value, this was not significant. However, the increase in pituitary FSH in +/- females after ovariectomy was significantly (P < 0.01) lower than that in the +/+ females. There was no difference between FSH content in +/+ and -/- pituitaries after ovariectomy.

Inhibin and ovarian steroids. Plasma levels of inhibin A and B in intact females from all genotypes are shown in Fig. 6Go, a and b. In -/- females, both inhibin A and B concentrations were extremely low, with some samples being below the minimum detectable limit of the assays (inhibin A, 3 of 10; inhibin B, 3 of 16). The remaining values were significantly (P < 0.01) lower than those detected in the other two groups. Although there was no difference in inhibin A between +/+ and +/- females, significantly (P < 0.05) higher levels of inhibin B were present in +/- females compared with those in both +/+ and -/- animals.



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  		Figure 6. Changes in mean (±SEM) plasma concentrations of inhibin A (a), inhibin B (b), estradiol (E2; c), and progesterone (d) in intact adult female +/+, +/-, and -/- Dazl mice. Different letters indicate significant differences between groups.

 
Despite the lack of oocytes in -/- females, there was no significant difference in the plasma concentrations of either estradiol or progesterone between the groups, with both steroids being detectable in all samples in intact females from all three genotypes (Fig. 6Go, c and d).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we have shown that in the complete absence of oocytes, there are cells present within the adult ovary that can produce estradiol and progesterone together with low measurable inhibin A and B and cause the proliferation of the uterine endometrium. This shows for the first time that some development of the granulosa and thecal-like cells can be maintained in the absence of factors from the oocyte, although no functional organization of the follicle occurs. In fact, it is possible that once these cells are differentiated they maintain their steroidogenic function until they die, as there appear to be fewer inhibin-positive cells in the adult ovary compared with those in day 16 females. In the Dazl -/- mouse, oocyte loss starts and chronologically would appear to be completed before the initiation of follicular growth. In the absence of Dazl protein expression, the developing oocyte on embryonic day 15 enters meiosis, but is unable to progress further, and most are lost by embryonic day 19 with no follicular development seen on postnatal day 4 (our unpublished observation). It should be noted that the Dazl gene is a member of the DAZ/SPGY family of genes, which, like the RBM genes, are located on the Y-chromosome in Old World primates and apes, but are represented by the autosomal gene Dazl in others mammals. The function of Dazl protein is unknown; however, as it is cytoplasmic and has RNA binding motifs it may be involved in translational control (19).

At birth in the mouse, only "naked" oocytes are present, which by 2 days postpartum associate with three to five pregranulosa cells and become primordial follicles (4, 5). Follicular growth proceeds with a change in the morphology of these somatic cells followed by an increase in the number of granulosa cells, leading to the formation of multiple layers. When there are two layers of follicle cells present, fibroblast-like cells are recruited from the interstitium to encircle the follicle and form the thecal layer. All of these events appear to be regulated primarily by intraovarian factors and require the presence of an oocyte (2), and this oocyte-granulosa cell interaction is exemplified by the expression of c-Kit ligand in granulosa cells and c-Kit receptor in the oocyte (12, 13, 32). The importance of the oocyte in the proliferation and differentiation of granulosa cells is illustrated in the GDF-9 knockout mouse, where follicles arrest after two mitotic divisions of granulosa cells but remain at this stage, with the oocyte overgrowing until cell death (33). Interestingly, although granulosa cells express FSH receptor, and thecal cells express LH receptor, there is no evidence for any gonadotropin requirement for follicle growth and development at this preantral stage (2, 34). However, in large preantral and small antral follicles, it is probably the presence of these functional receptors that marks the change from predominately intra- to both intra- and extraovarian gonadotropin regulation. The absence of normal large antral follicles and the dramatic reduction in the number of early antral follicles in both FSH-knockout and hpg mice support the concept that follicles do not require FSH for development to the early antral stage (35, 36). In addition, only those follicles able to respond to rising FSH levels continue to develop, while the majority of early antral follicles become atretic (2). In normal mice, all oocytes have entered prophase of meiosis 1 by postnatal day 2, and the minority that do not become atretic complete meiosis at ovulation. However, in the Dazl -/- mouse, oocytes enter meiosis and then arrest and are destroyed, possibly by atresia, due to the lack of Dazl protein, which is found in the cytoplasm of fetal ovarian pachytene cells of normal mice (19). Despite the early absence of oocytes, we have shown that in -/- mice there are steroidogenically competent cells in the -/- ovary expressing 3ßHSD, the enzyme necessary to convert pregnenolone to progesterone; 17{alpha}-hydroxylase, which is necessary to convert pregnenolone and progesterone to androgen precursors, both exclusively expressed in thecal and interstitial cells; as well as cells expressing aromatase, an exclusive granulosa cell enzyme that is essential for the conversion of aromatizable androgens to estrogen (16). In addition, the presence of some ERß-positive cells with a distribution similar to that of aromatase would indicate the presence of granulosa cells (37, 38, 39). Further confirmation that these are fully functional is shown by plasma concentrations of estradiol similar to those in the +/+ and +/- females. As all of these enzymes are induced by gonadotropins (16), it is suggested that the loss of the oocytes associated with these steroidogenic cells occurs at a stage when folliculogenesis is regulated by extraovarian factors. However, the lack of AMH/MIS in the Dazl -/- ovary suggests that no cells differentiated beyond the early preantral stage (40). Subsequently, the very low level of cell division in the adult ovary as demonstrated by few PCNA-positive cells suggests that no further steroidogenic cells differentiate, as there are no oocytes to initiate the growth and development of these granulosa and thecal type cells. Hence, as animals age the number of steroidogenically active cells appears to decrease. Furthermore, in the postnatal day 16 females, many cells positive for inhibin {alpha} and ßB-subunit form clusters, whereas sections from adult animals indicate a considerable decrease in inhibin-positive cells, with a more dispersed appearance.

Although the concentration of ovarian steroid in the -/- female is sufficient to cause substantial uterine endometrial proliferation, it has a minor effect on the regulation of LH secretion, as demonstrated by the slight increase in plasma LH after ovariectomy. Similarly, the high levels of both LH and FSH stored in the pituitaries of both intact and ovariectomized -/- females confirm the absence of any significant ovarian feedback. However, the significant increase in plasma FSH demonstrates the sensitivity of feedback. Although this study indicates that there are no differences in estradiol concentrations between genotypes, it should be noted that all animals were matched for estrus-type vaginal smears when estradiol is low in the cycling animals. Variation in estradiol in the -/- females, who do not have estrous cycles but secrete constant low levels, probably depends on the number and differentiation of the steroidogenic cells that surround those oocytes that persisted to form primordial or primary follicles before their demise.

There is an unexpected observation from this study. The heterozygous +/- females have normal estrous cycles, are fertile, and exhibit the gross morphology and histology of the +/+ females. Therefore, it was anticipated that there would be no differences in any of the gonadotropin parameters between these two genotypes. Although there were no apparent significant differences in either plasma or pituitary LH, surprisingly, both pituitary and plasma concentrations of FSH were significantly lower in +/- than +/+ animals. This was related to a large increase in plasma concentrations of inhibin B. As inhibin B is produced mainly by small follicles (41), the present results suggest that there may be an increase in the pool of small follicles in +/- ovaries. However, the lack of change in inhibin A, secreted preferentially by the larger follicles (41), suggests that the increased numbers of small follicles do not all progress to become preovulatory follicles. However, a significant (P < 0.05) increase in the ovulation rate in second litters from +/- compared with that in the +/+ females (McNeilly, J. R., unpublished data) indicates that more follicles may become ovulatory in +/- than +/+ animals. The reasons for the increase in inhibin B and the relation to the numbers of small follicles require further investigation.

As a model to investigate follicular steroidogenesis in the absence of oocytes, the Dazl -/- mouse is unique, in that there is maintenance in the adult of some thecal and granulosa steroidogenically and inhibin-active cells in the complete absence of oocytes from the early neonatal period. This contrasts with other mouse knockout and mutant models, which are associated with ovarian failure [Kit ligand-Steel1 (42), Kit ligand-Steel panda (12), connexin 43 knockout (43), ER{alpha} knockout (44), FSH knockout (35), FSH receptor knockout (45), and GDF-9 knockout (46)] in which oocytes remain present throughout adult life, and the cellular organization of small follicles remains intact. In contrast, in the absence of oocytes in the Dazl -/- mouse, the ovary becomes disorganized, and the steroidogenically and inhibin-active cells appear randomly distributed throughout the ovary.

In conclusion, the present study indicates, firstly, that despite the absence of factors from the oocyte, once steroidogenically active follicle cells have become differentiated to produce estrogen, they can maintain their steroid output throughout the lifetime of the cell. Furthermore, even though oocytes die from embryonic day 15 on, surrounding somatic cells differentiate sufficiently to become steroidogenically competent. Secondly, we have shown that low levels of Dazl protein in the +/- females are associated with the apparent survival of more small follicles. Additional studies to address this issue by counting the number of follicles are planned.


   Acknowledgments
 
The authors acknowledge the expert technical assistance of Ian Swanston, Ted Pinner, and Tom McFetters for graphics, and the Animal House staff, especially Denis Doogan and Maureen Ross. Some of the reagents for the RIAs were provided by the NIDDK through the National Hormone and Pituitary Program and Dr. A. F. Parlow.

Received April 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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