Histological analysis of the ‘critical point’ in follicular development in mice

Abstract

Aim:  To histologically examine ovarian follicular development in cycling mice.

Methods:   Mice were observed at 13:00 h at each stage of the estrous cycle. The ovaries were cut into complete serial sections. All sections were observed, and the size of each follicle was measured by using a micrometer. Follicles with advanced atresia were excluded and non‐atretic healthy follicles were differentiated from atretic follicles.

Results:  The measurement of the number of follicles in each stage of the estrous cycle, with reference to their size, showed that in each mouse, the number of small healthy follicles (100–249 µm in average diameter) was approximately 100 for all stages and the number of medium‐sized healthy follicles (250–349 µm) was close to 20 in only the metestrus and diestrus stages. In contrast, large healthy follicles (≥350 µm) showed marked changes throughout the estrous stage. Many healthy large follicles were observed in the proestrus stage, but they disappeared in the estrus stage, which suggests that they have ovulated. This was supported by observations of oocytes resuming meiosis in large healthy follicles.

Conclusion:  As follicular atresia was frequently observed in follicles of 250–399 µm diameter, this size range may be a ‘critical point’ for atresia. The results suggest that there is a ‘critical point’ in follicular development, and that only follicles that pass this point will ovulate, while those that do not will become atretic follicles. (Reprod Medical Biol 2004; 3: 141–145)

INTRODUCTION

IT IS UNCLEAR whether small ovarian follicles continue to grow to large follicles immediately before ovulation in the final stage of follicular development. Earlier studies suggested continuous growth of small follicles to large follicles; ¹ , ² , ³ however, Hirshfield and Midgley Jr showed that follicular growth is discontinuous. They mention that the development of large follicles occurs discontinuously and rhythmically in rats, which proposes a ‘critical point’ of follicular growth at which atresia tends to develop. ⁴ In rats, the number of large follicles changes more markedly than that of small follicles during the estrous cycle, suggesting that a regulation of large follicles occurs at the final stage of follicular growth.

To clarify the ‘critical point’ in follicular development and to determine the follicular size that results in ovulation, we serially observed follicular growth in mice.

MATERIALS AND METHODS

Experiment 1: Changes in the numbers of healthy follicles and early atretic follicles during the estrous cycle and the number of ova shed in mice

MATURE INSTITUTE OF Cancer Research (ICR) mice from Chiyoda Kaihatsu (Tokyo, Japan) were maintained at 23 ± 2°C with illumination from 07:00 to 21:00 h. To determine the estrous cycle, vaginal smears were obtained between 07:00 and 09:00 h. Only those mice that showed at least two consecutive normal 4‐day estrous cycles were used. Because nutrition affects follicular growth, ⁵ the mice were allowed free access to food and water. As a previous study with rats showed marked changes in the number of follicles with age (days), the vaginal smear check was initiated when the rats were aged 10 weeks, and observations were made when they were aged 12 weeks. ⁶

Five mice were observed at 13:00 h at each stage of the estrous cycle. Both the ovaries and oviducts were removed from rats, fixed for 24 h and dehydrated and embedded in paraffin using routine procedures. The ovaries were cut into complete serial sections (10 µm) and stained with Mayer's hematoxylin–eosin solution. All sections were observed, and the size of each follicle was measured by using a micrometer. In follicles with oocytes, the average of the major and minor axes was regarded as the follicle size.

In the present study, follicles with advanced atresia were excluded and non‐atretic healthy follicles were differentiated from atretic follicles according to the following criteria described by Braw and Tsafriri: ⁷

• • Non‐atretic healthy follicles (Fig. 1a): Oocytes are in the resting stage of prophase (dictyate), except immediately prior to ovulation; pyknosis of granulosa cells and cell debris in the antrum are absent.
• • Stage I atretic follicles (Fig. 1b): Oocytes are in the dictyate stage, and pyknosis of granulosa cells and cell debris in the follicular antrum are present.
• • Stage II atretic follicles (Fig. 1c): Chromosomes in many oocytes show meiosis‐like changes as signs of atresia, and pyknosis of granulosa cells and cell debris in the follicular cavity are present.
• • Stage III atretic follicles (Fig. 1d): Oocytes are fragmented or pseudocleaved and no granulosa cells are observed.

Figure 1.

Healthy and atretic follicles. (a) Non‐atretic healthy follicle. The oocyte nucleus is in the dictyate stage and there are no pyknotic granulosa cells (bar = 100 µm). (b) Stage I atresia. The oocyte is in the dictyate stage. Cell debris is present in the antrum (arrow) (bar = 50 µm). (c) Stage II atresia. In the oocyte, a meiosis‐like change occurs. Cell debris is present in the antrum (bar = 100 µm). (d) Stage III atresia. *The oocyte is fragmented (bar = 50 µm).

The number of ova in the fallopian tube at 13:00 h in the estrus stage was regarded as the ovulation number. Five mice were observed.

Experiment 2: Estimation of the follicular size of follicles that proceed to ovulate

To determine the size of follicles that ovulate, five mice were observed at 21:00 h on the day of proestrus, by which the lutenizing hormone surge had been completed. Complete serial sections were prepared. All sections were observed and the average diameter was regarded as the follicle size. Healthy follicles showing resumed meiosis of oocytes were considered to be follicles that ovulated (Fig. 2).

Figure 2.

Non‐atretic healthy follicle immediately before ovulation. The oocyte nucleus is in the resumed meiosis stage, and the cumulus oophorus is diffused. There are no pyknotic granulosa cells (bar = 100 µm).

In the present study, the size of follicles that ovulated was also estimated by using another approach. At 05:00 h on the day of proestrus, 5 IU human chorionic gonadotropin (hCG, Gonatoropin; Teikokuzoki, Tokyo, Japan) was administered to five mice, and similar histological observations (to that of the previous approach) were made after 8 h.

Experiment 3: Estimation of the ‘critical point’ in follicular development

To determine the follicular size associated with the development of atresia (‘critical point’), the percentage of early atretic follicles was calculated as follows:

The χ² test was used to analyze the percentage of atretic follicles. It was performed at P < 0.05 and P < 0.01.

RESULTS

Experiment 1: Changes in the numbers of healthy follicles and early atretic follicles during the estrous cycle and the number of ova shed in mice

T HE NUMBER OF small healthy follicles (100–249 µm) per mouse was in the range from 93.7 to 99.1 throughout the estrous cycle. The number of medium‐sized healthy follicles (250–349 µm) differed at each stage. There were only a few medium‐sized follicles in the proestrus and estrus stages, but their number increased to 26.2 and 17.6 in the metestrus and diestrus stages, respectively. The number of large healthy follicles (≥350 µm) was 21.8 in the proestrus stage, but no follicles were observed in the estrus stage. In the metestrus and diestrus stages, there were 4.9 and 11.0 large healthy follicles, respectively (Fig. 3a,b). The ovulation number was 14.5 ± 1.9 (mean ± SE).

Figure 3.

(a) Size distribution of healthy follicles in each stage of the estrous cycle. Mice were observed at 13:00 h in each stage of the estrous cycle. (b) Number of healthy follicles according to their size in each stage of the estrous cycle. Mice were observed at 13:00 h in each stage of the estrous cycle.

Experiment 2: Estimation of the follicular size of follicles that proceed to ovulate

In mice not treated with hCG, the size of follicles that showed meiosis of oocytes was observed. Meiosis results in ovulation of oocytes, which was observed only in follicles ≥350 µm in diameter (15.3% of follicles were 350–399 µm in diameter and generally 100% in those ≥400 µm in diameter; Fig. 4). Atretic follicles that are formed because of meiosis‐like division in oocytes were excluded.

Figure 4.

Relationship between the resumed maturation division in oocytes and follicular size in untreated mice ovaries. Mice were observed at 21:00 h on the day of proestrus.

In hCG‐treated mice, meiosis of oocytes, which resulted in ovulation, was observed only in follicles ≥350 µm in diameter (Fig. 5).

Figure 5.

Relationship between the resumed maturation division in oocytes and follicular size in human chorionic gonadotropin (hCG)‐treated mice ovaries. (□) Treated with 5 IU of hCG at 05:00 h on the day of proestrus and (▒) intact mice observed at 13:00 h on the same day.

Experiment 3: Estimation of the ‘critical point’ in follicular development

Early atresia of oocytes was frequently observed in follicles sized 250–399 µm in diameter throughout the estrous cycle. In particular, more than 80% of follicles in the proestrus stage were atretic (Fig. 6). Only low percentages of small and large follicles underwent early atresia.

Figure 6.

‘Critical point’ of follicular development in untreated mice. Animals were observed at 13:00 h at each of the estrous stages. The percentage of early atretic follicles calculated as follows:

DISCUSSION

CONTINUOUS FOLLICULAR GROWTH in mice has been reported. ⁸ It is generally considered that follicles grow until they ovulate or undergo atresia. ⁸ However, the present study showed that the number of small follicles does not markedly change during the estrous cycle while the number of medium‐large follicles does. In particular, marked changes in terms of the number of large healthy follicles were observed. These results suggest only slight changes in the number of follicles in the early growth stage but substantial changes in the number of follicles in the final growth and differentiation stages.

The results were similar to the findings found by the study conducted by Mandel and Zuckerman, ¹ where cyclic changes occurred in large follicles (≥350 µm in diameter) of rats. Peppler and Greenwald ³ and Hirshfield and Midgley Jr ⁴ reported that the number of follicles ≤352 µm in diameter remains almost constant throughout the estrous cycle and that some of them become larger in the move from the proestrus to estrus stages. Large follicles underwent cyclic changes immediately before ovulation. ¹

Hirshfield and Midgley Jr proposed the existence of a ‘critical point’ (follicular size related with atresia) in follicular growth and concluded that the ‘critical point’ of follicles in rats is approximately 349 µm. ⁴ That is, only the large follicles (≥550 µm) that continued to develop after the ‘critical point’ will ovulate. In the present study, the percentage of mice follicles 250–349 µm in diameter that showed early signs of atresia was more than 50% throughout the estrous cycle and exceeded 80% in the proestrus stage. In addition, the ‘critical point’ existed in the study mice.

During the proestrus stage, follicles ≥350 µm in diameter had already resumed meiosis, therefore confirming that follicles that do grow beyond the ‘critical point’ actually ovulate.

Follicles grow and undergo atresia when they near the ‘critical point’, and only those rescued from atresia by follicle‐stimulating hormone (FSH) will proceed to ovulate. ⁹ In addition, the healthy follicles in the small follicle group will continue to grow and edge towards the ‘critical point’.

Follicles that reach the ‘critical point’ are rescued from atresia by a FSH surge, overcome the ‘critical point’ and then ovulate. Many follicles undergo atresia and only a small number of follicles rescued by FSH proceed to ovulate. Ingram reported that most follicles undergo atresia, but only 0.1% ovulate. ¹⁰

In conclusion, the present study suggests that the proposed ‘critical point’ of follicular size for atresia exists in the follicular development of rats is also present in mice and that the ‘critical point’ for mice is approximately 250–349 µm.