A BINARY DECISION KEY FOR THE IDENTIFICATION OF STAGES IN THE CYCLE OF THE RAT SEMINIFEROUS EPITHELIUM

by Rex A. Hess

INDEX

(Click on the Stage of Interest)
[ I | II | III | IV | V | VI | VII | VIII | IX | X | XI | XII | XIII | XIV ]

First Questions

1. Are meiotic figures or large secondary spermatocytes present?

  	NO	(Stages I-XIII)
		YES	(Stage XIV)


2. If NO in #1, are there 2 steps of spermatids (round and elongated)?

		NO		(Stages IX-XIII)
		YES	(Stages I-VIII)


3. If YES in #2, are elongated spermatids located near the lumen?

		NO		(Late I-V)
		YES	(Late VI-VIII)


[Each stage should be confirmed by more than one correct response]

Stage I

1. Is there a faint PAS+ Golgi apparatus adjacent to the nucleus of the round spermatid?

		NO		(Newly formed step 1 spermatids)
		YES	(Mid to late step 1 spermatids)

2. Are nuclei of the round spermatids small and heterochromatic?

		NO		(Probably late Stage I)
		YES	(Early Stage I)

3. Are 1-6 small proacrosomic granules seen in the Golgi apparatus?

		NO	(Probably Stage I, unless out of plane of section)
		YES	(Stage II; if present in only a few spermatids then other characteristics must be considered)

4. Do the elongated spermatids have a prominent bulge of the dorsal-caudal aspect of the acrosome (caudal fin)? (difficult to see in epoxy)

		NO		(Stage II, although some overlap)
		YES	(Stage I)

5. Does the nucleus of the elongated spermatid have a pronounced hook? (rarely seen in epoxy)

		NO		(Late Stage I)
		YES	(Early Stage I)

6. Are the type A spermatogonia in mitosis?

		NO		(Sometimes overlap Stage II)
		YES	(Usually found in Stage I)

7. Are step 15 spermatid clusters located more toward the lumen?

		NO		(Not definitive)
		YES	(Stage I)

Stage II

1. Are 1-6 small proacrosomic granules (usually 1-2) present within the Golgi apparatus? (A surrounding vesicle may be visible only with the highest power).

		NO		(Stage I)
		YES	(Stage II)

2. If there is a single granule in the Golgi zone, is the granule large in size and surrounded by a large, distinct vesicle?

		NO		(Probably Stage II; very small pinpoint granules)
		YES	(Stage III; the vesicle is more distinct in epoxy)

3. Do the elongated spermatids have a prominent bulge of the dorsal-caudal aspect of the acrosome (caudal fin)?

		NO		(Stage II)
		YES	(Sometimes overlap Stage II; reconsider Stage I)

4. Are mitochondria distinctly aligned along the mid-piece?

		NO		(Reconsider Stage I; begins in late Stage I)
		YES	(Stage II; more distinct in epoxy than in methacrylate)

5. Are mitotic figures present?

		NO		(Not definitive)
		YES	(Sometimes overlap Stage II)

Stage III

1. Is there a single acrosomic granule surrounded by a vesicle (clear or slightly PAS+) in the Golgi zone located adjacent to the round spermatid nucleus? (more distinct in epoxy)

		NO	(Stage I or II or out of the plane of section)
		YES	(Stages II, III or IV)

2. Is the acrosomic granule large in size?

		NO		(Reconsider Stage II)
		YES	(Stage III or IV)

3. Does the acrosomic granule touch the nuclear membrane?

		NO		(Stage III)
		YES	(Consider Stage IV)

4. Is the acrosomic vesicle flattened against the nuclear membrane? (more distinct in epoxy)

		NO		(Reconsider Stage III)
		YES	(Probably Stage IV)

5. Does the apical acrosomal head cap extend beyond the tip of the late spermatid nucleus? (more distinct in methacrylate)

		NO		(Probably Stage III)
		YES	(Consider Stage IV)

6. Are mitotic figures present?

		NO	(Probably Stage III, if other features indicated)
		YES	(Consider Stage IV)

Stage IV

1. Does the acrosomic vesicle form a halo around the acrosomic granule?

		NO	(Consider Stage IV)
		YES	(Reconsider Stage III)

2. Does the acrosomic granule touch the nuclear membrane? (more distinct in epoxy)

		NO		(Stage III)
		YES	(Consider Stage IV)

3. Does the acrosomic vesicle form a PAS+ straight margin at the nucleus upon which the granule rests?

		NO		(Consider early Stage IV or late Stage III)
		YES	(Stage IV)

4. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Stage IV)

5. Does the apical acrosomal head cap extend beyond the tip of the nucleus in the elongated spermatid? (rarely seen in epoxy)

		NO		(Probably Stage III)
		YES	(Stage IV)

6. Does the PAS+ margin of the forming acrosomic head cap form lateral projections that curve slightly beneath the acrosomic granule?

		NO		(Stage IV)
		YES	(Consider Stage V)

7. Is the acrosomic granule located to one side of the forming acrosomic system?

		NO		(Not definitive)
		YES	(Probably late Stage IV)

8. Is the caudal portion of the acrosome retracted from the dorsal tip of the elongated spermatid nucleus? (rarely seen in epoxy)

		NO		(Reconsider Stage III)
		YES	(Stages IV-V)

9. Are spermatogonia of type B (large rounded nucleus containing thick clumps of chromatin) present?

		NO		(Stage IV)
		YES	(Consider Stage V)

10. Are clusters of elongate spermatids located adjacent to the Sertoli cell nuclei?

		NO		(Not definitive)
		YES	(More common in Stages IV-V)

Stage V

1. Are lateral projections present on both sides of the acrosomic granule?

		NO		(Reconsider IV) 
		YES	(Consider Stage V)

2. Do the lateral projections of the acrosomic system form a curvature (from a slight curve to less than 1/4 of the nucleus)?

		NO		(Reconsider Stage IV)
		YES	(Stage V)

3. Are the clusters of late spermatid nuclei buried deep within the epithelium, adjacent to the Sertoli cell nucleus?

		NO		(Consider Stages IV or VI)
		YES	(Probably Stage V)

4. Are most spermatogonia type B, rather than type A?

		NO		(Reconsider Stage IV)
		YES	(Stage V)

5. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Consider Stages IV or VI)

6. Is the Golgi apparatus still present in the elongated spermatid cytoplasm? (Primarily seen in epoxy)

		NO		(Consider Stage VI)
		YES	(Stage V)

Stage VI

1. Are the clusters of late spermatid nuclei buried deep within the epithelium, often adjacent to the Sertoli cell nucleus?

		NO		(Probably Stage VI)
		YES	(Reconsider Stage V, if the acrosomic system of round spermatids is small or if mitotic figures are absent)

2. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Stage VI)

3. Do the elongated spermatid nuclei line the luminal border?

		NO		(Probably Stage VI)
		YES	(Consider Stage VII)

4. Do some late spermatid nuclei occur in clusters?

		NO		(Consider Stage VII)
		YES	(Probably Stage VI)

5. Are type B spermatogonia predominant?

		NO		(Consider Stages VI or VII)
		YES	(Reconsider Stage V)

6. Are small basophilic granules located in the distal cytoplasm of elongated spermatids?

		NO	(Rarely seen in Stage V; large granules near the nucleus in Stage VII)
		YES	(Probably Stage VI)

7. Does the acrosomic head cap cover between 1/4 and 1/3 of the round spermatid nucleus?

		NO		(Reconsider Stage V)
		YES	(Stage VI or early VII)

Stage VII

1. Do the elongated spermatid nuclei line the luminal border?

		NO		(Reconsider Stage VI)
		YES	(Probably Stage VII or VIII)

2. Are basophilic granules large in size and located near the nucleus of elongated spermatids?

		NO		(Reconsider Stage VI)
		YES	(Probably Stage VII; located beneath the nuclei in Stage VIII)

3. Are large basophilic granules located beneath the ventral curvature of the elongated spermatid head?

		NO		(Within or adjacent to the ventral curvature in Stage VII)
		YES	(Consider Stage VIII)

4. Have type B spermatogonia been replaced by small preleptotene spermatocytes?

		NO		(Reconsider Stage VI)
		YES	(Stage VII)

5. Are mitotic figures present?

		NO		(Stage VII)
		YES	(Reconsider Stage VI)

6. Is the round spermatid nucleus covered by an extensive acrosomic head cap (1/3 or greater)?

		NO		(Reconsider Stage VI)
		YES	(Stages VII or VIII)

7. Are the round spermatid nuclei located centrally in the cytoplasm?

		NO		(Stage VIII; to one cytoplasmic pole)
		YES	(Stage VII)

Stage VIII

1. Do the late spermatid nuclei line the luminal border?

		NO		(Reconsider Stages VI-VII)
		YES	(Consider Stages VII or VIII)

2. Are large basophilic granules located between the curvature of the elongated spermatid nucleus and the round spermatids?

		NO		(Stage VII)
		YES	(Consider Stage VIII)

3. Are the nuclei of the round spermatids located to one pole of the cytoplasm?

		NO		(Reconsider Stage VII)
		YES	(Stage VIII)

4. Have some mature spermatids been released into the lumen?

		NO		(Stages VII-VIII)
		YES	(Stage VIII)

5. Do the nuclei of round spermatids stain less intensely and appear somewhat distorted?

		NO		(Reconsider Stage VII)
		YES	(Stages VIII-IX)

6. Does the Sertoli cell nucleus exhibit a perpendicular orientation that extends nearly half-way to the lumen?

		NO		(Not definitive)
		YES	(Probably Stage VIII)

7. Are a majority of the acrosomic systems in the round spermatids orientated toward the basement membrane?

		NO		(Reconsider Stage VII)
		YES	(Probably Stage VIII)

8. Are knob-like structures formed on the end of lateral acrosomic projections? (more distinct in epoxy)

		NO		(Reconsider Stage VII)
		YES	(Stage VIII)

9. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Consider Stage IX)

Stage IX

1. Are mature spermatids missing from the luminal side of the epithelium (a few step 19 spermatids may still be attached)?

		NO		(Reconsider Stage VIII)
		YES	(Consider Stages IX-X)

2. Are large residual bodies seen at various levels within the epithelium?

		NO		(If disappeared; then consider Stage X)
		YES	(Probably Stage IX)

3. Do the spermatid nuclei exhibit asymmetry and flattening?

		NO		(Reconsider Stage VIII)
		YES	(Probably Stage IX)

4. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Stage IX)

5. Does the acrosomic system extend the full length of the flattened nucleus?

		NO		(Reconsider Stage IX)
		YES	(Consider Stage X)

Stage X

1. Is there an angle formed on the ventral side of the acrosomic system and nucleus of the elongating spermatid? (more distinct in methacrylate)

		NO		(Reconsider Stage IX)
		YES	(Consider Stages X and XI)

2. Does the elongated acrosomic head cap extend the length of the nucleus?

		NO		(Reconsider Stage IX)
		YES	(Consider Stage X)

3. Does the head cap of the elongating spermatid form a blunt tip rather than a narrow tubular protrusion at a sharp angle to the nucleus?

		NO		(Consider Stage XI)
		YES	(Probably Stage X)

4. Is the narrow lateral width of the spermatid nucleus 1/2 that of step 9 spermatids? (at the acrosomic head cap position)

		NO		(Reconsider Stage IX)
		YES	(Stage X)

5. Have the residual bodies disappeared or relocated along the basement membrane?

		NO		(Reconsider Stage IX)
		YES	(Probably Stage X)

6. Are mitotic figures present?

		NO		(Not definitive)
		YES	(Reconsider Stage IX)

Stage XI

1. Does the head cap of the spermatid form a narrow tubular protrusion at a sharp angle to the nucleus?

		NO		(Reconsider Stage X)
		YES	(Stage XI)

2. Is the narrow lateral width of the spermatid nucleus approximately 2/3 that of step 10 spermatids? (at the acrosomic head cap junction)

		NO		(Reconsider Stage X)
		YES	(Stage XI)

3. Are there distinct bundles of elongated spermatids?

		NO		(Reconsider Stage X)
		YES	(Probably Stage XI)

4. Does the apex of the spermatid nucleus stain intensely?

		NO		(Consider Stage XI)
		YES	(Consider Stage XII)

5. Is the apex of the spermatid head long, narrow and nearly straight?

		NO		(Probably Stage XI)
		YES	(Consider Stage XII)

Stage XII

1. Does the apex of the acrosomic system form a blunt tip? (not always present in plastic sections)

		NO	(Reconsider Stage XI if the nucleus is less condensed)
			(Consider Stage XIII if the nucleus is more condensed)
		YES	(Stage XII)

2. Does the apex of the spermatid nucleus stain more intensely basophilic than the caudal portion?

		NO		(Reconsider Stage XI)
		YES	(Probably Stage XII)

3. Is the apex of the spermatid head long, narrow and slightly bent?

		NO		(Probably Stage XI)
		YES	(Probably Stage XII)

4. Is the lateral width of the spermatid nucleus thin (2/3 that of step 11)?

		NO		(Reconsider Stage XI)
		YES	(Stage XII)

5. Does the acrosomic head cap bulge slightly from the dorsal side of the nuclear apex? (rarely seen in epoxy)

		NO	(Consider Stage XIII; the bulge will be more ventral)
		YES	(Consider Stage XII)

6. Are large numbers of late diplotene and diakinetic spermatocytes present? (very large nuclei with dispersed clumps of chromatin and large prominent nucleoli with translucent cores)

		NO		(Reconsider XII)
		YES	(Stage XIII)

7. Are mitotic figures present?

		NO		(Not definitive) 
		YES	(Probably Stage XII)

Stage XIII

1. Does the spermatid nucleus stain intensely basophilic? (highly condensed)

		NO	(Stage XII)
		YES	(Stage XIII-XIV)

2. Is the lateral width of the spermatid nucleus very thin (2/3 that of step 12)?

		NO 	(Reconsider Stage XII)
		YES	(Stage XIII or XIV)

3. Does the acrosomic head cap extend beyond the apex or bulge from the ventral side of the nuclear apex?

		NO	(Reconsider Stage XII)
		YES	(Stage XIII-XIV)

4. Is the apex of the spermatid nucleus curved and thin? (rarely seen in epoxy)

		NO	(Stage XII)
		YES	(Late Stage XIII-XIV)

5. Are large numbers of late diplotene and diakinetic spermatocytes present? (very large nuclei with dispersed clumps of chromatin and large prominent nucleoli with a translucent cores)

		NO	(Reconsider XII)
		YES	(Probably Stage XIII)

6. Are mitotic figures present?

		NO	(Probably Stage XIII)
		YES	(Consider Stages XII or XIV)

Stage XIV

1. Are meiotic figures present (meiosis I or II)?

		NO	(Stages I-XIII or XIV with secondary spermatocytes)
		YES	(Stage XIV)

2. Are secondary spermatocytes present? (nuclear diameter is approximately the size of pachytene spermatocytes and larger than that of the round spermatid)

		NO	(Early or late Stage XIV)
		YES	(Mid Stage XIV)



Revised: July 2012
email: rexhess@illinois.edu