Fig 1.
Transmission electron micrographs of step 1 spermatids.
(A) Step 1 spermatids are ovoid with an oval nucleus (N) containing one or two eccentric nucleoli (Nu) and a large number of randomly dispersed small patches of chromatin. A well-developed Golgi complex (GC) is found in close proximity to the nucleus. A pair of centrioles (C) resides adjacent to both the Golgi complex and the nuclear membrane. Spherical mitochondria (M) are in clusters in the cytoplasm. (B) Numerous small membrane-bound vesicles (V) and multivesicular bodies (MB) are presented at the concave surface of the Golgi complex (GC). Centrioles (C) are also found next to the Golgi complex. Rough endoplasmic reticulum (RER) is randomly distributed in the cytoplasm. (C) Bundles of microtubules (Mt) are found tightly surrounding the nucleus (N), forming a circular manchette. M, mitochondria.
Fig 2.
Transmission electron micrographs of step 2 spermatids.
(A) Step 2 spermatids are oval with a spherical to oval nucleus (N) containing an eccentric nucleolus (Nu) and small clumps of chromatin. In the periphery of the nucleus, a distinct Golgi complex (GC) is observed adjacent to a spherical proacrosomal vacuole (PV) with an electron-lucent matrix, which originates from the fusion of small membrane-bound vesicles. Continuous profiles of rough endoplasmic reticulum (RER) are found running around the nucleus. Spherical mitochondria (M) are randomly dispersed throughout the cytoplasm. (B) The two centrioles in step 2 spermatids remain in close proximity to the Golgi complex (GC) but become relatively distant from the nucleus. While the distal centriole (DC) approaches the plasma membrane, the proximal centriole (PC) orientates parallel to the former and is relatively distant from the cell membrane.
Fig 3.
Transmission electron micrographs of step 3 spermatids.
(A) Similar to their immediate precursors, step 3 spermatids remain ovoid with a spherical to oval nucleus (N) containing rod-shaped chromatin blocks. Thick patches of chromatin also attach to the inner nuclear membrane. Next to the Golgi complex (GC), a spherical proacrosomal vacuole (PV) is observed containing a patch of electron-dense material. A portion of mitochondria (M) transform into rod-shape organelles in step 3 spermatids. (B) Both the proximal (PC) and distal centrioles (DC) remain in close proximity to the Golgi complex (GC) and start to orientate perpendicularly to each other. Rough endoplasmic reticulum (RER) is found surrounding the nucleus (N). M, mitochondria. (C) The step 3 spermatids are in pairs and interconnected by a cytoplasmic bridge (CB). Mitochondria (M) are clustered at the cytoplasm near the cytoplasmic bridge. N, nucleus.
Fig 4.
Transmission electron micrographs of step 4 spermatids.
(A) Chromatin blocks in the nucleus (N) of step 4 spermatids remain rod-shaped but become sparser than those in previous steps, indicating that chromosome decondensation commences at this stage. Mitochondria (M) and rough endoplasmic reticulum (RER) are randomly distributed throughout the cytoplasm. (B) The proacrosomal vacuole transforms into a proacrosome (P) in step 4 spermatids, with its matrix being mostly occupied by electron-dense material. N, nucleus. (C) The distal centriole (DC) next to the Golgi complex (GC) forms a flagellum (F) protruding out of step 4 spermatids. N, nucleus; RER, rough endoplasmic reticulum.
Fig 5.
Transmission electron micrographs of step 5 spermatids.
(A) As the nuclear size (N) of step 5 spermatids decreases, the decondensed chromosomes are aggregated into a retiform pattern. Large spherical mitochondria (M) are observed gathering at one end of the spermatids. RER, rough endoplasmic reticulum. (B) In the proacrosome (P) of step 5 spermatids, a ring-shaped structure (R) forms surrounding the sphere (S) at the centre. M, mitochondria; N, nucleus. (C) Step 5 spermatids remain interconnected by a cytoplasmic bridge (CB). A continuous profile of rough endoplasmic reticulum (RER) is found surrounding the nucleus.
Fig 6.
Transmission electron micrographs of step 6 spermatids.
(A) An implantation fossa (*), which appears as a shallow depression, forms at the posterior end of the nucleus (N). The proximal centriole (PC) is in close proximity to the implantation fossa while the distal centriole remains docked with the cytoplasmic membrane. Large spherical-shaped mitochondria (M) migrate towards the pole where the centrioles reside. (B) This electron micrograph represents a cross-section through a small portion of the proacrosome, showing fragments of both the ring-shaped structure (R) and the central sphere (S). The proacrosomal matrix becomes being occupied by coarse granules. A portion of the proacrosome adheres to the cytoplasmic membrane. Large spherical mitochondria are found next to the proacrosome. N, nucleus. (C) As the proximal centriole (PC) embeds into the implantation fossa, the flagellum (F) extends from the distal centriole and is further elongated. N, nucleus.
Fig 7.
Transmission electron micrographs of step 7 spermatids.
(A) The chromatin in the nucleus of step 7 spermatids exhibits a coarse granular form and aggregates into a large irregular-shaped block. The chromatin block attached to the posterior end of the nucleus (N), where an implantation fossa (*, white asterisk) has formed. The proximal centriole (PC) remains in close proximity to the fossa and orientates perpendicularly to the distal centriole (DC). The large spherical-shaped mitochondria (M) become surrounded the centrioles and start to form the mitochondrial sheath at the base of the flagellum. (B) The acrosome (A) transforms into an inverted-bowl shape with its anterior region tightly adherent to the cell membrane. In the acrosomal matrix, both the central sphere (S) and the ring-shaped structure (R) attach to the inner posterior membrane of the acrosome. The acrosome is located adjacent to large spherical mitochondria (M) which will ultimately form the mitochondrial sheath of the flagellum. (C) Where the mitochondria (M) approach the nuclear membrane (N), the nuclear envelope curves inward to form depressions (*, black asterisks).
Fig 8.
Transmission electron micrographs of step 8 spermatids.
(A) Step 8 spermatids are still interconnected by a cytoplasmic bridge (CB). The nucleus (N) remains spherical in shape while the chromatin block transforms into an oval shape with a highly condensed homogeneous appearance. The proximal centriole (PC) extends its microtubules into the implantation fossa (*) and orientates perpendicularly to the flagellum (F) which originates from the distal centriole (DC). The developing acrosome (A) remains adjacent to the flagellum at this stage. (B) The central sphere firmly attaches to the posterior membrane of the acrosome (A) and transforms into a hemisphere (Hs). The ring-like structure (R), with darker particles lining its interior border, also comes into direct contact with the acrosomal membrane on both sides. The acrosomal matrix is filled with densely packed coarse granules. M, mitochondria; N, nucleus. (C) As the morphogenesis of the acrosome proceeds, the central hemisphere (Hs) increases its volume and ascends towards the anterior membrane, resulting in the formation of an indentation at the posterior end of the acrosome. N, nucleus; R, ring-shaped structure.
Fig 9.
Transmission electron micrographs of step 9 spermatids.
(A) Step 9 spermatids are still in pairs and interconnected by a cytoplasmic bridge (CB). The central hemisphere (Hs) in the acrosome occupies the entire acrosomal matrix and connects to the ring-like structure (R) on both sides. The whole acrosome exhibits an identical electron density except for the darker particles lining the interior border of the ring structure. Multiple irregular-shaped residual bodies (RB) containing excess cytoplasm appear in step 9 spermatids. M, mitochondria; N, nucleus. (B) In step 9 spermatids, the acrosome (A) starts to migrate towards the anterior end of the cytoplasm, which is opposite to the pole where the flagellum (F) and mitochondria (M) reside. The mitochondria remain firmly adherent to depressions at the posterior end of the nucleus (N).
Fig 10.
Transmission electron micrographs of step 10 spermatids.
(A) The oval nucleus (N) is occupied by fully condensed homogeneous granular chromatin. The acrosome (A) reaches the anterior apex of the cytoplasm in step 10 spermatids, opposite to the pole where the mitochondria (M) and flagellum reside. As the dark particles spread throughout the ring-like structure, the ring appears darker than the central hemisphere. (B) Early step 10 spermatids remain in pairs and are interconnected by a cytoplasmic bridge (CB). The cytoplasm contains clusters of irregular-shaped vesicles, indicating the continuous formation and expulsion of residual bodies. The formation of mitochondrial sheath is accomplished, with four spherical mitochondria (M) surrounding the proximal centriole (PC). *, implantation fossa. (C) In late step 10 spermatids, numerous clusters of irregular-shaped vesicles are found inside and adjacent to the cytoplasm, suggesting the discharge of residual bodies (RB) occurs dramatically at this stage. With the elimination of excess cytoplasm, the cytoplasmic bridge connecting the spermatids disintegrates, resulting in the formation of two independent spermatozoa. A, acrosome; M, mitochondria; N, nucleus.
Fig 11.
Transmission electron micrographs of spermatozoa.
(A) Spermatozoa consist of a cap-like acrosome (A), an oval nucleus (N), a short midpiece containing four spherical mitochondria (M) and an elongated flagellum (F). A subacrosomal space (*, black asterisk) forms between the acrosome and the nucleus, containing evenly distributed electron-dense fibrillary subacrosomal material. The proximal centriole (PC) is inserted into the implantation fossa (*, white asterisk) formed at the posterior end of the nucleus. The four mitochondria lodge in concave recesses at the posterior nuclear margin and surround the base of the flagellum, where the annulus (An) forms. (B) A transverse section through the ring structure at the base of the acrosome shows the fibrillary content of the subacrosomal space (*, black asterisk). (C) A transverse section through the midpiece shows four spherical mitochondria (M) encircling the base of the flagellum.
Fig 12.
Illustrations showing the pattern of spermiogenesis in G. gemineoa.
Spermiogenesis can be temporally divided into (A—B) Golgi phase, (C—E) acrosomal phase and (F—G) maturation phase. (A) In early Golgi phase, a large number of single-membrane bound vesicles are observed adjacent to a conspicuous Golgi complex. These vesicles gradually merge into an electron-lucent proacrosomal vacuole. A pair of centrioles, which were initially located in close proximity to the nuclear membrane, migrate towards the cell membrane. (B) In the late Golgi phase, the fusion of proacrosomal vesicles ceases and a patch of electron-dense material forms in the matrix of the proacrosomal vacuole. The distal centriole docks to the cell membrane, where a formative flagellum subsequently protrudes out. (C) In early acrosomal phase, the Golgi complex becomes no longer involved in the differentiation of the acrosome and the proacrosomal vacuole transforms into a proacrosome containing a large sphere surrounded by a ring-like structure. The chromosomes decondense into rod-shaped chromatin blocks and occupy half volume of the nucleoplasm. (D) The proacrosome transforms into an inverted-bowl shaped acrosome, during which both the central sphere and the ring structure attach to the inner posterior membrane of the acrosome. The proximal centriole comes into intimate contact with the implantation fossa formed at the posterior end of the nucleus. After the fusion of mitochondria, they migrate to and adhere to concave recesses of the nucleus at the posterior pole. The acrosome starts to migrate towards the anterior pole of the cytoplasm after the adherence of the mitochondria. (E) In late acrosomal phase, the acrosome almost reaches the most anterior aspect of the spermatids. The chromatin condenses into a rough oval-shaped chromatin block. (F) In the early maturation phase, the acrosome has accomplished the majority of its differentiation, during which the central hemisphere comes to occupy the entire matrix of the acrosome. (G) In the late maturation phase, acrosome differentiation is completed and the nucleus is fully condensed. The excess cytoplasm is completely eliminated from the spermatid through the extrusion of residual bodies. The cytoplasmic bridge between the spermatids ruptures and the spermatids develop into individual spermatozoa.
Fig 13.
Representative types of spermatids and spermatozoa in the culture media.
(A) Step 1–3 spermatids. The spermatids at these steps are spherical to oval in shape. They are in pairs and interconnected by a cytoplasmic bridge (CB). N, nucleus. (B) Step 4–6 spermatids. Spermatids at these stages remain connected by a cytoplasmic bridge (CB) however a short flagellum (F) protrudes from the posterior end of each spermatid. N, nucleus. (C) Step 7–9 spermatids. The spermatids at these steps are spherical and contain a cap-like acrosome (A), a condensed nucleus (N) and an elongated flagellum (F). The acrosome resides at or near the posterior end of the spermatids. (D) Spermatozoa. Spermatozoa consist of a cap-like acrosome (A), an oval nucleus (N), a short midpiece (M) and a flagellum (F).
Fig 14.
The male germinal fluid is able to induce a substantial portion of spermatid differentiation in vitro.
After 36 h of incubation in the male germinal fluid, the average percentage of step 1–3 spermatids decreases from 11.3% to 0.7%, whereas that of step 7–9 increases from 27.3% to 44.7%. The percentages of step 4–6 slightly reduce from 52% to 47.3% in the first 12 h and become constant afterwards. The percentage of spermatozoa remains almost unchanged (~ 10%) during the entire 36 h-incubation, indicating no new spermatozoon has generated in this culture media.
Fig 15.
The boiled male germinal fluid loses its capacity to support the differentiation of spermatids in vitro.
During the 36 h of incubation in the boiled male germinal fluid, no significant change of the average percentages is detected in all four types of sperm cells (P > 0.05). It indicates that the boiled male germinal fluid cannot induce the young spermatids to differentiate in vitro, but can maintain the viability of the spermatids for at least 36 h.
Fig 16.
The female germinal fluid is unable to induce the differentiation of spermatids in vitro.
During the first 24 h of incubation, the percentages of step 1–3 spermatids, step 7–9 spermatids and spermatozoa are almost unchanged (P > 0.05). The spermatids degenerated after 24 h-incubation in the female germinal fluid; therefore, data are not obtainable at 36 h.
Fig 17.
10% foetal bovine serum in RPMI 1640 medium is able to induce a substantial portion of spermatid differentiation in vitro.
After 36 h of incubation in this culture medium, the percentage of step 1–3 spermatids decreases from 10% to 0% while that of step 7–9 spermatids increases from 28% to 39.3%. The average percentages of step 4–5 spermatids and spermatozoa exhibit no significant change, which remain about 55% and 7% respectively (P > 0.05).