Crucian carp (Carassius auratus L.,), which belongs to the genus of Carassius within the family of Cyprinidae, has a wide geographic distribution in the Eurasian continent. In China, the species has been found in most provinces, and the diverse genetic strains have been identified in different regions. Carassius auratus gibelio (C. gibelio Bloch) is originated from sympatric progenitor crucian carp (Carassius auratus) (Li et al., 2014), which is widely distributed in the Heilongjiang water system and many other natural habitats (Sheng and Wang, 1983; Gui and Zhou, 2010; Zhou et al., 2000). Other crucian carp have been reported in China since 1980s, including the Pengze crucian carp in the waters of Jiangxi province (Yang and Chen, 1992; Chen et al., 1996; Liu et al., 2004; Dong et al., 2004), the high-back crucian carp in the Dianchi lake of Yunnan and its affiliated waters, the Sogu crucian carp in Guangdong province (Yu et al., 1987), the Puan crucian carp in Guizhou province (Yu et al., 1992), and the wild crucian carp in the Dongting lake water system (Xiao et al., 2011; Liu et al., 2012; Qin et al., 2016) and so on. Most crucian carp in the abovementioned different natural waters have two sex ratio groups—bisexual and unisexual. The former always possesses a certain proportion of males, which can reproduce and undergo normal bisexual fertilization. The latter is a natural gynogenetic population, mostly being triploid, with no or a few males. In the natural gynogenetic monosexual population, there were many studies which showed that male individuals have the same genetic background as female individuals (Jiang et al., 1983; Liu et al., 2004), and the nature triploid population is generally propagated by gynogenesis. The spermatozoa of triploids mostly activate only triploid ova, without participating in the genetic makeup of the offspring (Li et al., 2016; Li et al., 2018; Zhu et al., 2018). The cause of spermatozoa with a normal genetic background not involving in the genetic composition of gynogenesis progeny liking in the bisexual fish deserves intensive investigation.

The natural crucian carp (Carassius auratus L.,) in the Dongting water system of China has been reported by our group. The population had both diploids (2nDTCC) and triploids (3nDTCC), in which the proportion of triploid (85.0%) was much higher than that in diploid (15.0%), and there was no significant difference in morphological characteristics between diploids and triploids. Among them, the proportion of male in diploid population was about 50.0%, and the proportion of male in triploid population was about 14.3% (Liu et al., 2012). The artificial breeding experiment showed that the male and female 3nDTCC were all fertile and produced triploid offspring when mating with each other (Xiao et al., 2011). The recent research studies have showed that 3nDTCC males presented a relative regular meiotic prophase I with complete conjugate chromosome pairs and finally formed mature sperms (Zhang et al., 2021). The 3nDTCC males are fertile and can produce spermatozoa with “normal morphology” and possess normal swimming ability, but the genetic material of them is rejected by gynogenetic progeny. How can the spermatozoa of 3nDTCC adapt to the propagating mode of gynogenesis? Whether there were structural differences between the spermatozoa of 3nDTCC and the spermatozoa of bisexual reproductive diploid species?

Considering the morphology of the spermatozoa is related to the semen quality; the ultrastructure of spermatozoa in 3nDTCC and 2nRCC was systematically studied to demonstrate their cytological structural differences. In addition, based on detecting the DNA content of semen in 2nDTCC and 3nDTCC by flow cytometric analysis, the artificial breeding tests of 3nDTCC(♀)×2nDTCC(♂) and 3nDTCC(♀)×3nDTCC(♂) were performed to verify if the spermatozoa produced by 3nDTCC do contribute to the gynogenesis progeny, with the ploidy of progeny being detected by the DNA content. Furthermore, the mRNA expression of centriole-related genes (i.e., cep57, cetn1, rootletin, and nek2) which were involved in spermatozoa packaging was also determined by quantitative real-time PCR (qPCR) to illustrate the molecular mechanism of the abnormal spermatozoa packaging process in 3nDTCC.

The study will initially reveal the adaptive structural features of spermatozoa in 3nDTCC not involving in the genetic composition of the gynogenesis offspring, which is of great significance in fish evolutionary research and aquatic breeding.

Gynogenesis fish often are not dependent on the involvement of sperm genetic material, in which the sperms are required to stimulate the eggs to initiate embryogenesis using only maternal genetic information (Gui and Zhou 2010). The introgression of paternal DNA might play an important role in the creation of genetic diversity in gynogenetic fish, for example, extra microchromosomes in males are closely related to male determination in gynogenetic gibel carp (Li et al., 2016; Zhao et al., 2021; Ding et al., 2021), and the insertion of paternal DNA was also reported in gynogenetic blunt snout bream (Gong et al., 2019).

3nDTCC is generally propagated by gynogenesis. The spermatozoa of the 3nDTCC mostly activate triploid ova and do not participate in the genetic makeup of offspring (Xiao et al., 2011). Furthermore, 3nDTCC possessed a complete spermatogenesis process to produce viable 1.5n sperm (Zhang et al., 2021), and the breeding test of 3nDTCC (♀) ×3nDTCC (♂) showed that the 1.5n spermatozoa only activated the eggs, not involved in the genetic composition of the offspring or only inserted a few of the paternal DNA fragments, which were speculated by the progeny were triploid and all-female. Do the spermatozoa of 3nDTCC possess the complete structure as that in bisexual reproductive diploid fish?

The typical fish spermatozoa consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum (Psenicka et al., 2009; Zhu et al., 2021). The basic task of the spermatozoon head is to transfer the genetic material localized in the nucleoplasma to the egg. The optimal shape and size of the spermatozoon head is a prerequisite for good penetration of the spermatozoon throughout the micropyle. In the study, ultrastructures of the testes have revealed some malformation in the spermatozoa formation process of 3nDTCC, while spermatids were packed slightly stained nuclei, some spermatozoa heads are concentrated lightly, some heavily vacuolated head, irregular shape head, and abnormal flagella are easily detected. Most important of all, the midpiece structure of 3nDTCC organized looser than that in 2nRCC. Expression validation of centriole-related genes indicate that the expression levels of cep57 gene and cetn1 gene, which are required for pericentriolar material (PCM) organization and the centriole cartwheel, are higher in 3nDTCC than in 2nRCC, and the expression levels of rootletin gene and nek2 gene, which are required for flagellum formation and linking the head to the tail, are lower in 3nDTCC than in 2nRCC. As the unnormal expression of centriole proteins, including up adjustment and down adjustment, directly affected recruiting the related proteins, which combined to affect the organization of the structure of PCM and linker (Watanabe et al., 2019; Avidor-Reiss et al., 2020), with both the PCM and linker being the important components of the midpiece, we conclude that the differential expression of centriole-related genes of 3nDTCC must be related to the malformation phenomenon in the midpiece of spermatozoa. The differential expression of centriole-related genes covering the molecular mechanisms needs further investigation.

As the midpiece has important functions in connecting the head to the flagellum (centriolar complex) and providing energy for sperm movement of the spermatozoon (mitochondria), and it has an influence on the movement and structure of the flagellum (Psenicka et al., 2009), we speculate that the motility of spermatozoa in 3nDTCC was different from that in bisexual diploid fish. As the lack of acrosome is compensated by the presence of a micropyle in the egg for penetration of the spermatozoon in fishes (Psenicka et al., 2006), we speculate that whether the nucleus of the spermatozoon that can go into the egg would be related to the motility of spermatozoa tails, which might push the sperm nucleus go into the egg. Thus, the dynamics of sperm tails is an important point which is worth further concerning in fish breeding. The breeding test of all the crossings having a high fertilization rate (>80.0%), but low hatching rate (28.0–46.0%) and survival rate after 1 month (<12.0%), might partly attribute to low incubation water temperature (18–20°C), which has affected the late development of the gynogenesis embryos. It is also possible that poor quality sperm leads to the low survival of gynogenesis offspring.

The malformation phenomenon in the spermatozoa formation process of 3nDTCC might be an adapted feature for the propagating mode of gynogenesis, which make the 3nDTCC spermatozoa maintain the basic structure and movement ability, to reach the egg surface, and to initiate egg development, but not enough ability to break through the egg surface obstacles and to transfer the genetic material into the fertilized egg. Of course, the triploid egg also has its special shielding system, the breeding test of 3nDTCC(♀)×2nDTCC(♂) showed that the haploid genome of bisexual spermatozoa were also found to be not able to incorporate into the 3nDTCC genome. Further research is needed on how the triploid eggs work and how the initiation process achieves the introgression of paternal DNA.

The spermatozoa of gynogenesis fish only start the egg development in the reproduction process. Its adaptive structure related to spermatozoa vitality and spermatozoa entry is worthy of deep understanding. It is not only of great significance in the aspect of biological evolution research, but also provides important theoretical guidance for the inactivation process of artificial gynogenetic induction.